U.S. patent application number 14/048636 was filed with the patent office on 2014-05-22 for engineered anti-tslp antibody.
This patent application is currently assigned to SCHERING CORPORATION. The applicant listed for this patent is RENE DE WAAL MALEFYT, LEONARD G. PRESTA. Invention is credited to RENE DE WAAL MALEFYT, LEONARD G. PRESTA.
Application Number | 20140141011 14/048636 |
Document ID | / |
Family ID | 39273256 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140141011 |
Kind Code |
A1 |
PRESTA; LEONARD G. ; et
al. |
May 22, 2014 |
ENGINEERED ANTI-TSLP ANTIBODY
Abstract
The invention relates to binding compounds that specifically
bind to human TSLP, as well as uses thereof, e.g., in the treatment
of inflammatory disorders.
Inventors: |
PRESTA; LEONARD G.; (SAN
FRANCISCO, CA) ; DE WAAL MALEFYT; RENE; (SUNNYVALE,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRESTA; LEONARD G.
DE WAAL MALEFYT; RENE |
SAN FRANCISCO
SUNNYVALE |
CA
CA |
US
US |
|
|
Assignee: |
SCHERING CORPORATION
KENILWORTH
NJ
|
Family ID: |
39273256 |
Appl. No.: |
14/048636 |
Filed: |
October 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13532131 |
Jun 25, 2012 |
8580938 |
|
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14048636 |
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12515915 |
Sep 17, 2009 |
8232372 |
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13532131 |
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Current U.S.
Class: |
424/158.1 ;
435/254.2; 435/320.1; 435/335; 435/69.6; 530/387.3; 530/389.2;
536/23.53 |
Current CPC
Class: |
A61P 17/00 20180101;
C07K 2317/567 20130101; C07K 2317/76 20130101; C07K 16/24 20130101;
C07K 2317/52 20130101; A61P 37/00 20180101; A61P 37/08 20180101;
A61P 11/06 20180101; A61K 2039/505 20130101; A61P 17/04 20180101;
A61P 29/00 20180101; C07K 2317/24 20130101; C07K 2317/565 20130101;
A61P 37/06 20180101; C07K 2317/56 20130101; C07K 2317/92 20130101;
C07K 16/244 20130101; A61P 27/02 20180101; A61P 11/02 20180101;
C07K 2317/73 20130101 |
Class at
Publication: |
424/158.1 ;
530/389.2; 536/23.53; 435/320.1; 435/69.6; 530/387.3; 435/335;
435/254.2 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2007 |
US |
PCT/US2007/025531 |
Claims
1. A binding compound that specifically binds human and cyno TSLP,
comprising: at least one antibody heavy chain variable region, or a
TSLP-binding fragment thereof, said heavy chain variable region
comprising at least one CDR sequence selected from the group
consisting of SEQ ID NOs: 1, 2 and 3; or at least one antibody
light chain variable region, or a TSLP-binding fragment thereof,
said light chain variable region comprising at least one CDR
sequence selected from the group consisting of SEQ ID NOs: 4, 5 and
6.
2. The binding compound of claim 1, wherein the binding compound
comprises: at least one antibody heavy chain variable region, or a
TSLP-binding fragment thereof, said heavy chain variable region
comprising at least one CDR sequence selected from the group
consisting of SEQ ID NOs: 1, 2 and 3; and at least one antibody
light chain variable region, or a TSLP-binding fragment thereof,
said light chain variable region comprising at least one CDR
sequence selected from the group consisting of SEQ ID NOs: 4, 5 and
6.
3. The binding compound of claim 2, wherein: the antibody heavy
chain variable region, or TSLP-binding fragment thereof, comprises
at least two CDR sequences selected from the group consisting of
SEQ ID NOs: 1, 2 an 3; and the antibody light chain variable
region, or TSLP-binding fragment thereof, comprises at least two
CDR sequences selected from the group consisting of SEQ ID NOs: 4,
5 and 6.
4. The binding compound of claim 2, wherein: the antibody heavy
chain variable region, or TSLP-binding fragment thereof, has the
three CDR sequences set forth in SEQ ID NOs: 1, 2 and 3; and the
antibody light chain variable region, or TSLP-binding fragment
thereof, has the three CDR sequences set forth in SEQ ID NOs: 4, 5
and 6.
5. A binding compound that specifically binds human and cyno TSLP,
comprising: at least one antibody heavy chain variable region, or a
TSLP-binding fragment thereof, said heavy chain variable region
comprising: the CDR-H1 of SEQ ID NO. 1, or a variant thereof; the
CDR-H2 of SEQ ID NO. 2, or a variant thereof; and the CDR-H3 of SEQ
ID NO. 3, or a variant thereof; or at least one antibody light
chain variable region, or a TSLP-binding fragment thereof, said
light chain variable region comprising: the CDR-L1 of SEQ ID NO. 4,
or a variant thereof; the CDR-L2 of SEQ ID NO. 5, or a variant
thereof; and the CDR-L3 of SEQ ID NO. 6, or a variant thereof.
6. The binding compound of claim 5, wherein said binding compound
comprises: at least one antibody heavy chain variable region, or a
TSLP-binding fragment thereof, said heavy chain variable region
comprising: the CDR-H1 of SEQ ID NO. 1, or a variant thereof; the
CDR-H2 of SEQ ID NO. 2, or a variant thereof; and the CDR-H3 of SEQ
ID NO. 3, or a variant thereof; and at least one antibody light
chain variable region, or a TSLP-binding fragment thereof, said
light chain variable region comprising: the CDR-L1 of SEQ ID NO. 4,
or a variant thereof; the CDR-L2 of SEQ ID NO. 5, or a variant
thereof; and the CDR-L3 of SEQ ID NO. 6, or a variant thereof.
7. A binding compound that specifically binds human and cyno TSLP,
comprising: a heavy chain variable region comprising residues 1-116
of SEQ ID NO: 10 or a variant thereof; and a light chain variable
region comprising residues 1-108 of SEQ ID NO: 12, or a variant
thereof, wherein the variant comprises up to 20 conservatively
modified amino acid residues.
8. The binding compound of claim 7, comprising: a heavy chain
variable region comprising residues 1-116 of SEQ ID NO: 10; and a
light chain variable region comprising residues 1-108 of SEQ ID NO:
12.
9. A binding compound that specifically binds human and cyno TSLP,
comprising: a heavy chain variable region having at least 90%
homology to residues 1-116 of SEQ ID NO: 10; and a light chain
variable region having at least 90% homology to residues 1-108 of
SEQ ID NO: 12.
10. An isolated nucleic acid encoding at least one of the heavy
chain variable region or light chain variable region of the binding
compound of claim 1.
11. An expression vector comprising the nucleic acid of claim 10
operably linked to control sequences that are recognized by a host
cell when the host cell is transfected with the vector.
12. A host cell comprising the expression vector of claim 11.
13. A method of producing a polypeptide comprising: culturing the
host cell of claim 8 in culture medium under conditions wherein the
nucleic acid sequence is expressed, thereby producing polypeptides
comprising the light and heavy chain variable regions; and
recovering the polypeptides from the host cell or culture
medium.
14. The binding compound of claim 1, further comprising: a human
heavy chain constant region or a variant thereof, wherein the
variant comprises up to 20 conservatively modified amino acid
substitutions; or a human light chain constant region or a variant
thereof, wherein the variant comprises up to 20 conservatively
modified amino acid substitutions.
15. The binding compound of claim 14, wherein the human heavy chain
constant region comprises a .gamma.4 or .gamma.1 human heavy chain
constant region or a variant thereof, wherein the variant comprises
up to 20 conservatively modified amino acid substitutions.
16. The binding compound of claim 2, further comprising: a human
heavy chain constant region or a variant thereof, wherein the
variant comprises up to 20 conservatively modified amino acid
substitutions; or a human light chain constant region or a variant
thereof, wherein the variant comprises up to 20 conservatively
modified amino acid substitutions.
17. The binding compound of claim 16, wherein the human heavy chain
constant region comprises a .gamma.4 or .gamma.1 human heavy chain
constant region or a variant thereof, wherein the variant comprises
up to 20 conservatively modified amino acid substitutions.
18. The binding compound of claim 1, wherein the binding compound
is a humanized antibody or a TSLP-binding fragment thereof.
19. The binding compound of claim 1, wherein the binding compound
is a TSLP-binding antibody fragment selected from the group
consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2, and a
diabody.
20. A method of suppressing an immune response in a human subject
comprising administering to a subject in need thereof the binding
compound of claim 1, or a TSLP-binding fragment thereof, in an
amount effective to block the biological activity of TSLP.
21. The method of claim 20, wherein the immune response is an
inflammatory response.
22. The method of claim 20, wherein the subject has a disorder
selected from the group consisting of allergic rhinosinusitis,
allergic asthma, allergic conjunctivitis, or atopic dermatitis.
23. The method of claim 20, wherein the subject has asthma.
24. A composition comprising the binding compound of claim 1 in
combination with a pharmaceutically acceptable carrier or
diluent.
25. An antibody that specifically binds to the epitope on human
TSLP that is bound by the antibody produced by the hybridoma
deposited as PTA-7951, wherein the antibody that specifically binds
to the epitope on human TSLP is not the antibody produced by the
hybridoma deposited as PTA-7951.
26. An antibody that competitively inhibits binding by the antibody
produced by the hybridoma deposited as PTA-7951 to human TSLP,
wherein the antibody that competitively inhibits binding is not the
antibody produced by the hybridoma deposited as PTA-7951.
27. The binding compound of claim 1, wherein the binding compound
blocks TSLP-mediated activity.
28. The binding compound of claim 1, wherein said binding compound
is able to block the binding of TSLP to TSLPR in a cross-blocking
assay.
29. The use of the binding compound of claim 1, or a TSLP-binding
fragment thereof, for the preparation of a medicament to suppress
an immune response.
30. The use of the binding compound of claim 1, or a TSLP-binding
fragment thereof, for the preparation of a medicament to treat
inflammation.
31. The use of the binding compound of claim 1, or a TSLP-binding
fragment thereof, for the preparation of a medicament to treat
allergic inflammation.
32. The use of the binding compound of claim 1, or a TSLP-binding
fragment thereof, for the preparation of a medicament to treat
allergic rhinosinusitis, allergic asthma, allergic conjunctivitis,
or atopic dermatitis.
33. The use of the binding compound of claim 1, or a TSLP-binding
fragment thereof, for the preparation of a medicament to treat
asthma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/532,131, filed Jun. 25, 2012, which is a continuation of
U.S. application Ser. No. 12/515,915, now U.S. Pat. No. 8,232,372,
filed Sep. 17, 2009, which is the national stage filing under 35
U.S.C. .sctn.371 of International Patent Application No.
PCT/US2007/025531, filed Dec. 13, 2007, which claims the benefit of
U.S. Provisional Application Ser. No. 60/869,974, filed Dec. 14,
2006; the disclosures of which are incorporated herein by reference
in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The sequence listing of the present application was
submitted electronically via EFS-Web as an ASCII formatted sequence
listing with a file name "BP06556SeqListing_ST25.txt" creation date
of Mar. 3, 2010 and a size of 20.9 KB. This sequence listing
submitted via EFS-Web is part of the specification and is herein
incorporated by reference in its entirely.
FIELD OF THE INVENTION
[0003] The present invention relates generally to a thymic stromal
lymphopoietin (TSLP) specific antibody, and uses thereof,
particularly in inflammatory, and allergic inflammatory
disorders.
BACKGROUND OF THE INVENTION
[0004] The immune system functions to protect individuals from
infective agents, e.g., bacteria, multi-cellular organisms, and
viruses, as well as from cancers. This system includes several
types of lymphoid and myeloid cells such as monocytes, macrophages,
dendritic cells (DCs), eosinophils, T cells, B cells, and
neutrophils. These lymphoid and myeloid cells often produce
signaling proteins known as cytokines. The immune response includes
inflammation, i.e., the accumulation of immune cells systemically
or in a particular location of the body. In response to an
infective agent or foreign substance, immune cells secrete
cytokines which, in turn, modulate immune cell proliferation,
development, differentiation, or migration. An immune response can
produce pathological consequences, e.g., when it involves excessive
inflammation, as in allergic inflammatory disorders.
[0005] TSLP is an immune cytokine that induces dendritic
cell-mediated CD4+ T cell responses with a proallogenic phenotype
(Gilliet et al., J. Exp. Medicine 197(8): 1059-1063 (2003). TSLP is
involved in the initiation of allergic inflammation (Watanabe et
al., Nature Immunology 5: 426-434 (2004); Soumelis et al., Nature
Immunology 3: 673-680 (2002)).
[0006] Antibodies are being developed against a number of antigen
targets that are involved in immune diseases. The most significant
limitation in using antibodies as a therapeutic agent in vivo is
the immunogenicity of the antibodies. As most monoclonal antibodies
are derived from rodents, repeated use in humans results in the
generation of an immune response against the therapeutic antibody.
Such an immune response results in a loss of therapeutic efficacy
at a minimum and a potential fatal anaphylactic response at a
maximum. Initial efforts to reduce the immunogenicity of rodent
antibodies involved the production of chimeric antibodies, in which
mouse variable regions were fused with human constant regions. Liu
et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43. However, mice
injected with hybrids of human variable regions and mouse constant
regions develop a strong anti-antibody response directed against
the human variable region, suggesting that the retention of the
entire rodent Fv region in such chimeric antibodies may still
result in unwanted immunogenicity in patients.
[0007] It is generally believed that complementarity determining
region (CDR) loops of variable domains comprise the binding site of
antibody molecules. Therefore, the grafting of rodent CDR loops
onto human frameworks (i.e., humanization) was attempted to further
minimize rodent sequences. Jones et al. (1986) Nature 321:522;
Verhoeyen et al. (1988) Science 239:1534. However, CDR loop
exchanges may not uniformly result in an antibody with the same
binding properties as the antibody of origin. Changes in framework
residues (FR), residues involved in CDR loop support, in humanized
antibodies may also be required to preserve antigen binding
affinity. Kabat et al. (1991) J. Immunol. 147:1709. While the use
of CDR grafting and framework residue preservation in a number of
humanized antibody constructs has been reported, it is difficult to
predict if a particular sequence will result in the antibody with
the desired binding, and sometimes biological, properties. See,
e.g., Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029,
Gorman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4181, and
Hodgson (1991) Biotechnology (NY) 9:421-5.
[0008] The present invention provides an engineered TSLP antibody
and uses thereof to treat inflammatory, and particularly allergic
inflammatory, disorders.
SUMMARY OF THE INVENTION
[0009] The present invention provides a binding compound that
specifically binds human and cyno TSLP, comprising: at least one
antibody heavy chain variable region, or a TSLP-binding fragment
thereof, said heavy chain variable region comprising at least one
CDR sequence selected from the group consisting of SEQ ID NOs: 1, 2
and 3; or at least one antibody light chain variable region, or a
TSLP-binding fragment thereof, said light chain variable region
comprising at least one CDR sequence selected from the group
consisting of SEQ ID NOs: 4, 5 and 6.
[0010] The present invention also provides a binding compound that
specifically binds to human and cyno TSLP comprising at least one
antibody heavy chain variable region, or a TSLP-binding fragment
thereof, said heavy chain variable region comprising at least one
CDR sequence selected from the group consisting of SEQ ID NOs: 1, 2
and 3; and at least one antibody light chain variable region, or a
TSLP-binding fragment thereof, said light chain variable region
comprising at least one CDR sequence selected from the group
consisting of SEQ ID NOs: 4, 5 and 6.
[0011] In some embodiments, the antibody heavy chain variable
region, or TSLP-binding fragment thereof, comprises at least two
CDR sequences selected from the group consisting of SEQ ID NOs: 1,
2 and 3. In other embodiments, the antibody heavy chain variable
region, or TSLP-binding fragment thereof, has the three CDR
sequences set forth in SEQ ID NOs: 1, 2 and 3.
[0012] In some embodiments, the antibody light chain variable
region, or TSLP-binding fragment thereof, comprises at least two
CDR sequences selected from the group consisting of SEQ ID NOs: 4,
5 and 6. In other embodiments, the antibody light chain variable
region, or TSLP-binding fragment thereof, has the three CDR
sequences set forth in SEQ ID NOs: 4, 5 and 6.
[0013] The present invention also provides a binding compound that
specifically binds human and cyno TSLP, comprising: at least one
antibody heavy chain variable region, or a TSLP-binding fragment
thereof, said heavy chain variable region comprising: the CDR-H1 of
SEQ ID NO. 1, or a variant thereof; the CDR-H2 of SEQ ID NO. 2, or
a variant thereof; and the CDR-H3 of SEQ ID NO. 3, or a variant
thereof; or at least one antibody light chain variable region, or a
TSLP-binding fragment thereof, said light chain variable region
comprising: the CDR-L1 of SEQ ID NO. 4, or a variant thereof; the
CDR-L2 of SEQ ID NO. 5, or a variant thereof; and the CDR-L3 of SEQ
ID NO. 6, or a variant thereof. The present invention also provides
a binding compound that specifically binds human and cyno TSLP,
comprising at least one antibody heavy chain variable region, or a
TSLP-binding fragment thereof, said heavy chain variable region
comprising: the CDR-H1 of SEQ ID NO. 1, or a variant thereof; the
CDR-H2 of SEQ ID NO. 2, or a variant thereof; and the CDR-H3 of SEQ
ID NO. 3, or a variant thereof; and at least one antibody light
chain variable region, or a TSLP-binding fragment thereof, said
light chain variable region comprising: the CDR-L1 of SEQ ID NO. 4,
or a variant thereof; the CDR-L2 of SEQ ID NO. 5, or a variant
thereof; and the CDR-L3 of SEQ ID NO. 6, or a variant thereof. In
one embodiment, the variant comprises up to 20 conservatively
modified amino acid residues. In one embodiment, the variant
comprises up to 10 conservatively modified amino acid residues. In
one embodiment, the variant comprises up to 5 conservatively
modified amino acid residues. In one embodiment, the variant
comprises up to 3 conservatively modified amino acid residues.
