U.S. patent application number 10/232187 was filed with the patent office on 2003-05-15 for sialoadhesin factor-2 antibodies.
Invention is credited to Abrahamson, Julie A., Bochner, Bruce, Erickson-Miller, Connie L., Kikly, Kristine K., Nutku, T. Esra, Schleimer, Robert.
Application Number | 20030092091 10/232187 |
Document ID | / |
Family ID | 26883185 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030092091 |
Kind Code |
A1 |
Abrahamson, Julie A. ; et
al. |
May 15, 2003 |
Sialoadhesin factor-2 antibodies
Abstract
Monoclonal antibodies have been generated that bind to human
sialoadhesion factor-2. These antibodies are useful as diagnostic
and therapeutic reagents
Inventors: |
Abrahamson, Julie A.;
(Harlow, GB) ; Erickson-Miller, Connie L.; (King
of Prussia, PA) ; Kikly, Kristine K.; (Fortville,
IN) ; Bochner, Bruce; (Lutherville, MD) ;
Schleimer, Robert; (Baltimore, MD) ; Nutku, T.
Esra; (Baltimore, MD) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW 2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Family ID: |
26883185 |
Appl. No.: |
10/232187 |
Filed: |
August 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10232187 |
Aug 29, 2002 |
|
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PCT/US01/07193 |
Mar 5, 2001 |
|
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60187595 |
Mar 7, 2000 |
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Current U.S.
Class: |
435/7.92 ;
435/320.1; 435/338; 435/69.1; 530/388.26; 536/23.53 |
Current CPC
Class: |
C07K 2317/56 20130101;
C07K 2317/73 20130101; C07K 16/2803 20130101; A61K 2039/505
20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/7.92 ;
435/69.1; 435/320.1; 530/388.26; 536/23.53; 435/338 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543; C07H 021/04; C12P 021/02; C12N 005/06; C07K
016/40 |
Goverment Interests
[0002] This invention resulted from research funded in whole or in
part by the National Institutes of Health, Grant No. A141472. The
Federal Government has certain rights in this invention.
Claims
What is claimed is:
1. An antibody that binds to human SAF-2.
2. The antibody of claim 1 wherein the antibody has the identifying
characteristics of monoclonal antibody 2C4.
3. The antibody of claim 2, wherein the antibody is monoclonal
antibody 2C4.
4. An isolated polypeptide comprising an immunoglobulin
complementarity determining region of the antibody of claim 1.
5. An isolated polypeptide comprising an immunoglobulin
complementarity determining region of the antibody of claim 2.
6. An isolated polypeptide comprising an immunoglobulin
complementarity determining region of the antibody of claim 3.
7. An isolated polynucleotide encoding the polypeptide of claim
4.
8. An isolated polynucleotide encoding the polypeptide of claim
5.
9. An isolated polynucleotide encoding the polypeptide of claim
6.
10. The polypeptide of claim 6 wherein the immunoglobulin
complementarity determining region that comprises the polypeptide
is set forth in a member of the group consisting of SEQ ID NO:5, 6,
7, 8, 9 and 10.
11. The polypeptide of claim 10 wherein the immunoglobulin
complementarity determining region comprises the polypeptides set
forth in SEQ ID NOs:3, 4 and 5.
12. The polypeptide of claim 10 wherein the immunoglobulin
complementarity determining region comprises the polypeptides set
forth in SEQ ID NOs:6, 7 and 8.
13. An isolated polynucleotide encoding the polypeptide of claim
10.
14. An isolated polynucleotide encoding the polypeptide of claim
11.
15. An isolated polynucleotide encoding thc polypeptide of claim
12.
16. The antibody of claim 1 wherein the immunoglobulin
complementarity determining region of the antibody comprises the
polypeptides set forth in SEQ ID NO:5, 6, 7, 8, 9 and 10.
17. The antibody of claim 16 comprising a heavy chain variable
region polypeptide as set forth in SEQ ID NO:2 and a kappa light
chain variable region polypeptide as set forth in SEQ ID NO:4.
18. An isolated polynucleotide encoding a polypeptide comprising a
member selected from the group consisting of SEQ ID NO:2 and SEQ ID
NO:4.
19. A hybridoma cell line that produces a monoclonal antibody
having the identifying characteristics the monoclonal antibody
2C4.
20. A pharmaceutical composition comprising the antibody of claim
1.
21. A pharmaceutical composition comprising the antibody of claim
2.
22. A pharmaceutical composition comprising the monoclonal antibody
of claim 3.
23. A method for detecting the presence of a cell in a sample
wherein the cell comprises an SAF-2 protein, the method comprising:
a) exposing the sample to an antibody that binds to SAF-2; and b)
detecting the antibody that is bound to SAF-2.
24. The method of claim 23 wherein the sample is treated before
exposure to the antibody such that the SAF-2 protein is accessible
to binding by the antibody.
25. The method of claim 23 wherein the cell is a member selected
from the group consisting of an eosinophil, a basophil and a mast
cell.
26. The method of claim 23 wherein the antibody has the identifying
characteristics of monoclonal antibody 2C4.
27. The method of claim 26 wherein the antibody is monoclonal
antibody 2C4.
28. A method for altering the function or viability of a cell
expressing SAF-2 comprising contacting the cell with a therapeutic
agent that binds to SAF-2.
29. The method of claim 28 wherein the cell is selected from the
group consisting of eosinophils, basophils and mast cells.
30. The method of claim 28 wherein the therapeutic agent is an
antibody.
31. The method of claim 28 wherein contact with the therapeutic
agent induces apoptosis of the cell.
32. A method for preventing or treating a disease or condition
mediated by cells expressing SAF-2, the method comprising
administering to a subject in need thereof an effective amount of a
pharmaceutical composition comprising a therapeutic agent that
binds to SAF-2.
33. The method of claim 32 wherein the disease is an allergic,
asthmatic or cancerous disease or a hypereosinophilic syndrome.
34. The method of claim 33 wherein the disease is a member selected
from the group consisting of asthma, allergic rhinitis, atopic
dermatitis, chronic urticaria, nasal polyposis, Churg-Strauss
Syndrome, allergic bronchopulmonary Aspergillosis or eosinophilic
leukemia, eczema, systemic mastocytosis, lymphoma, eosinophilic
leukemia and basophilic leukemia.
35. The method of claim 32 wherein the therapeutic agent is an
antibody.
36. The method of claim 35 wherein the antibody has the identifying
characteristics of monoclonal antibody 2C4.
37. The method of claim 32 wherein the pharmaceutical composition
further comprises a cytokine.
38. The method of claim 37 wherein the cytokine is a member
selected from the group consisting of IL-5 and GM-CSF.
39. A pharmaceutical composition comprising an effective amount of
a therapeutic agent that binds to SAF-2.
40. The pharmaceutical composition of claim 39 wherein the
therapeutic agent is an antibody.
41. The pharmaceutical composition of claim 40 wherein the antibody
has the identifying characteristics of monoclonal antibody 2C4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of application of PCT
International Application No. PCT/US01/07193, filed Mar. 5, 2001,
now pending, which claims benefit of U.S. Provisional Application
No. 60/187,595, filed Mar. 7, 2000, now abandoned. This
continuation-in-part application also claims the benefit of U.S.
Provisional Application No. 60/315, 943, filed Aug. 30, 2001, now
pending, 60/349,830, filed Jan. 18, 2002, now pending, and No.
60/394,741, filed Jul. 10, 2002, now pending.
FIELD OF THE INVENTION
[0003] This invention relates to monoclonal antibodies (mAbs) that
bind to sialoadhesin factor-2 (SAF-2) and to the use of such
antibodies for diagnostic and therapeutic purposes. This invention
also relates to the prevention and treatment of diseases and
conditions mediated by cells expressing SAF-2.
BACKGROUND OF THE INVENTION
[0004] Eosinophils, basophils and mast cells have been implicated
as the major cell types producing inflammatory mediators in
response to helminthic infections, as well as several diseases,
particularly asthma, rhinitis, and atopic dermatitis (Weller, P. F.
(1991) N. Engl. J. Med. 324:1110; Sur, S., C. et al. (1993) In
Allergy Principles and Practice. E. Middleton et al. eds. Mosby,
St. Louis, Mo., p. 169; Costa, J. J. et al. (1997) JAMA 278.1815).
In these situations, the preferential accumulation and activation
of these cells has been noted. Although considerable progress has
been made in our understanding of eosinophil recruitment to the
site of inflammation, a number of key points are still unclear,
including the exact mediators utilized for localization to these
sites during the migration process. For example, activation of
microvascular endothelial cells and expression of adhesion
molecules, notably VCAM-1, is felt to be a key event in this
process during allergic inflammation (Bochner, B. S. (1998) In
Allergy Principles and Practice. J. Middleton et al. eds. Mosby,
St. Louis). In addition, a number of chemokines and other
chemotactic factors, such as those acting via CCR3, have been
implicated because of their involvement in eosinophil, basophil and
mast cell chemotaxis (Dahinden, C. A. et al. (1994) J. Exp. Med.
179.751; Daffern, P. J. et al. (1995) J. Exp. Med. 181.2119;
Nickel, R., L. et al. (1999) J. Allergy Clin. Immunol. 104:723;
Romagnani, P. et al. (1999) Am. J. Pathol 155.1195; Rot, A. et al.
(1992) J. Exp. Med. 176:1489). Another possibility, however, is
that these cells are selectively recruited and activated in a
specific way due to a unique cell surface phenotype. While
eosinophils, basophils and mast cells are readily identifiable
based on their tinctorial properties, as yet there has been no cell
surface marker identified that is unique to these cell subsets
(Saito, H. et al. (1986) Blood 67:50; Bodger, M. P. et al. (1987)
Blood 69:1414).
