U.S. patent application number 12/843225 was filed with the patent office on 2011-04-14 for antibodies against il-13 receptor alpha 1 and uses thereof.
Invention is credited to Josef Endl, Maria Elena Fuentes, Yvo Graus, Adelbert Grossmann, Sebastian Neumann, Paul Parren, Frank Rebers, Joerg Thomas Regula, Ralf Schumacher, Stefan Seeber, Jan Olaf Stracke, Kay-Gunnar Stubenrauch, Jan Van De Winkel, Martine Van Vugt, Sandra Vereecken-Verploegen.
Application Number | 20110086026 12/843225 |
Document ID | / |
Family ID | 36199730 |
Filed Date | 2011-04-14 |
United States Patent
Application |
20110086026 |
Kind Code |
A1 |
Endl; Josef ; et
al. |
April 14, 2011 |
ANTIBODIES AGAINST IL-13 RECEPTOR ALPHA 1 AND USES THEREOF
Abstract
An antibody binding to IL-13R.alpha.1, inhibiting IL-13
bioactivity and comprising a variable heavy and a variable light
chain, characterized in that the variable heavy chain amino acid
sequence CDR3 of said antibody is selected from the group
consisting of the heavy chain CDR3 sequences of SEQ ID NO: 1, 3, 5,
7 or 9 is useful in the treatment of asthma and allergic
diseases.
Inventors: |
Endl; Josef; (Weilheim,
DE) ; Fuentes; Maria Elena; (Sunnyvale, CA) ;
Graus; Yvo; (Odijk, NL) ; Grossmann; Adelbert;
(Eglfing, DE) ; Neumann; Sebastian; (Weilheim,
DE) ; Parren; Paul; (Odijk, NL) ; Rebers;
Frank; (Utrecht, NL) ; Regula; Joerg Thomas;
(Muenchen, DE) ; Schumacher; Ralf; (Penzberg,
DE) ; Seeber; Stefan; (Penzberg, DE) ;
Stracke; Jan Olaf; (Penzberg, DE) ; Stubenrauch;
Kay-Gunnar; (Penzberg, DE) ; Van De Winkel; Jan;
(Zeist, NL) ; Van Vugt; Martine; (Houten, NL)
; Vereecken-Verploegen; Sandra; (Nieuwegein, NL) |
Family ID: |
36199730 |
Appl. No.: |
12/843225 |
Filed: |
July 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11325197 |
Jan 3, 2006 |
7807158 |
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12843225 |
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Current U.S.
Class: |
424/133.1 ;
435/243; 435/320.1; 435/334; 435/69.6; 530/389.1; 536/23.53 |
Current CPC
Class: |
A61P 37/02 20180101;
C07K 2317/21 20130101; C07K 2317/56 20130101; C07K 2317/76
20130101; A61P 37/00 20180101; A61P 37/08 20180101; A61P 11/06
20180101; C07K 16/2866 20130101 |
Class at
Publication: |
424/133.1 ;
536/23.53; 530/389.1; 435/334; 435/69.6; 435/320.1; 435/243 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/00 20060101 C07H021/00; C07K 16/00 20060101
C07K016/00; A61P 11/06 20060101 A61P011/06; A61P 37/02 20060101
A61P037/02; C12N 5/10 20060101 C12N005/10; C12P 21/02 20060101
C12P021/02; C12N 15/63 20060101 C12N015/63; C12N 1/00 20060101
C12N001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 3, 2005 |
EP |
EP05000003.3 |
Feb 3, 2005 |
EP |
EP05002229.2 |
Claims
1-10. (canceled)
11. A recombinant host cell capable that expresses an
anti-IL-13R.alpha.1 antibody, comprising variable heavy chain amino
acid sequences CDR1, CDR2, and CDR3, and variable light chain amino
acid sequences CDR1, CDR2 and CDR3, wherein said variable heavy
chain CDR1-3 sequences are selected from the group consisting of
the variable heavy chain amino acid sequence CDR1-3 of SEQ ID NO:
1, 3, 5, and 9 and wherein said variable light chain CDR1-3
sequences are selected from the group consisting of the variable
light chain amino acid sequence CDR1-3 of SEQ ID NO: 2, 4, 6 and
10; or wherein said variable heavy chain CDR1-3 sequences are
selected from the variable heavy chain amino acid sequence CDR1-3
of SEQ ID NO:7 and wherein said variable light chain CDR1-3
sequences are selected from the variable light chain amino acid
sequence CDR1-3 of SEQ ID NO:8.
12. A method for making said anti-IL-13R.alpha.1 antibody of claim
11, comprising culturing said recombinant host cell of claim
11.
13. A nucleic acid encoding a polypeptide selected from the group
consisting of heavy chain CDRs of SEQ ID NO:1, 3, 5, 7 and 9.
14. A nucleic acid encoding a polypeptide selected from the group
consisting of light chain CDRs of SEQ ID NO:2, 4, 6, 8 and 10
15. An expression vector comprising a nucleic acid to of claim 13,
capable of expressing said nucleic acid in a prokaryotic or
eukaryotic host cell.
16. An expression vector comprising a nucleic acid of claim 14,
capable of expressing said nucleic acid in a prokaryotic or
eukaryotic host cell.
17. An expression vector comprising a nucleic acid of claim 13 and
a nucleic acid of claim 14, capable of expressing said nucleic
acids in a prokaryotic or eukaryotic host cell.
18. A prokaryotic or eukaryotic host cell comprising the vector of
claim 17.
19. A method for producing a polypeptide capable of binding to
IL-13R.alpha.1 and inhibiting the binding of IL-13to
IL-13R.alpha.1, comprising: expressing a nucleic acid encoding a
heavy chain of claim 13 and a nucleic acid encoding a light chain
of claim 14 in a prokaryotic or eukaryotic host cell; and
recovering said polypeptide from said cell.
20. A method for treating a patient in need of an asthma or
antiallergic therapy, comprising: administering to the patient a
therapeutically effective amount of said antibody of claim 11.
21. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority from EP05000003.3 filed
Jan. 3, 2005 and EP05002229.2 filed Feb. 3, 2005, both incorporated
herein by reference in full.
FIELD OF THE INVENTION
[0002] This invention relates generally to human antibodies against
IL-13 receptor alpha1 (IL-13R.alpha. (alpha)1), methods for their
production, and uses.
BACKGROUND OF THE INVENTION
[0003] IL-13 is a secreted monomeric peptide produced mainly by Th2
cells but also by mast cells and NK cells. Biological functions of
IL-13 include regulation of IgE production and modulation of Th2
development. IL-13 binds to a receptor complex consisting of IL-13
receptor alpha1 (IL-13R.alpha.1) chain and IL-4 receptor alpha
(IL-4R.alpha.) chain. IL-13 binding triggers signal transduction
events mainly through STAT6. IL-13 binds with low affinity to the
IL-13R.alpha.1 alone and does not bind to IL-4R.alpha.1. Contrary
to this, IL-4 binds to IL-4R.alpha. alone and does not bind to
IL-13R.alpha.1 alone. Another receptor for IL-13 has been
described, the IL-13R.alpha.2. IL-13 binds with high affinity to
this receptor. Likely this receptor acts as a decoy receptor.
[0004] Inducible overexpression of IL-13 in transgenic mice results
in a phenotype that shares many characteristics with asthmatic
patients. They show mucus metaplasia, macrophage, lymphocyte and
eosinophil-rich inflammation, upregulation of proteases like MMP-9,
-12, -13, -2 and -14, cathepsin B, H, K and S and they also present
subepithelial fibrosis. Knockout mice for IL-13 show a significant
reduction in Th2 cytokine production due to impairment in Th2
development. These mice do not develop airway hyperreactivity (AHR)
in spite of the presence of eosinophil inflammation. The AHR was
restored by administration of IL-13, indicating that IL-13 is
necessary and sufficient for the induction of AHR in mouse. Other
important biological functions of IL-13 in relationship with asthma
include the induction of goblet cell metaplasia and mucus
production. It acts directly on airway epithelial cells,
fibroblasts and airway smooth muscle cells and induces different
transcriptional programs in each of this cell types. Interestingly,
IL-13 decreases the alpha-adrenergic response in smooth muscle
cells, contributing to airway narrowing. IL-13 promoter
polymorphism is associated with increased risk of allergic asthma.
Polymorphisms in the IL-13 gene are associated with high serum IgE
levels. Single nucleotide polymorphism in the intergene sequence
between the IL-4 and IL-13 genes is associated with atopic
asthma.
[0005] IL-13 antagonists have been utilized in animal models. For
example a soluble mouse IL-13R.alpha.2-IgGFc fusion protein has
been used to show efficacy in completely reversing
ovalbumin-induced AHR and the number of mucus containing cells. The
reversal was obtained even if the treatment is given after full
development of the phenotype. In addition, treatment of mice with
an IL-13 fusion cytotoxin molecule resulted in reduction of all
features of airway disease in a chronic fungal-induced allergic
inflammation. In conclusion, IL-13 is a critical mediator of the
effector arm of the allergic response.
[0006] IL-13R.alpha.1 is a member of the hemapoietin receptor
superfamily (type 1 cytokine receptor family) and identified and
described by Obiri N. I., et al., J. Biol. Chem., 270 (1995)
8797-8804) and WO 96/29417. It is a protein of 427 amino acids
including the signal sequence. Its DNA and protein sequences are
described in WO 97/15663 and SwissProt No. P78552. IL-13R.alpha.1
is a glycosylated protein binding to IL-13 with low affinity, but,
when linked with IL-4R.alpha. to a heterodimer, it binds IL-13 with
high affinity. This complex is also a receptor for IL-4.
[0007] Antibodies against IL-13R.alpha.1 are known from WO
96/29417, WO 97/15663, WO 03/080675, Graber P., et al., Eur. J.
Immunol., 28 (1998) 4286-4298; Poudrier J., et al., J. Immunol.,
163 (1999) 1153-1161; Poudrier J., et al., Eur. J. Immunol., 30
(2000) 3157-3164; Aikawa M., et al., Cytokine, 13 (2001) 75-84.
Antibodies against IL-13R.alpha.1 are commercially available from
R&D Systems Inc. USA.
SUMMARY OF THE INVENTION
[0008] The invention comprises an antibody binding to
IL-13R.alpha.1 and inhibiting IL-13 bioactivity, characterized in
that the variable heavy chain amino acid sequence CDR3 of said
antibody is selected from the group consisting of the heavy chain
CDR3 sequences of SEQ ID NO: 1, 3, 5, 7 or 9.
[0009] The antibody is preferably a human antibody.
[0010] The antibody is preferably characterized by an affinity of
10.sup.-9 M (K.sub.D) or less, preferably of 10.sup.-9 to
10.sup.-13 M for binding to IL-13R.alpha.1.
[0011] Preferably the antibody is characterized in that its heavy
chain CDR1, CDR2 and CDR3 sequences are selected from the group
consisting of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ
ID NO: 1, 3, 5, 7 or 9.
[0012] The antibody is preferably characterized in that the
variable light chain amino acid sequences CDR1, CDR2 and CDR3 of
said antibody are selected from the group consisting of the light
chain CDR sequences of SEQ ID NO: 2, 4, 6, 8 or 10.
[0013] The antibody is preferably characterized in that the
variable heavy chain amino acid sequences CDR1, CDR2 and CDR3 of
said antibody are selected from the group consisting of the heavy
chain CDR sequences of SEQ ID NO: 1, 3, 5, 7 or 9 and the variable
light chain amino acid sequences CDR1, CDR2 and CDR3, of said
antibody are selected from the group consisting of the light chain
CDR sequences of SEQ ID NO: 2, 4, 6, 8 or 10.
[0014] The CDR sequences are preferably selected independently of
each other and are separated by FR (framework) regions.