[0014] In some embodiments of the above described binding
compounds, all or substantially all of the remainder of the heavy
chain variable region is all or substantially all a human Ig
region; and all or substantially all of the remainder of the light
chain variable region variable region is all or substantially all a
human Ig region. In preferred embodiments, the remainder of the
heavy chain variable region is human heavy chain amino acid
sequence; and the remainder of the light chain variable region is
human light chain amino acid sequence.
[0015] The present invention also provides a binding compound that
specifically binds human and cyno TSLP, comprising: a heavy chain
variable region comprising residues 1-116 of SEQ ID NO: 10 or a
variant thereof; and a light chain variable region comprising
residues 1-108 of SEQ ID NO: 12, or a variant thereof. In one
embodiment, the variant comprises up to 20 conservatively modified
amino acid residues. In one embodiment, the variant comprises up to
10 conservatively modified amino acid residues. In one embodiment,
the variant comprises up to 5 conservatively modified amino acid
residues. In one embodiment, the variant comprises up to 3
conservatively modified amino acid residues. In one embodiment, the
light chain variable region comprises a variant wherein the amino
acid at position 49 of SEQ ID NO:12 has been changed from Y to
K.
[0016] The present invention also provides a binding compound that
specifically binds human and cyno TSLP, comprising: a heavy chain
variable region comprising residues 1-116 of SEQ ID NO: 10; and a
light chain variable region comprising residues 1-108 of SEQ ID NO:
12.
[0017] The present invention also provides a binding compound that
specifically binds human and cyno TSLP, comprising: a heavy chain
variable region consisting essentially of residues 1-116 of SEQ ID
NO: 10; and a light chain variable region consisting essentially of
residues 1-108 of SEQ ID NO: 12.
[0018] The present invention also provides a binding compound that
specifically binds human and cyno TSLP, comprising: a heavy chain
variable region having at least 95%, 90%, 85% or 80% homology to
residues 1-116 of SEQ ID NO: 10; and/or a light chain variable
region having at least 95%, 90%, 85% or 80% homology to residues
1-108 of SEQ ID NO: 12. In one embodiment, the invention provides a
binding compound that specifically binds human and cyno TSLP,
comprising: a heavy chain variable region having at least 90%
homology to residues 1-116 of SEQ ID NO: 10; and a light chain
variable region having at least 90% homology to residues 1-108 of
SEQ ID NO: 12. In some embodiments, the heavy chain variable region
will comprise at least 95% homology to residues 1-116 of SEQ ID NO:
10; and the light chain variable region will comprise at least 95%
homology to residues 1-108 of SEQ ID NO: 12.
[0019] In some embodiments, the binding compounds of the invention
also comprise a heavy chain constant region and/or a light chain
constant region. In one embodiment, the heavy chain constant region
comprises a .gamma.1, .gamma.2, .gamma.3, or .gamma.4 human heavy
chain constant region or a variant thereof. In various embodiments
the light chain constant region comprises a lambda or a kappa human
light chain constant region.
[0020] In some embodiments, the binding compound of the invention
is an antibody or an antigen binding fragment thereof. In various
embodiments the antibody or fragment thereof of the present
invention is polyclonal, monoclonal, chimeric, cyno-ized, humanized
or fully human. In a preferred embodiment, the antibody is a
humanized antibody or a fragment thereof.
[0021] The present invention also contemplates that the binding
fragment is an antibody fragment selected from the group consisting
of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, and a diabody. The
present invention also contemplates that the binding compound is a
nanobody, an avimer, or an aptimer.
[0022] In a preferred embodiment, the binding compound is the
antibody produced by the hybridoma deposited as PTA-7951. In
another embodiment, the binding compound is not the antibody
produced by the hybridoma deposited as PTA-7951.
[0023] The invention also encompasses an antibody or antigen biding
fragment thereof that specifically binds to human and cyno-TSLP
comprising the heavy chain amino acid sequence of SEQ ID NO: 18, or
a variant thereof; and/or a light chain amino acid sequence of SEQ
ID NO:17 or a variant thereof. The invention also encompasses an
antibody or antigen biding fragment thereof that specifically binds
to human and cyno-TSLP comprising amino acids 19 to 472 of SEQ ID
NO: 18, or a variant thereof; and/or amino acids 20 to 233 of SEQ
ID NO:17 or a variant thereof. In one embodiment, the variant
comprises up to 20 conservatively modified amino acid residues. In
one embodiment, the variant comprises up to 10 conservatively
modified amino acid residues. In one embodiment, the variant
comprises up to 5 conservatively modified amino acid residues. In
one embodiment, the variant comprises up to 3 conservatively
modified amino acid residues.
[0024] The present invention also comprises a binding compound that
specifically binds human and cyno TSLP, wherein said binding
compound has a KD of about 2.1 pM or less, as measured using KinExA
technology and human TSLP as the ligand. The present invention also
comprises a binding compound that specifically binds human and cyno
TSLP, wherein said binding compound has a KD of 2.1 pM (+/-
two-fold), as measured using KinExA technology and human TSLP as
the ligand. In one embodiment, the binding compound is a humanized
anti-TSLP antibody or an antigen binding fragment thereof.
[0025] The present invention also comprises a binding compound that
specifically binds human and cyno TSLP, wherein said binding
compound has a KD of about 111 pM or less, as measured using
surface plasmon resonance and human TSLP as the ligand. The present
invention also comprises a binding compound that specifically binds
human and cyno TSLP, wherein said binding compound has a KD of 111
pM (+/- two-fold), as measured using surface plasmon resonance and
human TSLP as the ligand. In one embodiment, the binding compound
is a humanized anti-TSLP antibody or an antigen binding fragment
thereof.
[0026] The present invention also comprises a binding compound that
specifically binds human and cyno TSLP, wherein said binding
compound has an EC50 of about 7.6 nM or less. The present invention
also comprises a binding compound that specifically binds human and
cyno TSLP, wherein said binding compound has an EC50 of about 7.6
nM (+/- two-fold). (The EC50 refers to the concentration of binding
compound required to neutralize human TSPL to 50% of the level
observed in the absence of the binding compound.) In one
embodiment, the binding compound is a humanized anti-TSLP antibody
or an antigen binding fragment thereof.
[0027] The present invention also provides an isolated nucleic acid
encoding at least one of the heavy chain variable region or light
chain variable region of the binding compound of the invention.
Also provided is an expression vector comprising the nucleic acid
operably linked to control sequences that are recognized by a host
cell when the host cell is transfected with the vector. Also
provided are a host cell comprising the expression vector.
[0028] Also provided is a method of producing a polypeptide
comprising a heavy chain variable region or a light chain variable
region of the invention comprising: culturing the host cell of in
culture medium under conditions wherein the nucleic acid sequence
is expressed, thereby producing polypeptides comprising the light
and heavy chain variable regions; and recovering the polypeptides
from the host cell or culture medium.
[0029] The invention also provides a binding compound (for example
an antibody or antigen binding fragment thereof) that specifically
binds to the epitope on human TSLP that is bound by the antibody
produced by the hybridoma deposited as PTA-7951, wherein the
antibody that specifically binds to the epitope on human TSLP is
not the antibody produced by the hybridoma deposited as
PTA-7951.
[0030] The invention also comprises a binding compound (for example
an antibody or antigen binding fragment thereof) that competitively
inhibits binding by the antibody produced by the hybridoma
deposited as PTA-7951 to human TSLP, wherein the antibody that
competitively inhibits binding is not the antibody produced by the
hybridoma deposited as PTA-7951.
[0031] The invention also comprises a binding compound (for example
an antibody or antigen binding fragment thereof) that blocks
TSLP-mediated activity. TSLP mediated activities include, but are
not limited to, binding to its receptor, promoting the activation
of dendritic cells leading to proliferation or survival of TH2
cells, secretion of TH2 attracting chemokines by dendritic cells
such as TARC and MDC, production of pro-allergic cytokines such as
IL-4, IL-5, IL-13 and TNF-alpha. A number of assays can be employed
to determine whether a binding compound blocks TSLP-mediated
activity. These include the assays described n the Examples and
other assays, including those described in the art. See, e.g.,
Reche et al., J. Immunol. 167:336-43 (2001); Isaksen et al., J.
Immunol. 168:3288-94 (2002).
[0032] In one embodiment, the binding compound is able to block the
binding of TSLP to TSLPR in a cross-blocking assay.
[0033] The present invention encompasses a method of suppressing an
immune response in a human subject comprising administering to a
subject in need thereof an a binding compound that specifically
binds human and cyno TSLP, in an amount effective to block the
biological activity of TSLP. The present invention also
contemplates administering an additional immunosuppressive or
anti-inflammatory agent. In a preferred embodiment, the immune
response is asthma. In another preferred embodiment, the immune
response is allergic inflammation. In another preferred embodiment,
the allergic inflammation is allergic rhinosinusitis, allergic
asthma, allergic conjunctivitis, or atopic dermatitis. In another
preferred embodiment, the immune response is fibrosis, inflammatory
bowel disease or Hodgkin's lymphoma. In another preferred
embodiment, the binding compound is administered in combination
with another immunomodulatory agent.
[0034] The antibody or fragment thereof of the present invention
can be in a composition comprising the binding compound of the
invention, in combination with a pharmaceutically acceptable
carrier or diluent. In a further embodiment, the composition
further comprises an immunosuppressive or anti-inflammatory
agent.
[0035] The present invention also encompasses a composition
comprising a binding compound of the invention and a
pharmaceutically acceptable carrier or diluent.
[0036] The present invention encompasses an isolated nucleic acid
encoding the polypeptide sequence of the antibody or fragment
thereof of the present invention. The nucleic acid can be in an
expression vector operably linked to control sequences recognized
by a host cell transfected with the vector. Also encompassed is a
host cell comprising the vector, and a method of producing a
polypeptide comprising culturing the host cell under conditions
wherein the nucleic acid sequence is expressed, thereby producing
the polypeptide, and recovering the polypeptide from the host cell
or medium.
[0037] In various embodiments, the invention relates to medicaments
comprising the antibody or fragment thereof of the present
invention. For example, the invention encompasses the use of a
binding compound that specifically binds human and cyno TSLP (for
example, any one of the binding compounds according to the
invention) for the preparation of a medicament to treat suppress an
immune response. The present invention encompasses the use of a
binding compound that specifically binds human and cyno TSLP (for
example, any one of the binding compounds according to the
invention) for the preparation of a medicament to treat asthma. The
present invention encompasses the use of a binding compound that
specifically binds human and cyno TSLP (for example, any one of the
binding compounds according to the invention) for the preparation
of a medicament to treat an inflammatory disorder. In one
embodiment, the inflammatory disorder is an allergic inflammatory
disorder. In one embodiment, the allergic inflammatory disorder is
allergic rhinosinusitis, allergic asthma, allergic conjunctivitis,
or atopic dermatitis.
DETAILED DESCRIPTION
[0038] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise. All references cited herein are incorporated by
reference to the same extent as if each individual publication,
patent application, or patent, was specifically and individually
indicated to be incorporated by reference.
I. DEFINITIONS
[0039] "Activation," "stimulation," and "treatment," as it applies
to cells or to receptors, may have the same meaning, e.g.,
activation, stimulation, or treatment of a cell or receptor with a
ligand, unless indicated otherwise by the context or explicitly.
"Ligand" encompasses natural and synthetic ligands, e.g.,
cytokines, cytokine variants, analogues, muteins, and binding
compositions derived from antibodies. "Ligand" also encompasses
small molecules, e.g., peptide mimetics of cytokines and peptide
mimetics of antibodies. "Activation" can refer to cell activation
as regulated by internal mechanisms as well as by external or
environmental factors. "Response," e.g., of a cell, tissue, organ,
or organism, encompasses a change in biochemical or physiological
behavior, e.g., concentration, density, adhesion, or migration
within a biological compartment, rate of gene expression, or state
of differentiation, where the change is correlated with activation,
stimulation, or treatment, or with internal mechanisms such as
genetic programming.
[0040] "Activity" of a molecule may describe or refer to the
binding of the molecule to a ligand or to a receptor, to catalytic
activity; to the ability to stimulate gene expression or cell
signaling, differentiation, or maturation; to antigenic activity,
to the modulation of activities of other molecules, and the like.
"Activity" of a molecule may also refer to activity in modulating
or maintaining cell-to-cell interactions, e.g., adhesion, or
activity in maintaining a structure of a cell, e.g., cell membranes
or cytoskeleton. "Activity" can also mean specific activity, e.g.,
[catalytic activity]/[mg protein], or [immunological activity]/[mg
protein], concentration in a biological compartment, or the like.
"Proliferative activity" encompasses an activity that promotes,
that is necessary for, or that is specifically associated with,
e.g., normal cell division, as well as cancer, tumors, dysplasia,
cell transformation, metastasis, and angiogenesis.
[0041] "Administration" and "treatment," as it applies to an
animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid. "Administration"
and "treatment" can refer, e.g., to therapeutic, pharmacokinetic,
diagnostic, research, and experimental methods. Treatment of a cell
encompasses contact of a reagent to the cell, as well as contact of
a reagent to a fluid, where the fluid is in contact with the cell.
"Administration" and "treatment" also means in vitro and ex vivo
treatments, e.g., of a cell, by a reagent, diagnostic, binding
composition, or by another cell. "Treatment," as it applies to a
human, veterinary, or research subject, refers to therapeutic
treatment, prophylactic or preventative measures, to research and
diagnostic applications.
[0042] As used herein, the term "antibody" refers to any form of
antibody or fragment thereof that exhibits the desired biological
activity. Thus, it is used in the broadest sense and specifically
covers monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies), and antibody fragments so long as they
exhibit the desired biological activity.
[0043] As used herein, the term "TSLP binding fragment" or "binding
fragment thereof" encompasses a fragment or a derivative of an
antibody that still substantially retain its biological activity of
inhibiting TSLP activity. Therefore, the term "antibody fragment"
or TSLP binding fragment refers to a portion of a full length
antibody, generally the antigen binding or variable region thereof.
Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv
fragments; diabodies; linear antibodies; single-chain antibody
molecules, e.g., sc-Fv; domain antibodies; and multispecific
antibodies formed from antibody fragments. Typically, a binding
fragment or derivative retains at least 10% of its TSLP inhibitory
activity. Preferably, a binding fragment or derivative retains at
least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of
its TSLP inhibitory activity, although any binding fragment with
sufficient affinity to exert the desired biological effect will be
useful. It is also intended that a TSLP binding fragment can
include conservative amino acid substitutions that do not
substantially alter its biologic activity.
[0044] The term "monoclonal antibody", as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic epitope. In contrast, conventional (polyclonal) antibody
preparations typically include a multitude of antibodies directed
against (or specific for) different epitopes. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., (1975) Nature
256: 495, or may be made by recombinant DNA methods (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., (1991) Nature 352: 624-628 and Marks
et al., (1991) J. Mol. Biol. 222: 581-597, for example.
[0045] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; and Morrison
et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).
[0046] A "domain antibody" is an immunologically functional
immunoglobulin fragment containing only the variable region of a
heavy chain or the variable region of a light chain. In some
instances, two or more VH regions are covalently joined with a
peptide linker to create a bivalent domain antibody. The two VH
regions of a bivalent domain antibody may target the same or
different antigens.
[0047] A "bivalent antibody" comprises two antigen binding sites.
In some instances, the two binding sites have the same antigen
specificities. However, bivalent antibodies may be bispecific (see
below).
[0048] As used herein, the term "single-chain Fv" or "scFv"
antibody refers to antibody fragments comprising the VH and VL
domains of antibody, wherein these domains are present in a single
polypeptide chain. Generally, the Fv polypeptide further comprises
a polypeptide linker between the VH and VL domains which enables
the sFv to form the desired structure for antigen binding. For a
review of sFv, see Pluckthun (1994) The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New
York, pp. 269-315.
[0049] The monoclonal antibodies herein also include camelized
single domain antibodies. See, e.g., Muyldermans et al. (2001)
Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol.
Methods 231:25; WO 94/04678; WO 94/25591; U.S. Pat. No. 6,005,079,
which are hereby incorporated by reference in their entireties). In
one embodiment, the present invention provides single domain
antibodies comprising two VH domains with modifications such that
single domain antibodies are formed.
[0050] As used herein, the term "diabodies" refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy chain variable domain (VH) connected to a light
chain variable domain (VL) in the same polypeptide chain (VH-VL or
VL-VH). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc.
Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered
antibody variants generally see Holliger and Hudson (2005) Nat.
Biotechnol. 23:1126-1136.
[0051] As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from non-human (e.g.,
murine) antibodies as well as human antibodies. Such antibodies
contain minimal sequence derived from non-human immunoglobulin. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. The prefix "hu" or "hum" is added to antibody clone
designations when necessary to distinguish humanized antibodies
(e.g., "hu23B12") from parental rodent antibodies (e.g., rat 23B12,
or "r23B12"). The humanized forms of rodent antibodies will
generally comprise the same CDR sequences of the parental rodent
antibodies, although certain amino acid substitutions may be
included to increase affinity or increase stability of the
humanized antibody.
[0052] The antibodies of the present invention also include
antibodies with modified (or blocked) Fc regions to provide altered
effector functions. See, e.g., U.S. Pat. No. 5,624,821;
WO2003/086310; WO2005/120571; WO2006/0057702; Presta (2006) Adv.
Drug Delivery Rev. 58:640-656. Such modification can be used to
enhance or suppress various reactions of the immune system, with
possible beneficial effects in diagnosis and therapy. Alterations
of the Fc region include amino acid changes (substitutions,
deletions and insertions), glycosylation or deglycosylation, and
adding multiple Fc. Changes to the Fc can also alter the half-life
of antibodies in therapeutic antibodies, and a longer half-life
would result in less frequent dosing, with the concomitant
increased convenience and decreased use of material. See Presta
(2005) J. Allergy Clin. Immunol. 116:731 at 734-35.