[0005] Sialoadhesin factor-2, or SAF-2 (European Patent Publication
No. EP 0 924 297 A1), is a member of the sialoadhesin family of
proteins also known as the I-type lectins and recently renamed the
siglec family (sialic acid-binding Ig-like lectins) (Kelm, S. et
al. (1996) Glycoconjugate Journal 13.913). The family members
include sialoadhesin (siglec-1), CD22 (siglec-2), CD33 (siglec-3),
myelin associated glycoprotein (MAG or siglec-4), siglec-5
(Cornish, A. L. et al. (1998) Blood 92.2123), OB-BP-1/siglec-6
(Patel, N. et al. (1999) J. Biol. Chem. 274:22729) and AIRM1 or
siglec-7 (Falco, M. et al. (1999) J. Exp. Med. 190:793; Nicoll, G.
et al. (1999) J. Biol. Chem. 274:34089). With the exception of
siglec-4, all are expressed on various subsets of hematopoietic
cells. Siglecs belong to the immunoglobulin (Ig) supergene family
and have an N-terminal V-set Ig domain followed by 1-16 C2 set Ig
domains. Siglecs mediate sialic acid-dependent adhesion with other
cells generally preferring either .alpha.2,3 linkages (siglec-1,
-3, and -4) or a2,6 linkages (siglec-2) (Kelm et al. supra). Most
family members have either immunoreceptor tyrosine-based inhibition
motifs (ITIM) or activation motifs (ITAM) that participate in
signaling through Src homology 2 (SH2) domain binding to the
phosphotyrosine of the ITIM or ITAM. This has been demonstrated for
CD22, CD33 and AIRM1 (Falco et al., supra; Freeman, S. D. et al.
(1995) Blood 85:2005; Blasioli, J. et al. (1999) J. Biol. Chem.
274:2302).
[0006] SAF-2, now known as Siglec-8, exists in two isoforms with
identical extracellular and transmembrane sequences. One isoform
has a short cytoplasmic tail with no known signaling sequences
(Siglec-8), while the other, Siglec-8 long form (Siglec-8L), has a
longer cytoplasmic tail containing two tyrosine-based signaling
motifs (Foussias, G. et al. (2000) Biochem Biophys Res Commun
278:775; Munday, J. et al. (2001) Biochem. J 355:489). Although the
function of Siglec-8 and Siglec-8L, and indeed most Siglecs, is
unknown, the cytoplasmic region of Siglec-8L contains one consensus
immunoreceptor tyrosine-based inhibitory motif (ITIM) and a
signaling lymphocyte activation molecule (SLAM)-like motif,
suggesting that Siglec-8L may possess signal transduction
activity.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention includes an antibody
that binds to human SAF-2. More specifically, the present invention
includes a monoclonal antibody having the identifying
characteristics of monoclonal antibody 2C4. A specific embodiment
of this aspect of the present invention is an antibody comprising a
heavy chain variable region polypeptide as set forth in SEQ ID NO:2
and a kappa light chain variable region polypeptide as set forth in
SEQ ID NO:4.
[0008] The present invention also includes an immunoglobulin heavy
chain complementarity determining region comprising any of the
polypeptides set forth in SEQ ID NOs:5, 6 or 7 or any combination
thereof, and an immunoglobulin kappa light chain complementarity
determining region comprising any of the polypeptides set forth in
SEQ ID NOs:8, 9 or 10 or any combination thereof. A preferred
embodiment of the present invention is a polypeptide comprising an
immunoglobulin complementarity determining region comprising the
polypeptides set forth in SEQ ID NOs:5, 6, 7, 8, 9 and 10. The
present invention also includes an isolated polynucleotide encoding
any of the forgoing polypeptides.
[0009] An additional embodiment of the present invention is a
method for detecting the presence of a cell in a sample wherein the
cell comprises an SAF-2 protein, the method comprising a) exposing
the sample to an antibody that binds to SAF-2 and b) detecting the
antibody that is bound to SAF-2. The sample suspected of containing
the cell can optionally be treated before exposure to the antibody
in order to render the SAF-2 susceptible to binding by the
antibody. The preferred utility for this embodiment is the
detection of eosinophils.
[0010] Another aspect of the instant invention is a method for the
prevention and treatment of a disease and condition mediated by
cells expressing SAF-2, the method comprising administering to a
subject in need thereof an effective amount of a pharmaceutical
composition that comprises a therapeutic agent that binds to SAF-2.
Preferred is a method for treating or preventing an allergic,
asthmatic or cancerous disease state, as well as hypereosinophilic
syndromes. Most preferred is a method for preventing or treating
asthma, allergic rhinitis, nasal polyposis, atopic dermatitis,
chronic urticaria, mastocytosis or eosinophilic or basophilic
leukemias. A preferred therapeutic agent that comprises the
composition for use in the method is a monoclonal antibody or
fragment thereof which binds to human SAF-2 and has the identifying
characteristics of monoclonal antibody 2C4.
[0011] Yet another aspect of the present invention includes a
pharmaceutical composition comprising an effective amount of a
therapeutic agent that binds to SAF-2. A preferred therapeutic
agent is a monoclonal antibody against human SAF-2 having the
identifying characteristics of monoclonal antibody 2C4.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the V.sub.H cDNA sequence and the deduced amino
acid sequence of a monoclonal antibody that binds to SAF-2, mAb 2C4
(SEQ ID NOs: 1 and 2, respectively). The bolded residues indicate
the three CDRs (SEQ ID NOs:5, 6, and 7).
[0013] FIG. 2 shows the V.sub.K cDNA sequence and the deduced amino
acid sequence of a monoclonal antibody that binds to SAF-2, 2C4
(SEQ ID NOs:3 and 4, respectively). The bolded residues indicate
the three CDRs (SEQ ID NOs:8, 9, and 10).
[0014] FIG. 3 presents data on expression of SAF-2 on human
peripheral blood eosinophils, basophils and 16 week old cord
blood-derived cultured mast cells. Histograms shown are
representative of 3-4 experiments with virtually identical results
for each cell type. Monoclonal reagents used as positive and
negative controls are also shown.
[0015] FIG. 4 shows the effect of Siglec-8 crosslinking on
eosinophil death. Purified peripheral blood eosinophils were
cultured under the indicated conditions. Viability was assessed
using erythrosin-B dye exclusion. Data are from six
experiments.
[0016] FIG. 5 demonstrates that Siglec-8 ligation induces
eosinophil apoptosis. Eosinophils were cultured as indicated,
harvested and analyzed by flow cytometry for annexin-V labeling.
Data are from six experiments.
[0017] FIG. 6 demonstrates the effect of IL-5 and GM-CSF on
Siglec-8 crosslinking-induced eosinophil death. In panel a, IL-5 (1
ng/ml) was added simultaneously at the beginning of the cell
culture and viability determined at various time points as
indicated. Data are from 4-6 experiments. In panel b, IL-5 or
GM-CSF (each used at 30 ng/ml) reduces the concentration of
Siglec-8 mAb needed to induce maximal eosinophil apoptosis.
Eosinophils were initially cultured with IL-5 or GM-CSF in the
presence of secondary Ab and the indicated concentrations of 2E2.
After 24 h, apoptosis was analyzed using annexin-V staining. Data
are presented as mean.+-.SD, n=2.
[0018] FIG. 7 demonstrates that IL-5 or GM-CSF priming enhances
eosinophil apoptosis in response to Siglec-8 mAb. Eosinophils were
preincubated with or without IL-5 or GM-CSF (each at 30 ng/ml) for
24 h. Antibodies were then added to the cultures, as indicated, and
apoptosis was analyzed using annexin-V staining 24 h later. Data
are presented as mean.+-.SD of two experiments.
[0019] FIG. 8 demonstrates that monoclonal antibodies to Siglec-8
either alone or in the presence of a secondary, crosslinking
antibody (pc) induces cellular death in eosinophils obtained from
bronchoalveolar lavage fluid after segmental allergen challenge.
Siglec-8 antibody (2E2 Ab) was used at two different
concenterations: 2.5 and 10 ug/ml (n=1, data presented as mean
+/-SEM of a duplicate set of experiments).
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides a variety of antibodies,
including altered antibodies and fragments thereof, directed
against SAF-2, which are characterized by their ability to bind to
human SAF-2 polypeptide or polypeptides derived therefrom.
Exemplary of this class of antibodies is monoclonal antibody 2C4.
These antibody products are useful in the detection of cells
comprising SAF-2 polypeptide including the specific detection of
eosinophils. These antibody products are also useful in therapeutic
and pharmaceutical compositions for treating allergic rhinitis,
allergies, asthma, eczema, or diseases such as lymphoma, leukemia,
or systemic mastocytosis. Alternatively, the antibodies of the
invention can be coupled to toxins, antiproliferative drugs or
radionuclides to kill cells in areas of excessive SAF-2 expression,
thereby treating allergic rhinitis, allergies, asthma, eczema, or
diseases such as lymphoma, leukemia, or systemic mastocytosis.
[0021] The instant invention also provides a novel means to treat
or prevent various disease states that are mediated by cells (or
molecules by such cells) expressing SAF-2. These disease states
include various allergies, asthma and cancers. The instant
invention pertains to the findings that SAF-2 represents a unique
cell surface marker for a circumscribed set of cells (eosinophils,
basophils and mast cells), and that binding of therapeutic agent,
such as an antibody or an altered antibody (as defined herein),
results in the specific reduction in such cells that mediate such
disease states.
[0022] "Therapeutic agent" refers to a prophylactically or
therapeutically effective molecule, including a polypeptide, an
antibody or altered antibody, and an agonist/antagonist peptide or
small molecule compound.