[0015] The antibody is preferably characterized in comprising as
heavy chain CDRs the CDRs of SEQ ID NO: 1 and as light chain CDRs
the CDRs of SEQ ID NO: 2, as heavy chain CDRs the CDRs of SEQ ID
NO: 3 and as light chain CDRs the CDRs of SEQ ID NO: 4, as heavy
chain CDRs the CDRs of SEQ ID NO: 5 and as light chain CDRs the
CDRs of SEQ ID NO: 6, as heavy chain CDRs the CDRs of SEQ ID NO: 7
and as light chain CDRs the CDRs of SEQ ID NO: 8 or as heavy chain
CDRs the CDRs of SEQ ID NO: 9 and as light chain CDRs the CDRs of
SEQ ID NO: 10.
[0016] The CDR sequences can be determined according to the
standard definition of Kabat et al., Sequences of Proteins of
Immunological Interest, 5th ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). On this basis, the
complementarity determining regions (CDRs) of SEQ ID NO: 1-8 have
the following sequences:
[0017] Heavy chain CDRs: CDR1 (aa 31-35) of SEQ ID NO: 1, 3, 5, 7,
9, CDR2 (aa 50-66) of SEQ ID NO: 1, 3, 5, 7, 9, CDR3 (aa 99-108) of
SEQ ID NO: 1, 3, 9, CDR3 (aa 99-107) of SEQ ID NO: 5, CDR3 (aa
99-112) of SEQ ID NO: 7;
[0018] Light chain CDRs: CDR1 (aa 24-34) of SEQ ID NO: 2, 4, 6, 10,
CDR1 (aa 24-35) of SEQ ID NO: 8, CDR2 (aa 50-56) of SEQ ID NO: 2,
4, 6, 10, CDR2 (aa 51-57) of SEQ ID NO:8 and CDR3 (aa 89-97) of SEQ
ID NO: 2, 4, 6, 10, CDR3 (aa 90-97) of SEQ ID NO: 8.
[0019] Preferably, the invention provides an antibody comprising as
complementarity determining regions (CDRs) the following sequences:
[0020] a) an antibody heavy chain comprising heavy chain CDRs of
SEQ ID NO:1, 3, 5, 7 or 9; [0021] b) an antibody light chain
comprising light chain CDRs of SEQ ID NO:2, 4, 6, 8 or 10, wherein
the CDRs are selected independently of each other.
[0022] The antibody is preferably characterized in comprising as
heavy chain variable region SEQ ID NO: 1 and as light chain
variable region SEQ ID NO: 2, as heavy chain variable region SEQ ID
NO: 3 and as light chain variable region of SEQ ID NO: 4, as heavy
chain variable region SEQ ID NO: 5 and as light chain variable
region SEQ ID NO: 6, as heavy chain variable region SEQ ID NO: 7
and as light chain variable region SEQ ID NO: 8 or as heavy
variable region SEQ ID NO: 9 and as light chain variable region SEQ
ID NO: 10.
[0023] The antibody is preferably characterized in comprising
[0024] a) as heavy chain variable region SEQ ID NO: 1, as light
chain variable region SEQ ID NO: 2, as .kappa. light chain constant
region SEQ ID NO: .gamma.1 and as .gamma.1 heavy chain constant
region SEQ ID NO: 12 optionally with mutations L234A and L235A or
D265A and N297A,
[0025] b) as heavy chain variable region SEQ ID NO: 3 and as light
chain variable region of SEQ ID NO: 4, as .kappa. light chain
constant region SEQ ID NO: .gamma.1 and as .gamma.1 heavy chain
constant region SEQ ID NO: 12 optionally with mutations L234A and
L235A or D265A and N297A,
[0026] c) as heavy chain variable region SEQ ID NO: 5 and as light
chain variable region SEQ ID NO: 6, as .kappa. light chain constant
region SEQ ID NO: .gamma.1 and as .gamma.1 heavy chain constant
region SEQ ID NO: 12 optionally with mutations L234A and L235A or
D265A and N297A,
[0027] d) as heavy chain variable region SEQ ID NO: 7 and as light
chain variable region SEQ ID NO: 8, as .kappa. light chain constant
region SEQ ID NO: 11 and as .gamma.1 heavy chain constant region
SEQ ID NO: 12 optionally with mutations L234A and L235A or D265A
and N297A, or
[0028] e) as heavy variable region SEQ ID NO: 9 and as light chain
variable region SEQ ID NO: 10, as .kappa. light chain constant
region SEQ ID NO: 11 and as .gamma.1 heavy chain constant region
SEQ ID NO: 12 optionally with mutations L234A and L235A or D265A
and N297A.
[0029] Preferably the antibody is characterized in binding to
IL-13R.alpha.1 in competition to antibody LC5002-002, LC5002-003,
LC5002-005, LC5002-007 and/or LC5002-018.
[0030] Preferably the antibody is characterized in comprising as
variable regions the variable regions of LC5002-002, LC5002-003,
LC5002-005, LC5002-007 or LC5002-018. The variable regions of these
antibodies are shown in SEQ ID NO: 1-10. Useful constant regions
are well known in the state of the art. Examples are shown in SEQ
ID NO: 11-12.
[0031] The antibody is preferably a monoclonal or a recombinantly
produced antibody.
[0032] In one embodiment of the invention the antibody is a
class-altered human antibody.
[0033] In a preferred embodiment of the invention the antibody
contains a human .gamma.1 heavy chain comprising [0034] a) amino
acid sequence Pro.sub.233Val.sub.234Ala.sub.235 with deletion of
Gly.sub.236 and/or amino acid sequence
Gly.sub.327Leu.sub.328Pro.sub.329Ser.sub.330Ser.sub.331, [0035] b)
amino acid sequence Ala.sub.234Ala.sub.235 or [0036] c) amino acids
Ala.sub.265 and Ala.sub.297.
[0037] Preferably the antibody according to the invention inhibits
IL-13 induced Stat-6 phosphorylation with an IC.sub.50 value of 6
nM or lower, inhibits IL-13 induced eotaxin production with an
IC.sub.50 value of 20 nM or lower and/or inhibits IL-13 or IL-4
induced cell proliferation, preferably of TF-1 cells (ATCC CRL
2003) with an IC.sub.50 value of 10 nM or lower (IL-13) and 60 nM
or lower (IL-4). IL-13 induced Stat-6 phosphorylation, eotaxin
production and induction of cell proliferation are determined
according to examples 6 to 8.
[0038] The antibody according to the invention preferably does not
bind to denatured IL-13R.alpha.1(K.sub.D for binding affinity
10.sup.-6M or higher). The antibody is preferably characterized by
showing substantially no crossreactivity with IL-13R.alpha.2 and
IL-4R.alpha. (K.sub.D for binding affinity 10.sup.-6M or
higher).
[0039] The invention further provides hybridoma cell lines which
produce antagonistic monoclonal antibodies against
IL-13R.alpha.1.
[0040] The preferred hybridoma cell lines according to the
invention (hu-MAB<h-IL-13R alpha>LC.5002-002 (DSM ACC2709),
hu-MAB<h-IL-13Ralpha>LC.5002-003 (DSM ACC2710),
hu-MAB<h-IL-13Ralpha>LC.5002-005 (DSM ACC2711),
hu-MAB<h-IL-13R alpha>LC.5002-007 (DSM ACC2712)) were
deposited Jan. 13, 2005 with Deutsche Sammlung von Mikroorganismen
and Zellkulturen GmbH (DSMZ), Germany.
[0041] The antibodies obtainable from said cell lines are
embodiments of the invention.
[0042] The invention further provides nucleic acids encoding
polypeptides of which antibodies according to the invention are
comprised, expression vectors comprising said nucleic acids, and
host cells for the recombinant production of such antibodies. The
invention further provides methods for the recombinant production
of such antibodies.
[0043] The polypeptides encoded by the nucleic acids according to
the invention are [0044] a) an antibody heavy chain comprising
heavy chain CDRs of SEQ ID NO: NO:1, 3, 5, 7 or 9 and [0045] b) an
antibody light chain comprising light chain CDRs of SEQ ID NO: 2,
4, 6, 8 or 10.
[0046] These polypeptides are capable of assembling together with
the respective other antibody chain to generate an antibody.
[0047] Antibodies according to the invention show benefits for
patients in need of corticosteroid therapy. The antibodies
according to the invention have new and inventive properties
causing a benefit for a patient suffering from asthma or an
allergic disease.
[0048] The invention further provides methods for treating asthma
and allergic diseases.
[0049] The invention further comprises the use of an antibody
according to the invention for asthma treatment and for the
manufacture of a pharmaceutical composition according to the
invention. In addition, the invention comprises a method for the
manufacture of a pharmaceutical composition according to the
invention.
[0050] The invention further comprises a pharmaceutical composition
comprising an antibody according to the invention with a
pharmaceutically effective amount, optionally together with a
buffer and/or an adjuvant useful for the formulation of antibodies
for pharmaceutical purposes.
[0051] The invention further provides pharmaceutical compositions
comprising such antibodies in a pharmaceutically acceptable
carrier. In one embodiment, the pharmaceutical composition may be
included in an article of manufacture or kit.
[0052] The invention further comprises a vector comprising a
nucleic acid according to the invention, capable of expressing said
nucleic acid in a prokaryotic or eukaryotic host cell.
[0053] The invention further comprises a prokaryotic or eukaryotic
host cell comprising a vector according to the invention.
[0054] The invention further comprises a method for the production
of a recombinant human antibody according to the invention,
characterized by expressing a nucleic acid according to the
invention in a prokaryotic or eukaryotic host cell and recovering
said antibody from said cell. The invention further comprises the
antibody obtainable by such a recombinant method.
[0055] The invention further comprises a method for the preparation
of a pharmaceutical composition characterized in selecting an
antibody against IL-13R.alpha.1 from a plurality of antibodies
against IL-13R.alpha.1 when compared to such an assay without said
antibody, producing said antibody by means of recombinant
expression, recovering said antibody and combining said antibody
with a pharmaceutical acceptable buffer and/or adjuvant. Preferably
the antibody has one or more of the above mentioned additional
properties.
DETAILED DESCRIPTION OF THE INVENTION
[0056] The terms "IL-13R.alpha.1, murine IL-13R.alpha.1, IL-13,
IL-13R.alpha.2 and IL-4R.alpha." and their domains are well known
in the state of the art and e.g. defined by SwissProt P78552,
O09030, P35225, Q14627 and P24394. If not otherwise stated, the
terms "IL-13R.alpha.1, IL-13, IL-13R.alpha.2 and IL-4R.alpha."
therefore denotes the human polypeptides IL-13R.alpha.1, IL-13,
IL-13R.alpha.2 and IL-4R.alpha..
[0057] The term "human antibody", as used herein, includes
antibodies having variable and constant regions (domains) which can
be assigned to defined human germ line immunoglobulin sequences
because of their high sequence similarity or identity with these
germ line sequences. Human antibodies are well-known in the state
of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.
Chem. Biol. 5 (2001) 368-374). Human antibodies can also be
produced in transgenic animals (e.g. mice) that are capable, upon
immunization, of producing a full repertoire or a selection of
human antibodies in the absence of endogenous immunoglobulin
production. Transfer of the human germ-line immunoglobulin gene
array in such germ-line mutant mice results in the production of
human antibodies upon antigen challenge (see, e.g., Jakobovits, A.,
et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits,
A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year
Immunol. 7 (1993) 33-40). Human antibodies can also be produced in
phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol.
Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol. 222
(1991) 581-597). The techniques of Cole et al. and Boerner et al.
are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol.
147 (1991) 86-95). A human antibody encompasses the various forms
of antibodies, preferably monoclonal antibodies including but not
being limited to whole antibodies, antibody fragments,
class-altered antibodies and genetically engineered antibodies
(variant or mutant antibodies) as long as the characteristic
properties according to the invention are retained. Especially
preferred are recombinant human antibodies. The term "monoclonal
antibody" as used herein refers to a preparation of antibody
molecules all having substantially the same amino acid
sequence.