[0053] The term "fully human antibody" refers to an antibody that
comprises human immunoglobulin protein sequences only. A fully
human antibody may contain murine carbohydrate chains if produced
in a mouse, in a mouse cell, or in a hybridoma derived from a mouse
cell. Similarly, "mouse antibody" refers to an antibody which
comprises mouse immunoglobulin sequences only.
[0054] As used herein, the term "hypervariable region" refers to
the amino acid residues of an antibody that are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (e.g.
residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the
light chain variable domain and residues 31-35 (CDRH1), 50-65
(CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain;
Kabat et al., (1991) Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md.) and/or those residues from a "hypervariable
loop" (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the
light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101
(H3) in the heavy chain variable domain; Chothia and L, (1987) J.
Mol. Biol. 196: 901-917). As used herein, the term "framework" or
"FR" residues refers to those variable domain residues other than
the hypervariable region residues defined herein as CDR residues.
The residue numbering above relates to the Kabat numbering system
and does not necessarily correspond in detail to the sequence
numbering in the accompanying Sequence Listing. See Tables 2 and 3,
in which sequence numbering is with reference to the Sequence
Listing.
[0055] "Binding" refers to an association of the binding
composition with a target where the association results in
reduction in the normal Brownian motion of the binding composition,
in cases where the binding composition can be dissolved or
suspended in solution.
[0056] "Binding compound" refers to a molecule that comprises one
or more amino acid sequences that specifically bind to human TSLP.
In one preferred embodiment, the binding compound is an antibody.
In another preferred embodiment, the binding compound comprises an
antibody fragment.
[0057] "Binding composition" refers to a TSLP-binding compound in
combination with a stabilizer, excipient, salt, buffer, solvent, or
additive, capable of binding to a target.
[0058] "Conservatively modified variants" or "conservative
substitution" refers to substitutions of amino acids are known to
those of skill in this art and may be made generally without
altering the biological activity of the resulting molecule. Those
of skill in this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson, et al.,
Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.
224 (4th Edition 1987)). Such exemplary substitutions are
preferably made in accordance with those set forth in Table 1 as
follows:
TABLE-US-00001 TABLE 1 Exemplary Conservative Amino Acid
Substitutions Original residue Conservative substitution Ala (A)
Gly; Ser Arg (R) Lys, His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C)
Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln
Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu;
Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser
Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0059] The terms "consists essentially of," or variations such as
"consist essentially of" or "consisting essentially of," as used
throughout the specification and claims, indicate the inclusion of
any recited elements or group of elements, and the optional
inclusion of other elements, of similar or different nature than
the recited elements, that do not materially change the basic or
novel properties of the specified dosage regimen, method, or
composition. As a nonlimiting example, an antibody or fragment
thereof that consists essentially of a recited amino acid sequence
may also include one or more amino acids, including substitutions
of one or more amino acid residues, that do not materially affect
the properties of the binding compound.
[0060] "Effective amount" encompasses an amount sufficient to
ameliorate or prevent a symptom or sign of the medical condition.
Effective amount also means an amount sufficient to allow or
facilitate diagnosis. An effective amount for a particular patient
or veterinary subject may vary depending on factors such as the
condition being treated, the overall health of the patient, the
method route and dose of administration and the severity of side
affects (see, e.g., U.S. Pat. No. 5,888,530 issued to Netti, et
al.). An effective amount can be the maximal dose or dosing
protocol that avoids significant side effects or toxic effects. The
effect will result in an improvement of a diagnostic measure or
parameter by at least 5%, usually by at least 10%, more usually at
least 20%, most usually at least 30%, preferably at least 40%, more
preferably at least 50%, most preferably at least 60%, ideally at
least 70%, more ideally at least 80%, and most ideally at least
90%, where 100% is defined as the diagnostic parameter shown by a
normal subject (see, e.g., Maynard, et al. (1996) A Handbook of
SOPs for Good Clinical Practice, Interpharm Press, Boca Raton,
Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch
Publ., London, UK).
[0061] "Exogenous" refers to substances that are produced outside
an organism, cell, or human body, depending on the context.
[0062] "Endogenous" refers to substances that are produced within a
cell, organism, or human body, depending on the context.
[0063] As used herein, the term "isolated nucleic acid molecule"
refers to a nucleic acid molecule that is identified and separated
from at least one contaminant nucleic acid molecule with which it
is ordinarily associated in the natural source of the antibody
nucleic acid. An isolated nucleic acid molecule is other than in
the form or setting in which it is found in nature. Isolated
nucleic acid molecules therefore are distinguished from the nucleic
acid molecule as it exists in natural cells. However, an isolated
nucleic acid molecule includes a nucleic acid molecule contained in
cells that ordinarily express the antibody where, for example, the
nucleic acid molecule is in a chromosomal location different from
that of natural cells.
[0064] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0065] 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 contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. 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.
[0066] 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. Mutant 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.
[0067] As used herein, "polymerase chain reaction" or "PCR" refers
to a procedure or technique in which minute amounts of a specific
piece of nucleic acid, RNA and/or DNA, are amplified as described
in, e.g., U.S. Pat. No. 4,683,195. Generally, sequence information
from the ends of the region of interest or beyond needs to be
available, such that oligonucleotide primers can be designed; these
primers will be identical or similar in sequence to opposite
strands of the template to be amplified. The 5' terminal
nucleotides of the two primers can coincide with the ends of the
amplified material. PCR can be used to amplify specific RNA
sequences, specific DNA sequences from total genomic DNA, and cDNA
transcribed from total cellular RNA, bacteriophage or plasmid
sequences, etc. See generally Mullis et al. (1987) Cold Spring
Harbor Symp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR
Technology (Stockton Press, N.Y.) As used herein, PCR is considered
to be one, but not the only, example of a nucleic acid polymerase
reaction method for amplifying a nucleic acid test sample
comprising the use of a known nucleic acid as a primer and a
nucleic acid polymerase to amplify or generate a specific piece of
nucleic acid.
[0068] As used herein, the term "germline sequence" refers to a
sequence of unrearranged immunoglobulin DNA sequences. Any suitable
source of unrearranged immunoglobulin DNA may be used.
[0069] "Inhibitors" are compounds that decrease, block, prevent,
delay activation, inactivate, desensitize, or down regulate, e.g.,
a gene, protein, ligand, receptor, or cell. An inhibitor may also
be defined as a composition that reduces, blocks, or inactivates a
constitutive activity. An "antagonist" is a compound that opposes
the actions of an agonist. An antagonist prevents, reduces,
inhibits, or neutralizes the activity of an agonist. An antagonist
can also prevent, inhibit, or reduce constitutive activity of a
target, e.g., a target receptor, even where there is no identified
agonist.
[0070] To examine the extent of inhibition, for example, samples or
assays comprising a given, e.g., protein, gene, cell, or organism,
are treated with a potential activating or inhibiting agent and are
compared to control samples without the agent. Control samples,
i.e., not treated with agent, are assigned a relative activity
value of 100% Inhibition is achieved when the activity value
relative to the control is about 90% or less, typically 85% or
less, more typically 80% or less, most typically 75% or less,
generally 70% or less, more generally 65% or less, most generally
60% or less, typically 55% or less, usually 50% or less, more
usually 45% or less, most usually 40% or less, preferably 35% or
less, more preferably 30% or less, still more preferably 25% or
less, and most preferably less than 25%.
[0071] Endpoints in inhibition can be monitored as follows
Inhibition, and response to treatment, e.g., of a cell,
physiological fluid, tissue, organ, and animal or human subject,
can be monitored by an endpoint. The endpoint may comprise a
predetermined quantity or percentage of, e.g., an indicia of
inflammation, oncogenicity, or cell degranulation or secretion,
such as the release of a cytokine, toxic oxygen, or a protease. The
endpoint may comprise, e.g., a predetermined quantity of ion flux
or transport; cell migration; cell adhesion; cell proliferation;
potential for metastasis; cell differentiation; and change in
phenotype, e.g., change in expression of gene relating to
inflammation, apoptosis, transformation, cell cycle, or metastasis
(see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158; Hood and
Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme, et al. (2003)
Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.
Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu Rev.
Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia
36:235-243; Stanimirovic and Satoh (2000) Brain Pathol.
10:113-126).
[0072] An endpoint of inhibition is generally 75% of the control or
less, preferably 50% of the control or less, more preferably 25% of
the control or less, and most preferably 10% of the control or
less. Generally, an endpoint of activation is at least 150% the
control, preferably at least two times the control, more preferably
at least four times the control, and most preferably at least 10
times the control.
[0073] "Specifically" or "selectively" binds, when referring to a
ligand/receptor, antibody/antigen, or other binding pair, indicates
a binding reaction which is determinative of the presence of the
protein, e.g., TSLP, in a heterogeneous population of proteins
and/or other biologics. Thus, under designated conditions, a
specified ligand/antigen binds to a particular receptor/antibody
and does not bind in a significant amount to other proteins present
in the sample.
[0074] The antibody, or binding composition derived from the
antigen-binding site of an antibody, of the contemplated method
binds to its antigen with an affinity that is at least two fold
greater, preferably at least ten times greater, more preferably at
least 20-times greater, and most preferably at least 100-times
greater than the affinity with unrelated antigens. In a preferred
embodiment the antibody will have an affinity that is greater than
about 109 liters/mol, as determined, e.g., by Scatchard analysis
(Munsen, et al. (1980) Analyt. Biochem. 107:220-239).
[0075] As used herein, the term "inflammatory disorder" refers to
any disease or disorder characterized by local inflammation at a
site of injury or infection and includes, without limitation,
allergic inflammation, autoimmune diseases, and other disorders
characterized by undesired immune cell accumulation at a local
tissue site.
[0076] As used herein, the term "immunomodulatory agent" refers to
natural or synthetic agents that suppress or modulate an immune
response. The immune response can be a humoral or cellular
response. Immunomodulatory agents encompass immunosuppressive or
anti-inflammatory agents.
[0077] "Immunosuppressive agents," "immunosuppressive drugs," or
"immunosuppressants" as used herein are therapeutics that are used
in immunosuppressive therapy to inhibit or prevent activity of the
immune system. Clinically they are used to prevent the rejection of
transplanted organs and tissues (e.g. bone marrow, heart, kidney,
liver), and/or in the treatment of autoimmune diseases or diseases
that are most likely of autoimmune origin (e.g. rheumatoid
arthritis, myasthenia gravis, systemic lupus erythematosus,
ulcerative colitis, multiple sclerosis). Immunosuppressive drugs
can be classified into four groups: glucocorticoids cytostatics;
antibodies (including Biological Response Modifiers or DMARDs);
drugs acting on immunophilins; other drugs, including known
chemotherpeutic agents used in the treatment of proliferative
disorders. For multiple sclerosis, in particular, the antibodies of
the present invention can be administered in conjunction with a new
class of myelin binding protein-like therapeutics, known as
copaxones.
[0078] "Anti-inflammatory agents" or "anti-inflammatory drugs", is
used to represent both steroidal and non-steroidal therapeutics.
Steroids, also known as corticosteroids, are drugs that closely
resemble cortisol, a hormone produced naturally by adrenal glands.
Steroids are used as the main treatment for certain inflammatory
conditions, such as: Systemic vasculitis (inflammation of blood
vessels); and Myositis (inflammation of muscle). Steroids might
also be used selectively to treat inflammatory conditions such as:
rheumatoid arthritis (chronic inflammatory arthritis occurring in
joints on both sides of the body); systemic lupus erythematosus (a
generalized disease caused by abnormal immune system function);
Sjogren's syndrome (chronic disorder that causes dry eyes and a dry
mouth).
[0079] Non-steroidal anti-inflammatory drugs, usually abbreviated
to NSAIDs, are drugs with analgesic, antipyretic and
anti-inflammatory effects--they reduce pain, fever and
inflammation. The term "non-steroidal" is used to distinguish these
drugs from steroids, which (amongst a broad range of other effects)
have a similar eicosanoid-depressing, anti-inflammatory action.
NSAIDs are generally indicated for the symptomatic relief of the
following conditions: rheumatoid arthritis; osteoarthritis;
inflammatory arthropathies (e.g. ankylosing spondylitis, psoriatic
arthritis, Reiter's syndrome); acute gout; dysmenorrhoea;
metastatic bone pain; headache and migraine; postoperative pain;
mild-to-moderate pain due to inflammation and tissue injury;
pyrexia; and renal colic. NSAIDs include salicylates, arlyalknoic
acids, 2-arylpropionic acids (profens), N-arylanthranilic acids
(fenamic acids), oxicams, coxibs, and sulphonanilides.
II. GENERAL
[0080] The present invention provides engineered anti-TSLP
antibodies and uses thereof to treat inflammatory, and particularly
allergic inflammatory, disorders. In a preferred embodiment, the
inflammatory disorder is asthma. In a preferred embodiment, the
allergic inflammatory disorder is allergic rhinosinusitis, allergic
asthma, allergic conjunctivitis, or atopic dermatitis. The present
invention also provides engineered anti-TSLP antibodies to treat
fibrosis, inflammatory bowel disease or Hodgkin's lymphoma.
[0081] TSLP is a member of the `long chain` family of hematopoetic
cytokines Insights into the structural basis of `long chain`
cytokine/receptor recognition have shown that although large areas
of protein surface are buried in formation of cytokine-receptor
complexes, the affinity of the interaction is dominated by a few,
often tightly clustered amino acid residues forming an energetic
`hot spot` in the center of the binding interface. The identity of
the residues that dominate the binding energy of a large
protein-protein interface has been termed the `functional epitope`.
The affinity of the interaction (and hence biological specificity)
is consequently defined by the structural complementarity of the
functional epitopes of ligand and receptor. Detailed mutagenesis
studies have shown that the most significant residues that make up
the functional epitopes of cytokines and receptors are hydrophobic
contacts involving either non-polar side chains such as tryptophan,
the aliphatic components of non-polar side chains or the
polypeptide backbone. The non-polar `core` is surrounded by a halo
of polar residues of lesser importance for binding energy. Kinetic
studies indicate that the primary role of the functional epitopes
is to stabilize protein-protein interaction by decreasing the
dissociation rate of the complex. It has been suggested that the
initial contact between cytokine and receptor is dominated by
random diffusion or `rolling` of protein surfaces producing many
unstable contacts. The complex is then stabilized when the
functional epitopes of the receptor and ligand engage (see, e.g.,
Bravo and Heath, supra).
III. GENERATION OF TSLP SPECIFIC ANTIBODIES
[0082] Any suitable method for generating monoclonal antibodies may
be used. For example, a recipient may be immunized with a linked or
unlinked (e.g. naturally occurring) form of the TSLP heterodimer,
or a fragment thereof. Any suitable method of immunization can be
used. Such methods can include adjuvants, other immunostimulants,
repeated booster immunizations, and the use of one or more
immunization routes.
[0083] Any suitable source of TSLP can be used as the immunogen for
the generation of the non-human antibody, of the compositions and
methods disclosed herein. Such forms include, but are not limited
to whole protein, including linked and naturally occurring
heterodimers, peptide(s), and epitopes, generated through
recombinant, synthetic, chemical or enzymatic degradation means
known in the art.
[0084] Any form of the antigen can be used to generate the antibody
that is sufficient to generate a biologically active antibody.
Thus, the eliciting antigen may be a single epitope, multiple
epitopes, or the entire protein alone or in combination with one or
more immunogenicity enhancing agents known in the art. The
eliciting antigen may be an isolated full-length protein, a cell
surface protein (e.g., immunizing with cells transfected with at
least a portion of the antigen), or a soluble protein (e.g.,
immunizing with only the extracellular domain portion of the
protein). The antigen may be produced in a genetically modified
cell. The DNA encoding the antigen may genomic or non-genomic
(e.g., cDNA) and encodes at least a portion of the extracellular
domain. As used herein, the term "portion" refers to the minimal
number of amino acids or nucleic acids, as appropriate, to
constitute an immunogenic epitope of the antigen of interest. Any
genetic vectors suitable for transformation of the cells of
interest may be employed, including but not limited to adenoviral
vectors, plasmids, and non-viral vectors, such as cationic
lipids.
[0085] Any suitable method can be used to elicit an antibody with
the desired biologic properties to inhibit TSLP. It is desirable to
prepare monoclonal antibodies (mAbs) from various mammalian hosts,
such as mice, rodents, primates, humans, etc. Description of
techniques for preparing such monoclonal antibodies may be found
in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th
ed.) Lange Medical Publications, Los Altos, Calif., and references
cited therein; Harlow and Lane (1988) Antibodies: A Laboratory
Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principles
and Practice (2d ed.) Academic Press, New York, N.Y. Thus,
monoclonal antibodies may be obtained by a variety of techniques
familiar to researchers skilled in the art. Typically, spleen cells
from an animal immunized with a desired antigen are immortalized,
commonly by fusion with a myeloma cell. See Kohler and Milstein
(1976) Eur. J. Immunol. 6:511-519. Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods known in the art. See,
e.g., Doyle, et al. (eds. 1994 and periodic supplements) Cell and
Tissue Culture: Laboratory Procedures, John Wiley and Sons, New
York, N.Y. Colonies arising from single immortalized cells are
screened for production of antibodies of the desired specificity
and affinity for the antigen, and yield of the monoclonal
antibodies produced by such cells may be enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate host. Alternatively, one may isolate DNA sequences which
encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according, e.g., to the
general protocol outlined by Huse, et al. (1989) Science
246:1275-1281.
[0086] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse et al.,
Science 246:1275-1281 (1989); and Ward et al., Nature 341:544-546
(1989). The polypeptides and antibodies of the present invention
may be used with or without modification, including chimeric or
humanized antibodies. Frequently, the polypeptides and antibodies
will be labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly U.S. Pat. No.
4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033; or made in transgenic mice, see Mendez et al.
(1997) Nature Genetics 15:146-156; also see Abgenix and Medarex
technologies.