[0023] "Antibodies" refers to immunoglobulins which can be prepared
by conventional hybridoma techniques, phage display combinatorial
libraries, immunoglobulin chain shuffling and humanization
techniques. Also included are fully human monoclonal antibodies. As
used herein, "antibody" also includes "altered antibody" which
refers to a protein encoded by an altered immunoglobulin coding
region, which may be obtained by expression in a selected host
cell. Such altered antibodies are engineered antibodies (e.g.,
chimeric or humanized antibodies) or antibody fragments lacking all
or part of an immunoglobulin constant region, e.g., Fv, Fab, Fab'
or F(ab').sub.2 and the like. The terms Fv, Fe, Fd, Fab, Fab' or
F(ab').sub.2 are used with their standard meanings. See, e.g.,
Harlow et al. in "Antibodies A Laboratory Manual", Cold Spring
Harbor Laboratory, (1988).
[0024] "CDRs" are defined as the complementarity determining region
amino acid sequences of an antibody which are the hypervariable
regions of immunoglobulin heavy and light chains. See, e.g., Kabat
et al., Sequences of Proteins of Immunological Interest, 4th Ed.,
U.S. Department of Health and Human Services, National Institutes
of Health (1987). There are three heavy chain and three light chain
CDRs or CDR regions in the variable portion of an immunoglobulin.
Thus, "CDRs" as used herein refers to all three heavy chain CDRs,
or all three light chain CDRs or both all heavy and all light chain
CDRs, if appropriate.
[0025] CDRs provide the majority of contact residues for the
binding of the antibody to the antigen or epitope. CDRs of interest
in this invention are derived from donor antibody variable heavy
and light chain sequences, and include analogs of the naturally
occurring CDRs, which analogs share or retain the same antigen
binding specificity and/or antagonist ability as the donor antibody
from which they were derived, yet may exhibit increased affinity
for the antigen. An exemplary process for obtaining analogs is
affinity maturation by means of phage display technology as
reviewed by Hoogenboom (1997) Trends in Biotechnology 15:62; Barbas
et al. (1996) Trends in Biotechnology 14:230; and Winter et al.
(1994) Ann. Rev. Immunol. 12:433 and described by Irving et al.
(1996) Immunotechnology 2:127.
[0026] "Altered immunoglobulin coding region" refers to a nucleic
acid sequence encoding an altered antibody of the invention. When
the altered antibody is a complementarity determining
region-grafted (CDR-grafted) or humanized antibody, the sequences
that encode the CDRs from a non-human immunoglobulin are inserted
into a first immunoglobulin partner comprising human variable
framework sequences. Optionally, the first immunoglobulin partner
is operatively linked to a second immunoglobulin partner.
[0027] "First immunoglobulin partner" refers to a nucleic acid
sequence encoding a human framework or human immunoglobulin
variable region in which the native (or naturally-occurring)
CDR-encoding regions are replaced by the CDR-encoding regions of a
donor antibody. The human variable region can be an immunoglobulin
heavy chain, a light chain (or both chains), an analog or
functional fragments thereof. Such CDR regions, located within the
variable region of antibodies (immunoglobulins) can be determined
by known methods in the art. For example Kabat et al. in "Sequences
of Proteins of Immunological Interest", 4th Ed., U.S. Department of
Health and Human Services, National Institutes of Health (1987)
disclose rules for locating CDRs. In addition, computer programs
are known which are useful for identifying CDR
regions/structures.
[0028] "Second immunoglobulin partner" refers to another nucleotide
sequence encoding a protein or peptide to which the first
immunoglobulin partner is fused in frame or by means of an optional
conventional linker sequence (i.e., operatively linked).
Preferably, it is an immunoglobulin gene. The second immunoglobulin
partner may include a nucleic acid sequence encoding the entire
constant region for the same (i.e., homologous, where the first and
second altered antibodies are derived from the same source) or an
additional (i.e., heterologous) antibody of interest. It may be an
immunoglobulin heavy chain or light chain (or both chains as part
of a single polypeptide). The second immunoglobulin partner is not
limited to a particular immunoglobulin class or isotype. In
addition, the second immunoglobulin partner may comprise part of an
immunoglobulin constant region, such as found in a Fab, or
F(ab').sub.2 (i.e., a discrete part of an appropriate human
constant region or framework region). Such second immunoglobulin
partner may also comprise a sequence encoding an integral membrane
protein exposed on the outer surface of a host cell, e.g., as part
of a phage display library, or a sequence encoding a protein for
analytical or diagnostic detection, e.g., horseradish peroxidase,
.beta.-galactosidase, etc.
[0029] As used herein, an "engineered antibody" describes a type of
altered antibody, i.e., a full-length synthetic antibody (e.g., a
chimeric or humanized antibody as opposed to an antibody fragment)
in which a portion of the light and/or heavy chain variable domains
of a selected acceptor antibody are replaced by analogous parts
from one or more donor antibodies which have specificity for the
selected epitope. For example, such molecules may include
antibodies characterized by a humanized heavy chain associated with
an unmodified light chain (or chimeric light chain), or vice versa.
Engineered antibodies may also be characterized by alteration of
the nucleic acid sequences encoding the acceptor antibody light
and/or heavy variable domain framework regions in order to retain
donor antibody binding specificity. These antibodies can comprise
replacement of one or more CDRs (preferably all) from the acceptor
antibody with CDRs from a donor antibody described herein.
[0030] The term "donor antibody" refers to a monoclonal or
recombinant antibody which contributes the nucleic acid sequences
of its variable regions, CDRs or other functional fragments or
analogs thereof to a first immunoglobulin partner, so as to provide
the altered immunoglobulin coding region and resulting expressed
altered antibody with the antigenic specificity and neutralizing
activity characteristic of the donor antibody. Donor antibodies
suitable for use in this invention is a murine monoclonal antibody
designated as 2C4.
[0031] The term "acceptor antibody" refers to monoclonal or
recombinant antibodies heterologous to the donor antibody, which
contributes all, or a portion, of the nucleic acid sequences
encoding its heavy and/or light chain framework regions and/or its
heavy and/or light chain constant regions or V region subfamily
consensus sequences to the first immunoglobulin partner.
Preferably, a human antibody is the acceptor antibody.
[0032] A "chimeric antibody" refers to a type of engineered
antibody which contains a naturally-occurring variable region
(light chain and heavy chains) derived from a donor antibody in
association with light and heavy chain constant regions derived
from an acceptor antibody.
[0033] A "humanized antibody" refers to a type of engineered
antibody having its CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one or more human immunoglobulins. In
addition, framework support residues may be altered to preserve
binding affinity. See, e.g., Queen et al. (1089) Proc. Natl Acad
Sci USA 86:10029; Hodgson et al. (1991) Bio/Technology 9:421).
Furthermore, as described herein, additional residues may be
altered to preserve the activity of the donor antibody.
[0034] By "sharing the antigen binding specificity" is meant, for
example, that although mAb 2C4 may be characterized by a certain
level of binding activity, a polypeptide encoding a CDR derived
from mAb 2C4 in any appropriate structural environment may have a
lower or higher activity. It is expected that CDRs of mAb 2C4 in
such environments will nevertheless recognize the same epitope(s)
as mAb 2C4.
[0035] The phrase "having the identifying characteristics of" as
used herein indicates that such antibodies or polypeptides share
the same antigen binding specificity as the antibodies exemplified
herein, and bind to the specific antigen with a substantially
similar affinity as the antibodies exemplified herein as measured
by methods well known to those skilled in this art.
[0036] A "functional fragment" is a partial heavy or light chain
variable sequence (e.g., minor deletions at the amino or carboxy
terminus of the immunoglobulin variable region) which shares the
same antigen binding specificity as the antibody from which the
fragment was derived.
[0037] An "analog" is an amino acid sequence modified by at least
one amino acid, wherein said modification can be chemical or a
substitution or a rearrangement of a few amino acids (i.e., no more
than 10) and corresponding nucleic acid sequences, which
modification permits the amino acid sequence to retain the
biological characteristics, e.g., antigen specificity and high
affinity, of the unmodified sequence. Exemplary nucleic acid
analogs include silent mutations which can be constructed, via
substitutions, to create certain endonuclease restriction sites
within or surrounding CDR-encoding regions.
[0038] Analogs may also arise as allelic variations. An "allelic
variation or modification" is an alteration in the nucleic acid
sequence encoding the amino acid or peptide sequences of the
invention. Such variations or modifications may be due to
degeneracy in the genetic code or may be deliberately engineered to
provide desired characteristics. These variations or modifications
may or may not result in alterations in any encoded amino acid
sequence.
[0039] The term "effector agents" refers to non-protein carrier
molecules to which the altered antibodies, and/or natural or
synthetic light or heavy chains of the donor antibody or other
fragments of the donor antibody may be associated by conventional
means. Such non-protein carriers can include conventional carriers
used in the diagnostic field, e.g., polystyrene or other plastic
beads, polysaccharides, e.g., as used in the BIAcore (Pharmacia)
system, or other non-protein substances useful in the medical field
and safe for administration to humans and animals. Other effector
agents may include a macrocycle, for chelating a heavy metal atom
or radioisotopes. Such effector agents may also be useful to
increase the half-life of the altered antibodies, e.g.,
polyethylene glycol.
[0040] As used herein, the term "treating" and derivatives thereof
means prophylactic, palliative or therapeutic therapy.
[0041] For use in constructing the antibodies, altered antibodies
and fragments of this invention, a non-human species such as
bovine, ovine, monkey, chicken, rodent (e.g., murine and rat) may
be employed to generate a desirable immunoglobulin upon presentment
with human SAF-2 or a peptide epitope therefrom. Conventional
hybridoma techniques are employed to provide a hybridoma cell line
secreting a non-human-mAb to SAF-2. Such hybridomas are then
screened for binding activity as described in the Examples section.
Alternatively, fully human mAbs can be generated by techniques
known to those skilled in the art.