[0058] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies isolated from a host cell such as a NS0 or CHO cell or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into such a host cell. Such
recombinant human antibodies have variable and constant regions in
a rearranged form. The recombinant human antibodies according to
the invention have been subjected to in vivo somatic hypermutation.
Thus, the amino acid sequences of the VH and VL regions of the
recombinant antibodies are sequences that can be assigned to
defined human germ line VH and VL sequences, but may not naturally
exist within the human antibody germ line repertoire in vivo.
[0059] The term "class-altered antibody" refers to a monoclonal
antibody, preferably a human antibody, comprising a variable
region, i.e., binding region, from one source or germ line and at
least a portion of a constant region that matches a constant region
of an antibody from a different source or germ line, usually
prepared by recombinant DNA techniques. Such class-altered
antibodies are not naturally occurring and therefore not available
directly from xenograft mice. Forms of "class-altered antibodies"
encompassed by the present invention are those in which the
constant region has differences from the wild-type constant region
sequence that result in an antibody having different properties
according to the invention, especially in regard to C1q binding
and/or Fc receptor (FcR) binding, i.e. by change or mutation of Fc.
Class-altered antibodies are the product of expressed
immunoglobulin genes comprising DNA segments encoding
immunoglobulin variable regions and DNA segments encoding
immunoglobulin constant regions. Methods for producing
class-altered antibodies involve conventional recombinant DNA and
gene transfection techniques are well known in the art (see, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244).
[0060] The "variable region" (variable region of a light chain
(VL), variable region of a heavy chain (VH)) as used herein denotes
the part of each of the pair of light and heavy chains which is
involved directly in binding the antibody to the antigen. The
domains of variable human light and heavy chains have the same
general structure and each domain comprises four framework (FR)
regions whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a .beta.(beta)-sheet
conformation and the CDRs may form loops connecting the
.beta.-sheet structure. The CDRs in each chain are held in their
three-dimensional structure by the framework regions and form
together with the CDRs from the other chain the antigen binding
site. The antibody heavy and light chain CDR3 regions, preferably
the heavy chain CDR3, play a particularly important role in the
binding specificity/affinity of the antibodies according to the
invention and therefore provide a further object of the
invention.
[0061] The terms "hypervariable region" or "antigen-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for antigen-binding. The
hypervariable region comprises amino acid residues from the
"complementarity determining regions" or "CDRs". "Framework" or
"FR" regions are those variable domain regions other than the
hypervariable region residues as herein defined. Therefore, the
light and heavy chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
CDR and FR regions are determined according to the standard
definition of Kabat et al., Sequences of Proteins of Immunological
Interest, 5th ed., Public Health Service, National Institutes of
Health, Bethesda, Md. (1991).
[0062] The "constant domains" are not involved directly in binding
of an antibody to an antigen, but exhibit various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins are
divided into the classes: IgA, IgD, IgE, IgG and IgM, and several
of these may be further divided into subclasses (isotypes), e.g.
IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavy chain constant
regions that correspond to the different classes of immunoglobulins
are called .mu., .delta., .gamma., .alpha., and .epsilon.,
respectively. The antibodies according to the invention are
preferably of IgG1 type.
[0063] The Fc part of an antibody is directly involved in
complement activation, C1q binding, C3 activation and Fc receptor
binding. Binding to C1q is caused by defined binding sites in the
Fc part. Such binding sites are known in the state of the art and
described e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)
2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979)
907-917; Burton, D. R., et al., Nature 288 (1980) 338-344;
Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004;
Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh,
M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al.,
Immunology 86 (1995) 319-324; and EP 0 307 434. Such binding sites
are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329
(numbering according to EU index of Kabat, see below). Antibodies
of subclass IgG1, IgG2 and IgG3 usually show complement activation,
C1q binding and C3 activation, whereas IgG4 antibodies do not
activate the complement system, do not bind C1q and do not activate
C3. As used herein the term "Fc part derived from human origin"
denotes a Fc part which preferably has an amino acid sequence of a
Fc part of a human antibody of the subclass IgG1 modified in such a
way that no C1q binding, C3 activation and/or FcR binding can be
detected or binding is at least reduced for 50%, preferably 70%,
compared to a human IgG1 antibody. An "Fc part of an antibody" is a
term well known to the skilled artisan and defined on the basis of
papain cleavage of antibodies. The antibodies according to the
invention contain as Fc part, preferably with an amino acid
sequence of a Fc part derived from human origin and preferably all
other parts of the human constant regions. Preferably the Fc part
is a mutated human Fc part from human IgG1 subclass. Mostly
preferred are Fc parts comprising a .gamma.1-heavy chain constant
region (an example is shown in SEQ ID NO: 11) with mutations L234A
and L235A or D265A and N297A (WO99/51642).
[0064] Human constant chains, e.g. .gamma.1-heavy chains are
described in detail by Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), and by Bruggemann, M.,
et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T. W., et al.,
Methods Enzymol. 178 (1989) 515-527. The constant domains preferred
in the invention provide no complement binding. The "variable
region" (variable region of a light chain (VL), variable region of
a heavy chain (VH)) as used herein denotes each of the pair of
light and heavy chains which is involved directly in binding the
antibody to the antigen.
[0065] The term nucleic acid or nucleic acid molecule, as used
herein, is intended to include DNA molecules and RNA molecules. A
nucleic acid molecule may be single-stranded or double-stranded,
but preferably is double-stranded DNA.
[0066] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are cis, and, in the case of a secretory
leader, contiguous and in reading frame. However, enhancers do not
have to be contiguous Linking is accomplished by ligation at
convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in
accordance with conventional practice.
[0067] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Variant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included. Where
distinct designations are intended, it will be clear from the
context.
[0068] The term "binding to IL-13R.alpha.1" as used herein means
the binding of the antibody to IL-13R.alpha.1 in an in vitro assay,
preferably in a binding assay in which the antibody is bound to a
surface and binding of IL-13R.alpha.1 is measured by Surface
Plasmon Resonance (SPR). Binding means a binding affinity (K.sub.D)
of 10.sup.-8 M or less, preferably 10.sup.-13 to 10.sup.-9 M. "No
binding" means a K.sub.D of 10.sup.-6 M or more. The antibodies
according to the invention bind to the extracellular domain of
human IL-13R.alpha.1 and preferably also of mouse
IL-13R.alpha.1.
[0069] Binding to IL-13R.alpha.1 can be investigated by a BIAcore
assay (Pharmacia Biosensor AB, Uppsala, Sweden). The affinity of
the binding is defined by the terms ka (rate constant for the
association of the antibody from the antibody/antigen complex), kd
(dissociation rate), and K.sub.D (kd/ka).
[0070] The binding of IL-13 to IL-13R.alpha.1 is inhibited by the
antibodies according to the invention. The inhibition is measured
as IC.sub.50 in an ELISA for binding of IL-13 to
IL-13R.alpha.1/IL-4R.alpha. heterodimer. For performing such an
assay IL-13R.alpha.1 is immobilized and IL-13 and IL-4R.alpha. are
added. The IC.sub.50 values of the antibodies according to the
invention for the binding of IL-13 to IL-3R.alpha.1 are no more
than 6 nM. IC.sub.50 values are measured as average or median
values of at least three independent measurements. Single IC.sub.50
values may be out of the scope.
[0071] The antibodies according to the invention show preferably a
binding to the same epitopes of IL-13R.alpha.1 as an antibody
selected from the group consisting of antibodies LC5002-002,
LC5002-003, LC5002-005, LC5002-007 or LC5002-018 or are inhibited
in binding to IL-13R.alpha.1 due to steric hindrance of binding by
these antibodies. Binding inhibition can be detected by an SPR
assay using an immobilized antibody selected from the group
consisting of antibodies LC5002-002, LC5002-003, LC5002-005,
LC5002-007 or LC5002-018 and IL-13R.alpha.1 at a concentration of
20-50 nM and the antibody to be detected at a concentration of 100
nM. A signal reduction of 50% or more shows that the antibody
competes with an antibody selected from the group consisting of
antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007 or
LC5002-018. The term "epitope" means a protien determinant capable
of specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents. The invention
comprises also a human antibody binding to IL-13R.alpha.1 and
inhibiting IL-13 bioactivity, characterized by an affinity of
10.sup.-9 M (K.sub.D) or less, preferably of 10.sup.-9 to
10.sup.-13 M for binding to IL-13R.alpha.1 and by an affinity of
10.sup.-7 M (K.sub.D) or less, preferably of 10.sup.-8 to
10.sup.-9M for binding to murine IL-13R.alpha.1.
[0072] In a preferred embodiment of the invention, the antibodies
according to the invention are further characterized by one or more
of the characteristics selected from the group selected from the
binding parameters ka, kd and K.sub.D, binding to the same epitope
to which an antibody selected from the group consisting of
antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007 or
LC5002-018 binds.
[0073] The antibodies according to the invention are preferably
produced by recombinant means. Such methods are widely known in the
state of the art and comprise protein expression in prokaryotic and
eukaryotic cells with subsequent isolation of the antibody
polypeptide and usually purification to a pharmaceutically
acceptable purity. For the protein expression, nucleic acids
encoding light and heavy chains or fragments thereof are inserted
into expression vectors by standard methods. Expression is
performed in appropriate prokaryotic or eukaryotic host cells like
CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, yeast,
or E. coli cells, and the antibody is recovered from the cells
(supernatant or cells after lysis).
[0074] Recombinant production of antibodies is well-known in the
state of the art and described, for example, in the review articles
of Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202; Geisse,
S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J.,
Mol. Biotechnol. 16 (2000) 151-161; Werner, R. G., Drug Res. 48
(1998) 870-880.
[0075] The antibodies may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form.
Purification is performed in order to eliminate other cellular
components or other contaminants, e.g. other cellular nucleic acids
or proteins, by standard techniques, including alkaline/SDS
treatment, CsC1 banding, column chromatography, agarose gel
electrophoresis, and others well known in the art. See Ausubel, F.,
et al., ed. Current Protocols in Molecular Biology, Greene
Publishing and Wiley Interscience, New York (1987).
[0076] Expression in NS0 cells is described by, e.g., Barnes, L.
M., et al., Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et
al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is
described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30
(2002) E9. Cloning of variable domains is described by Orlandi, R.,
et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P.,
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and
Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A
preferred transient expression system (HEK 293) is described by
Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999)
71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)
191-199.
[0077] The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, enhancers and polyadenylation signals.
[0078] The monoclonal antibodies are suitably separated from the
culture medium by conventional immunoglobulin purification
procedures such as, for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography. DNA and RNA encoding the monoclonal
antibodies is readily isolated and sequenced using conventional
procedures. The hybridoma cells can serve as a source of such DNA
and RNA. Once isolated, the DNA may be inserted into expression
vectors, which are then transfected into host cells such as CHO
cells, HEK 293 cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, to obtain the synthesis of
recombinant monoclonal antibodies in the host cells.
[0079] The antibodies according to the invention include, in
addition, such antibodies having "conservative sequence
modifications", nucleotide and amino acid sequence modifications
which do not affect or alter the above-mentioned characteristics of
the antibody according to the invention. Modifications can be
introduced by standard techniques known in the art, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions include ones in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g. glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a human
anti-IL-13R.alpha.1 antibody can be preferably replaced with
another amino acid residue from the same side chain family.
[0080] Amino acid substitutions can be performed by mutagenesis
based upon molecular modeling as described by Riechmann, L., et
al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl.
Acad. Sci. USA 86 (1989)10029-10033.
[0081] Amino acid sequence variants of human IL-13R.alpha.1
antibody are prepared by introducing appropriate nucleotide changes
into the antibody DNA, or by peptide synthesis. Such modifications
can be performed, however, only in a very limited range, e.g. as
described above. For example, the modifications do not alter the
abovementioned antibody characteristics such as the IgG isotype and
epitope binding, but may improve the yield of the recombinant
production, protein stability or facilitate the purification.