[0087] Antibodies or binding compositions against predetermined
fragments of TSLP can be raised by immunization of animals with
conjugates of the polypeptide, fragments, peptides, or epitopes
with carrier proteins. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be
screened for binding to normal or defective TSLP. These monoclonal
antibodies will usually bind with at least a Kd of about 1 .mu.M,
more usually at least about 300 nM, typically at least about 30 nM,
preferably at least about 10 nM, more preferably at least about 3
nM or better, usually determined by ELISA.
IV. HUMANIZATION OF TSLP SPECIFIC ANTIBODIES
[0088] Any suitable non-human antibody can be used as a source for
the hypervariable region. Sources for non-human antibodies include,
but are not limited to, murine, Lagomorphs (including rabbits),
bovine, and primates. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which hypervariable
region residues of the recipient are replaced by hypervariable
region residues from a non-human species (donor antibody) such as
mouse, rat, rabbit or nonhuman primate having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance of
the desired biological activity. For further details, see Jones et
al. (1986) Nature 321:522-525; Reichmann et al. (1988) Nature
332:323-329; and Presta (1992) Curr. Op. Struct. Biol.
2:593-596.
[0089] Methods for recombinantly engineering antibodies have been
described, e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly
et al. (U.S. Pat. No. 4,816,567), Law et al. (European Patent
Application Publication No. 438 310) and Winter (European Patent
Application Publication No. 239400).
[0090] Amino acid sequence variants of humanized anti-TSLP antibody
are prepared by introducing appropriate nucleotide changes into the
humanized anti-TSLP antibody DNA, or by peptide synthesis. Such
variants include, for example, deletions from, and/or insertions
into and/or substitutions of, residues within the amino acid
sequences shown for the humanized anti-TSLP F(ab). Any combination
of deletion, insertion, and substitution is made to arrive at the
final construct, provided that the final construct possesses the
desired characteristics. The amino acid changes also may alter
post-translational processes of the humanized anti-TSLP antibody,
such as changing the number or position of glycosylation sites.
[0091] A useful method for identification of certain residues or
regions of the humanized anti-TSLP antibody polypeptide that are
preferred locations for mutagenesis is called "alanine scanning
mutagenesis," as described by Cunningham and Wells (1989) Science
244: 1081-1085. Here, a residue or group of target residues are
identified (e.g., charged residues such as Arg, Asp, His, Lys, and
Glu) and replaced by a neutral or negatively charged amino acid
(most preferably alanine or polyalanine) to affect the interaction
of the amino acids with TSLP antigen. The amino acid residues
demonstrating functional sensitivity to the substitutions then are
refined by introducing further or other variants at, or for, the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to analyze
the performance of a mutation at a given site, Ala scanning or
random mutagenesis is conducted at the target codon or region and
the expressed humanized anti-TSLP antibody variants are screened
for the desired activity.
[0092] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include humanized anti-TSLP
antibody with an N-terminal methionyl residue or the antibody fused
to an epitope tag. Other insertional variants of the humanized
anti-TSLP antibody molecule include the fusion to the N- or
C-terminus of humanized anti-TSLP antibody of an enzyme or a
polypeptide which increases the serum half-life of the
antibody.
[0093] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
humanized anti-TSLP antibody molecule removed and a different
residue inserted in its place. The sites of greatest interest for
substitutional mutagenesis include the hypervariable loops, but FR
alterations are also contemplated. Hypervariable region residues or
FR residues involved in antigen binding are generally substituted
in a relatively conservative manner.
[0094] 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 either N-linked or O-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. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.
[0095] 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). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0096] Yet another type of amino acid variant is the substitution
of residues to provide for greater chemical stability of the final
humanized antibody. For example, an asparagine (N) residue may be
changed to reduce the potential for formation of isoaspartate at
any NG sequences within a rodent CDR. In one embodiment, the
asparagine is changed to glutamine (Q). Isoaspartate formation may
debilitate or completely abrogate binding of an antibody to its
target antigen. Presta (2005) J. Allergy Clin. Immunol. 116:731 at
734. In addition, methionine residues in rodent CDRs may be changed
to reduce the possibility that the methionine sulfur would oxidize,
which could reduce antigen binding affinity and also contribute to
molecular heterogeneity in the final antibody preparation. Id. In
one embodiment, the methionine is changed to alanine (A).
Antibodies with such substitutions are subsequently screened to
ensure that the substitutions do not decrease TSLP binding affinity
to unacceptable levels.
[0097] Nucleic acid molecules encoding amino acid sequence variants
of humanized TSLP specific 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 humanized anti-TSLP
antibody.
[0098] Ordinarily, amino acid sequence variants of the humanized
anti-TSLP antibody will have an amino acid sequence having at least
75% amino acid sequence identity with the original humanized
antibody amino acid sequences of either the heavy or the light
chain more preferably at least 80%, more preferably at least 85%,
more preferably at least 90%, and most preferably at least 95%.
Identity or homology with respect to this sequence is defined
herein as the percentage of amino acid residues in the candidate
sequence that are identical with the humanized anti-TSLP residues,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
None of N-terminal, C-terminal, or internal extensions, deletions,
or insertions into the antibody sequence shall be construed as
affecting sequence identity or homology.
[0099] The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably,
the antibody is an IgG antibody. Any isotype of IgG can be used,
including IgG1, IgG2, IgG3, and IgG4. Variants of the IgG isotypes
are also contemplated. The humanized antibody may comprise
sequences from more than one class or isotype. Optimization of the
necessary constant domain sequences to generate the desired
biologic activity is readily achieved by screening the antibodies
in the biological assays described below.
[0100] Likewise, either class of light chain can be used in the
compositions and methods herein. Specifically, kappa, lambda, or
variants thereof are useful in the present compositions and
methods.
[0101] Any suitable portion of the CDR sequences from the non-human
antibody can be used. The CDR sequences can be mutagenized by
substitution, insertion or deletion of at least one residue such
that the CDR sequence is distinct from the human and non-human
antibody sequence employed. It is contemplated that such mutations
would be minimal. Typically, at least 75% of the humanized antibody
residues will correspond to those of the non-human CDR residues,
more often 90%, and most preferably greater than 95%.
[0102] Any suitable portion of the FR sequences from the human
antibody can be used. The FR sequences can be mutagenized by
substitution, insertion or deletion of at least one residue such
that the FR sequence is distinct from the human and non-human
antibody sequence employed. It is contemplated that such mutations
would be minimal. Typically, at least 75% of the humanized antibody
residues will correspond to those of the human FR residues, more
often 90%, and most preferably greater than 95%.
[0103] CDR and FR residues are determined according to the standard
sequence definition of Kabat. Kabat et al., Sequences of Proteins
of Immunological Interest, National Institutes of Health, Bethesda
Md. (1987). Table 2 provides sequence identifier information for
the rat (r23B12) and human (hu23B12) variable heavy chain CDRs.
Table 3 provides sequence identifier information for the r23B12 and
hu23B12 variable light chain CDRs.
TABLE-US-00002 TABLE 2 Heavy Chain Sequences and Domains ANTIBODY
SEQ ID V.sub.H HEAVY CHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-H1
CDR-H2 CDR-H3 r23B12 7 1-116 26-35 50-65 95-105 hu23B12 10 1-116
26-35 50-65 95-105
TABLE-US-00003 TABLE 3 Light Chain Sequences and Domains ANTIBODY
SEQ ID V.sub.L LIGHT CHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-L1
CDR-L2 CDR-L3 r23B12 8 1-108 24-34 50-56 89-97 hu23B12 12 1-108
24-34 50-56 89-97
[0104] The r23B12 and hu23B12 CDR-H1 sequence is GYIFTDYAMH (SEQ ID
NO. 1). The r23B12 and hu23B12 CDR-H2 sequence is TFIPLLDTSDYNQNFKG
(SEQ ID NO. 2). The r23B12 and hu23B12 CDR-H3 sequence is
MGVTHSYVMDA (SEQ ID NO. 3).
[0105] The r23B12 and hu23B12 CDR-L1 sequence is RASQPISISVH (SEQ
ID NO. 4). The r23B12 and hu23B12 CDR-L2 sequence is FASQSIS (SEQ
ID NO. 5). The r23B12 and hu23B12 CDR-L3 sequence is QQTFSLPYT (SEQ
ID NO. 6).
[0106] The r23B12 variable heavy chain amino acid sequence is
EEKLQQSGDD LVR PGAAVKMSCKASGYIFTDYAMHWVKQRPGQGLEWIGTFIPLLDTSDYNQN
FKGRATLTADKSSNTAYMELSRLTSEDSAVYYCARMGVTHSYVMDAWGQGASVT VSS (SEQ ID
NO. 7).
[0107] The r23B12 variable light chain amino acid sequence is
DIVLTQSPATLSV
TPGESVSLSCRASQPISISVHWFQQKSNESPRLLIKFASQSISGIPSRFSGSGSGTDFTL
NINRVESEDFSVYYCQQTFSLPYTFGTGTKLELKR (SEQ ID NO. 8).
[0108] The nucleic acid sequence for the hu23B12 variable heavy
chain is CAG GTG CAG CTG GTG CAG TCT GGC GCT GAG GTG AAG AAG CCT
GGC GCC TCC GTG AAG GTC TCC TGC AAG GCT TCT GGC TAC ATC TTC ACC GAC
TAC GCC ATG CAC TGG GTG CGG CAG GCC CCT GGC CAG GGG CTG GAG TGG ATG
GGT ACC TTC ATC CCT CTG CTG GAC ACC AGC GAC TAC AAC CAG AAC TTC AAG
GGC AGA GTC ACC ATG ACC ACA GAC ACA TCC ACC AGC ACA GCC TAC ATG GAG
CTG AGG AGC CTG AGA TCT GAC GAC ACC GCC GTG TAT TAC TGT GCC AGA ATG
GGA GTG ACC CAC AGC TAC GTG ATG GAT GCA TGG GGC CAG GGC ACC CTG GTC
ACC GTC TCC AGC (SEQ ID NO: 9), which encodes the hu23B12 variable
heavy chain amino acid sequence
QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYAMHWVRQAPGQGLEWMGTFIPLL
DTSDYNQNFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARMGVTHSYVM DAWGQGTLVTVSS
(SEQ ID NO. 10).
[0109] The nucleic acid sequence for the hu23B12 variable light
chain is GAA ATT GTG CTG ACT CAG AGC CCA GGC ACC CTG TCT CTG TCT
CCA GGC GAG AGA GCC ACC CTC TCC TGC CGG GCC AGC CAG CCC ATC TCC ATC
AGC GTG CAC TGG TAC CAG CAG AAA CCA GGA CAG GCT CCA AGG CTG CTG ATC
TAC TTT GCC TCC CAG AGC ATC TCC GGG ATC CCC GAT AGG TTC AGC GGA TCC
GGA TCT GGG ACA GAT TTC ACC CTC ACC ATC AGC AGA CTG GAG CCT GAA GAT
TTC GCA GTG TAT TAC TGT CAG CAG ACC TTC AGC CTG CCT TAC ACT TTC GGC
CAA GGG ACC AAG GTG GAG ATC AAG CGT (SEQ ID NO: 11), which encodes
the hu23B12 variable light chain amino acid sequence
EIVLTQSPGTLSLSPGERATLSCRASQPISISVHWYQQKPGQA
PRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTFSLPYTFGQG TKVEIKR
(SEQ ID NO. 12).
[0110] The nucleic acid sequence for the hu23B12 heavy chain is CAG
GTG CAG CTG GTG CAG TCT GGC GCT GAG GTG AAG AAG CCT GGC GCC TCC GTG
AAG GTC TCC TGC AAG GCT TCT GGC TAC ATC TTC ACC GAC TAC GCC ATG CAC
TGG GTG CGG CAG GCC CCT GGC CAG GGG CTG GAG TGG ATG GGT ACC TTC ATC
CCT CTG CTG GAC ACC AGC GAC TAC AAC CAG AAC TTC AAG GGC AGA GTC ACC
ATG ACC ACA GAC ACA TCC ACC AGC ACA GCC TAC ATG GAG CTG AGG AGC CTG
AGA TCT GAC GAC ACC GCC GTG TAT TAC TGT GCC AGA ATG GGA GTG ACC CAC
AGC TAC GTG ATG GAT GCA TGG GGC CAG GGC ACC CTG GTC ACC GTC TCC AGC
GCT AGC ACC AAG GGC CCA TCG GTC TTC CCC CTG GCA CCC TCC TCC AAG AGC
ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG GTC AAG GAC TAC TTC CCC
GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC AGC GGC GTG CAC
ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC TCC CTC AGC AGC GTG
GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG ACC TAC ATC TGC AAC GTG
AAT CAC AAG CCC AGC AAC ACC AAG GTG GAC AAG AAA GTT GAG CCC AAA TCT
TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAA CTC CTG GGG
GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC
TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC
CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC
AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC
GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC
AAG GTC TCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA
GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG
GAT GAG CTG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC
TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC
AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC
TAC AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC
TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC
CTC TCC CTG TCT CCG GGT AAA (SEQ ID NO: 13), which encodes the
hu23B12 heavy chain amino acid sequence
QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYAMHWVRQAPGQGLEWMGTFIPLL
DTSDYNQNFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARMGVTHSYVMDA
WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 14).
[0111] The nucleic acid sequence for the hu23B12 light chain is GAA
ATT GTG CTG ACT CAG AGC CCA GGC ACC CTG TCT CTG TCT CCA GGC GAG AGA
GCC ACC CTC TCC TGC CGG GCC AGC CAG CCC ATC TCC ATC AGC GTG CAC TGG
TAC CAG CAG AAA CCA GGA CAG GCT CCA AGG CTG CTG ATC TAC TTT GCC TCC
CAG AGC ATC TCC GGG ATC CCC GAT AGG TTC AGC GGA TCC GGA TCT GGG ACA
GAT TTC ACC CTC ACC ATC AGC AGA CTG GAG CCT GAA GAT TTC GCA GTG TAT
TAC TGT CAG CAG ACC TTC AGC CTG CCT TAC ACT TTC GGC CAA GGG ACC AAG
GTG GAG ATC AAG CGT ACG GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA
TCT GAT GAG CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT
AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC
CAA TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC
ACC TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA
CAC AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC
ACA AAG AGC TTC AAC AGG GGA GAG TGT (SEQ ID NO: 15), which encodes
the hu23B12 light chain amino acid sequence
EIVLTQSPGTLSLSPGERATLSCRASQPISISVHWYQQKPGQAPRLLI
YFASQSISGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTFSLPYTFGQGTKVEIKR
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:
16).
[0112] Also contemplated are chimeric antibodies. As noted above,
typical chimeric antibodies comprise a portion of the heavy and/or
light chain identical with or homologous to corresponding sequences
in antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl.
Acad. Sci. USA 81: 6851-6855).
[0113] Bispecific antibodies are also useful in the present methods
and compositions. As used herein, the term "bispecific antibody"
refers to an antibody, typically a monoclonal antibody, having
binding specificities for at least two different antigenic
epitopes. In one embodiment, the epitopes are from the same
antigen. In another embodiment, the epitopes are from two different
antigens. Methods for making bispecific antibodies are known in the
art. For example, bispecific antibodies can be produced
recombinantly using the co-expression of two immunoglobulin heavy
chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature
305: 537-39. Alternatively, bispecific antibodies can be prepared
using chemical linkage. See, e.g., Brennan, et al. (1985) Science
229: 81. Bispecific antibodies include bispecific antibody
fragments. See, e.g., Hollinger, et al. (1993) Proc. Natl. Acad.
Sci. U.S.A. 90: 6444-48, Gruber, et al., J. Immunol. 152: 5368
(1994).
[0114] In yet other embodiments, different constant domains may be
appended to the humanized VL and VH regions provided herein. For
example, if a particular intended use of an antibody (or fragment)
of the present invention were to call for altered effector
functions, a heavy chain constant domain other than IgG1 may be
used. Although IgG1 antibodies provide for long half-life and for
effector functions, such as complement activation and
antibody-dependent cellular cytotoxicity, such activities may not
be desirable for all uses of the antibody. In such instances an
IgG4 constant domain, for example, may be used.
V. BIOLOGICAL ACTIVITY OF HUMANIZED ANTI-TSLP
[0115] Antibodies having the characteristics identified herein as
being desirable in a humanized anti-TSLP antibody can be screened
for inhibitory biologic activity in vitro or for suitable binding
affinity. To screen for antibodies that bind to the epitope on
human TSLP bound by an antibody of interest (e.g., those which
block binding of the cytokine to its receptor), a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Antibodies that bind to the
same epitope are likely to cross-block in such assays, but not all
cross-blocking antibodies will necessarily bind at precisely the
same epitope since cross-blocking may result from steric hindrance
of antibody binding by antibodies bound at nearby, or even
overlapping, epitopes.
[0116] Alternatively, epitope mapping, e.g., as described in Champe
et al. (1995) J. Biol. Chem. 270:1388-1394, can be performed to
determine whether the antibody binds an epitope of interest.
"Alanine scanning mutagenesis," as described by Cunningham and
Wells (1989) Science 244: 1081-1085, or some other form of point
mutagenesis of amino acid residues in human TSLP may also be used
to determine the functional epitope for an anti-TSLP antibody of
the present invention. Mutagenesis studies, however, may also
reveal amino acid residues that are crucial to the overall
three-dimensional structure of TSLP but that are not directly
involved in antibody-antigen contacts, and thus other methods may
be necessary to confirm a functional epitope determined using this
method.
[0117] The epitope bound by a specific antibody may also be
determined by assessing binding of the antibody to peptides
comprising fragments of human TSLP. The amino acid sequence of
human TSLP is set forth in SEQ ID NO: 4 in WO 00/17362. A series of
overlapping peptides encompassing the sequence of TSLP may be
synthesized and screened for binding, e.g. in a direct ELISA, a
competitive ELISA (where the peptide is assessed for its ability to
prevent binding of an antibody to TSLP bound to a well of a
microtiter plate), or on a chip. Such peptide screening methods may
not be capable of detecting some discontinuous functional epitopes,
i.e. functional epitopes that involve amino acid residues that are
not contiguous along the primary sequence of the TSLP polypeptide
chain.