[0042] An exemplary mAb of the present invention is mAb 2C4, a
murine antibody which can be used for the development of a chimeric
or humanized molecule. The 2C4 mAb is characterized by specific
binding activity on human SAF-2. This mAb is produced by the
hybridoma cell line 2C4.
[0043] The present invention also includes the use of Fab fragments
or F(ab').sub.2 fragments derived from mAbs directed against SAF-2
as bivalent fragments. These fragments are useful as agents having
binding activity to SAF-2. A Fab fragment contains the entire light
chain and amino terminal portion of the heavy chain. An
F(ab').sub.2 fragment is the fragment formed by two Fab fragments
bound by disulfide bonds. The mAb 2C4 and other similar high
affinity antibodies provide sources of Fab fragments and
F(ab').sub.2 fragments which can be obtained by conventional means,
e.g., cleavage of the mAb with the appropriate proteolytic enzymes,
papain and/or pepsin, or by recombinant methods. These Fab and
F(ab').sub.2 fragments are useful themselves as therapeutic,
prophylactic or diagnostic agents, and as donors of sequences
including the variable regions and CDR sequences useful in the
formation of recombinant or humanized antibodies as described
herein.
[0044] The Fab and F(ab').sub.2 fragments can be constructed via a
combinatorial phage library (see, e.g., Winter et al. (1994) Ann.
Rev. Immunol. 12:433) or via immunoglobulin chain shuffling (see,
e.g., Marks et al. (1992) Bio/Technology 10:779), wherein the Fd or
V.sub.H immunoglobulin from a selected antibody (e.g., 2C4) is
allowed to associate with a repertoire of light chain
immunoglobulins, V.sub.L (or V.sub.K), to form novel Fabs.
Conversely, the light chain immunoglobulin from a selected antibody
may be allowed to associate with a repertoire of heavy chain
immunoglobulins, V.sub.H (or Fd), to form novel Fabs. Anti-SAF-2
mAbs can be obtained by allowing the Fd of mAb 2C4 to associate
with a repertoire of light chain immunoglobulins. Hence, one is
able to recover Fabs with unique sequences (nucleotide and amino
acid) from the chain shuffling technique.
[0045] The mAb 2C4 may contribute sequences, such as variable heavy
and/or light chain peptide sequences, framework sequences, CDR
sequences, functional fragments, and analogs thereof, and the
nucleic acid sequences encoding them, useful in designing and
obtaining various altered antibodies which are characterized by the
antigen binding specificity of the donor antibody.
[0046] The nucleic acid sequences of this invention, or fragments
thereof, encoding the variable light chain and heavy chain peptide
sequences are also useful for mutagenic introduction of specific
changes within the nucleic acid sequences encoding the CDRs or
framework regions, and for incorporation of the resulting modified
or fusion nucleic acid sequence into a plasmid for expression. For
example, silent substitutions in the nucleotide sequence of the
framework and CDR-encoding regions can be used to create
restriction enzyme sites which facilitate insertion of mutagenized
CDR and/or framework regions. These CDR-encoding regions can be
used in the construction of the humanized antibodies of the
invention.
[0047] The nucleic and amino acid sequences of the heavy chain
variable region of mAb 2C4 is set forth in SEQ ID NO:1. The CDR
amino acid sequences from this region are set forth in SEQ ID NOs:
5, 6 and 7.
[0048] The nucleic and amino acid sequences of the light chain
variable region of mAb 2C4 set forth in SEQ ID NO:3. The CDR amino
acid sequences from this region are set forth in SEQ ID NOs: 8, 9
and 10.
[0049] Taking into account the degeneracy of the genetic code,
various coding sequences may be constructed which encode the
variable heavy and light chain amino acid sequences and CDR
sequences of the invention as well as functional fragments and
analogs thereof which share the antigen specificity of the donor
antibody. The isolated nucleic acid sequences of this invention, or
fragments thereof, encoding the variable chain peptide sequences or
CDRs can be used to produce altered antibodies, e.g., chimeric or
humanized antibodies or other engineered antibodies of this
invention when operatively combined with a second immunoglobulin
partner.
[0050] It should be noted that in addition to isolated nucleic acid
sequences encoding portions of the altered antibody and antibodies
described herein, other such nucleic acid sequences are encompassed
by the present invention, such as those complementary to the native
CDR-encoding sequences or complementary to the modified human
framework regions surrounding the CDR-encoding regions. Useful DNA
sequences include those sequences which hybridize under stringent
hybridization conditions to the DNA sequences. See, T. Maniatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory (1982), pp. 387-389. An example of one such stringent
hybridization condition is hybridization at 4.times.SSC at
65.degree. C., followed by a washing in 0.1.times.SSC at 65.degree.
C. for one hour. Alternatively, an exemplary stringent
hybridization condition is 50% formamide, 4.times.SSC at 42.degree.
C. Preferably, these hybridizing DNA sequences are at least about
18 nucleotides in length, i.e., about the size of a CDR.
[0051] Altered immunoglobulin molecules can encode altered
antibodies which include engineered antibodies such as chimeric
antibodies and humanized antibodies. A desired altered
immunoglobulin coding region contains CDR-encoding regions that
encode peptides having the antigen specificity of an anti-SAF-2
antibody, preferably a high-affinity antibody such as provided by
the present invention, inserted into a first immunoglobulin partner
such as a human framework or human immunoglobulin variable
region.
[0052] Preferably, the first immunoglobulin partner is operatively
linked to a second immunoglobulin partner. The second
immunoglobulin partner is defined above, and may include a sequence
encoding a second antibody region of interest, for example an Fc
region. Second immunoglobulin partners may also include sequences
encoding another immunoglobulin to which the light or heavy chain
constant region is fused in frame or by means of a linker sequence.
Engineered antibodies directed against functional fragments or
analogs of human SAF-2 may be designed to elicit enhanced binding
with the same antibody.
[0053] The second immunoglobulin partner may also be associated
with effector agents as defined above, including non-protein
carrier molecules, to which the second immunoglobulin partner may
be operatively linked by conventional means.
[0054] Fusion or linkage between the second immunoglobulin
partners, e.g., antibody sequences, and the effector agent, may be
by any suitable means, e.g., by conventional covalent or ionic
bonds, protein fusions, or hetero-bifunctional cross-linkers, e.g.,
carbodiimide, glutaraldehyde and the like. Such techniques are
known in the art and are described in conventional chemistry and
biochemistry texts.
[0055] Additionally, conventional linker sequences which simply
provide for a desired amount of space between the second
immunoglobulin partner and the effector agent may also be
constructed into the altered immunoglobulin coding region. The
design of such linkers is well known to those of skill in the
art.
[0056] In addition, signal sequences for the molecules of the
invention may be modified by techniques known to those skilled in
the art to enhance expression and intra- and intercellular
trafficing.
[0057] A preferred altered antibody contains a variable heavy
and/or light chain peptide or protein sequence having the antigen
specificity of mAb 2C4, e.g., the V.sub.H and V.sub.L chains. Still
another desirable altered antibody of this invention is
characterized by the amino acid sequence containing at least one,
and preferably all of the CDRs of the variable region of the heavy
and/or light chains of the murine antibody molecule 2C4 with the
remaining sequences being derived from a human source, or a
functional fragment or analog thereof.
[0058] In a further embodiment, the altered antibody of the
invention may have attached to it an additional agent. For example,
recombinant DNA technology may be used to produce an altered
antibody of the invention in which the Fc fragment or CH2 CH3
domain of a complete antibody molecule has been replaced by an
enzyme or other detectable molecule, i.e., a polypeptide effector
or reporter molecule. Other additional agents include toxins,
antiproliferative drugs and radionuclides.
[0059] The second immunoglobulin partner may also be operatively
linked to a non-immunoglobulin peptide, protein or fragment thereof
heterologous to the CDR-containing sequence having antigen
specificity to human SAF-2. The resulting protein may exhibit both
antigen specificity and characteristics of the non-immunoglobulin
upon expression. That fusion partner characteristic may be, for
example, a functional characteristic such as another binding or
receptor domain or a therapeutic characteristic if the fusion
partner is itself a therapeutic protein or additional antigenic
characteristics.
[0060] Another desirable protein of this invention may comprise a
complete antibody molecule, having full length heavy and light
chains or any discrete fragment thereof, such as the Fab or
F(ab').sub.2 fragments, a heavy chain dimer or any minimal
recombinant fragments thereof such as an Fv or a single-chain
antibody (SCA) or any other molecule with the same specificity as
the selected donor monoclonal antibody, e.g., mAb 2C4. Such protein
may be used in the form of an altered antibody or may be used in
its unfused form.
[0061] Whenever the second immunoglobulin partner is derived from
an antibody different from the donor antibody, e.g., any isotype or
class of immunoglobulin framework or constant regions, an
engineered antibody results. Engineered antibodies can comprise
immunoglobulin constant regions and variable framework regions from
one source, e.g., the acceptor antibody, and one or more
(preferably all) CDRs from the donor antibody, e.g., mAb 2C4. In
addition, alterations, e.g., deletions, substitutions, or
additions, of the acceptor mAb light and/or heavy variable domain
framework region at the nucleic acid or amino acid levels, or the
donor CDR regions may be made in order to retain donor antibody
antigen binding specificity.
[0062] Such engineered antibodies are designed to employ one (or
both) of the variable heavy and/or light chains of an anti-SAF-2
mAb (optionally modified as described) or one or more of the heavy
or light chain CDRs. The engineered antibodies of the invention
exhibit binding activity.