[0082] Any cysteine residue not involved in maintaining the proper
conformation of the anti-IL-13R.alpha.1 antibody also may be
substituted, generally with serine, to improve the oxidative
stability of the molecule and prevent aberrant crosslinking
Conversely, cysteine bond(s) may be added to the antibody to
improve its stability (particularly where the antibody is an
antibody fragment such as an Fv fragment).
[0083] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody. Glycosylation of antibodies is
typically N-linked. N-linked refers to the attachment of the
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. Addition of glycosylation
sites to the antibody is conveniently accomplished by altering the
amino acid sequence such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation
sites).
[0084] Nucleic acid molecules encoding amino acid sequence variants
of anti-IL-13R.alpha.1 antibody are prepared by a variety of
methods known in the art. These methods include, but are not
limited to, isolation from a natural source (in the case of
naturally occurring amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of anti-IL-13R.alpha.1
antibody.
[0085] Another type of covalent modification involves chemically or
enzymatically coupling glycosides to the antibody. These procedures
are advantageous in that they do not require production of the
antibody in a host cell that has glycosylation capabilities for N-
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 sulfhydryl groups such as those of
cysteine, (d) free hydroxyl groups such as those of serine,
threonine, or hydroxyproline, (e) aromatic residues such as those
of phenylalanine, tyrosine, or tryptophan, or (f) the amide group
of glutamine. These methods are described in WO 87/05330, and in
Aplin, J. D., and Wriston, J. C. Jr., CRC Crit. Rev. Biochem.
(1981) 259-306.
[0086] Removal of any carbohydrate moieties present on the antibody
may be accomplished chemically or enzymatically. Chemical
deglycosylation can be accomplished by exposing the antibody to
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the antibody intact. Chemical deglycosylation is described
by Sojahr, H. T., and Bahl, O. P., Arch. Biochem. Biophys. 259
(1987) 52-57 and by Edge, A. S., et al. Anal. Biochem. 118 (1981)
131-137. Enzymatic cleavage of carbohydrate moieties on antibodies
can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura, N. R., and Bahl, O. P.,
Meth. Enzymol. 138 (1987) 350-359.
[0087] 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 polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0088] In yet another aspect, the invention provides isolated
B-cells from a transgenic non-human animal, e.g. a transgenic
mouse, which express the human anti-IL-13R.alpha.1 antibodies
according to the invention. Preferably, the isolated B cells are
obtained from a transgenic non-human animal, e.g., a transgenic
mouse, which has been immunized with a purified or enriched
preparation of IL-13R.alpha.1 antigen and/or cells expressing
IL-13R.alpha.1. Preferably, the transgenic non-human animal, e.g. a
transgenic mouse, has a genome comprising a human heavy chain
transgene and a human light chain transgene encoding all or a
portion of an antibody of the invention. The isolated B-cells are
then immortalized to provide a source (e.g. a hybridoma) of human
anti-IL-13R.alpha.1 antibodies. Accordingly, the present invention
also provides a hybridoma capable of producing human monoclonal
antibodies according to the invention. In one embodiment, the
hybridoma includes a B cell obtained from a transgenic non-human
animal, e.g., a transgenic mouse having a genome comprising a human
heavy chain transgene and a human light chain transgene encoding
all or a portion of an antibody of the invention, fused to an
immortalized cell.
[0089] In a particular embodiment, the transgenic non-human animal
is a transgenic mouse having a genome comprising a human heavy
chain transgene and a human light chain transgene encoding all or a
portion of an antibody of the invention. The transgenic non-human
animal can be immunized with a purified or enriched preparation of
IL-13R.alpha.1 antigen and/or cells expressing IL-13R.alpha.1.
Preferably, the transgenic non-human animal, e.g. the transgenic
mouse, is capable of producing IgG1 isotypes of human monoclonal
antibodies to IL-13R.alpha.1.
[0090] The human monoclonal antibodies according to the invention
can be produced by immunizing a transgenic non-human animal, e.g. a
transgenic mouse, having a genome comprising a human heavy chain
transgene and a human light chain transgene encoding all or a
portion of an antibody of the invention, with a purified or
enriched preparation of IL-13R.alpha.1 antigen and/or cells
expressing IL-13R.alpha.1. B cells (e.g. splenic B cells) of the
animal are then obtained and fused with myeloma cells to form
immortal, hybridoma cells that secrete human monoclonal antibodies
against IL-13R.alpha.1.
[0091] In a preferred embodiment, human monoclonal antibodies
directed against IL-13R.alpha.1 can be generated using transgenic
mice carrying parts of the human immune system rather than the
mouse system. These transgenic mice, referred to herein as "HuMab"
mice, contain a human immunoglobulin gene miniloci that encodes
unrearranged human immunoglobulin genes which include the heavy
(.mu. and .gamma.) and .kappa. light chain (constant region genes),
together with targeted mutations that inactivate the endogenous
.mu. and .kappa. chain loci (Lonberg, N., et al., Nature 368 (1994)
856-859). Accordingly, the mice exhibit reduced expression of mouse
IgM or K, and in response to immunization, the introduced human
heavy and light chain transgenes undergo class switching and
somatic mutation to generate high affinity human IgG monoclonal
antibodies (Lonberg, N., et al., Nature 368 (1994) 856-859;
reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113
(1994) 49-101; Lonberg, N., and Huszar, D., Intern. Rev. Immunol.
25 (1995) 65-93; and Harding, F., and Lonberg, N., Ann. N. Acad.
Sci 764 (1995) 536-546). The preparation of HuMab mice is described
in Taylor, L., et al., Nucleic Acids Res 20 (1992) 6287-6295; Chen,
J., et al., Int'l Immunol 5 (1993) 647-656; Tuaillon, N., et al.,
Proc. Natl. Acad. Sci USA 90 (1993) 3720-3724; Choi, T. K., et al.,
Nature Genetics 4 (1993) 117-123; Chen, J., et al., EMBO J. 12
(1993) 821-830; Tuaillon, N., et al., Immunol. 152 (1994)
2912-2920; Lonberg, N., et al., Nature 368 (1994) 856-859; Lonberg,
N., Handbook of Experimental Pharmacology 113 (1994) 49-101;
Taylor, L., et al., Int. Immunol. 6 (1994) 579-591; Lonberg, N.,
and Huszar, D., Intern. Rev. Immunol. 25 (1995) 65-93; Harding, F.,
and Lonberg, N., Ann. N. Acad. Sci 764 (1995) 536-546; Fishwild, D.
M., et al., Nat. Biotechnol. 14 (1996) 845-851, the contents of all
of which are hereby incorporated by reference in their entirety.
See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299;
5,545,807; 5,770,429; WO 98/24884; WO 94/25585; WO 93/1227; WO
92/22645; and WO 92/03918.
[0092] To generate fully human monoclonal antibodies to
IL-13R.alpha.1, HuMab mice can be immunized with a purified or
enriched preparation of IL-13R.alpha.1 antigen and/or cells
expressing IL-13R.alpha.1 in accordance with the general method, as
described by Lonberg, N., et al., Nature 368 (1994) 856-859;
Fishwild, D. M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO
98/24884. Preferably, the mice will be 6-16 weeks of age upon the
first immunization. For example, a purified or enriched preparation
of soluble IL-13R.alpha.1 antigen (e.g. purified from
IL-13R.alpha.1-expressing cells) can be used to immunize the HuMab
mice intraperitoneally. In the event that immunizations using a
purified or enriched preparation of IL-13R.alpha.1 antigen do not
result in antibodies, mice can also be immunized with cells
expressing IL-13R.alpha.1, e.g., a tumor cell line, to promote
immune responses. Cumulative experience with various antigens has
shown that the HuMab transgenic mice respond best when initially
immunized intraperitoneally (i.p.) with antigen in complete
Freund's adjuvant, followed by every other week alternatingly i.p.
or s.c. immunizations (for example, up to a total of 6) with
antigen in incomplete Freund's adjuvant. The immune response can be
monitored over the course of the immunization protocol with plasma
samples being obtained by retroorbital bleeds. The plasma can be
screened by ELISA, and mice with sufficient titers of
anti-IL-13R.alpha.1 human immunoglobulin can be used for
immortalization of corresponding B cells. Mice can be boosted
intravenously with antigen 3 to 4 days before sacrifice and removal
of the spleen and lymph nodes. It is expected that 2-3 fusions for
each antigen may need to be performed. Several mice will be
immunized for each antigen. For example, a total of five to twelve
HuMab mice of the HCo7 and HCo12 strains can be immunized.
[0093] The HCo7 mice have a JKD disruption in their endogenous
light chain (kappa) genes (as described in Chen, J., et al., EMBO
J. 12 (1993) 821-830), a CMD disruption in their endogenous heavy
chain genes (as described in Example 1 of WO 01/14424), a KCo5
human kappa light chain transgene (as described in Fishwild, D. M.,
et al., Nat. Biotechnol. 14 (1996) 845-851), and a HCo7 human heavy
chain transgene (as described in U.S. Pat. No. 5,770,429).
[0094] The HCo12 mice have a JKD disruption in their endogenous
light chain (kappa) genes (as described in Chen, J., et al., EMBO
J. 12 (1993) 821-830), a CMD disruption in their endogenous heavy
chain genes (as described in Example 1 of WO 01/14424), a KCo5
human kappa light chain transgene (as described in Fishwild, D. M.,
et al., Nat. Biotechnol. 14 (1996) 845-851), and a HCo12 human
heavy chain transgene (as described in Example 2 of WO
01/14424).
[0095] The mouse lymphocytes can be isolated and fused with a mouse
myeloma cell line using PEG based on standard protocols to generate
hybridomas. The resulting hybridomas are then screened for the
production of antigen-specific antibodies. For example, single cell
suspensions of splenic and lymph node-derived lymphocytes from
immunized mice are fused to one-sixth the number of SP 2/0
nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG.
Cells are plated at approximately 2.times.10.sup.5 in flat bottom
microtiter plate, followed by about two weeks incubation in
selective medium.
[0096] Individual wells are then screened by ELISA for human
anti-IL-13R.alpha.1 monoclonal IgM and IgG antibodies. Once
extensive hybridoma growth occurs, medium is analyzed, usually
after 10-14 days. The antibody secreting hybridomas are replated,
screened again, and if still positive for human IgG,
anti-IL-13R.alpha.1 monoclonal antibodies, can be subcloned at
least twice by limiting dilution. The stable subclones are then
cultured in vitro to produce antibody in tissue culture medium for
characterization.
[0097] Because CDR sequences are responsible for antibody-antigen
interactions, it is possible to express recombinant antibodies
according to the invention by constructing expression vectors that
include the CDR sequences according to the invention onto framework
sequences from a different human antibody (see, e.g., Riechmann,
L., et al., Nature 332 (1998) 323-327; Jones, P., et al., Nature
321 (1986) 522-525; and Queen, C., et al., Proc. Natl. Acad. See.
USA 86 (1989)10029-10033). Such framework sequences can be obtained
from public DNA databases that include germline human antibody gene
sequences. These germline sequences will differ from mature
antibody gene sequences because they will not include completely
assembled variable genes, which are formed by V(D)J joining during
B cell maturation. Germline gene sequences will also differ from
the sequences of a high affinity secondary repertoire antibody at
individual evenly across the variable region.
[0098] The invention preferably comprises a nucleic acid fragment
encoding a polypeptide binding to IL-13R.alpha.1, whereby said
polypeptide inhibits the binding of IL-13to IL-13R.alpha.1,
selected from the group consisting of [0099] a) an antibody heavy
chain comprising heavy chain CDRs of SEQ ID NO: 1, 3, 5, 7 or 9;
[0100] b) an antibody light chain comprising light chain CDRs of
SEQ ID NO: 2, 4, 6, 8 or 10.