[0118] The epitope bound by antibodies of the present invention may
also be determined by structural methods, such as X-ray crystal
structure determination (e.g., WO2005/044853), molecular modeling
and nuclear magnetic resonance (NMR) spectroscopy, including NMR
determination of the H-D exchange rates of labile amide hydrogens
in TSLP when free and when bound in a complex with an antibody of
interest (Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347;
Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).
[0119] With regard to X-ray crystallography, crystallization may be
accomplished using any of the known methods in the art (e.g. Giege
et al. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur.
J. Biochem. 189:1-23), including microbatch (e.g. Chayen (1997)
Structure 5:1269-1274), hanging-drop vapor diffusion (e.g.
McPherson (1976) J. Biol. Chem. 251:6300-6303), seeding and
dialysis. It is desirable to use a protein preparation having a
concentration of at least about 1 mg/mL and preferably about 10
mg/mL to about 20 mg/mL. Crystallization may be best achieved in a
precipitant solution containing polyethylene glycol 1000-20,000
(PEG; average molecular weight ranging from about 1000 to about
20,000 Da), preferably about 5000 to about 7000 Da, more preferably
about 6000 Da, with concentrations ranging from about 10% to about
30% (w/v). It may also be desirable to include a protein
stabilizing agent, e.g. glycerol at a concentration ranging from
about 0.5% to about 20%. A suitable salt, such as sodium chloride,
lithium chloride or sodium citrate may also be desirable in the
precipitant solution, preferably in a concentration ranging from
about 1 mM to about 1000 mM. The precipitant is preferably buffered
to a pH of from about 3.0 to about 5.0, preferably about 4.0.
Specific buffers useful in the precipitant solution may vary and
are well-known in the art (Scopes, Protein Purification: Principles
and Practice, Third ed., (1994) Springer-Verlag, New York).
Examples of useful buffers include, but are not limited to, HEPES,
Tris, MES and acetate. Crystals may be grow at a wide range of
temperatures, including 2.degree. C., 4.degree. C., 8.degree. C.
and 26.degree. C.
[0120] Antibody:antigen crystals may be studied using well-known
X-ray diffraction techniques and may be refined using computer
software such as X-PLOR (Yale University, 1992, distributed by
Molecular Simulations, Inc.; see e.g. Blundell & Johnson (1985)
Meth. Enzymol. 114 & 115, H. W. Wyckoff et al., eds., Academic
Press; U.S. Patent Application Publication No. 2004/0014194), and
BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997)
Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et
al. (2000) Acta Cryst. D56:1313-1323), the disclosures of which are
hereby incorporated by reference in their entireties.
[0121] Additional antibodies binding to the same epitope as an
antibody of the present invention may be obtained, for example, by
screening of antibodies raised against TSLP for binding to the
epitope, or by immunization of an animal with a peptide comprising
a fragment of human TSLP comprising the epitope sequence.
Antibodies that bind to the same functional epitope might be
expected to exhibit similar biological activities, such as blocking
receptor binding, and such activities can be confirmed by
functional assays of the antibodies.
[0122] Antibody affinities (e.g. for human TSLP) may be determined
using standard analysis. Preferred humanized antibodies are those
which bind human TSLP with a KD value of no more than about
1.times.10-7; preferably no more than about 1.times.10-8; more
preferably no more than about 1.times.10-9; and most preferably no
more than about 1.times.10-10 M.
[0123] The antibodies and fragments thereof useful in the present
compositions and methods are biologically active antibodies and
fragments. As used herein, the term "biologically active" refers to
an antibody or antibody fragment that is capable of binding the
desired the antigenic epitope and directly or indirectly exerting a
biologic effect. Typically, these effects result from the failure
of TSLP to bind its receptor. In one embodiment, the antibody and
fragments thereof useful in the present compositions and methods
inhibit: hTSLP induced proliferation of a Baf-3 cell line
transfected with hTSLP-receptor and IL-7Ralpha; hTSLP induced
luciferase expression from a Baf-3 cell line transfected with the
TSLP-receptor and a luciferase reporter system; hTSLP induced TARC
secretion from human primary monocytes isolated from PBMCs; and
induction of Th2 differentiation.
[0124] As used herein, the term "specific" refers to the selective
binding of the antibody to the target antigen epitope. Antibodies
can be tested for specificity of binding by comparing binding to
TSLP to binding to irrelevant antigen or antigen mixture under a
given set of conditions. If the antibody binds to TSLP at least 10,
and preferably 50 times more than to irrelevant antigen or antigen
mixture then it is considered to be specific. An antibody that
"specifically binds" to TSLP does not bind to proteins that do not
comprise the TSLP-derived sequences, i.e. "specificity" as used
herein relates to TSLP specificity, and not any other sequences
that may be present in the protein in question. For example, as
used herein, an antibody that "specifically binds" to TSLP will
typically bind to FLAG-h TSLP, which is a fusion protein comprising
TSLP and a FLAG.RTM. peptide tag, but it does not bind to the
FLAG.RTM. peptide tag alone or when it is fused to a protein other
than TSLP.
VI. PHARMACEUTICAL COMPOSITIONS
[0125] To prepare pharmaceutical or sterile compositions, the
antibody or fragment thereof is admixed with a pharmaceutically
acceptable carrier or excipient, see, e.g., Remington's
Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary,
Mack Publishing Company, Easton, Pa. (1984). Formulations of
therapeutic and diagnostic agents may be prepared by mixing with
physiologically acceptable carriers, excipients, or stabilizers in
the form of, e.g., lyophilized powders, slurries, aqueous solutions
or suspensions (see, e.g., Hardman, et al. (2001) Goodman and
Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill,
New York, N.Y.; Gennaro (2000) Remington: The Science and Practice
of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;
Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral
Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman,
et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems,
Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity
and Safety, Marcel Dekker, Inc., New York, N.Y.).
[0126] Toxicity and therapeutic efficacy of the antibody
compositions, administered alone or in combination with an
immunosuppressive agent, can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50%
of the population). The dose ratio between toxic and therapeutic
effects is the therapeutic index and it can be expressed as the
ratio between LD50 and ED50. Antibodies exhibiting high therapeutic
indices are preferred. The data obtained from these cell culture
assays and animal studies can be used in formulating a range of
dosage for use in humans. The dosage of such compounds lies
preferably within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage may vary
within this range depending upon the dosage form employed and the
route of administration utilized.
[0127] Suitable routes of administration include parenteral
administration, such as intramuscular, intravenous, or subcutaneous
administration. Administration of antibody used in the
pharmaceutical composition or to practice the method of the present
invention can be carried out in a variety of conventional ways,
such as oral ingestion, inhalation, topical application or
cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial
or intravenous injection. In one embodiment, the binding compound
of the invention is administered intravenously. In another
embodiment, the binding compound of the invention is administered
subcutaneously.
[0128] Alternately, one may administer the antibody in a local
rather than systemic manner, for example, via injection of the
antibody directly into an arthritic joint or pathogen-induced
lesion characterized by immunopathology, often in a depot or
sustained release formulation. Furthermore, one may administer the
antibody in a targeted drug delivery system, for example, in a
liposome coated with a tissue-specific antibody, targeting, for
example, arthritic joint or pathogen-induced lesion characterized
by immunopathology. The liposomes will be targeted to and taken up
selectively by the afflicted tissue.
[0129] Selecting an administration regimen for a therapeutic
depends on several factors, including the serum or tissue turnover
rate of the entity, the level of symptoms, the immunogenicity of
the entity, and the accessibility of the target cells in the
biological matrix. Preferably, an administration regimen maximizes
the amount of therapeutic delivered to the patient consistent with
an acceptable level of side effects. Accordingly, the amount of
biologic delivered depends in part on the particular entity and the
severity of the condition being treated. Guidance in selecting
appropriate doses of antibodies, cytokines, and small molecules are
available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios
Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)
Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New
York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide
Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.;
Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom, et al.
(1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New
Engl. J. Med. 344:783-792; Beniaminovitz, et al. (2000) New Engl.
J. Med. 342:613-619; Ghosh, et al. (2003) New Engl. J. Med.
348:24-32; Lipsky, et al. (2000) New Engl. J. Med.
343:1594-1602).
[0130] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced. Preferably, a biologic that will be used is
derived from the same species as the animal targeted for treatment,
thereby minimizing an inflammatory, autoimmune, or proliferative
response to the reagent.
[0131] Antibodies, antibody fragments, and cytokines can be
provided by continuous infusion, or by doses at intervals of, e.g.,
one day, one week, or 1-7 times per week. Doses may be provided
intravenously, subcutaneously, topically, orally, nasally,
rectally, intramuscular, intracerebrally, intraspinally, or by
inhalation. A preferred dose protocol is one involving the maximal
dose or dose frequency that avoids significant undesirable side
effects. A total weekly dose is generally at least 0.05 .mu.g/kg
body weight, more generally at least 0.2 .mu.g/kg, most generally
at least 0.5 .mu.g/kg, typically at least 1 .mu.g/kg, more
typically at least 10 .mu.g/kg, most typically at least 100
.mu.g/kg, preferably at least 0.2 mg/kg, more preferably at least
1.0 mg/kg, most preferably at least 2.0 mg/kg, optimally at least
10 mg/kg, more optimally at least 25 mg/kg, and most optimally at
least 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J. Med.
349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;
Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456;
Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144).
The desired dose of a small molecule therapeutic, e.g., a peptide
mimetic, natural product, or organic chemical, is about the same as
for an antibody or polypeptide, on a moles/kg basis.
[0132] As used herein, "inhibit" or "treat" or "treatment" includes
a postponement of development of the symptoms associated with
autoimmune disease or pathogen-induced immunopathology and/or a
reduction in the severity of such symptoms that will or are
expected to develop. The terms further include ameliorating
existing uncontrolled or unwanted autoimmune-related or
pathogen-induced immunopathology symptoms, preventing additional
symptoms, and ameliorating or preventing the underlying causes of
such symptoms. Thus, the terms denote that a beneficial result has
been conferred on a vertebrate subject with an inflammatory
disease.
[0133] As used herein, the term "therapeutically effective amount"
or "effective amount" refers to an amount of an anti-TSLP antibody
or fragment thereof, that when administered alone or in combination
with an additional therapeutic agent to a cell, tissue, or subject
is effective to prevent or ameliorate the autoimmune disease or
pathogen-induced immunopathology associated disease or condition or
the progression of the disease. A therapeutically effective dose
further refers to that amount of the compound sufficient to result
in amelioration of symptoms, e.g., treatment, healing, prevention
or amelioration of the relevant medical condition, or an increase
in rate of treatment, healing, prevention or amelioration of such
conditions. When applied to an individual active ingredient
administered alone, a therapeutically effective dose refers to that
ingredient alone. When applied to a combination, a therapeutically
effective dose refers to combined amounts of the active ingredients
that result in the therapeutic effect, whether administered in
combination, serially or simultaneously. An effective amount of
therapeutic will decrease the symptoms typically by at least 10%;
usually by at least 20%; preferably at least about 30%; more
preferably at least 40%, and most preferably by at least 50%.
[0134] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are well known in the art, see,
e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New
York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics
for Advanced Practice: A Practical Approach, Lippincott, Williams
& Wilkins, Phila., PA; Chabner and Longo (eds.) (2001) Cancer
Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins,
Phila., PA. The pharmaceutical composition of the invention may
also contain other immunosuppressive or immunomodulating agents.
Any suitable immunosuppressive agent can be employed, including but
not limited to anti-inflammatory agents, corticosteroids,
cyclosporine, tacrolimus (i.e., FK-506), sirolimus, interferons,
soluble cytokine receptors (e.g., sTNRF and sIL-1R), agents that
neutralize cytokine activity (e.g., inflixmab, etanercept),
mycophenolate mofetil, 15-deoxyspergualin, thalidomide, glatiramer,
azathioprine, leflunomide, cyclophosphamide, methotrexate, and the
like. The pharmaceutical composition can also be employed with
other therapeutic modalities such as phototherapy and
radiation.
[0135] Typical veterinary, experimental, or research subjects
include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs,
horses, and humans
VII. ANTIBODY PRODUCTION
[0136] For recombinant production of the antibodies of the present
invention, the nucleic acids encoding the two chains are isolated
and inserted into one or more replicable vectors for further
cloning (amplification of the DNA) or for expression. DNA encoding
the monoclonal antibody is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of the antibody). Many vectors are available. The
vector components generally include, but are not limited to, one or
more of the following: a signal sequence, an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a
transcription termination sequence. In one embodiment, both the
light and heavy chains of the humanized anti-TSLP antibody of the
present invention are expressed from the same vector, e.g. a
plasmid or an adenoviral vector.
[0137] Antibodies of the present invention may be produced by any
method known in the art. In one embodiment, antibodies are
expressed in mammalian or insect cells in culture, such as chinese
hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells,
mouse myeloma NSO cells, baby hamster kidney (BHK) cells,
Spodoptera frugiperda ovarian (Sf9) cells. In one embodiment,
antibodies secreted from CHO cells are recovered and purified by
standard chromatographic methods, such as protein A, cation
exchange, anion exchange, hydrophobic interaction, and
hydroxyapatite chromatography. Resulting antibodies are
concentrated and stored in 20 mM sodium acetate, pH 5.5.
[0138] In another embodiment, the antibodies of the present
invention are produced in yeast according to the methods described
in WO2005/040395. Briefly, vectors encoding the individual light or
heavy chains of an antibody of interest are introduced into
different yeast haploid cells, e.g. different mating types of the
yeast Pichia pastoris, which yeast haploid cells are optionally
complementary auxotrophs. The transformed haploid yeast cells can
then be mated or fused to give a diploid yeast cell capable of
producing both the heavy and the light chains. The diploid strain
is then able to secret the fully assembled and biologically active
antibody. The relative expression levels of the two chains can be
optimized, for example, by using vectors with different copy
number, using transcriptional promoters of different strengths, or
inducing expression from inducible promoters driving transcription
of the genes encoding one or both chains.
[0139] In one embodiment, the respective heavy and light chains of
the anti-TSLP antibody are introduced into yeast haploid cells to
create a library of haploid yeast strains of one mating type
expressing a plurality of light chains, and a library of haploid
yeast strains of a different mating type expressing a plurality of
heavy chains. These libraries of haploid strains can be mated (or
fused as spheroplasts) to produce a series of diploid yeast cells
expressing a combinatorial library of antibodies comprised of the
various possible permutations of light and heavy chains. The
combinatorial library of antibodies can then be screened to
determine whether any of the antibodies has properties that are
superior (e.g. higher affinity for TSLP) to those of the original
antibodies. See. e.g., WO2005/040395.
[0140] In another embodiment, antibodies of the present invention
are human domain antibodies in which portions of an antibody
variable domain are linked in a polypeptide of molecular weight
approximately 13 kDa. See, e.g., U.S. Pat. Publication No.
2004/0110941. Such single domain, low molecular weight agents
provide numerous advantages in terms of ease of synthesis,
stability, and route of administration.
VIII. USES
[0141] The present invention provides methods for using engineered
anti-TSLP for the treatment and diagnosis of inflammatory
disorders.
[0142] In a preferred embodiment, the inflammatory disorder is
asthma.
[0143] In another preferred embodiment, the inflammatory disorder
is an allergic inflammatory disorder. In a preferred embodiment,
the allergic inflammatory disorder is allergic rhinosinusitis,
allergic asthma, allergic conjunctivitis, or atopic dermatitis.
[0144] The present invention provides methods for using engineered
anti-TSLP for the treatment and diagnosis of fibrosis, inflammatory
bowel disease, Hodgkin's lymphoma, respiratory viral infections or
other viral infections, rheumatoid arthritis, or any other disorder
characterized by inflammation at the site of injury.
[0145] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to the specific embodiments.
[0146] All citations herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0147] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
Example 1
General Methods
[0148] Standard methods in molecular biology are described
(Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;
Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993)
Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).
Standard methods also appear in Ausbel et al. (2001) Current
Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York, N.Y., which describes cloning in bacterial cells and
DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast
(Vol. 2), glycoconjugates and protein expression (Vol. 3), and
bioinformatics (Vol. 4).
[0149] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies are described (Coligan et al. (2001) Current
Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane,
supra). Standard techniques for characterizing ligand/receptor
interactions are available (see, e.g., Coligan et al. (2001)
Current Protcols in Immunology, Vol. 4, John Wiley, Inc., New
York).
[0150] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2nd
ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow
Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probes
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0151] Standard methods of histology of the immune system are
described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt, et al. (2000) Color Atlas of Histology, Lippincott,
Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic
Histology:Text and Atlas, McGraw-Hill, New York, N.Y.).
[0152] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence alignments, are
available (see, e.g., GenBank, Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San
Diego, Calif.); DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.);
Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000)
Bioinformatics Applications Note 16:741-742; Wren et al. (2002)
Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur.
J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.
14:4683-4690).
Example 2
Humanization of Anti-Human TSLP Antibodies
[0153] Rat anti-human TSLP antibody 23B12 is produced by the
hybridoma deposited at the American Type Culture Collection (10801
University Blvd., Manassas, Va. 20110-2209 USA) ("ATCC") with the
patent deposit designation "PTA-7951." The hybridoma was deposited
on Oct. 26, 2006 under the conditions of the Budapest Treaty, and
received accession number PTA-7951. The humanization of rat
anti-human TSLP antibody 23B12 was performed as essentially as
described in PCT patent application publications WO 2005/047324 and
WO 2005/047326, which are incorporated by reference.
[0154] Variable light and heavy domains of the anti-TSLP monoclonal
antibody (23B12) were cloned and fused to a human kappa light chain
(CL domain) and human IgG1 heavy chain (CH1-hinge-CH2-CH3),
respectively.
[0155] The amino acid sequence of the non-human VH domain was
compared to a group of three human VH germline amino acid
sequences; one representative from each of subgroups IGHV1, IGHV3
and IGHV4. The VH subgroups are listed in M.-P. Lefranc,
"Nomenclature of the Human Immunoglobulin Heavy (IGH) Genes",
Experimental and Clinical Immunogenetics, 18:100-116, 2001. Rat
23B12 antibody scored highest against human heavy chain germline
DP-14 in subgroup VH1.