[0063] Such engineered antibodies may include a humanized antibody
containing the framework regions of a selected human immunoglobulin
or subtype or a chimeric antibody containing the human heavy and
light chain constant regions fused to the anti-SAF-2 mAb functional
fragments. A suitable human (or other animal) acceptor antibody may
be one selected from a conventional database, e.g., the KABAT.RTM.
database, Los Alamos database, and Swiss Protein database, by
homology to the nucleotide and amino acid sequences of the donor
antibody. A human antibody characterized by a homology to the V
region frameworks of the donor antibody or V region subfamily
consensus sequences (on an amino acid basis) may be suitable to
provide a heavy chain variable framework region for insertion of
the donor CDRs. A suitable acceptor antibody capable of donating
light chain variable framework regions may be selected in a similar
manner. It should be noted that the acceptor antibody heavy and
light chains are not required to originate from the same acceptor
antibody.
[0064] Preferably, the heterologous framework and constant regions
are selected from human immunoglobulin classes and isotypes, such
as IgG (subtypes 1 through 4), IgM, IgA, and IgE. IgG1, k and IgG4,
k are preferred. Particularly preferred is IgG 4, k. Most
particularly preferred is the IgG4 subtype variant containing the
mutations S228P and L235E (PE mutation) in the heavy chain constant
region which results in reduced effector function. This IgG4
subtype variant is known herein as IgG4PE. See U.S. Pat. Nos.
5,624,821 and 5,648,260.
[0065] The acceptor antibody need not comprise only human
immunoglobulin protein sequences. For instance, a gene may be
constructed in which a DNA sequence encoding part of a human
immunoglobulin chain is fused to a DNA sequence encoding a
non-immunoglobulin amino acid sequence such as a polypeptide
effector or reporter molecule.
[0066] A particularly preferred humanized antibody contains CDRs of
mAb 2C4 inserted into the framework regions of a selected human
antibody sequence. For humanized antibodies, one, two or preferably
three CDRs from mAb 2C4 heavy chain and/or light chain variable
regions are inserted into the framework regions of the selected
human antibody sequence, replacing the native CDRs of the human
antibody.
[0067] Preferably, in a humanized antibody, the variable domains in
both human heavy and light chains have been engineered by one or
more CDR replacements. It is possible to use all six CDRs, or
various combinations of less than the six CDRs. Preferably all six
CDRs are replaced. It is possible to replace the CDRs only in the
human heavy chain, using as light chain the unmodified light chain
from the human acceptor antibody. Still alternatively, a compatible
light chain may be selected from another human antibody by recourse
to conventional antibody databases. The remainder of the engineered
antibody may be derived from any suitable acceptor human
immunoglobulin.
[0068] The engineered humanized antibody thus preferably has the
structure of a natural human antibody or a fragment thereof, and
possesses the combination of properties required for effective
therapeutic use such as the treatment of allergic rhinitis,
allergies, asthma, eczema, or diseases such as lymphoma, leukemia,
or systemic mastocytosis.
[0069] It will be understood by those skilled in the art that an
engineered antibody may be further modified by changes in variable
domain amino acids without necessarily affecting the specificity
and high affinity of the donor antibody (i.e., an analog). It is
anticipated that heavy and light chain amino acids may be
substituted by other amino acids either in the variable domain
frameworks or CDRs or both. These substitutions could be supplied
by the donor antibody or consensus sequences from a particular
subgroup.
[0070] In addition, the constant region may be altered to enhance
or decrease selective properties of the molecules of this
invention. For example, dimerization, binding to Fc receptors, or
the ability to bind and activate complement (see, e.g., Angal et
al. (1993) Mol. Immunol. 30:105; Xu et al. (1994) J. Biol. Chem.
269: 3469; European Patent Publication No. EP 0 307 434 B1).
[0071] An altered antibody which is a chimeric antibody differs
from the humanized antibodies described above by providing the
entire non-human donor antibody heavy chain and light chain
variable regions, including framework regions, in association with
human immunoglobulin constant regions for both chains. It is
anticipated that chimeric antibodies which retain additional
non-human sequence relative to humanized antibodies of this
invention may be useful for treating allergic rhinitis, allergies,
asthma, eczema, or diseases such as lymphoma, leukemia, or systemic
mastocytosis.
[0072] Preferably, the variable light and/or heavy chain sequences
and the CDRs of mAb 2C4 or other suitable donor mAbs and their
encoding nucleic acid sequences, are utilized in the construction
of altered antibodies, preferably humanized antibodies, of this
invention, by the following process. The same or similar techniques
may also be employed to generate other embodiments of this
invention.
[0073] A hybridoma producing a selected donor mAb, e.g., the murine
antibody 2C4, is conventionally cloned and the DNA of its heavy and
light chain variable regions obtained by techniques known to one of
skill in the art, e.g., the techniques described in Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold
Spring Harbor Laboratory (1989). The variable heavy and light
regions containing at least the CDR-encoding regions and those
portions of the acceptor mAb light and/or heavy variable domain
framework regions required in order to retain donor mAb binding
specificity, as well as the remaining immunoglobulin-derived parts
of the antibody chain derived from a human immunoglobulin, are
obtained using polynucleotide primers and reverse transcriptase.
The CDR-encoding regions are identified using a known database and
by comparison to other antibodies.
[0074] A mouse/human chimeric antibody may then be prepared and
assayed for binding ability. Such a chimeric antibody contains the
entire non-human donor antibody V.sub.H and V.sub.L regions, in
association with human Ig constant regions for both chains.
[0075] Homologous framework regions of a heavy chain variable
region from a human antibody are identified using computerized
databases, e.g., KABAT.RTM., and a human antibody characterized by
homology to the V region frameworks of the donor antibody or V
region subfamily consensus sequences (on an amino acid basis) to
mAb 2C4 is selected as the acceptor antibody. The sequences of
synthetic heavy chain variable regions containing the CDR-encoding
regions within the human antibody frameworks are designed with
optional nucleotide replacements in the framework regions to
incorporate restriction sites. This designed sequence is then
synthesized using long synthetic oligomers. Alternatively, the
designed sequence can be synthesized by overlapping
oligonucleotides, amplified by polymerase chain reaction (PCR), and
corrected for errors. A suitable light chain variable framework
region can be designed in a similar manner.
[0076] A humanized antibody may be derived from the chimeric
antibody, or preferably, made synthetically by inserting the donor
mAb CDR-encoding regions from the heavy and light chains
appropriately within the selected heavy and light chain framework.
Alternatively, a humanized antibody of the invention may be
prepared using standard mutagenesis techniques. Thus, the resulting
humanized antibody contains human framework regions and donor mAb
CDR-encoding regions. There may be subsequent manipulation of
framework residues. The resulting humanized antibody can be
expressed in recombinant host cells, e.g., COS, CHO or myeloma
cells.
[0077] A conventional expression vector or recombinant plasmid is
produced by placing these coding sequences for the altered antibody
in operative association with conventional regulatory control
sequences capable of controlling the replication and expression in,
and/or secretion from, a host cell. Regulatory sequences include
promoter sequences, e.g., CMV or Rous Sarcoma virus promoter, and
signal sequences, which can be derived from other known antibodies.
Similarly, a second expression vector can be produced having a DNA
sequence which encodes a complementary antibody light or heavy
chain. Preferably, this second expression vector is identical to
the first except with respect to the coding sequences and
selectable markers, in order to ensure, as much as possible, that
each polypeptide chain is functionally expressed. Alternatively,
the heavy and light chain coding sequences for the altered antibody
may reside on a single vector.
[0078] A selected host cell is co-transfected by conventional
techniques with both the first and second vectors (or simply
transfected by a single vector) to create the transfected host cell
of the invention comprising both the recombinant or synthetic light
and heavy chains. The transfected cell is then cultured by
conventional techniques to produce the engineered antibody of the
invention. The humanized antibody which includes the association of
both the recombinant heavy chain and/or light chain is screened
from culture by an appropriate assay such as ELISA or RIA. Similar
conventional techniques may be employed to construct other altered
antibodies and molecules of this invention.
[0079] Suitable vectors for the cloning and subcloning steps
employed in the methods and construction of the compositions of
this invention may be selected by one of skill in the art. For
example, the pUC series of cloning vectors, such as pUC19, which is
commercially available from vendors such as Amersham or Pharmacia,
may be used. Additionally, any vector which is capable of
replicating readily, has an abundance of cloning sites and
selectable genes (e.g., antibiotic resistance), and is easily
manipulated may be used for cloning. Thus, the selection of the
cloning vector is not a limiting factor in this invention.
[0080] Similarly, the vectors employed for expression of the
engineered antibodies according to this invention may be selected
by one of skill in the art from any conventional vector. The
vectors also contain selected regulatory sequences (such as CMV or
Rous Sarcoma virus promoters) which direct the replication and
expression of heterologous DNA sequences in selected host cells.
These vectors contain the above-described DNA sequences which code
for the engineered antibody or altered immunoglobulin coding
region. In addition, the vectors may incorporate the selected
immunoglobulin sequences modified by the insertion of desirable
restriction sites for ready manipulation.
[0081] The expression vectors may also be characterized by genes
suitable for amplifying expression of the heterologous DNA
sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR).
Other preferable vector sequences include a poly A signal sequence,
such as from bovine growth hormone (BGH) and the betaglobin
promoter sequence (betaglopro). The expression vectors useful
herein may be synthesized by techniques well known to those skilled
in this art.
[0082] The components of such vectors, e.g., replicons, selection
genes, enhancers, promoters, signal sequences and the like, may be
obtained from commercial or natural sources or synthesized by known
procedures for use in directing the expression and/or secretion of
the product of the recombinant DNA in a selected host. Other
appropriate expression vectors of which numerous types are known in
the art for mammalian, bacterial, insect, yeast and fungal
expression may also be selected for this purpose.
[0083] The present invention also encompasses a cell line
transfected with a recombinant plasmid containing the coding
sequences of the engineered antibodies or altered immunoglobulin
molecules thereof. Host cells useful for the cloning and other
manipulations of these cloning vectors are also conventional.
However, most desirably, cells from various strains of E. coli are
used for replication of the cloning vectors and other steps in the
construction of altered antibodies of this invention.