[0101] The reconstructed heavy and light chain variable regions are
combined with sequences of promoter, translation initiation,
constant region, 3' untranslated, polyadenylation, and
transcription termination to form expression vector constructs. The
heavy and light chain expression constructs can be combined into a
single vector, co-transfected, serially transfected, or separately
transfected into host cells which are then fused to form a single
host cell expressing both chains.
[0102] Accordingly, the invention provides a method for the
production of a recombinant human antibody according to the
invention, comprising expressing a nucleic acid encoding [0103] a)
an antibody heavy chain comprising heavy chain CDRs of SEQ ID NO:1,
3, 5, 7 or 9; [0104] b) an antibody light chain comprising light
chain CDRs of SEQ ID NO: 2, 4, 6, 8 or 10.
[0105] The invention further comprises the use of an antibody
according to the invention for the detection of IL-13R.alpha.1 in
vitro, preferably by an immunological assay determining the binding
between IL-13R.alpha.1 of a sample and the antibody according to
the invention.
[0106] In another aspect, the present invention provides a
composition, e.g. a pharmaceutical composition, comprising one or a
combination of human monoclonal antibodies, or the antigen-binding
portion thereof, of the present invention, formulated together with
a pharmaceutically acceptable carrier.
[0107] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.
by injection or infusion).
[0108] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the antibody and does
not impart any undesired toxicological effects (see e.g. Berge, S.
M., et al., J. Pharm. Sci. 66 (1977) 1-19). Such salts are included
in the invention. Examples of such salts include acid addition
salts and base addition salts. Acid addition salts include those
derived from nontoxic inorganic acids, such as hydrochloric
salts.
[0109] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results.
[0110] To administer a compound of the invention by certain routes
of administration, it may be necessary to coat the compound with,
or coadminister the compound with, a material to prevent its
inactivation. For example, the compound may be administered to a
subject in an appropriate carrier, for example, liposomes, or a
diluent. Pharmaceutically acceptable diluents include saline and
aqueous buffer solutions.
[0111] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art.
[0112] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0113] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0114] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0115] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0116] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier can be an isotonic buffered saline solution, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), and suitable mixtures
thereof.
[0117] Proper fluidity can be maintained, for example, by use of
coating such as lecithin, by maintenance of required particle size
in the case of dispersion and by use of surfactants. In many cases,
it is preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition. Long-term absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0118] The following examples, references, sequence listing and
figures are provided to aid the understanding of the present
invention, the true scope of which is set forth in the appended
claims. It is understood that modifications can be made in the
procedures set forth without departing from the spirit of the
invention.
DESCRIPTION OF THE SEQUENCE LISTING
[0119] SEQ ID NO:1 heavy chain variable domain of HuMab
LC5002-002
[0120] SEQ ID NO:2 light chain variable domain of HuMab
LC5002-002
[0121] SEQ ID NO:3 heavy chain variable domain of HuMab
LC5002-003
[0122] SEQ ID NO:4 light chain variable domain of HuMab
LC5002-003
[0123] SEQ ID NO:5 heavy chain variable domain of HuMab
LC5002-005
[0124] SEQ ID NO:6 light chain variable domain of HuMab
LC5002-005
[0125] SEQ ID NO:7 heavy chain variable domain of HuMab
LC5002-007
[0126] SEQ ID NO:8 light chain variable domain of HuMab
LC5002-007
[0127] SEQ ID NO:9 heavy chain variable domain of HuMab
LC5002-018
[0128] SEQ ID NO:10 light chain variable domain of HuMab
LC5002-018
[0129] SEQ ID NO:11 .kappa. light chain constant region
[0130] SEQ ID NO:12 .gamma.1 heavy chain constant region
DESCRIPTION OF THE FIGURES
[0131] FIG. 1 shows binding of anti-IL-13R.alpha.1 antibodies to
immobilized recombinant human IL-13R.alpha.1 polypeptide. Included
are polyclonal rabbit-anti-human IL-13R.alpha.1 antibody AF152
(R&D systems) and anti-KLH as a negative control HuMab.
[0132] FIG. 2 shows inhibition of IL-13 binding to immobilized
IL-13R.alpha.1/IL-4R.alpha. receptor by anti-IL-13R.alpha.1
antibodies.
[0133] FIG. 3 shows the blockade of IL-13 binding to CHO cells
(expressing IL-13Ralpha1 and IL-4R.alpha.2) by anti-IL-13R.alpha.1
antibodies. As a positive control, a commercially available
polyclonal anti-IL-13R.alpha.1 antibody (AF152, R&D Systems,
Minneapolis, Minn.) was included.
[0134] FIG. 4 shows binding of anti-IL-13R.alpha.1 antibodies to
hIL-13R.alpha.1 and binding properties to functionally related
receptors hIL-13R.alpha.2 and hIL-4R.alpha..
[0135] FIG. 5 shows the capacity of anti-IL-13R.alpha.1 antibodies
to bind to immobilized recombinant murine IL-13R.alpha.1
polypeptide. Included are polyclonal goat-anti-human IL-13R.alpha.1
antibody AF152 (R&D Systems) and anti-KLH as a negative control
HuMab.
EXAMPLES
Example 1
Generation of Hybridomas
[0136] The human monoclonal antibodies according to the invention
can be produced by immunizing a transgenic non-human animal, e. g.
a transgenic mouse having a genome comprising a human heavy chain
transgene and a human light chain transgene encoding all or a
portion of an antibody of the invention, with cells expressing
human IL-13R.alpha.1. B cells (e.g. splenic B cells) of the animal
are then obtained and fused with myeloma cells to form immortal,
hybridoma cells that secrete human monoclonal antibodies against
IL-13R.alpha.1. Human monoclonal antibodies directed against human
IL-13R.alpha.1 can be generated using transgenic mice carrying
parts of the human immune system rather than the mouse system.
These transgenic mice, referred to herein as "HuMab" mice, contain
a human immunoglobulin gene minilocus that encodes unrearranged
human immunoglobulin genes which include the heavy (.mu. and
.gamma.) and .kappa. (kappa) light chain (constant region genes),
together with targeted mutations that inactivate the endogenous
.mu. and kappa chain loci (Lonberg N., et al., Nature 368 (1994)
856-859). Accordingly, the mice exhibit reduced expression of mouse
IgM or .kappa., and in response to immunization, the introduced
human heavy and light chain transgenes undergo class switching and
somatic mutation to generate high affinity human IgG monoclonal
antibodies. To generate fully human monoclonal antibodies to human
IL-13R.alpha.1, HuMab mice can be immunized with cells expressing
human IL-13R.alpha.1 in accordance with the general method, as
described by Lonberg, N., et al., Nature 368 (1994) 856-859;
Fishwild, D. M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO
98/24884. Preferably, the mice will be 6-16 weeks of age upon the
first immunization. For example, IL-13R.alpha.1 transfected cells
can be used to immunize the HuMab mice intraperitoneally. The
immune response can be monitored over the course of the
immunization protocol with plasma samples being obtained by
retroorbital bleeds. The plasma can be screened by ELISA and/or
FACS. Mice with sufficient titers of anti-human IL-13R.alpha.1
human immunoglobulin can be used for immortalization of
corresponding B cells. Mice can be boosted intravenously with
antigen 3 to 4 days before sacrifice and removal of the spleen and
lymph nodes. For example, HuMab mice of the HCo7 or HCo12 strain
can be immunized. The HCo7 mice have a JKD disruption in their
endogenous light chain (kappa) genes (as described in Chen et al.
(1993) EMBO J. 12: 821-830), a CMD disruption in their endogenous
heavy chain genes (as described in Example 1 of WO 01/14424), a
KCo5 human kappa light chain transgene (as described in Fishwild,
D. M., et al. (1996) Nature Biotechnol 14:845-851), and a HCo7
human heavy chain transgene (as described in U.S. Pat. No.
5,770,429). The HCo12 mice have a JKD disruption in their
endogenous light chain (kappa) genes (as described in Chen, J., et
al., EMBO J. 12 (1993) 821-830), a CMD disruption in their
endogenous heavy chain genes (as described in Example 1 of WO
01/14424)) a KCo5 human kappa light chain transgene (as described
in Fishwild, D. M., et al., Nat. Biotechnol. 14 (1996) 845-851, and
a HCo12 human heavy chain transgene (as described in Example 2 of
WO 01/14424). The mouse lymphocytes can be isolated and fused with
a mouse myeloma cell line using PEG based on standard protocols to
generate hybridomas. The resulting hybridomas are then screened for
the production of antigen-specific antibodies. For example, single
cell suspensions of splenic and lymph node derived lymphocytes from
immunized mice are fused to SP 2/0 nonsecreting mouse myeloma cells
(ATCC, CRL 1581) with 50% PEG. Cells are plated at approximately
0.75.times.10.sup.7 in flat bottom micro titer plate, followed by
about two weeks incubation in selective medium.
[0137] Individual wells are then screened by ELISA and/or FACS for
human anti-IL-13R.alpha.1 monoclonal IgM and IgG antibodies. Once
extensive hybridoma growth occurs, the antibody secreting
hybridomas are replated, screened again, and if still positive for
human IgG, anti-IL-13R.alpha.1 monoclonal antibodies, can be
subcloned at least twice by limiting dilution. The stable subclones
are then cultured in vitro to produce antibody in tissue culture
medium for characterization.
[0138] Immunization procedure of transgenic mice: Three HCo7 mice
(3 males), strain GG2201 (Medarex, San Jose, Calif., USA) and 2
HCo12 mice (1 male and 1 female), strain GG2198 (Medarex, San Jose,
Calif., USA) were immunized with 1.times.10.sup.6 HEK293 cells,
transfected with an expression vector for IL-13R.alpha.1. In total
eight immunizations were given alternating intraperitoneally (i.p.)
and subcutaneous (s.c.) at the tail base. For the first
immunization, 100 .mu.l of 1.times.10.sup.6 HEK293: IL-13R.alpha.1
cells, was mixed with 100 .mu.l complete Freund's adjuvant (CFA;
Difco Laboratories, Detroit, USA). For all other immunizations, 100
.mu.l of cells in PBS was mixed with 100 .mu.l incomplete Freund's
adjuvant (ICFA; Difco).
[0139] Boosting of mice: When serum titers of anti-IL-13R.alpha.1
were found to be sufficient, mice were additionally boosted twice
with 1.times.10.sup.6 HEK293: IL-13R.alpha.1 cells in 200 .mu.l PBS
intravenously (i.v.) 4 and 3 days before fusion.
Example 2
Testing the Binding of HuMab to Immobilized IL-13R.alpha.1 by
ELISA
[0140] To determine the ability of the antibodies of the invention
to bind to recombinant IL-13R.alpha.1, the extracellular domain of
IL-13R.alpha.1 (R&D Systems, UK) was dissolved in PBS (1
.mu.g/ml) and allowed to adsorb to microtiter plates (NUNC
Maxisorb) by incubation over night at 4.degree. C. After washing
the plates with washing buffer (WB=0.9% NaCl; 0.1% Tween.RTM. 20)
unspecific binding sites were blocked by addition of 100 .mu.l
incubation buffer (IB=PBS with 1% crotein C and 0.1% Tween.RTM. 20)
and incubation for 30 min at room-temperature (RT). Then, serially
diluted HuMab and control antibodies (100 .mu.l/well; dilutions in
IB) were added and incubated for 1 hour at RT. The plates were
again washed, and bound human antibodies were detected by
incubation with peroxidase-conjugated rabbit anti-human kappa
(DAKO, Denmark) in a final dilution of 1:500 in IB. Polyclonal goat
anti-hIL-13R.alpha.1 antibodies were detected with
peroxidase-conjugated polyclonal donkey anti-goat IgG (Santa Cruz;
dilution 1:1000 in IB). After incubation for 1 h at RT and a
subsequent washing step, the plates were developed with
ready-to-use ABTS.RTM. solution (Roche Diagnostics GmbH) at RT in
the dark. Absorbance was measured at 405 nm after absorbance of the
highest concentration reached a sufficient OD.