[0156] For the rat 23B12 antibody, the VL sequence was of the kappa
subclass of VL. The amino acid sequence of the non-human VL domain
was compared to a group of four human VL kappa germline amino acid
sequences. The group of four is comprised of one representative
from each of four established human VL subgroups listed in V.
Barbie & M.-P. Lefranc, "The Human Immunoglobulin Kappa
Variable (IGKV) Genes and Joining (IGKJ) Segments", Experimental
and Clinical Immunogenetics, 15:171-183, 1998 and M.-P. Lefranc,
"Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes",
Experimental and Clinical Immunogenetics, 18:161-174, 2001. The
four subgroups also correspond to the four subgroups listed in
Kabat et al. "Sequences of Proteins of Immunological Interest",
U.S. Department of Health and Human Services, NIH Pub. 91-3242, 5th
Ed., 1991, pp. 103-130. Rat 23B12 antibody scored highest against
human light chain germline Z-A27 in subgroup VLkIII.
[0157] Once the target amino acid sequences of the variable heavy
and light chains were determined, plasmids encoding the full-length
humanized antibody were generated. Starting with a plasmid encoding
a humanized anti-IL-23 antibody having VH1 DP-14 germline framework
and a separate plasmid encoding a humanized anti-IGFR antibody
having VLkIII Z-A27 germline framework, the plasmids were altered
using Kunkel mutagenesis (see, e.g., Kunkel T A. (1985) Proc. Natl.
Acad. Sci. U.S.A 82:488-492) to change the DNA sequence to the
target humanized 23B12 sequence. Simultaneously, codon optimization
was incorporated into the changes to provide for potentially
optimal expression. The humanized heavy and light variable chain
amino acid sequences, are set forth in SEQ ID NOs: 10 and 12. The
full-length humanized heavy and light chain amino acid sequences,
are set forth in SEQ ID NOs: 14 and 16. A variant of the light
chain was also created, wherein the variant comprised a K (rather
than a Y) at amino acid position 49 of SEQ ID NO:12 or SEQ ID
NO:16.
Example 3
Determining the Equilibrium Dissociation Constant (K.sub.D) for
Humanized Anti-Human TSLP Using KinExA Technology
[0158] The equilibrium dissociation constant (K.sub.D) was
determined using the KinExA 3000 instrument (Sapidyne Instruments
Inc., www.sapidyne.com). The KinExA uses the principle of the
Kinetic Exclusion Assay method based on measuring the concentration
of uncomplexed antibody in a mixture of antibody, antigen and
antibody-antigen complex. The concentration of free antibody is
measured by exposing the mixture to a solid-phase immobilized
antigen for a very brief period of time. In practice, this is
accomplished by flowing the solution phase antigen-antibody mixture
past antigen-coated particles trapped in a flow cell. Data
generated by the instrument are analyzed using custom software.
Equilibrium constants are calculated using a mathematical theory
based on the following assumptions:
[0159] 1. The binding follows the reversible binding equation for
equilibrium:
k.sub.on[Ab][Ag]=k.sub.off[AbAg]
[0160] 2. Antibody and antigen bind 1:1 and total antibody equals
antigen-antibody complex plus free antibody.
[0161] 3. Instrument signal is linearly related to free antibody
concentration.
[0162] Materials
[0163] Antibodies:
[0164] Rat anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330)
[0165] Rat anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330A)
[0166] Humanized anti hu TSLP mAb 23B12 (VL Y49)
[0167] Humanized anti hu TSLP mAb 23B12 (VL K49)
[0168] Humanized anti hu TSLP mAb 23B12 (VL Y49)
[0169] Antigens:
[0170] Recombinant human TSLP (SPB Lot P345)
[0171] Recombinant human TSLP (SPB Lot P367)
[0172] Recombinant human TSLP (R&D, Cat.N. 1398-TS/CF, Lot IDK
015031)
[0173] Biotinylated Antigens:
[0174] Biotinylated human TSLP (SPB Lot p367AC)
[0175] Biotinylated human TSLP (SPB Lot p367AA)
[0176] Biotinylated human TSLP (SPB Lot 38ABMA)
[0177] Other Reagents:
[0178] PMMA particles, 98 micron (Sapidyne, Cat No. 440198)
[0179] Neutravidin (Pierce, Cat No. 31000)
[0180] Cy5-conjugated Goat anti-rat IgG (H+ L) (Jackson
Immunoresearch Laboratories Cat. No 112-175-167, Lot 60306)
[0181] Cy5-conjugated Goat anti-HuIgG (H+L) (Jackson Immunoresearch
Laboratories Cat. No 109-175-088, lot 49069 and lot 58552)
[0182] Experimental Conditions:
[0183] PMMA particles were coated with biotinylated human TSLP
according to Sapidyne "Protocol for coating PMMA particles with
biotinylated ligands having short or nonexistent linker arms". All
experimental procedures were done according to the KinExA 3000
manual. All runs were done in duplicate.
[0184] For Rat anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330) the
following conditions were used:
[0185] Sample volume: 2 mL
[0186] Sample flow rate: 0.25 mL/min
[0187] Label volume: 1 mL
[0188] Label flow rate: 0.25 mL/min
[0189] Antibody conc.: 0.1 nM
[0190] Highest antigen conc.: 10 nM
[0191] Lowest antigen conc.: 10 pM
[0192] For Rat anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330A) the
following conditions were used:
[0193] Sample volume: 4 mL
[0194] Sample flow rate: 0.25 mL/min
[0195] Label volume: 1 mL
[0196] Label flow rate: 0.25 mL/min
[0197] Antibody conc.: 0.05 nM
[0198] Highest antigen conc.: 0.5 nM
[0199] Lowest antigen conc.: 0.5 pM
[0200] For Humanized anti hu TSLP mAbs the following conditions
were used:
[0201] Sample volume: 2 mL
[0202] Sample flow rate: 0.25 mL/min
[0203] Label volume: 1 mL
[0204] Label flow rate: 0.25 mL/min
[0205] Antibody conc.: 0.02 nM
[0206] Highest antigen conc.: 0.2 nM
[0207] Lowest antigen conc.: 0.2 pM
[0208] Two-fold serial dilutions of the antigen were prepared and
mixed with the antibody at constant concentration. The mixture was
incubated for 2 hours at 25.degree. C. to equilibrate.
[0209] Table 4 shows the results of the KinExA analysis.
TABLE-US-00004 TABLE 4 K.sub.D Values Determined by KinExa mAb TSLP
Expression K.sub.D (pM) rat 23B12.H8.H4 human HEK293 0.22 rat
23B12.H8.H4 human E. coli 0.47 hu23B12(VL Y49) human HEK293 2.1
hu23B12(VL K49) human HEK293 1.0 hu23B12(VL Y49) human E. coli
1.7
Example 4
Determining the EC.sub.50 for Humanized Anti-Human TSLP Using
ELISA
[0210] The ELISA measures the EC50 of rat 23B12 purified from
hybridoma supernatant or recombinant humanized 23B12 IgG1 binding
to either adenovirus-derived human TSLP (S-P Biopharma) or E.
coli-derived human TSLP (S-P Biopharma or R&D 1398-TS).
[0211] Materials:
[0212] Nunc Maxisorb 96 well Immunoplate cert. (Nunc #439454)
[0213] 0.times. phosphate-buffered saline (PBS), pH 7.4 (Fisher #
BP399-20)
[0214] 20.times. Tris Buffered Saline (TBS), pH 7.4 (Technova
#1680)
[0215] Tween-20, enzyme grade (Fisher # BP337-500)
[0216] 500 mM EDTA (Technova # E0306)
[0217] Albumin, bovine serum RIA grade (Sigma # A7888)
[0218] Coating Buffer: 1 .mu.g/mL TSLP in PBS at 100 .mu.L/well
[0219] Detection Reagent:
[0220] HRP-F(ab)'2 goat anti-human IgG H+L (Jackson
#109-036-088);
[0221] HRP-F(ab)'2 goat anti-rat IgG H+L (Jackson #112-032-072)
[0222] Substrate & Stop Solutions:
[0223] TMB Microwell Peroxidase Substrate System 2C (Kirkegaard
& Perry Labs #50-76-00) 1:1; 1M H.sub.3PO.sub.4 0.1 mL/well
[0224] ABTS (Kirkegaard & Perry Labs #50-66-06) 100
.mu.L/well
[0225] ABTS Peroxidase stop solution (Kirkegaard & Perry Labs
#50-085-02) 5.times. concentrate diluter 1:5 in MIlli-Q water, 100
.mu.L/well
[0226] ELISA Diluent and Assay Buffer:
[0227] 50 mM TBS or PBS; 0.5% BSA; 0.05% Tween-20; 4 mM EDTA
[0228] ELISA Wash Buffer:
[0229] 50 mM TBS or PBS; 0.05% Tween-20; 4 mM EDTA
[0230] Equipment:
[0231] Skatron Scanwasher300.TM.
[0232] Molecular Devices VersaMax.TM. microplate reader
[0233] Protocol
[0234] Coating of plates was performed as follows: TSLP (100 or 200
ng per well) in PBS was incubated at 40 C overnight. Plates were
washed with 1 cycle (4 washes/cycle) on a Skatron plate washer,
blocked by addition of 0.2 mL/well ELISA assay buffer, incubated
for 60 min at 25.degree. C. on an orbital shaker and then washed
for 1 cycle. Antibody was then titrated across a row of eight wells
in the range of 1000 ng/mL to 0.4572 ng/mL using serial 3-fold
dilutions and incubated for 90 min. at 25.degree. C. on an orbital
shaker. Plates were washed for 1 cycle, HRP-goat F(ab')2 anti-human
or anti-rat IgG (H+L) (1:5,000 dilution) was added at 0.1 mL/well
and incubated for 60 min at 25.degree. C. on an orbital shaker.
Plates were washed for 2 cycles with plate rotation between cycles.
TMB or ABTS substrate was added at 0.1 mL/well and incubated 5 min
on orbital shaker. Stop solution was then added at 0.1 mL/well and
the plates read at A450-570 nm (TMB) or A405 nm (ABTS).
[0235] Table 5 shows the results of the ELISA analysis.
TABLE-US-00005 TABLE 5 EC.sub.50 Values Determined By ELISA TSLP
TSLP mAb Species Expression.sup.1 EC.sub.50 (nM) rat 23B12.H8.H4
human HEK293-B 0.79 rat 23B12.H8.H4 human HEK293 0.37 rat
23B12.H8.H4 human HEK293-F 0.26 0.47 .+-. 0.28(n = 3) rat
23B12.H8.H4 human E. coli-B 0.40 rat 23B12.H8.H4 human E. coli-B
0.39 0.44 .+-. 0.20(n = 5) hu23B12(VL Y49) human HEK293-B 0.22
hu23B12(VL Y49) human HEK293 0.029 hu23B12(VL Y49) human HEK293-F
0.018 hu23B12(VL Y49) human HEK293 0.17 hu23B12(VL Y49) human
HEK293 0.21 hu23B12(VL Y49) human HEK293 0.11 hu23B12(VL Y49) human
HEK293 0.13 hu23B12(VL Y49) human HEK293 0.15 hu23B12(VL Y49) human
HEK293 0.24 hu23B12(VL Y49) human HEK293 0.18 hu23B12(VL Y49) human
HEK293 0.11 hu23B12(VL Y49) human HEK293.sup.2 0.073 0.14 .+-.
0.07(n = 12) hu23B12(VL Y49) human E. coli-B 0.064 hu23B12(VL Y49)
human E. coli-B 0.085 hu23B12(VL Y49) human E. coli 0.11 hu23B12(VL
Y49) human E. coli 0.11 hu23B12(VL Y49) human E. coli 0.060
hu23B12(VL Y49) human E. coli.sup.2 0.061 hu23B12(VL Y49) human E.
coli.sup.2 0.029 0.07 .+-. 0.03(n = 7) 0.11 .+-. 0.07(n = 19)
hu23B12(VL Y49) cyno HEK293 0.45 hu23B12(VL Y49) cyno HEK293 0.52
hu23B12(VL Y49) cyno HEK293 0.29 hu23B12(VL Y49) cyno HEK293 0.15
hu23B12(VL Y49) cyno HEK293 0.19 hu23B12(VL Y49) cyno HEK293 0.27
0.31 .+-. 0.15(n = 6) hu23B12(VL K49) human HEK293-B 0.12
hu23B12(VL K49) human E. coli-B 0.038 hu23B12(VL K49) human E.
coli-B 0.050 hu23B12(VL K49) human HEK293 0.034 hu23B12(VL K49)
human HEK293-F 0.021 0.05 .+-. 0.04(n = 5) .sup.1B = TSLP
biotinylated F = TSLP removal of furin cleavage site via
K101A/R102A .sup.2direct coat of 200 ng TSLP instead of 100 ng
Example 5
Affinity of Rat 23B12 and Humanized 23B12 Antibodies for Human and
Cyno TSLP
[0236] The kinetic binding activities of the parental rat and its
humanized derivative anti human TSLP antibody 23B12 against both
human (hu) and cynomolgus monkey (cyno) TSLP were measured by
surface plasmon resonance using a BIAcore T100 system (BIAcore AB,
Upsalla, Sweden). Approximately 100RUs of human TSLP or cyno TSLP
were immobilized via amine coupling chemistry onto a Sensor Chip
CM5 (Research grade, BR-1006-68). HBS-EP buffer (BR-1006-69) was
used as the running buffer with a flow rate of 30 .mu.L/min. rat
and humanized 23B12 antibodies at varying concentrations ranging
from 0.82 to 600 nM were injected over the immobilized hu or cyno
TSLP surfaces at a flow rate of 30 .mu.L/min. Following each
injection cycle the CM5 chip surface was regenerated using a series
of solutions (10 mM Glycine pH 1.5 and 25 mM NaOH repectively) at a
flow rate of 75 .mu.L/min.
[0237] Background subtraction binding sensorgrams were used for
analyzing the rate constant of association (ka) and dissociation
(kd), and the equilibrium dissociation constant KD. The resulting
data sets were fitted with a bivalent analyte model using the
BIAevaluation software (version 1.0). The KD determined for the
parental rat 23B12 antibody against human TSLP was 64 pM, while the
respective value against the cyno TSLP ligand was 86 pM (Table 6).
The KD determined for the humanized 23B12 antibody against human
TSLP was 111 pM, while the respective value against the cyno TSLP
ligand was 132 pM (Table 6), indicating a less than two fold loss
of affinity upon humanization of 23B12 mAb.
TABLE-US-00006 TABLE 6 BIAcore Analysis K.sub.D Antibody Ligand ka
(1/Ms) kd (1/s) (pM) rat 23B12 huTSLP 3.18E+05 2.1E-05 64 cynoTSLP
1.86E+05 1.6E-05 86 hu 23B12 huTSLP 5.00E+05 5.6E-05 111 cynoTSLP
3.57E+05 4.7E-05 132
Example 6
Proliferation Bioassay for the Assessment of Neutralizing Anti-TSLP
Antibody
[0238] The ability of a monoclonal antibody to biologically
neutralize TSLP was assessed by the application of short-term
proliferation bioassays that utilize cells which express
recombinant TSLP receptors. The transfectant Ba/F3-TSLPR-IL7Ra
cells proliferate in response to TSLP and the response can be
inhibited by a neutralizing anti-TSLP antibody. Each antibody was
titrated against a concentration of TSLP chosen within the linear
region of the TSLP dose-response curve, near plateau and above the
TSLP EC50. Proliferation, or lack thereof, is measured by
colorimetric means using Alamar Blue, a growth indicator dye based
on detection of metabolic activity. The ability of an antibody to
neutralize TSLP is assessed by its EC50 value, or concentration of
antibody that induces half-maximal inhibition of TSLP
proliferation.
[0239] Ba/F3 transfectants are maintained in RPMI-1640 medium, 10%
fetal calf serum, 50 .mu.M 2-mercaptoethanol, 2 mM L-Glutamine, 50
.mu.ug/mL penicillin-streptomycin, and 10 ng/mL mouse IL-3.
[0240] Ba/F3 proliferation bioassays are performed in RPMI-1640
medium, 10% fetal calf serum, 50 .mu.M 2-mercaptoethanol, 2 mM
L-Glutamine, and 50 .mu.g/mL penicillin-streptomycin.
[0241] The assay is performed in 96-well flat bottom plates (Falcon
3072 or similar). All preparations of reagents and cell suspensions
utilize the appropriate bioassay medium. The assay volume is 150
.mu.L per well. Titrations of an anti-TSLP antibody are
pre-incubated with TSLP for 30-60 minutes at room temperature,
during which time cells are prepared. Cells are added to plates
following the antibody-cytokine pre-incubation. Bioassay plates are
incubated in a humidified tissue culture chamber (37 C, 5% CO2) for
40-48 hours. At the end of the culture time, Alamar Blue (Biosource
Cat #DAL1100) is added and allowed to develop for 8-12 hours.
Absorbance is then read at 570 nm and 600 nm (VERSAmax Microplate
Reader, Molecular Probes), and an OD570-600 is obtained. Duplicates
or triplicates are recommended.
[0242] Cells are used in a healthy growth state, generally at
densities of 3-8.times.105/mL. Cells are counted, pelleted, washed
twice in bioassay medium, and suspended to the appropriate density
for plating.
[0243] TSLP was prepared to working concentration and added to
first well at 75 .mu.L. Serial dilutions of 1:3 were made by
titrating 25:50 .mu.L in bioassay medium across wells, leaving 50
.mu.L/well. Cells were suspended to the appropriate density for
plating at 100 .mu.L per well.
[0244] The antibody was prepared to working concentration and added
to the first well at 75 .mu.L. Serial dilutions of 1:3 were made by
titrating 25:50 .mu.L in bioassay medium across wells, leaving 50
.mu.L per well. TSLP at the appropriate concentration was added at
50 .mu.L per well to the wells containing the titrated antibody.