[0084] Suitable host cells or cell lines for the expression of the
engineered antibody or altered antibody of the invention are
preferably mammalian cells such as CHO, COS, a fibroblast cell
(e.g., 3T3) and myeloid cells, and more preferably a CHO or a
myeloid cell. Human cells may be used, thus enabling the molecule
to be modified with human glycosylation patterns. Alternatively,
other eukaryotic cell lines may be employed. The selection of
suitable mammalian host cells and methods for transformation,
culture, amplification, screening and product production and
purification are known in the art. See, e.g., Sambrook et al.,
supra.
[0085] Bacterial cells may prove useful as host cells suitable for
the expression of the recombinant Fabs of the present invention
(see, e.g., Pluckthun, A., Immunol. Rev., 130, 151-188 (1992)).
However, due to the tendency of proteins expressed in bacterial
cells to be in an unfolded or improperly folded form or in a
non-glycosylated form, any recombinant Fab produced in a bacterial
cell would have to be screened for retention of antigen binding
ability. If the molecule expressed by the bacterial cell was
produced in a properly folded form, that bacterial cell would be a
desirable host. For example, various strains of E. coli used for
expression are well-known as host cells in the field of
biotechnology. Various strains of B. subtilis, Streptomyces, other
bacilli and the like may also be employed.
[0086] Where desired, strains of yeast cells known to those skilled
in the art are also available as host cells, as well as insect
cells, e.g. Drosophila and Lepidoptera, and viral expression
systems. See, e.g. Miller et al., Genetic Engineering, 8, 277-298,
Plenum Press (1986) and references cited therein.
[0087] The general methods by which the vectors of the invention
may be constructed, the transfection methods required to produce
the host cells of the invention, and culture methods necessary to
produce the altered antibody of the invention from such host cell
are all conventional techniques. Likewise, once produced, the
altered antibodies of the invention may be purified from the cell
culture contents according to standard procedures of the art,
including ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and the like. Such techniques
are within the skill of the art and do not limit this
invention.
[0088] Yet another method of expression of the humanized antibodies
may utilize expression in a transgenic animal, such as described in
U.S. Pat. No. 4,873,316. This relates to an expression system using
the animal's casein promoter which when transgenically incorporated
into a mammal permits the female to produce the desired recombinant
protein in its milk.
[0089] Once expressed by the desired method, the engineered
antibody is then examined for in vitro activity by use of an
appropriate assay.
[0090] Following the procedures described for humanized antibodies
prepared from mAb 2C4, one of skill in the art may also construct
humanized antibodies from other donor antibodies, variable region
sequences and CDR peptides described herein. Engineered antibodies
can be produced with variable region frameworks potentially
recognized as "self" by recipients of the engineered antibody.
Modifications to the variable region frameworks can be implemented
to effect increases in antigen binding and antagonist activity
without appreciable increased immunogenicity for the recipient.
Such engineered antibodies may effectively treat a human for
ischemic diseases such as myocardial infarction or cerebral stroke
or treatment of vascular insufficiency diseases, such as diabetes.
Such antibodies may also be useful in the diagnosis of those
conditions.
[0091] This invention also relates to a method for treating
allergic rhinitis, allergies, asthma, eczema, or diseases such as
lymphoma, leukemia, or systemic mastocytosis in a mammal,
particularly a human, which comprises administering an effective
dose of a therapeutic agent that binds to SAF-2. Preferred is an
anti-SAF-2 monoclonal antibody. The mAb can include one or more of
the antibodies or altered antibodies described herein or fragments
thereof. Thus, the therapeutic agents of the present invention,
when in preparations and formulations appropriate for therapeutic
use, are highly desirable for persons susceptible to or
experiencing allergic rhinitis and other allergic diseases, asthma,
nasal polyposis, urticaria, hypereosinophilic syndromes (including
Churg-Strauss Syndrome and allergic bronchopulmonary
Aspergillosis), eczema, or diseases such as lymphoma, leukemia
(including cosinophilic and basophilic leukemias) or systemic
mastocytosis.
[0092] The monoclonal antibodies used in the methods of the
invention can include one or more of the antibodies or altered
antibodies described herein or fragments thereof. Preferably, the
anti-SAF-2 antibody used in the methods of the invention has the
identifying characteristics of mAb 2C4.
[0093] The altered antibodies, antibodies and fragments thereof of
this invention may also be used in conjunction with other
antibodies, particularly human mAbs reactive with other markers
(epitopes) responsible for the condition against which the
engineered antibody of the invention is directed.
[0094] The antibodies of the present invention can be formulated
into pharmaceutical compositions and administered in the same
manner as described for mature proteins. See, e.g., International
Patent Application, Publication No. WO90/02762. Generally, these
compositions contain a therapeutically effective amount of an
antibody of this invention and an acceptable pharmaceutical
carrier. Suitable carriers are well known to those of skill in the
art and include, for example, saline. Alternatively, such
compositions may include conventional delivery systems into which
protein of the invention is incorporated. Optionally, these
compositions may contain other active ingredients.
[0095] The therapeutic agents of this invention may be administered
by any appropriate internal route, and may be repeated as needed,
e.g., as frequently as one to three times daily for between 1 day
to about three weeks to once per week or once biweekly. Preferably,
the antibody is administered less frequently than is the ligand,
when it is used therapeutically. The dose and duration of treatment
relates to the relative duration of the molecules of the present
invention in the human circulation, and can be adjusted by one of
skill in the art depending upon the condition being treated and the
general health of the patient.
[0096] As used herein, the term "pharmaceutical" includes
veterinary applications of the invention. The term "therapeutically
effective amount" refers to that amount of therapeutic agent, which
is useful for alleviating a selected condition. These therapeutic
compositions of the invention may be administered to mimic the
effect of the normal receptor ligand.
[0097] This invention provides for a pharmaceutical composition
which comprises a therapeutic agent of this invention and a
pharmaceutically acceptable carrier, diluent or excipient.
Accordingly, the therapeutic agent may be used in the manufacture
of a medicament. Pharmaceutical compositions of the therapeutic
agent may be formulated as solutions or lyophilized powders for
parenteral administration. Powders may be reconstituted by addition
of a suitable diluent or other pharmaceutically acceptable carrier
prior to use. The liquid formulation may be a buffered, isotonic,
aqueous solution. Examples of suitable diluents are normal isotonic
saline solution, standard 5% dextrose in water or buffered sodium
or ammonium acetate solution. Such formulation is especially
suitable for parenteral administration, but may also be used for
oral administration or contained in a metered dose inhaler or
nebulizer for insufflation. It may be desirable to add excipients
such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
polyethylene glycol, mannitol, sodium chloride or sodium
citrate.
[0098] Alternately, the therapeutic agent may be encapsulated,
tableted or prepared in an emulsion or syrup for oral
administration. Pharmaceutically acceptable solid or liquid
carriers may be added to enhance or stabilize the composition, or
to facilitate preparation of the composition. Solid carriers
include starch, lactose, calcium sulfate dihydrate, terra alba,
magnesium stearate or stearic acid, talc, pectin, acacia, agar or
gelatin. Liquid carriers include syrup, peanut oil, olive oil,
saline and water. The carrier may also include a sustained release
material such as glyceryl monostearate or glyceryl distearate,
alone or with a wax. The amount of solid carrier varies but,
preferably, will be between about 20 mg to about 1 g per dosage
unit. The pharmaceutical preparations are made following the
conventional techniques of pharmacy involving milling, mixing,
granulating, and compressing, when necessary, for tablet forms; or
milling, mixing and filling for hard gelatin capsule forms. When a
liquid carrier is used, the preparation will be in the form of a
syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
Such a liquid formulation may be administered directly p.o. or
filled into a soft gelatin capsule.
[0099] The mode of administration of the therapeutic agent of the
invention may be any suitable route which delivers the agent to the
host. The altered antibodies, antibodies, engineered antibodies,
and fragments thereof, and pharmaceutical compositions of the
invention are particularly useful for parenteral administration,
i.e., subcutaneously, intramuscularly, intravenously or
intranasally.
[0100] Therapeutic agents of the invention may be prepared as
pharmaceutical compositions containing an effective amount of the
engineered (e.g., humanized) antibody of the invention as an active
ingredient in a pharmaceutically acceptable carrier. In the
compositions of the invention, an aqueous suspension or solution
containing the engineered antibody, preferably buffered at
physiological pH, in a form ready for injection is preferred. The
compositions for parenteral administration will commonly comprise a
solution of the engineered antibody of the invention or a cocktail
thereof dissolved in an pharmaceutically acceptable carrier,
preferably an aqueous carrier. A variety of aqueous carriers may be
employed, e.g., 0.4% saline, 0.3% glycine and the like. These
solutions are sterile and generally free of particulate matter.
These solutions may be sterilized by conventional, well known
sterilization techniques (e.g., filtration). The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, etc. The concentration of the
antibody of the invention in such pharmaceutical formulation can
vary widely, i.e., from less than about 0.5%, usually at or at
least about 1% to as much as 15 or 20% by weight and will be
selected primarily based on fluid volumes, viscosities, etc.,
according to the particular mode of administration selected.
[0101] Thus, a pharmaceutical composition of the invention for
intramuscular injection could be prepared to contain 1 mL sterile
buffered water, and between about 1 ng to about 100 mg, e.g. about
50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg,
of an engineered antibody of the invention. Similarly, a
pharmaceutical composition of the invention for intravenous
infusion could be made up to contain about 250 mL of sterile
Ringer's solution, and about 1 mg to about 30 mg and preferably 5
mg to about 25 mg of an engineered antibody of the invention.
Actual methods for preparing parenterally administrable
compositions are well known or will be apparent to those skilled in
the art and are described in more detail in, for example,
"Remington's Pharmaceutical Science", 15th ed., Mack Publishing
Company, Easton, Pa.