[0141] All antibodies against IL-13Ralpha1 tested were able to bind
to immobilized extracelluar domains of human IL-13R.alpha.1. The
EC50 values determined were in the range of 0.5-2 nM for the
various LC antibodies tested. The negative control HuMab anti-KLH
did not bind to the immobilized extracellular domains of
IL-13R.alpha.1. Polyclonal goat-anti human IL-13R.alpha.1 antibody,
included as positive control, also bound efficiently to the
immobilized extracellular domains of IL-13R.alpha.1 (FIG. 1).
Example 3
Inhibition of IL-13 binding to IL-13R.alpha.1/IL-4R.alpha.
Heterodimer (ELISA)
[0142] Microtiter plates were coated with 100 .mu.l
hIL-13R.alpha.1:hFc chimeric protein (R&D Systems, UK) in PBS
at 3 .mu.g/ml at 4.degree. C. overnight on a shaker. After washing
the plates with WB, serially diluted HuMab and control antibodies
(100 .mu.l/well; dilutions in IB) were added and incubated for 30
min at RT. The plates were again washed, and then a mixture of
IL-13 (R&D Systems, UK; 0.5 .mu.g/ml; dilution with IB) and
IL-4R.alpha. (R&D Systems, UK; 0.75 .mu.g/ml; dilution with IB)
were added and incubated for 1 h at RT. After washing the plates
100 .mu.l biotinylated anti IL-13 antibody (BAF213; R&D
Systems, UK) in a concentration of 0.4 .mu.g/ml was added and
incubated for 1 h at RT. After washing the plates, bound IL-13 was
detected by peroxidase-coupled streptavidin (Roche Diagnostics
GmbH, DE) in a dilution of 1:5000 in IB (incubation period 1 h at
RT). Finally, plates were washed and developed with ready-to-use
ABTS.RTM. solution (Roche Diagnostics GmbH, DE) at room temperature
(RT) in the dark. Absorbance was measured at 405 nm after 45 to 60
minutes.
[0143] Antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007
and LC5002-018 were able to inhibit binding of IL-13 to the
heterodimeric receptor with maximal inhibition values ranging from
approximately 50% to 80-85%. Positive control was AF152 (polyclonal
rabbit antibody). As expected, the negative control anti-KLH did
not inhibit binding of IL-13 to the heterodimeric receptor. The
IC.sub.50 values obtained were between 1.5 nM and 10.1 nM for
LC5002-002, LC5002-003, LC5002-005, LC5002-007 and LC5002-018 (FIG.
2).
Example 4
Radioligand Binding Assay
[0144] .sup.125I-IL-13 binding assay was performed using CHO cells
expressing human IL-13R.alpha.1 and human IL-4R.alpha. in binding
buffer (25 mM HEPES, 150 mM NaCl, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2,
and 0.5% bovine serum albumin, adjusted to pH 7.2).
1.times.10.sup.5 cells per well were mixed with the antibodies and
preincubated for 15 minutes to 1 hour. 0.1 nM .sup.125I-IL-13 was
added, and the mix was incubated at 4.degree. C. for 4 hours. The
concentration of .sup.125I-IL-13 used in the assay was determined
from saturation binding analysis, competition analysis and
determination of input .sup.125I-IL-13 to reach equilibrium binding
with the cell line. Samples were harvested onto a GF/C filter plate
pretreated with 1% PEI/0.5% BSA and counted on Packard TopCount
Scintillation counter. Data analysis was performed in PRISM using
nonlinear regression curve fit (GraphPad Software, San Diego,
Calif.).
[0145] All antibodies against IL-13Ralphal1 tested block binding of
labeled IL-13 to the IL-13R.alpha.1/IL-4R.alpha. complex. The
calculated IC.sub.50 values for antibodies LC5002-002, LC5002-003,
LC5002-005, LC5002-007 and LC5002-018 were between 0.09 nM and 0.32
nM and 84.8 nM for AF152 (FIG. 3).
Example 5
Inhibition of IL-13 Induced Upregulation of CD23 on Human B Cells
and Monocytes by HuMab
[0146] Peripheral blood mononuclear cells (PBMC) were isolated by a
Ficoll Hypaque density gradient. After washing the cells with RPMI
they were resuspended in RPMI/10% FCS and distributed at
3.times.10.sup.5 PBMC/well (volume 50 .mu.l) in 96 well flat bottom
microtiter plates (Corning Incorporated Costar). Then, 25 .mu.l of
an anti-human CD40 antibody (Immunotech) in a final concentration
0.5 .mu.g/ml in RPMI/10% FCS and 25 .mu.l of an anti-human
IgA+IgG+IgM antibody (Immunoresearch) in a final concentration of
10 .mu.g/ml in RPMI/10% FCS were added. Then serially diluted HuMab
and control antibodies (50 .mu.l/well; dilutions in RPMI/10% FCS)
were added and the cells incubated for 30 min in the incubator
(37.degree. C.; 5% CO.sub.2). Then recombinant human IL-13 (R&D
Systems) in a final concentration of 0.67 ng/ml was added (50
.mu.l/well) and the cells incubated for 72 h at 37.degree. C./5%
CO.sub.2. After this incubation the plates were centrifuged and the
medium aspirated. For detachment of adherent cells 200 .mu.l of
Accutase (PAA) was added and the cells incubated for approximately
5 min at 37.degree. C.; 5% CO.sub.2. The cells were detached by
repeated flushing and transferred to a round bottom plate. After
centrifugation and aspiration of the supernatants the cells were
incubated with 200 .mu.l of a mixture of anti-CD23-PE,
anti-CD20-FITC and anti-CD14-APC (all from BD Biosciences
Pharmingen, San Diego, Calif.). The cells were incubated for 30 min
at 4.degree. C., then centrifuged and the supernatants aspirated.
This washing step was repeated once, and finally the cells were
resupended in 200 .mu.l of PBS/0.1% human serum albumin and
analysed in a FACS Calibur flow cytometer (BD Biosciences
Pharmingen, San Diego, Calif.) using the CellQuest software. In
most cases, 10 000 events were acquired and gated on a light
scatter gate to include only viable lymphocytes and monocytes. The
cells were pregated on a CD19 positive cluster for B lymphocytes or
a CD14 positive cluster for monocytes and analyzed further for CD23
expression.
[0147] The observed IC.sub.50 values for inhibiting of CD23
upregulation on B-lymphocytes were between 0.5 nM and >70 nM for
antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007 and
LC5002-018, and 13.6 nM for AF152. A similar profile was found for
inhibition of IL-13 induced CD23 upregulation on human monocytes.
On monocytes the IC.sub.50 values were between 0.1 nM and 62.8 nM
for antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007 and
LC5002-018, and 62.9 nM for AF152.
Example 6
TF-1 Proliferation Assay in Response to IL-13 or IL-4 as
Stimulus
[0148] TF-1 cells (ATCC #CRL 2003) were grown in media containing
ATCC modified RPMI, 10% FBS, 1.times. Pencillin/Streptomycin, 2
ng/ml GM-CSF. A day prior to the assay the cells were maintained in
GM-CSF free media. 5.times.10.sup.3 cells per well were incubated
with appropriate concentrations of anti-IL-13R.alpha.1 antibodies
at 37.degree. C. for 1 hour. Then the cells were stimulated with 2
ng/ml of human IL-13 (R&D Systems, Minneapolis, Minn.) or 0.1
ng/ml of human IL-4 (R&D Systems, Minneapolis, Minn.) and
incubated at 37.degree. C. for 48 hours. The cells were pulsed with
0.5 .mu.Ci .sup.3H-Thymidine and incubated at 37.degree. C. for
16-18 hours. Samples were harvested onto GFC plates pretreated with
1% PEI/0.25% BSA using Perkin Elmer Filtermate 96 harvester. The
GFC plates were counted on a Perkin Elmer Top count Scintillation
counter. Data analysis was performed in PRISM using nonlinear
regression curve fit (GraphPad Software, San Diego, Calif.).
[0149] Anti-KLH antibody did not show any inhibition in this assay.
The same was true for LC5002-007. All other antibodies inhibited
the response, even though LC5002-007 inhibited the response with
higher IC value than the other antibodies. The observed IC.sub.50
values for the different antibodies were: 13.50 nM for AF152, 9.21
nM for LC5002-002, 3.07 nM for LC5002-003 and 0.39 nM for
LC5002-005. A similar profile was found for IL-4-induced TF-1 cell
proliferation, however the potency of the antibodies was decreased
compared to IL-13-induced responses. IC.sub.50 values for
IL-4-induced proliferation were 0.02 nM for anti-IL4R antibody,
74.37 nM for AF152 and for antibodies LC5002-002, LC5002-003,
LC5002-005, LC5002-007 and LC5002-018 between 4.68 nM and 60
nM.
Example 7
Inhibition of Eotaxin Production in Response to IL-13 by Human Lung
Fibroblast
[0150] The assay was performed using HFL-1 cells (Human Lung
Fibroblast, ATCC #CCL-153). Cells were plated at a density of
100,000 cells per well in a 12-well plate and incubated at
37.degree. C. for 72 hours to reach confluency. Cells were then
starved in serum free medium for 24 h and treated with
anti-IL-13R.alpha.1 antibody at 37.degree. C. for 1 h. Following
this treatment cells were stimulated with 10 ng/ml IL-13 (R&D
Systems, Minneapolis, Minn.) at 37.degree. C. for 48 h.
Supernatants were collected and eotaxin determinations were done
using commercially available ELISA from R&D Systems (Cat. No.
DTX00). Absorbance was read using Spectromax microplate reader and
the data was analyzed using PRISM (GraphPad Software, San Diego,
Calif.).
[0151] The antibodies tested showed different capability to inhibit
eotaxin release. With the exeption of LC5002-007 all other
antibodies tested showed some inhibition. The calculated average
IC.sub.50 values from 3 to 4 different experiments were 11.5 nM for
AF152 and between 2.45 nM and 19.8 nM for antibodies LC5002-002,
LC5002-003, LC5002-005, LC5002-007 and LC5002-018.
Example 8
Inhibition of IL-13-Induced Stat-6 Phosphorylation in Human
Bronchial Smooth Muscle Cells
[0152] Human Bronchial Smooth Muscle Cells (BSMC; Clonetics, Cat.
No CC-2576) were grown following the manufacturer's instructions.
Cells were grown in 12-well tissue culture plates until they
reached confluency. Cells were starved for 24 h in serum-free
medium and variable amounts of antibody were added. Plates were
incubated for 1 h and then stimulated with 2.5 ng/ml IL-13 (R&D
System). After 15 min incubation, supernatant was removed, cells
were washed with phosphate buffer and 100 .mu.l of lysis buffer was
added. The mix was briefly sonicated on ice and centrifuged. Lysate
was used for Western Blot detection of phophorylated Stat-6. Equal
amounts of protein were loaded onto an SDS-gel, run and transferred
to a membrane. Anti-Stat-6 antibody was from Santa Cruz
Biotechnology (Cat. No. SC-11762R) and a secondary antibody coupled
to peroxidase was used. Detection was done using the ECL Plus
System from Amersham (Cat No RPN 2132). Quantitation was done in a
Typhoon 9400 Imager.
[0153] Both HuMabs tested in this assay (LC50002-003 and
LC5002-005) inhibited IL-13-induced Stat-6 phosphorylation. The
potency found in this assay was similar to that of other functional
assays. The calculated IC.sub.50 values were 18.64 nM for AF152,
5.98 nM for LC5002-003 and 1.18 nM for LC5002-005.