Cells were suspended to the appropriate density for plating at 50
.mu.L per well, and added following the antibody-cytokine
pre-incubation.
[0245] Using GraphPad Prism 3.0 software, absorbance was plotted
against cytokine or antibody concentration and EC50 values were
determined using non-linear regression (curve fit) of sigmoidal
dose-response.
[0246] The assay results are shown in Table 7.
TABLE-US-00007 TABLE 7 Inhibition Of Proliferation TSLP TSLP mAb
Species Expression EC50 (nM) rat 23B12.H8.H4 human HEK293 0.093 rat
23B12.H8.H4 human HEK293 0.085 rat 23B12.H8.H4 human HEK293 0.23
rat 23B12.H8.H4 human HEK293 0.040 rat 23B12.H8.H4 human HEK293
0.10 0.11 .+-. 0.07(n = 5) rat 23B12.H8.H4 human E. coli 2.16 rat
23B12.H8.H4 human E. coli 2.78 rat 23B12.H8.H4 human E. coli 4.15
rat 23B12.H8.H4 human E. coli 3.81 rat 23B12.H8.H4 human E. coli
1.83 rat 23B12.H8.H4 human E. coli 3.46 rat 23B12.H8.H4 human E.
coli 2.77 rat 23B12.H8.H4 human E. coli 3.10 3.01 .+-. 0.79(n = 8)
rat 23B12.H8.H4 cyno HEK293 0.45 rat 23B12.H8.H4 cyno HEK293 0.42
rat 23B12.H8.H4 cyno HEK293 0.61 rat 23B12.H8.H4 cyno HEK293 0.77
0.56 .+-. 0.16(n = 4)
Example 7
Neutralizing Activity of Anti-TSLP mAb r23B12 on TSLP Induced TARC
Production by Human Primary Dendritic Cells
[0247] Peripheral blood mononuclear cells (PBMCs) were isolated
from buffy coats obtained from healthy blood donors (Stanford
Medical School Blood Center, Stanford, Calif.) by Ficoll
centrifugation and CD11c+Dendritic Cells were obtained by MACS
(Miltenyi Biotech, Auburn, Calif.) using negative selection
followed by cell sorting using a FACS. Lineage negative (Lin-)
cells were obtained by MACS depletion of T cells, B cells, NK
cells, red blood cells and monocytes form PBMC using mouse
anti-human CD3 mAb (OKT3, DNAX) and mouse anti-CD16 mAb and goat
anti-mouse IgG coated magnetic beads (Miltenyi Biotech), and using
magnetic beads directly coated with anti-CD19, CD56 and CD14 mAbs
(Miltenyi Biotech). Subsequently, Lin- cells were stained with
TC-anti-CD4 (Caltag, Burlingame, Calif.), PE-anti-CD11c and
FITC-anti-CD3, -CD14, -CD19, -CD56, -CD16, and -CD20 (all BD
Biosciences, San Diego, Calif.) and CD11c+ DC sorted on a Vantage
FACsorter.TM. (BD Biosciences) to a purity >99% of CD11c+ CD4+
Lin- cells.
[0248] CD11c+ CD4+ DCs were cultured immediately after sorting in
RPMI (Mediatech, Herndon, Va.) containing 10% FCS and 1% pyruvate
(Mediatech), HEPES (Invitrogen, Grand Island, N.Y.) and
penicillin-streptomycin (Mediatech). Cells were seeded at
0.5.times.106/ml in flat-bottomed 96-well plates in the presence of
medium alone, TSLP (15 ng/ml, DNAX), or in a combination of TSLP
and the neutralizing anti-TSLP mAb (clone 23B12) or an anti-TSLPR
monoclonal antibody or an isotype control rat IgG2a (R&D
Systems, Minneapolis, Minn.). DC culture supernatants were
collected after 24 h of culture, stored frozen at -20.degree. C.
and analyzed for TARC protein levels by ELISA (R&D
Systems).
[0249] The results are provided in Table 8.
TABLE-US-00008 TABLE 8 TARC (pg/ml) DC 5 TSLP-DC 1400.5 TSLP + 5
.mu.g/ml r23B12 antibody 41.5 TSLP + 0.5 .mu.g/ml r23B12 antibody
146 TSLP + 0.05 .mu.g/ml r23B12 antibody 570.5 TSLP + 20 .mu.g/ml
r23B12 antibody 199
[0250] CD11c+ DC cultured in media alone do not produce significant
levels of TARC. The addition of TSLP (15 ng/ml) to CD11c+ DC
induced significant levels of TARC production up to .about.1500
pg/ml. This TSLP mediated induction of TARC was blocked in a dose
dependent manner by the simultaneous addition of anti-TSLP mAb
23B12.
Example 8
Neutralizing Activity of Anti-TSLP mAb r23B12 on TSLP Induced Th2
Differentiation by Human Primary Dendritic Cells
[0251] Peripheral blood mononuclear cells (PBMCs) were isolated
from buffy coats obtained from healthy blood donors (Stanford
Medical School Blood Center, Stanford, Calif.) by Ficoll
centrifugation and CD11c+ Dendritic Cells were obtained by MACS
(Miltenyi Biotech, Auburn, Calif.) using negative selection
followed by cell sorting using a FACS. Lineage negative (Lin-)
cells were obtained by MACS depletion of T cells, B cells, NK
cells, red blood cells and monocytes form PBMC using mouse
anti-human CD3 mAb (OKT3, DNAX) and mouse anti-CD16 mAb and goat
anti-mouse IgG coated magnetic beads (Miltenyi Biotech), and using
magnetic beads directly coated with anti-CD19, CD56 and CD14 mAbs
(Miltenyi Biotech). Subsequently, Lin- cells were stained with
TC-anti-CD4 (Caltag, Burlingame, Calif.), PE-anti-CD11c and
FITC-anti-CD3, -CD14, -CD19, -CD56, -CD16, and -CD20 (all BD
Biosciences, San Diego, Calif.) and CD11c+ DC sorted on a Vantage
FACsorter.TM. (BD Biosciences) to a purity >99% of CD11c+ CD4+
Lin- cells.
[0252] CD11c.sup.+ CD4.sup.+ DCs were cultured immediately after
sorting in RPMI (Mediatech, Herndon, Va.) containing 10% FCS and 1%
pyruvate (Mediatech), HEPES (Invitrogen, Grand Island, N.Y.) and
penicillin-streptomycin (Mediatech). Cells were seeded at
0.5.times.10.sup.6/ml in flat-bottomed 96-well plates in the
presence of medium alone, TSLP (15 ng/ml, DNAX), or in a
combination of TSLP and the neutralizing anti-TSLP mAb (clone
23B12) or the anti-TSLPR monoclonal antibody or an isotype control
rat IgG2a (R&D Systems, Minneapolis, Minn.). CD11c.sup.+ DCs
were collected after 24 h of culture under the different
conditions, washed twice and recultured with allogeneic CD4.sup.+
CD45RA' naive T cells.
[0253] CD4.sup.+ CD45RA.sup.+ naive T cells were isolated by cell
sorting after negative depletion of CD8, CD16, CD20, CD19, CD56 and
CD14 cells using magnetic beads (Myltenyi Biotech). After 24 h of
culture under different conditions, CD11c.sup.+ DCs were collected,
washed twice, and co-cultured with 5.times.10.sup.4 allogeneic
naive CD4.sup.+ T cells in round-bottomed 96-well plates at a ratio
of 1:5 DC:T cells. After 6 d. of culture, supernatants were
collected and frozen at -20.degree. C. and numbers of viable cells
determined by trypan blue exclusion. To test their capacity to
secrete cytokines, DC-primed CD4.sup.+ T cells (10.sup.6/ml) were
restimulated with biotinylated anti-CD3 (10 ng/ml) mAbs crosslinked
to streptavidin coated tissue culture plates in the presence of
soluble anti-CD28 mAbs (1000 ng/ml). Culture supernatants were
collected after 24 hrs of culture, frozen at -20.degree. C. or
analyzed by Luminex assay for IFN.gamma.-, TNF.alpha.-, IL-2, IL-4,
IL-5, IL-10 and IL-13 (Linco Research Inc., St Charles, Mo.).
[0254] The results are provided in Table 9.
TABLE-US-00009 TABLE 9 TSLP-DC + TSLP-DC + TSLP-DC + TSLP- 5
.mu.g/ml 0.5 .mu.g/ml 0.05 .mu.g/ml Cytokine DC + DC + r23B12 +
r23B12 + r23B12 + Production CD4 CD4 CD4 CD4 CD4 IL-5 (pg/ml) 115
777 24 70.6 32 IL-4 (pg/ml) 14 89 14.5 21.3 27.3 IL-13 136 1290
55.5 182 43.3 (pg/ml)
[0255] The coculture of naive CD4+ CD45RA+ T cells with CD11c+ DC
that had been cultured in media alone resulted in a T cell
population, that upon reactivation by anti-CD3+anti-CD28 mAbs,
produced low levels of Th2 cytokines Addition of TSLP (15 ng/ml) to
the primary CD11c+ DC culture induced the production of significant
levels of IL-4, IL-5 and IL-13 by the responding T cells,
suggesting that TSLP-DC induced the differentiation of naive T
cells towards Th2 cells. This TSLP mediated induction of Th2
differentiation was blocked in a dose dependent manner by the
simultaneous addition of anti-TSLP mAb 23B12 to the primary DC
cultures.
Example 9
Cyno-Ization of Anti-Human TSLP Antibodies
[0256] Two studies have shown that cynomolgus monkeys (Macaca
fascicularis) VL are similar to human VL.kappa.-I and that
cynomolgus VH are similar to human VH-III (41%), VH-IV (39%), and
VH-I (14%). (Lewis et al., Dev. Comp. Immunol. 17:549-560 (1993);
and Druar et al., Immunogentics 57:730-738 (2005.) In order to
minimize potential immunogenicity of hu23B12 in cymomolgus monkeys,
the rat 23B12 CDRs were transferred onto human VL.kappa.-I and
VH-III frameworks; these were then fused onto cynomolgus IgG
constant domains.
[0257] The amino acid sequence of the cyno-ized light chain is
MAPVQLLGLLVLFLPAMRCDIQMTQSPSSLSASVGDRVTITCRASQPISISVHWYQQK
PGKAPKLLIYFASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTFSLPYTFG
QGTKVEIKRTVAAPSVFIFPPSEDQVKSGTVSVVCLLNNFYPREASVKWKVDGVLKT
GNSQESVTEQDSKDNTYSLSSTLTLSSTDYQSHNVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID
NO: 17). The signal sequence is underlined.
[0258] The amino acid sequence of the cyno-ized heavy chain is
MAVLGLLFCLVTFPSCVLSQVQLVESGGGVVQPGRSLRLSCAASGYIFTDYAMHWV
RQAPGKGLEWVATFIPLLDTSDYNQNFKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCARMGVTHSYVMDAWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK
DYFPEPVTVSWNSGSLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYVCNVNH
KPSNTKVDKRVEIKTCGGGSKPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSQEDPDVKFNWYVNGAEVHHAQTKPRETQYNSTYRVVSVLTVTHQDWL
NGKEYTCKVSNKALPAPIQKTISKDKGQPREPQVYTLPPSREELTKNQVSLTCLVKGF
YPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK (SEQ ID NO:18). The signal sequence is
underlined.
[0259] Cyno-ized anti-human TSLP 23B12 antibodies were then
recombinantly produced in CHO cells.
Example 10
Proliferation Bioassay for the Assessment of the Neutralizing
Activity of Cyno-Ized Anti-Human TSLP Antibodies
[0260] The ability of cyno-ized anti-human TSLP 23B12 antibodies to
biologically neutralize human or cyno TSLP was assessed by the
application of short-term proliferation bioassays that utilize
cells which express recombinant TSLP receptors. The transfectant
Ba/F3-huTSLPR/huIL-7Ra and Ba/F3-cyTSLPR/cyIL-7Ra cells proliferate
in response to human TSLP and cyno TSLP, and the response can be
inhibited by a neutralizing anti-TSLP antibody. An antibody is
titrated against a concentration of human or cyno TSLP chosen
within the linear region of the dose-response curve, near plateau
and above EC50. Proliferation, or lack thereof, is measured by
colorimetric means using Alamar Blue, a growth indicator dye based
on detection of metabolic activity. The ability of an antibody to
neutralize TSLP is assessed by its EC50 value, or concentration of
antibody that induces half-maximal inhibition of TSLP
proliferation.
[0261] Ba/F3 transfectants are maintained in RPMI-1640 medium, 10%
fetal bovine serum, 50 uM 2-mercaptoethanol, 2 mM L-Glutamine, 50
ug/mL penicillin-streptomycin, 10 ng/mL mouse IL-3, 1 mg/ml G418,
and 2 ug/ml puromycin.
[0262] Ba/F3 proliferation bioassays are performed in RPMI-1640
medium, 10% fetal bovine serum, 50 uM 2-mercaptoethanol, 2 mM
L-Glutamine, and 50 ug/mL penicillin-streptomycin.
[0263] The assays were performed in 96-well flat bottom plates
(Falcon 3072 or similar). All preparations of reagents and cell
suspensions utilized the appropriate bioassay medium. The assay
volume was 150 uL per well. Titrations of an anti-TSLP antibody
were pre-incubated with huTSLP or cyTSLP for approximately 30
minutes at room temperature, during which time cells were prepared.
Cells were added to plates following the antibody-cytokine
pre-incubation. Bioassay plates were incubated in a humidified
tissue culture chamber (37 C, 5% CO2) for 40-48 hr. At the end of
the culture time, Alamar Blue (Biosource Cat #DAL1100) was added at
16.5 uL/well and allowed to develop for 5-12 hours. Absorbance was
then read at 570 nm and 600 nm (VERSAmax Microplate Reader,
Molecular Devices), and an OD570-600 was obtained. Duplicates were
run for each sample.
[0264] Cells are used in a healthy growth state, generally at
densities of 7-9.times.105/mL. Cells are washed twice in bioassay
medium, counted, and suspended to the appropriate density for
plating at 7500 cells/50 ul per well.
[0265] Human or cyno TSLP was prepared to working concentration
(600 ng/mL) and added to first well at 75 uL. Serial dilutions of
1:3 were made by titrating 25:50 uL in bioassay medium across
wells, leaving 50 uL/well. Cells were suspended to the appropriate
density for plating at 7500 cells/50 uL per well. To substitute the
addition of antibody, 50 ul of bioassay medium was added to these
wells to bring the final volume to 150 ul.
[0266] The antibody was prepared to working concentration (3.times.
the final concentration; the final starting concentration of each
antibody varied) and added to first well at 75 uL. Serial dilutions
of 1:3 were made by titrating 25:50 uL in bioassay medium across
wells, leaving 50 uL per well. TSLP (at working concentration of 9
ng/ml for HuTSLP, 3 ng/ml for CyTSLP) was added at 50 uL per well
to the wells containing the titrated antibody. Cells were then
suspended to the appropriate density for plating at 7500 cells/50
uL per well, and added following the antibody-cytokine
pre-incubation.
[0267] EC50 values are determined by non-linear regression (curve
fit) of sigmoidal dose-response using GraphPad Prism 4 software.
For TSLP dose response, absorbance is plotted against cytokine
concentration. For neutralization activity, percentage inhibition
is plotted against antibody concentration.
[0268] The assay results are shown in Table 10.
TABLE-US-00010 TABLE 10 Ba/F3 Cell-based Assay of Human or
Cynomolgus TSLPR-Transfected Cells EC50 nM TSLP TSLPR rat23B12
hu23B12 hu-cy* cy-hu** cy23B12 hu hu 0.6 7.6 5.3 3.9 9.4 hu hu 1.2
9.0 hu hu 0.1 1.2 hu cyno 0.02 0.03 hu cyno 0.08 0.05 cyno cyno 0.2
0.5 0.5 2.8 4.7 cyno cyno 0.4 2.2 3.2 16 26.19 cyno cyno 0.7 3.0
22.17 *hu-cy = humanized 23B12 VL/VH on cynomolgus constant domains
**cy-hu = cyno-ized 23B12 VL/VH on human constant domains
Example 11
BIAcore and KinExA Affinity Measurements for Cyno-Ized Anti-TSLP
Antibodies
[0269] The affinity of cyno-ized anti-human TSLP 23B12 antibodies
towards human and cyno TSLP ligand was determined by surface
plasmon resonance using the BIAcore T100 system as described in
Example 5.
[0270] The equilibrium disassociation constant for the anti-TSLP
antibodies was determined using the KinExA 3000 instrument
(Sapidyne Instruments Inc.). as described in Example 3.