[0102] It is preferred that the therapeutic agent of the invention,
when in a pharmaceutical preparation, be present in unit dose
forms. The appropriate therapeutically effective dose can be
determined readily by those of skill in the art. Such dose may, if
necessary, be repeated at appropriate time intervals selected as
appropriate by a physician during the response period.
[0103] The present invention may be embodied in other specific
forms, without departing from the spirit or essential attributes
thereof, and, accordingly, reference should be made to the appended
claims, rather than to the foregoing specification or following
examples, as indicating the scope of the invention.
[0104] All publicatio indicated to be incorporated by reference
herein as though fully set forth.
EXAMPLES
[0105] The present invention will now be described with reference
to the following specific, non-limiting examples.
Example 1
Generation of Monoclonal Antibodies
[0106] A. Preparation of Recombinant SAF-2
[0107] The full length coding region of SAF-2 (see European Patent
Publication No. EP 0 924 297 A1, incorporated herein by reference)
was subcloned into the mammalian expression vector pCDN (see Aiyar
et al. (1994) Mol. Cell Biochem. 131:75-86) using PCR. The sequence
of the insert was confirmed before being transfected into HEK293
cells using Ca.sup.++PO.sub.4. Clones were selected in 500 .mu.g/mL
G418 and evaluated for expression using Northern blot analysis
followed by FACS analysis. The extracellular domain of SAF-2 was
subcloned by PCR and inserted in frame with a Factor Xa cleavage
site and the Fc portion of human IgG1. The sequence was confirmed
before the vector was electroporated into CHOEA1 cells. Stably
expressing clones were selected, expanded, evaluated for Fe
expression and scaled up. The SAF-2/Fc fusion protein was purified
from supernate using Protein A Sepherose and an aliquot was cleaved
with Factor Xa to generate the SAF-2 polypeptide used for antibody
generation.
[0108] B. Monoclonal Antibody Generation
[0109] Mice were initially immunized with SAF-2 (25 .mu.g) in
Freund's complete adjuvant and then received two booster injections
(25 .mu.g) 2 and 4 weeks later. On the basis of a good serum
antibody titer to SAF-2, one mouse received a further immunization
of 20 .mu.g of SAF-2 i.v. in PBS. The spleen was harvested four
days later and fused with myeloma cells according to the method
described in Zola (Zola, H. (1987) Monoclonal antibodies: A manual
of techniques. CRC Press, Boca Raton, Fla.).
[0110] C. Hybridoma Screening Assay
[0111] Positive hybridomas were tested for binding in 96 well
microtiter plates coated with SAF-2/Fc at 0.5 .mu.g/mL and detected
with europium conjugated anti-mouse IgG. Specifically, 96-well
plates were coated with SAF-2/Fc (100 .mu.L/well in PBS) by
incubation overnight at 4.degree. C. The solution was then
aspirated and non-specific binding sites were blocked with 250
.mu.L/well of 1% bovine serum albumin (BSA) in TBS buffer (50 mM
Tris, 150 mM NaCl, 0.02% Kathon, pH 7.4) for 5-60 minutes at RT.
Following this and each of the following steps, the plate was
washed 4 times in wash buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween
20, 0.02% Kathon, pH 7.4). To each well, 50 .mu.L hybridoma medium
and 50 .mu.l assay buffer (0.5% BSA, 0.05% bovine gamma globulin,
0.01% Tween 40, 20 .mu.M diethylenetriaminepentaacetic acid in TBS
buffer) was added and incubated for 60 minutes at RT in a
shaker-incubator. To each well was then added 100 .mu.L 0.5
.mu.g/mL Eu3+ labeled anti-mouse antibody in assay buffer. Finally,
200 .mu.L/well of enhancer (Wallac, Tuku, Finland) was added and
incubated for 5 minutes at RT, and the time-resolved fluorescence
measured. Positives were rescreened by immunoassay and BIAcore and
then cloned by the limiting dilution method. Antibodies produced by
cloned cell lines were confirmed to be specific for SAF-2 by ELISA,
BIAcore and flow cytometry using transfected cell lines.
[0112] D. Purification of mAbs
[0113] Monoclonal antibodies were purified by ProsepA (Bio
Processing, Consett, UK) chromatography, respectively, using the
manufacturer's instructions. Monoclonal antibodies were >95%
pure by SDS-PAGE.
Example 2
Characterization of Monoclonal Antibodies
[0114] A. Isotyping of Monoclonal Antibodies
[0115] Monoclonal antibody 2C4 used in this study was isotyped as
IgG1 kappa using commercially available reagents (Pharmingen, San
Diego, Calif.).
[0116] B. Affinity Measurements of Monoclonal Antibodies
[0117] The affinity of monoclonal antibody 2C4 was determined using
a BIAcore optical biosensor (Pharmacia Biosensor, Uppsala, Sweden)
using a flow rate of 30 .mu.L/min. Kinetic data was evaluated using
relationships described previously (Karlsson, et al. (1991) J.
Immunol. Meth. 145:229). The mAb (diluted in HBS buffer, 10 mM
HEPES, 150 mM NaCl, 0.01% Tween-20, pH 7.4) was injected over a
rabbit anti-mouse IgG Fe surface, followed by buffer flow, and the
RU was recorded. SAF-2 (diluted in HBS buffer) was then injected
for 180 seconds, followed by a buffer flow for 300 seconds, and the
RU was recorded. The sensor chip surface was regenerated by an
injection of 0.1 M phosphoric acid. The on-rates (K.sub.a) and
off-rates (K.sub.d) of binding were calculated using BIAcore
(Uppswala, Sweden) software. The data from this analysis indicated
that monoclonal antibody 2C4 displayed an on-rate of (K.sub.a)
2.2.times.10.sup.5 M.sup.-1s.sup.-1 and an off-rate of (K.sub.d)
4.3.times.10.sup.-5 s.sup.-1, giving a calculated equilibrium
constant (KD) of 2.0.times.10.sup.-10M.
[0118] C. Purification and Culture of Cells
[0119] Eosinophils were purified from peripheral blood following
Percoll removal of PBMC, lysis of RBC and immunomagnetic negative
selection of neutrophils (Hansel, T. T. et al. (1991) J. Immunol.
Methods 145.105). The resulting population was >95% eosinophils.
In some experiments, purified eosinophils were cultured for up to
two days in complete RPMI containing 10% FCS and 1 or 10 ng/mL
IL-5, or 10 or 50 ng/mL eotaxin (Peprotech, Rocky Hill, N.J.), C3a,
or C5a (Advanced Research Technologies) (Matsumoto, K. et al.
(1998) Am. J. Respir. Cell Mol. Biol. 18.860). Viability after 2
days or less of culture was >80%. Enrichment of peripheral blood
for basophils was performed using a double-Percoll density gradient
separation, increasing the number of basophils to 3-10% of the
total leukocyte count (Bochner, B. S. et al. (1989) J. Immunol.
Methods 125:265) or with further immunomagnetic negative selection
to at least 50%. Human cord blood-derived mast cells were generated
as previously reported (Tachimoto, H. et al. (1997) Int. Arch.
Allergy Immunol. 113:293; Saito, H. et al. (1996) J. Immunol.
157.343). The purified CD34+ cells were cultured in IMDM
supplemented with 10 .mu.g/mL insulin, 5.5 .mu.g/mL transferrin,
6.7 ng/mL selenium, 5.times.10-5 M 2-mercaptoethanol, 5% fetal
bovine serum, 100 U/mL penicillin, 100 .mu.g/mL streptomycin, 100
ng/mL stem cell factor (generously provided by Amgen, Thousand
Oaks, Calif.) and 50 ng/mL IL-6 (Biosource, Camarillo, Calif.) for
at least 10 weeks and 1 ng/mL IL-3 (Biosource) for the first 7
days. The purity of mast cells was determined by staining with
May-Grunwald and Giemsa reagents, and routinely reached 99-100% by
14-16 weeks of culture. For these experiments, cells used were
harvested at 16-17 weeks of culture. Bone marrow derived
eosinophils were cultured as follows: light density cells of
Ficolled human bone marrow were cultured in IMDM/20% FCS with 20
ng/mL rhGM-CSF and 20 ng/mL rhIL-5 (R&D Systems) at
1.5.times.106 cells/mL at 37.degree. C., 5% CO2. The cell lines
HL-60 and EOL3 were treated with sodium butyrate to differentiate
them to a more eosinophil-like phenotype (Collins, S. (1987) Blood
70:1233).
[0120] D. Expression of SAF-2 on Human Eosinophils, Basophils and
Mast Cells
[0121] Expression of integrins or SAF-2 was evaluated in
anticoagulated whole blood or in enriched cells using single color
indirect immunofluoresence and flow cytometry as previously
described (Matsumoto, K. et al. (1998) Am. J. Respir. Cell Mol.
Biol. 18:860; Bochner, B. S. et al. (1989) J. Immunol. Methods
125:265). Dual color detection of basophils was also performed
(Bochner, B. S. et al. (1989) J. Immunol. Methods 125:265).
Monoclonal antibodies used included the following: control IgG1,
CD18 (7E4), CD51 (AMF7, all Coulter-Immunotech, Hialeah, Fla.), CD9
(Immunotech) and mAb 2C4. Also used was R-phycoerythrin
(PE)-conjugated or FITC-conjugated F(ab')2 goat-anti-mouse IgG
(Biosource) and FITC-conjugated polyclonal goat anti-human IgE
(Kierkegaard and Perry, Gaithersburg, Md.). All samples were fixed
in 0.1% paraformaldehyde (Sigma) and analyzed using a
FACSCalibur.TM. flow cytometer (Becton Dickinson, Mountainview,
Calif.). At least 1,000 events were collected and displayed on a
4-log scale yielding values for mean fluorescence intensity
(MFI).