Example 9
Cloning and Sequence Analysis of Anti-hIL-13R.alpha.1 HuMab
Variable Domains (.kappa.-Light and .gamma.1-Heavy Chains)
[0154] The nucleotide sequences coding for the light chain variable
region V.sub.L and the heavy chain variable region V.sub.H of the
anti hIL-13R.alpha.1 HuMabs were isolated by a standard cDNA
synthesis/PCR procedure. Total RNA was prepared from
1.times.10.sup.6-1.times.10.sup.7 hybridoma cells using the
GeneRacer.TM. Kit (Invitrogen). Hybridoma derived RNA was used as a
template for the 1.sup.st strand cDNA synthesis and ligation of the
GeneRacer.TM. Oligo-dT Primer. 2.sup.nd-strand cDNA synthesis and
further PCR amplification of V.sub.L and V.sub.H encoding cDNA
fragments were performed with reverse light and heavy chain primers
complementary to nucleotide sequences of the .kappa.-light and
.gamma.1-heavy chain constant region and 5'-specific GeneRacer.TM.
primers, respectively. The PCR products were cloned using the
TOPO.TM. TA cloning kit from Invitrogen.TM. Life Technologies and
pCR4-TOPO.TM. as a cloning vector. Cloned PCR products were
identified by restriction mapping of the appropriate plasmids using
EcoRI for digestion and expected/calculated DNA fragment sizes of
about 740 and 790 bp for V.sub.L and V.sub.H, respectively. The DNA
sequence of cloned PCR fragments was determined by double strand
sequencing. The GCG (Genetics Computer Group, Madison, Wis.)
software package version 10.2 and Vector-NTI 8 (InforMax, Inc) was
used for general data processing. DNA and protein sequences were
aligned using the GCG modul CLUSTALW. Sequence alignments were made
using the program GENEDOC (version 2.1).
Example 10
Construction of Expression Plasmids for an Anti-hIL-13R.alpha.1
IgG1 HuMab
[0155] The anti-hIL-13R.alpha.1 HuMab light and heavy chain
encoding genes were separately assembled in mammalian cell
expression vectors. Thereby the gene segments encoding the
anti-hIL-13R.alpha.1 HuMab light chain variable region (V.sub.L)
and the human K-light chain constant region (C.sub.L, SEQ ID NO:
11) were joined as were gene segments for the anti-hIL-13R.alpha.1
HuMab heavy chain variable region (V.sub.H) and the human
.gamma.1-heavy chain constant region
(C.sub.H1-Hinge-C.sub.H2-C.sub.H3, SEQ ID NO: 12). General
information regarding the nucleotide sequences of human light and
heavy chains from which the codon usage is given in: Kabat, E. A.,
Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C., (1991)
Sequences of Proteins of Immunological Interest, Fifth Ed., NIH
Publication No. 91-3242. The transcription unit of the
anti-hIL-13R.alpha.1 HuMab .kappa.-light chain is composed of the
following elements: [0156] The immediate early enhancer and
promoter from the human cytomegalovirus (HCMV), [0157] A synthetic
5'-UT including a Kozak sequence, [0158] A murine immunoglobulin
heavy chain signal sequence including the signal sequence intron,
[0159] The cloned anti-hIL-13R.alpha.1 HuMab variable light chain
cDNA arranged with a unique BsmI restriction site at the 5' end and
a splice donor site and a unique NotI restriction site at the 3'
end, [0160] The genomic human K-gene constant region, including the
intron 2 mouse Ig-.kappa. enhancer [Picard, D., and Schaffner, W.,
Nature 307 (1984) 80-82] and [0161] The human immunoglobulin
.kappa.-polyadenylation ("poly A") signal sequence.
[0162] The transcription unit of the anti-hIL-13R.alpha.1 HuMab
.gamma.1-heavy chain is composed of the following elements: [0163]
The immediate early enhancer and promoter from the human
cytomegalovirus (HCMV), [0164] A synthetic 5'-UT including a Kozak
sequence, [0165] A modified murine immunoglobulin heavy chain
signal sequence including the signal sequence intron, [0166] The
cloned anti-hIL-13R.alpha.1 HuMab variable heavy chain cDNA
arranged with a unique BsmI restriction site at the 5' and a splice
donor site and a unique NotI restriction site at the 3' end, [0167]
The genomic human .gamma.1-heavy gene constant region, including
the mouse Ig .mu.-enhancer (Neuberger, M. S., EMBO J. 2 (1983)
1373-1378), [0168] The human .gamma.1-immunoglobulin
polyadenylation ("poly A") signal sequence.
[0169] Functional elements of the anti-hIL-13R.alpha.1 HuMab
.kappa.-light chain and .gamma.1-heavy chain expression
plasmids:
[0170] Besides the anti-hIL-13R.alpha.1 HuMab .kappa.-light chain
or .gamma.1-heavy chain expression cassette these plasmids contain
[0171] A hygromycin resistance gene [0172] An origin of
replication, oriP, of Epstein-Barr virus (EBV) [0173] An origin of
replication from the vector pUC18 which allows replication of this
plasmid in E. coli, and [0174] A .beta.-lactamase gene which
confers ampicillin resistance in E. coli.
Example 11
Construction of Expression Plasmids for Mutant (Variant)
Anti-hIL-13R.alpha.1 IgG1
[0175] Expression plasmids encoding mutant anti-hIL-13R.alpha.1
.gamma.1-heavy chains can be created by site-directed mutagenesis
of the wild type expression plasmids using the QuickChange.TM.
Site-Directed mutagenesis Kit (Stratagene) and are described in
table 1. Amino acids are numbered according to EU numbering
(Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969)
78-85; Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and
Foeller, C., (1991) Sequences of Proteins of Immunological
Interest, Fifth Ed., NIH Publication No. 91-3242).
TABLE-US-00001 TABLE 1 Mutation Description PVA-236; GLPSS331 as
The amino acid sequence specified by E233P;
Glu.sub.233Leu.sub.234Leu.sub.235Gly.sub.236 of the human .gamma.1-
L234V; L235A; delta heavy chain is replaced by the amino acid G236;
sequence Pro.sub.233Val.sub.234Ala.sub.235 of the human
.gamma.2-heavy chain. A327G; A330S; The amino acid sequence P331S
Ala.sub.327Leu.sub.328Pro.sub.329Ala.sub.330Pro.sub.331 of the
human .gamma.1-heavy chain is replaced by the amino acid sequence
Gly.sub.327Leu.sub.328Pro.sub.329Ser.sub.330Ser.sub.331 of the
human .gamma.4-heavy chain L234A; L235A The amino acid sequence
Leu.sub.234Leu.sub.235 of the human .gamma.1-heavy chain is
replaced by the amino acid sequence Ala.sub.234Ala.sub.235
Example 12
Production of Recombinant Anti-hIL-13R.alpha.1 HuMabs
[0176] Recombinant HuMabs were generated by transient transfection
of adherent HEK293-EBNA cells (ATTC CRL-10852) cultivated in DMEM
(Gibco) supplemented with 10% ultra-low IgG FCS (Gibco), 2 mM
Glutamine (Gibco), 1% v/v nonessential aminoacids (Gibco) and 250
.mu.g/ml G418 (Roche Diagnostics GmbH, DE). For transfection
Fugene.TM. 6 (Roche Diagnostics GmbH, DE) transfection reagent was
used in a ratio of reagent (.mu.l) to DNA (.mu.g) ranging from 3:1
to 6:1. Immunoglobulin light and heavy chains were expressed from
two different plasmids using a molar ratio of light chain to heavy
chain encoding plasmid from 1:2 to 2:1. HuMab containing cell
culture supernatants were harvested at day 4 to 11 after
transfection.
[0177] General information regarding the recombinant expression of
human antibody in e.g. HEK293 is given in: Meissner, P., et al.,
Biotechnol Bioeng 75 (2001) 197-203.
Example 13
[0178] a) Affinity Analysis of HuMabs LC5002-003, -005, and -007
Using Chimeric hIL-13R.alpha.1:hFc
[0179] For interaction analysis a Biacore 3000 instrument was used.
As running and reaction buffer, HBS-P (10 mM HEPES, 150 mM NaCl,
0.005% polysurfactant P, pH 7.4) at 25.degree. C. was used.
Capturing molecules (goat anti-human-IgG, Fc.gamma. specifc) were
amine-coupled at a concentration of 20 .mu.g/ml at a flow rate of 5
.mu.l/min for 20 minutes. HuMabs were injected at a concentration
of 1 .mu.g/ml at a flow rate of 10 .mu.l/min for 1 minute. Blocking
of the free goat anti human IgG, Fc.gamma. was achieved by
injecting human gamma globulin at 500 nM and 30 .mu.l/min for 3
minutes. Analyte (hIL-13R.alpha.1:hFc chimeric protein) was
injected for two minutes at five concentrations between 5.63 nM and
90 nM and washed with HBS-P for five minutes. Regeneration of the
surface was accomplished by two injections of 100 mM HCl for 1 min
each. The chip, assay format and sequence of injections and kinetic
data correspond to the description in table 2. Negative control
data (e.g. buffer curves) were subtracted from sample curves for
correction of system intrinsic baseline drift and for noise signal
reduction. BiaEvaluation version 4.01 was used for analysis of
sensorgrams and for calculation of affinity data. Kinetic data were
calculated by fitting kinetic data to a 1:1 Langmuir binding model
(Table 2).
TABLE-US-00002 TABLE 2 Affinity analysis of HuMabs using
hIL-13R.alpha.1:hFc. Data analysis based on 1:1 Langmuir binding
model. Chip Capturing Ligand Analyte k.sub.a (1/Ms) k.sub.d (1/s)
K.sub.D (M) CM5 Anti-hFc.gamma. LC5002-003 hIL-13 2.1 .times.
10.sup.5 1.4 .times. 10.sup.-6 <6.5 .times. 10.sup.-12
R.alpha.1:hFc CM5 Anti-hFc.gamma. LC5002-005 hIL-13 1.73 .times.
10.sup.5 3.12 .times. 10.sup.-6 1.8 .times. 10.sup.-11
R.alpha.1:hFc CM5 Anti-hFc.gamma. LC5002-007 hIL-13 1.19 .times.
10.sup.5 1 .times. 10.sup.-6 <8.4 .times. 10.sup.-12
R.alpha.1:hFc
[0180] b) Affinity Analysis of HuMabs LC5002-003, -005 and -007
Using the Extracellular Domain of hIL-13R.alpha.1 Cleaved from of
hIL-13R.alpha.1:hFc Chimeric Protein
[0181] For interaction analysis, a Biacore 3000 instrument was
used. Running and reaction buffer was HBS-P (10 mM HEPES, 150 mM
NaCl, 0.005% polysurfactant P, pH 7.4) at 25.degree. C. Capturing
antibody molecules (anti-hFc.gamma.) were amine-coupled at a
concentration of 100 .mu.g/ml at a flow rate of 5 .mu.l/min for 20
minutes. HuMabs were injected at concentrations of 10 .mu.g/ml at a
flow rate of 10 .mu.l/min for 30 seconds. Cleaved hIL-13R.alpha.1
molecules (M.sub.W 40 kDa) were injected for 200 seconds at seven
concentrations between 1.56 nM and 100 nM and washed with HBS-P for
five minutes. Regeneration of the surface was accomplished by two
injections of 100 mM HCl for 1 min each at a flow rate of 10
.mu.g/ml. The chip, assay format and sequence of injections and
kinetic data correspond to the description in the following Table
3. Kinetic data were calculated by fitting kinetic data to a 1:1
Langmuir binding model.