[0271] The following materials were used:
[0272] Antibodies: [0273] Rat anti hTSLP GNE01.23B12.H8.A4 (SPB Lot
pab 330A) [0274] Humanized anti hTSLP mAb 23B12(621HC/780LC) [0275]
Cynoized anti hTSLP mAb 23B12 (782+MAFA19/781MAFA7) [0276] Cynoized
anti hTSLP mAb 23B12 (782+hIgG1/781hukappa) [0277] Cynoized anti
hTSLP mAb 23B12 (huV-CynoC chimera)
[0278] Antigens: [0279] Recombinant human TSLP, R&D Systems
(Cat. No. 1398-TS/CF, Lot. IDK 015031) [0280] Recombinant human
TSLP, R&D Systems (Cat. No. 1398-TS, Lot. IDK 026031) [0281]
Biotinylated human TSLP (SPB Lot 38ABMA)
[0282] Other Reagents: [0283] PMMA particles, 98 micron (Sapidyne,
Cat No. 440198) [0284] Neutravidin (Pierce, Cat No. 31000) [0285]
Cy5 conjugated Goat anti-rat IgG (H+L) (Jackson Immunoresearch
Laboratories Cat. No 112-175-167, Lot 60306) [0286] Cy5 conjugated
Goat anti-huIgG (H+L) (Jackson Immunoresearch Laboratories Cat. No
109-175-088, lot 58552)
[0287] For r23B12 the following conditions were used:
[0288] Sample volume: 2 ml
[0289] Sample flow rate: 0.25 ml/min
[0290] Label volume: 1 ml
[0291] Label flow rate: 0.25 ml/min
[0292] mAb conc.: 0.05 nM
[0293] Highest Ag (TSLP) conc.: 0.5 nM
[0294] Lowest Ag (TSLP) conc.: 0.5 .mu.M
[0295] For hu23B12 the following conditions were used:
[0296] Sample volume: 2 ml
[0297] Sample flow rate: 0.25 ml/min
[0298] Label volume: 1 ml
[0299] Label flow rate: 0.25 ml/min
[0300] mAb conc.: 0.02 nM
[0301] Highest Ag (TSLP) conc.: 0.4 nM
[0302] Lowest Ag (TSLP) conc.: 0.4 .mu.M
[0303] For cy23B12 the following conditions were used:
[0304] Sample volume: 2 ml
[0305] Sample flow rate: 0.25 ml/min
[0306] Label volume: 1 ml
[0307] Label flow rate: 0.25 ml/min
[0308] mAb conc.: 0.1 nM
[0309] Highest Ag (TSLP) conc.: 1 nM
[0310] Lowest Ag (TSLP) conc.: 1 pM
[0311] For hu-cy23B12* and cy-hu23B12** the following conditions
were used:
[0312] Sample volume: 2 ml
[0313] Sample flow rate: 0.25 ml/min
[0314] Label volume: 1 ml
[0315] Label flow rate: 0.25 ml/min
[0316] mAb conc.: 0.05 nM
[0317] Highest Ag (TSLP) conc.: 1 nM
[0318] Lowest Ag (TSLP) conc.: 1 pM
[0319] For all experiments two-fold serial dilutions of the antigen
were prepared and mixed with the antibody at constant
concentration. The mixture was incubated for 2 hours at RT to
equilibrate. The results of the BIAcore and KinExA experiments
described above are summarized on Table 11.
TABLE-US-00011 TABLE 11 Biacore and Kinexa Binding Affinity
Measurements KD (pM) TSLP r23B12 hu23B12 hu-cy* cy-hu** cy23B12
KinExA hu 0.47 1.7 1.4 63 52 BIAacore hu 64 111 106 556 620 hu 126,
114 1548, 2066 cy 86, 112 132 114 1203 1159, 2508 *hu-cy =
humanized 23B12 VL/VH on cynomolgus constant domains **cy-hu =
cynoized 23B12 VL/VH on human constant domains
[0320] In summary, the humanized anti-human TSLP 23B12 antibody
showed approximately 5-fold reduced binding compared to the
parental rat antibody based on BIAcore and KinExA measurements
(Table 11). Replacing the humanized 23B12 frameworks
(VL.kappa.-III/VH-I) with those less potentially immunogenic in
cynomolgus monkeys (VL.kappa.-I/VH-III) effected a 10-fold
reduction in binding compared to parental rat 23B12 and a 5-fold
reduction compared to hu23B12 (Tables 10, 11).
Example 12
Pharmacokinetic Studies of Cyno-Ized Anti-TSLP 23B12 Antibodies
[0321] An ELISA assay was designed to measure the amount of
cyno-ized anti-TSLP antibody reaching the plasma, serum or
bronchoalveolar lavage (BAL) fluid of an animal inoculated with
such an antibody.
[0322] Reagents and Buffers: [0323] Solid Support: Nunc Maxisorp
96-Well plate (cat#439454) [0324] Coating Buffer: 50 mM Sodium
carbonate/bicarbonate pH9.6 [0325] Blocking Buffer: 0.5% BSA in PBS
[0326] Assay Diluent Buffer 0.5% BSA [wt/v], 0.05% Tween 20 [v/v],
0.25% CHAPS [wt/v], 5 mM EDTA, 0.35M NaCl in PBS (AD), pH7.4 [0327]
Wash Buffer: 0.05% Tween 20 in PBS [0328] Capturing molecule:
huTSLP, 38ABM, 2497 .mu.g/mL [0329] Detection molecules: [0330] QED
R799, 3600 ug/mL (a rabbit polyclonal anti-cy23B12 antibody) [0331]
anti rabbit-HRP, JIR cat#711-036-152 [0332] Substrate: TMB
(Kirkegaard & Perry, cat#50-76-03) [0333] Stop solution: 1M
H3PO4 [0334] Plate Washer SkanWasher 300 Model 12010 (Molecular
Devices Cat. No. 0200-3903) [0335] Stop solution: SpectraMax Plus
384 Microtiterplate Spectrophotometer (Molecular Devices Part No.
0112-0056
Protocol:
[0336] Coating of plates was performed as follows: huTSLP (100 ng
per well) in coating buffer was incubated at 40 C overnight. Plates
were washed with 1 cycle (3 washes/cycle) on a Skatron plate
washer, blocked by addition of 150 .mu.L/well blocking buffer,
incubated for 60 min at room temperature on an orbital shaker and
then washed for 1 cycle. The cyno-ized anti-TSLP 23B12 antibody
standard was titrated across a row of eight wells (replicates) in
the range of 200 ng/mL to 1.56 ng/mL using serial 2-fold dilutions.
Samples are serially diluted respect to their expected levels. 100
.mu.L of standards, controls, and samples were added to the coated
plate and incubated for 120 minutes at room temperature on an
orbital shaker. Plates were washed for 2 cycles and the rabbit
polyclonal anti-cy23B12 antibody was added at 100 .mu.A/well and
incubated for 60 min at room temperature on an orbital shaker.
Plates were washed for 2 cycles, HRP-donkey anti-rabbit IgG (H+L)
(1:10,000 dilution) was added at 100 .mu.L/well and incubated for
60 min at room temperature on an orbital shaker. Plates were washed
for 2 cycles with plate rotation between cycles. TMB substrate was
added at 100 .mu.A/well and incubated approximately 5 min on an
orbital shaker. Stop solution was then added at 100 .mu.L/well and
the plates read at A450-650 nm (TMB).
[0337] This assay can detect as low as 156 ng/mL of cyno-ized
anti-TSLP antibodies in plasma (5% dilution) and serum; and as low
as 3.2 ng/mL in BAL fluid.
[0338] This ELISA assay was used to measure the pharmacokinetics of
cyno-ized anti-TSLP 23B12 antibodies after administration to mice
and monkeys.
[0339] A single dose PK study was conducted in normal CD-1 mice. In
this study, ten mice received 10 mg/kg of the antibody by
intravenous (IV) administration; and ten mice received 10 mg/kg of
the antibody by subcutaneous (SC) administration. The results of
the study are summarized in Table 12.
TABLE-US-00012 TABLE 12 Clearance Vss AUC 0-last t1/2 terminal Tmax
Cmax F Route (mL/day/kg) (mL/kg) (.mu.g * day/mL) (day) (day)
(.mu.g/mL) (%) IV 24.8 220 366 6.69 -- -- -- IV (w/o 22.8 198 404
6.36 Day 10) SC -- -- 370 3.13 0.667 73.7 95.6
[0340] Table 13 summarizes the percentage of cyno-ized anti-TSLP
antibody found in the BAL fluid versus in the serum at various time
points after IV or SC administration.
TABLE-US-00013 TABLE 13 IV SC Time point (day) BAL/Serum Mean (%)
BAL/Serum Mean (%) 0.250 1.686 4.080 1.000 2.811 5.880 3.000 5.677
7.301 7.000 13.740 16.945 10.000 14.319 5.064 14.000 6.517
16.912
[0341] Two single dose PK studies were also conducted in cynomolgus
monkeys. The two studies used different formulations of the
cyno-ized anti-TSLP 23B12 antibody: one containing 0.05% of Triton
X-100 and one without Triton X-100. Each study contained three
monkeys. The dose used in each study is indicated in Table 14. The
antibody was administered subcutaneously. The results of the
studies are summarized in Tables 14 and 15.
TABLE-US-00014 TABLE 14 t1/2 Animal Dose CL/F AUC 0-last terminal
Tmax Cmax Formulation ID (mg/kg) (mL/day/kg) (mg * day/mL) (day)
(day) (mg/mL) no Triton Cyno 5.6 15.1 326 (600) 9.18 1 26.4 M2-04
Cyno 5.6 7.96 594 (1090) 10 1 48.3 M7-05 Cyno 5.7 8.82 491 (887)
12.4 2 34 M21-05 0.05% Cyno 10.7 10.3 666 8.15 4 61.6 Triton 2172
X100 Cyno 10.3 8.27 778 8.86 2 88.8 3048 Cyno 9.8 8.14 753 9.08 1
89.9 5102
TABLE-US-00015 TABLE 15 Formulation BAL/Serum Ratio (%) Time point
(day) M2-04 M7-05 M21-05 Mean No Triton 3 0.657 0.712 0.335 0.568
10 1.83 1.15 1.81 1.60 Time point (day) 2172 3048 5102 Mean +0.05%
Triton 3 0.313 0.492 1.39 0.733 X100 10 0.090 0.620 0.693 0.467
Example 13
Administration of Cyno-Ized Anti-TSLP 23B12 Antibodies to
Cynomolgus Monkeys
[0342] Cyno-ized anti-TSLP 23B12 antibodies were produced from a
stably transfected CHO cell line in suspension culture. The
supernantant was harvested, contreated, and purified using several
standard chromatogrphic steps to achieve a low endotoxin, >95%
pure preparation. The purified antibody was formulated for
stability during handling and use, including multiple freeze-thaws,
in 20 mM sodium acetate, pH 5.5, 7% (w/v) sucrose, and 0.05% Triton
X-100. This antibody is to be administered to house dust mite (HDM)
allergic cynomolgus monkeys to demonstrate the effectiveness of an
anti-TSLP antibodies to treat allergic lung inflammation. This
animal model will make possible the collection of airway tissues,
BAL fluid, and associated PBMC's harvested from the control and
cy23B12 treated animals; and will provide the ability to access
efficacy in Early Allergic Reactions (EAR) and Late Allergic
Reactions (LAR). Further information regarding non-primate models
of chronic allergic asthma are well known in the art. See, e.g.,
Schelegle et al., Am. J. Pathology 158(1):333-341 (2001); Avdalovic
et al., Am. J. Respir. Crit. Care Med. 174:1069-74 (2006) Care and
Van Scott et al., J. Appl. Physiol. 99(6):2080-2086 (2005).
[0343] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
[0344] Citation of the above publications or documents is not
intended as an admission that any of the foregoing is pertinent
prior art, nor does it constitute any admission as to the contents
or date of these publications or documents. U.S. patents and other
publications referenced herein are hereby incorporated by
reference.
Sequence CWU 1
1
18110PRTRattus norvegicus 1Gly Tyr Ile Phe Thr Asp Tyr Ala Met His
1 5 10 217PRTRattus norvegicus 2Thr Phe Ile Pro Leu Leu Asp Thr Ser
Asp Tyr Asn Gln Asn Phe Lys 1 5 10 15 Gly 311PRTRattus norvegicus
3Met Gly Val Thr His Ser Tyr Val Met Asp Ala 1 5 10 411PRTRattus
norvegicus 4Arg Ala Ser Gln Pro Ile Ser Ile Ser Val His 1 5 10
57PRTRattus norvegicus 5Phe Ala Ser Gln Ser Ile Ser 1 5 69PRTRattus
norvegicus 6Gln Gln Thr Phe Ser Leu Pro Tyr Thr 1 5 7120PRTRattus
norvegicus 7Glu Glu Lys Leu Gln Gln Ser Gly Asp Asp Leu Val Arg Pro
Gly Ala 1 5 10 15 Ala Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile
Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Phe Ile Pro Leu Leu Asp
Thr Ser Asp Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Arg Ala Thr Leu
Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Met Gly Val Thr His Ser Tyr Val Met Asp Ala Trp Gly Gln 100 105 110
Gly Ala Ser Val Thr Val Ser Ser 115 120 8108PRTRattus norvegicus
8Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1
5 10 15 Glu Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Pro Ile Ser Ile
Ser 20 25 30 Val His Trp Phe Gln Gln Lys Ser Asn Glu Ser Pro Arg
Leu Leu Ile 35 40 45 Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Asn Ile Asn Arg Val Glu Ser 65 70 75 80 Glu Asp Phe Ser Val Tyr Tyr
Cys Gln Gln Thr Phe Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Thr Gly
Thr Lys Leu Glu Leu Lys Arg 100 105 9360DNAArtificialHumanized
antibody sequence 9caggtgcagc tggtgcagtc tggcgctgag gtgaagaagc
ctggcgcctc cgtgaaggtc 60tcctgcaagg cttctggcta catcttcacc gactacgcca
tgcactgggt gcggcaggcc 120cctggccagg ggctggagtg gatgggtacc
ttcatccctc tgctggacac cagcgactac 180aaccagaact tcaagggcag
agtcaccatg accacagaca catccaccag cacagcctac 240atggagctga
ggagcctgag atctgacgac accgccgtgt attactgtgc cagaatggga
300gtgacccaca gctacgtgat ggatgcatgg ggccagggca ccctggtcac
cgtctccagc 36010120PRTArtificialHumanized antibody sequence 10Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr
20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Thr Phe Ile Pro Leu Leu Asp Thr Ser Asp Tyr
Asn Gln Asn Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr
Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Gly Val Thr
His Ser Tyr Val Met Asp Ala Trp Gly Gln 100 105 110 Gly Thr Leu Val
Thr Val Ser Ser 115 120 11324DNAArtificialHumanized antibody
sequence 11gaaattgtgc tgactcagag cccaggcacc ctgtctctgt ctccaggcga
gagagccacc 60ctctcctgcc gggccagcca gcccatctcc atcagcgtgc actggtacca
gcagaaacca 120ggacaggctc caaggctgct gatctacttt gcctcccaga
gcatctccgg gatccccgat 180aggttcagcg gatccggatc tgggacagat
ttcaccctca ccatcagcag actggagcct 240gaagatttcg cagtgtatta
ctgtcagcag accttcagcc tgccttacac tttcggccaa 300gggaccaagg
tggagatcaa gcgt 32412108PRTArtificialHumanized antibody sequence
12Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Pro Ile Ser Ile
Ser 20 25 30 Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45 Tyr Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro
Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Thr Phe Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 131350DNAArtificialHumanized
antibody sequence 13caggtgcagc tggtgcagtc tggcgctgag gtgaagaagc
ctggcgcctc cgtgaaggtc 60tcctgcaagg cttctggcta catcttcacc gactacgcca
tgcactgggt gcggcaggcc 120cctggccagg ggctggagtg gatgggtacc
ttcatccctc tgctggacac cagcgactac 180aaccagaact tcaagggcag
agtcaccatg accacagaca catccaccag cacagcctac 240atggagctga
ggagcctgag atctgacgac accgccgtgt attactgtgc cagaatggga
300gtgacccaca gctacgtgat ggatgcatgg ggccagggca ccctggtcac
cgtctccagc 360gctagcacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 660aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 720ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggatgag 1080ctgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1200ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa
gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc tccgggtaaa
135014450PRTArtificialHumanized antibody sequence 14Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr 20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Thr Phe Ile Pro Leu Leu Asp Thr Ser Asp Tyr Asn Gln Asn
Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Gly Val Thr His Ser Tyr
Val Met Asp Ala Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165
170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445 Gly Lys 450 15642DNAArtificialHumanized
antibody sequence 15gaaattgtgc tgactcagag cccaggcacc ctgtctctgt
ctccaggcga gagagccacc 60ctctcctgcc gggccagcca gcccatctcc atcagcgtgc
actggtacca gcagaaacca 120ggacaggctc caaggctgct gatctacttt
gcctcccaga gcatctccgg gatccccgat 180aggttcagcg gatccggatc
tgggacagat ttcaccctca ccatcagcag actggagcct 240gaagatttcg
cagtgtatta ctgtcagcag accttcagcc tgccttacac tttcggccaa
300gggaccaagg tggagatcaa gcgtacggtg gctgcaccat ctgtcttcat
cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg
gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt
64216214PRTArtificialHumanized antibody sequence 16Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Pro Ile Ser Ile Ser 20 25 30
Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35
40 45 Tyr Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Asp Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr
Phe Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
17233PRTArtificialCyonized antibody sequence with signal peptide
17Met Ala Pro Val Gln Leu Leu Gly Leu Leu Val Leu Phe Leu Pro Ala 1
5 10 15 Met Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Pro Ile 35 40 45 Ser Ile Ser Val His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys 50 55 60 Leu Leu Ile Tyr Phe Ala Ser Gln Ser
Ile Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Phe Ser 100 105 110 Leu Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Glu Asp Gln Val 130 135
140 Lys Ser Gly Thr Val Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
145 150 155 160 Arg Glu Ala Ser Val Lys Trp Lys Val Asp Gly Val Leu
Lys Thr Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Asn Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser
Ser Thr Asp Tyr Gln Ser His 195 200 205 Asn Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn
Arg Gly Glu Cys 225 230 18472PRTArtificialCyonized antibody
sequence with signal peptide 18Met Ala Val Leu Gly Leu Leu Phe Cys
Leu Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe 35 40 45 Thr Asp Tyr
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu
Trp Val Ala Thr Phe Ile Pro Leu Leu Asp Thr Ser Asp Tyr Asn 65 70
75 80 Gln Asn Phe Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Met Gly Val Thr His Ser
Tyr Val Met Asp Ala 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val
Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe 180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195
200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Val Cys Asn
Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ile Lys 225 230 235 240 Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr
Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser
Gln Glu Asp Pro Asp Val Lys Phe Asn Trp Tyr Val 290 295 300 Asn Gly
Ala Glu Val His His Ala Gln Thr Lys Pro Arg Glu Thr Gln 305 310 315
320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Thr His Gln
325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Thr Cys Lys Val Ser Asn
Lys Ala 340 345 350 Leu Pro Ala Pro Ile Gln Lys Thr
Ile Ser Lys Asp Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Leu Thr 370 375 380 Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp
Ile Val Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Thr Tyr 405 410
415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr
420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys 465
470
* * * * *
References