TABLE-US-00003 TABLE 3 Chip Capturing Ligand Analyte k.sub.a (1/Ms)
k.sub.d (1/s) K.sub.D (M) C1 Anti-hFc.gamma. LC5002-
hIL-13R.alpha.1 1.1 .times. 10.sup.6 6.5 .times. 10.sup.-4 6.2
.times. 10.sup.-10 002 C1 Anti-hFc.gamma. LC5002- hIL-13R.alpha.1
1.3 .times. 10.sup.6 5.1 .times. 10.sup.-4 3.9 .times. 10.sup.-10
003 C1 Anti-hFc.gamma. LC5002- hIL-13R.alpha.1 1.4 .times. 10.sup.6
3.0 .times. 10.sup.-4 2.2 .times. 10.sup.-10 005 C1 Anti-hFc.gamma.
LC5002- hIL-13R.alpha.1 1.9 .times. 10.sup.5 8.3 .times. 10.sup.-4
4.4 .times. 10.sup.-9 007 C1 Anti-hFc.gamma. LC5002-005,
hIL-13R.alpha.1 1.4 .times. 10.sup.6 2.9 .times. 10.sup.-4 2.1
.times. 10.sup.-10 mutant L234A; L235A
[0182] c) Comparative Affinity Analysis of Recombinant Variants of
LC5002-005 Using the Extracellular Domain of hIL-13R.alpha.1
Cleaved from hIL-13R.alpha.1:hFc Chimeric Protein
[0183] In these experiments, the affinity of the original IgG1
derived from hybridoma was compared with the affinities of the
recombinant variant IgG1-Ala-Ala. For interaction analysis, a
Biacore 3000 instrument was used. Running and reaction buffer was
HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% polysurfactant P, pH 7.4)
at 25.degree. C. Capturing antibody molecules (anti-hFc.gamma.)
were amine-coupled at a concentration of 20 .mu.g/ml at a flow rate
of 5 .mu.l/min for 20 minutes. HuMabs were injected at
concentrations of 10 .mu.g/ml at a flow rate of 10 .mu.l/min for 1
minute. Cleaved hIL-13 R.alpha.1 molelcules (analyte) were injected
for five minutes at eight concentrations between 1.56 nM and 200 nM
and washed with HBS-P for five minutes. Regeneration of the surface
was accomplished by two injections of 100 mM HCl for 1 min each.
The chip, assay format and sequence of injections and kinetic data
correspond to the description in Table 4. Kinetic data were
calculated by fitting kinetic data to a bivalent analyte binding
model.
TABLE-US-00004 TABLE 4 k.sub.a1 k.sub.a2 k.sub.d2 Chip Capturing
Ligand Analyte (1/Ms) k.sub.d1 (1/s) (1/RUs) (1/s) K.sub.D (M) CM5
Anti-hFc.gamma. LC5002- hIL-13 1.33 .times. 10.sup.5 3.6 .times.
10.sup.-4 5.8 .times. 10.sup.-3 0.06 2.7 .times. 10.sup.-9 005
R.alpha.1 CM5 Anti-hFc.gamma. IgG1 ala- hIL-13 1.53 .times.
10.sup.5 4.2 .times. 10.sup.-4 4.1 .times. 10.sup.-3 0.04 2.8
.times. 10.sup.-9 ala R.alpha.1 LC5002- 005
Example 14
Testing the Crossreactivity of HuMab with hIL-13R.alpha.2 and
hIL-4R.alpha. by ELISA
[0184] Chimeric proteins hIL-13R.alpha.2:hFc and hIL-4R.alpha.:hFc
(R&D Systems, UK) were dissolved in PBS (1 .mu.g/ml) and
allowed to adsorb to microtiter plates (NUNC Maxisorb) by
incubation over night at 4.degree. C. After washing the plates with
washing buffer (WB=0.9% NaCl; 0.1% Tween.RTM. 20) unspecific
binding sites were blocked by addition of 100 .mu.l incubation
buffer (IB=PBS with 1% crotein C and 0.1% Tween.RTM. 20) and
incubation for 30 min at room-temperature (RT). Then serially
diluted HuMab and control antibodies (100 .mu.l/well; dilutions in
IB) were added and incubated for 1 hour at RT. The plates were
again washed and bound antibody was detected by incubation with
peroxidase conjugated rabbit anti-human kappa (DAKO, Denmark) in a
final dilution of 1:500 in IB. After incubation for 1 h at RT and a
subsequent washing step, the plates were developed with
ready-to-use ABTS.RTM. solution (Roche Diagnostics GmbH, DE) at RT
in the dark. Absorbance was measured at 405 nm after absorbance of
the highest concentration reached a sufficient OD.
[0185] All antibodies against IL-13Ralpha1 tested were able to bind
to immobilized extracelluar domains of human IL-13R.alpha.1, but
not neither to hIL-13R.alpha.2 nor to hIL-4R.alpha. (FIG. 4).
Example 15
Crossreactivity of HuMab with Murine IL-13R.alpha.1
[0186] Chimeric protein murine IL-13R.alpha.1:hFc (R&D Systems,
UK) was dissolved in PBS (1 .mu.g/ml) and allowed to adsorb to
microtiter plates (NUNC Maxisorb) by incubation over night at
4.degree. C. After washing the plates with washing buffer (WB=0.9%
NaCl; 0.1% Tween.RTM. 20), unspecific binding sites were blocked by
addition of 100 .mu.l incubation buffer (IB=PBS with 1% Crotein C
and 0.1% Tween.RTM. 20) and incubation for 30 min at
room-temperature (RT). Then, serially diluted HuMab and control
antibodies (HuMab anti-KLH and polyclonal goat anti-hIL-13R.alpha.1
(R&D Systems)) were added to the wells (100 .mu.l/well;
dilutions in IB) and incubated for 1 hour at RT. The plates were
again washed and bound human antibodies were detected by incubation
with peroxidase conjugated rabbit anti-human kappa (DAKO, Denmark)
in a final dilution of 1:500 in IB. Goat anti-hIL-13R.alpha.1
antibodies bound to the plates were detected by
peroxidase-conjugated donkey anti-goat IgG (Santa Cruz; 1:1000 in
IB). After incubation for 1 h at RT and a subsequent washing step,
the plates were developed with ready-to-use ABTS.RTM. solution
(Roche Diagnostics GmbH, DE) at RT in the dark. Absorbance was
measured at 405 nm after absorbance of the highest concentration
reached a sufficient OD (FIG. 5).
Example 16
Crossreactivity of HuMab with Cynomolgus IL-13R.alpha.1
[0187] The gene coding for IL-13R.alpha.1 was isolated by RT-PCR
from Cynomolgus tissue and transfected into in the murine cell line
Ba/F3. In order to test whether the HuMabs crossreact with
Cynomolgus IL-13R.alpha.1, the stably transfected Ba/F3 cells as
well as the parental Ba/F3 cells were incubated with 10 .mu.g/ml of
HuMab and control antibodies. As positive control, a polyclonal
goat-anti hIL-13R.alpha.1 (R&D Systems) was used. Negative
controls included were: a human IgG1 myeloma protein (Nordic) and
normal goat serum. Bound antibodies were detected by FACS analysis
using an antibody directed against human IgG conjugated with FITC
to detect HuMabs and an antibody directed against goat IgG
conjugated with FITC to detect goat antibodies. Mean fluorescence
intensities (MFI) were compared for the individual antibodies
tested on the transfected Ba/F3 line versus the parental line.
[0188] All HuMabs of the invention were able to bind to Cynomolgus
IL-13R.alpha.1 expressed in transfected Ba/F3 cells. As expected
from the close homology between human and Cynomolgus
IL-13R.alpha.1, the polyclonal AF152 antibody also bound to the
Cynomolgus IL-13R.alpha.1. The negative control antibodies showed
only a marginal increase in MFI when tested with the transfected
Ba/F3 cell line (Table 5).
TABLE-US-00005 TABLE 5 Fold Increase MFI MFIBa/ in MFI in Ba/
F3_Cyno_IL- the presence of Antibody F3 13R.alpha.1
Cyno_IL-13R.alpha.1 HuMabs LC5002-002 3.9 83.7 79.8 LC5002-003 3.4
82.4 79.0 LC5002-005 14.8 101.5 86.7 LC5002-007 4.1 19 14.9
Controls AF152 3.2 21.2 18.0 Normal goat IgG 3.3 5.7 2.4 Normal
human 3.5 10.2 6.7 IgG1 (Nordic) Anti human IgG- 3.3 5.5 2.2 FITC
only
Example 17
Binding of IL-13R.alpha.1 HuMabs to Fc.gamma. Receptors (Binding to
Fc.gamma.RIIIa on NK Cells)
[0189] To determine the ability of the antibodies of the invention
to bind to Fc.gamma.RIIIa (CD16) on Natural Killer (NK) cells,
Peripheral Blood Mononuclear Cells (PBMCs) were isolated and
incubated with 20 .mu.g/ml of HuMab antibody and control antibodies
in the presence or absence of 20 .mu.g/ml of a blocking mouse
antibody to Fc.gamma.RIIIa (anti-CD16, clone 3G8, RDI, Flanders,
N.J.), to verify binding via Fc.gamma.RIIIa. As negative controls,
human IgG2 and IgG4 (The Binding Site), that do not bind
Fc.gamma.RIIIa, were used. Human IgG1 and IgG3 (The Binding Site)
were included as positive controls for Fc.gamma.RIIIa binding.
Bound antibodies on NK cells were detected by FACS analysis using a
PE-labeled mouse anti-human CD56 (NK-cell surface marker) antibody
(BD Biosciences Pharmingen, San Diego, Calif.) in combination with
a FITC-labeled goat F(ab).sub.2 anti-human IgG (Fc) antibody
(Protos immunoresearch, Burlingame, Calif.). Maximum binding at 20
.mu.g/ml (Bmax: MFI.+-.st.dev) of the HuMab tested was
determined.
[0190] LC5002-005 was able to bind to Fc.gamma.RIIIa efficiently
(comparable to the control IgG1 antibody) as indicated by a Bmax
(MFI) value of 580.6.+-.245.8. Addition of a blocking antibody
against Fc.gamma.RIIIa dramatically reduced binding of LC5002-005
to NK cells (Bmax (MFI) value of 260.4.+-.95.90) indicating
specific binding to Fc.gamma.RIIIa.
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Sequence CWU 1
1
151119PRTHomo sapiens 1Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Ile Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Gly Arg Gly Ile
Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Ser
Ser Ser Trp Thr Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 1152107PRTHomo sapiens 2Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1053119PRTHomo sapiens 3Glu Val Gln Leu Leu Glu Ser Gly Gly Asp Leu
Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Ile Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Gly Arg Gly Ile
Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asp Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Ser
Ser Tyr Trp Thr Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 1154107PRTHomo sapiens 4Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1055118PRTHomo sapiens 5Glu Val Gln Val Leu Asp Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly
Phe Thr Phe Arg Leu Tyr 20 25 30Thr Met Ser Trp Val Arg Gln Thr Pro
Gly Arg Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Gly Ser Gly Leu
Ser Thr Tyr Phe Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Glu Gly
Asp Trp Ile Tyr Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110Leu Val
Ile Val Ser Ser 1156107PRTHomo sapiens 6Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser His Pro Pro 85 90
95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 1057123PRTHomo
sapiens 7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Ala Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Pro Ile Leu Gly Arg Thr Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Val Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Glu Thr Leu
Asp Tyr Phe Tyr Tyr Gly Met Asp Val 100 105 110Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 1208107PRTHomo sapiens 8Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile
Tyr Gly Ala Ser Ser Arg Ala Ile Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Ser Ser
Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
1059119PRTHomo sapiens 9Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Ile Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Gly Ser Gly Val
Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Ser
Ser Trp Tyr Val Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11510107PRTHomo sapiens 10Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10511107PRTHomo sapiens 11Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 10512330PRTHomo sapiens 12Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330135PRTHomo sapiens 13Gly Leu Pro Ser Ser1 5144PRTHomo sapiens
14Glu Leu Leu Gly1155PRTHomo sapiens 15Ala Leu Pro Ala Pro1 5
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