[0122] SAF-2 was localized to eosinophils (FIG. 3) and was absent
from other purified cell populations including neutrophils,
monocytes, B cells and T cells (data not shown). Activation of
purified eosinophils with optimal concentrations of eotaxin, C5a,
C3a or IL-5 for 1 hour, 24 or 48 hours before analysis did not
alter the levels of SAF-2 expression on the cell surface (data not
shown). Two cells lines, HL-60 and EOL3, which have been reported
to become more eosinophil-like following differentiation with
Na-butyrate for 5 days, were examined for the expression of SAF-2
(Mayumi, M. (1992) Leukemia & Lymphoma 7:243). Under these
culture conditions, HL-60 and EOL3 failed to express SAF-2 (data
not shown). Interestingly, when eosinophils are generated in vitro
from bone marrow with IL-5, no SAF-2 expression was noted.
Eosinophils could be identified by day 14 by staining with CD9
(3-12% of the cells) and Wright stain (data not shown). It thus
appears that SAF-2 expression may be a later marker for eosinophil
differentiation.
[0123] Low, but consistently detectable levels of SAF-2 were found
on basophils (FIG. 3; for mAb 2C4, 21.1.+-.4.0 percent positive;
mean.+-.SEM, n=4). Mature human cord blood-derived mast cells also
strongly expressed SAF-2, although the pattern of expression was
somewhat more heterogeneous than for blood leukocytes in that the
peaks were not perfectly unimodal (FIG. 3).
Example 3
Cloning and Sequencing of Heavy and Light Chain Antigen Binding
Regions
[0124] Full-length V.sub.H and V.sub.K region sequences were
obtained for monoclonal antibody 2C4 using the following cloning
strategy. The N-terminal amino acid sequences of the mAb 2C4
V.sub.H and V.sub.K were determined. In the event that the
N-terminal V region residue was blocked with pyroglutamic acid,
enzymatic de-blocking was performed by means of pyroglutamate
aminopeptidase.
[0125] Total hybridoma RNA was purified, reverse transcribed and
PCR amplified. For the heavy chains, the RNA/DNA hybrid was PCR
amplified using a mouse IgG CH1-specific primer and a degenerate
primer based on the N-terminal protein sequence. Similarly, for the
light chains, the RNA/DNA hybrid was PCR amplified using a mouse C
kappa primer and a degenerate primer based on the N-terminal
protein sequence. PCR products of the appropriate size, i.e.,
.about.350 were cloned into a plasmid vector, and sequenced by a
modification of the Sanger method (Sanger et al. (1977) PNAS USA
74:5463). In each case, the sequences of multiple V.sub.H clones
and the sequences of multiple V.sub.K clones were compared to
generate a consensus heavy chain variable region sequence and
consensus light chain variable region sequence, respectively. The
nucleotide and deduced amino acid sequences of the V.sub.H and
V.sub.K regions of monoclonal antibody 2C4 are shown in FIGS. 1 and
2, respectively.
Example 4
Expression of Short (Siglec-8) and Long (Siglec-8L) Forms of SAF-2
on Eosinophils, Basophils and Mast Cells
[0126] To clarify whether human eosinophils contain Siglec-8 or
Siglec-8L, RT-PCR was performed with Siglec-8-specific or
Siglec-8L-specific primers on mRNA from eosinophils purified as
described previously. Total RNA was prepared with Trizol.TM. (Life
Technologies, Gaithersburg, Md.) according to the manufacturers'
instructions. After DNase treatment of total RNA (DNA-free.TM.,
Ambion, Austin, Tex.), cDNAs were synthesized by extension of
oligo(dT) primers (Roche Diagnostics, Indianapolis, Ind.) using the
GeneAmp.TM. RNA PCR kit (Perkin Elmer, Foster City, Calif.).
[0127] Siglec-8 Primers:
1 5'-CTGCAGGAAGAAATCGGCA-3' (SEQ ID NO:11)
5'-ATGCTCGGTGTGGAGAAGC-3' (SEQ ID NO:12)
[0128] Siglec-8L Primers:
2 5'-CTGCAGGAAGAAATCGGCA-3' (SEQ ID NO:13)
5'-TGTGATTCCTCAAACAGGCCT-3' (SEQ ID NO:14)
[0129] The amplification cycles were 94.degree. C. for 30 seconds,
60.degree. C. for 45 seconds, and 72.degree. C. for 1 minute. After
35 cycles, PCR products were separated by 3% agarose gel
electrophoresis and stained with ethidium bromide.
[0130] Bands for both Siglec-8 and Siglec-8L were detected from
eosinophils. Sequence analysis of these PCR products revealed a
100% match with those in public databases.
[0131] Using similar RT-PCR methods, human basophils and HMC-cells
also expressed both Siglec-8 and Siglec-8L mRNA.
Example 5
Functional Analysis
[0132] Calcium Flux and Chemotaxis
[0133] Initial functional assays (Ca++ and chemotaxis) were
performed as previously described (Macphee, C. H. et al. (1998) J.
Immunol. 161:6273). To determine the role of SAF-2 in eosinophil
biology, anti-SAF-2 mAbs were analyzed for their ability to affect
eosinophil function. First, the antibodies were tested for their
ability to cause a Ca++ flux in purified eosinophils either on
their own or following crosslinking with a second antibody.
Compared with eotaxin, which gave a robust Ca++ response, none of
the mAbs to SAF-2 caused a Ca++ flux in eosinophils over a 15 min.
time course (data not shown). The mAbs were then tested for the
ability to modulate the Ca++ response to eotaxin in purified
eosinophils. The eosinophils were preincubated with anti-SAF-2 with
or without a crosslinking antibody and then simulated with eotaxin.
Again, the mAbs did not influence the Ca++ flux in response to
eotaxin. In addition, the mAbs were also evaluated in an eosinophil
chemotaxis assay using eotaxin as the chemotactic agent; again the
mAbs failed to modulate eosinophil function.
[0134] Viability Assays
[0135] Viability assays were performed in the presence of
monoclonal antibodies described herein. Eosinophils from normal,
allergic, and hypereosinophilic donors were purified from
peripheral blood as described. Eosinophil purity was consistently
>98%, with neutrophils being the only contaminating cells. The
viability of freshly isolated eosinophils was >99% as determined
by erythrosin-B dye exclusion.
[0136] Polyclonal intact and F(ab').sub.2 goat anti-mouse IgG
(heavy and light chain) were purchased from Caltag Laboratories
(Burlingame, Calif.). Recombinant human IL-5 and GM-CSF were from
R&D Systems (Minneapolis, Minn.). Mouse anti-human CD44 mAb
(clone J-173, IgG1), anti-Fas/CD95 (clone 7C11, IgM) and anti-CD18
mAb (clone 195N, IgG1) were from Beckman-Coulter (Hialeah, Fla.).
Rabbit polyclonal IgG polyhistidine His-1 Ab (mHis6 Ab) was from
Santa-Cruz Biotechnology (Santa-Cruz, Calif.). IgG and IgM isotype
control Abs were from Sigma-Aldrich (St. Louis, Mo.).
[0137] Eosinophils were harvested at different time points over
2-72 h after co-culture with the monoclonal antibodies described
herein in the presence or absence of polyclonal goat anti-mouse IgG
Ab used for secondary cross-linking. In some experiments, intact
versus F(ab').sub.2 goat anti-mouse IgG were compared in order to
elucidate any effect of Fc on eosinophil apoptosis. For controls,
cells were incubated with medium alone or CD44 mAb in the presence
or absence of secondary crosslinking Ab and with or without IL-5 or
GM-CSF (1-30 ng/ml). In priming experiments, eosinophils were first
preincubated with IL-5 or GM-CSF (30 ng/ml) for 24 h, then various
Ab were added for an additional 24 h of culture before analysis of
apoptosis.
[0138] In certain experiments, viability of cultured eosinophils
was determined by erythrosin dye exclusion as assessed by light
microscopy (Matsumoto, K., et al. (1995) Blood 86:1437; Walsh, G.
M., et al. (1998) J. Immunol. Methods 217:153). For other
experiments, morphological analysis using established light
microscopic criteria was performed. Briefly, cytocentrifugation
preparations were stained with Leukostat (Fisher Diagnostics,
Pittsburgh, Pa.) to reveal nuclear morphology. Apoptotic cells were
detected by the condensed and rounded appearance of their nuclei
under light microscopy. Cells exhibiting apoptotic nuclei were
enumerated in different fields in a blinded manner using a random
coded order. At least 500 total cells were counted per slide. Cells
were then photographed using a Zeiss Axioscope microscope
(Oberkochen, Germany) at 400.times. magnification. In addition to
light microscopic techniques, cell cycle analysis was performed
using PI staining (50 mg/ml) of fixed, permeabilized (70% EtOH,
4.sub.iC, 30 min), and RNase treated (RNase A, 0.05 mg/ml,
37.sub.iC, 30 min) eosinophils. Stained cells were then analyzed by
flow cytometry (FACS Calibur, Becton-Dickinson, San Jose, Calif.)
as described previously. Finally, annexin-V labeling was used to
detect apoptosis in eosinophils (15, 17).
[0139] Using specific murine monoclonal antibodies against Siglec-8
(note that all subsequent uses of the term Siglec-8 will refer to
both isoforms unless specified otherwise) and a secondary
polyclonal anti-mouse antibody to enhance crosslinking, we
determined whether Siglec-8 ligation inhibited eosinophil survival
in vitro. As shown in FIG. 4, Siglec-8 crosslinking with 2E2 mAb
plus a secondary polyclonal Ab induced a significant increase in
eosinophil death. Determined at 24 h of culture, for example, the
percentage of eosinophil death induced by Siglec-8 crosslinking
(68.+-.4%), was significantly higher then medium alone (23.+-.4%,
p<0.05) or CD44 control crosslinking conditions (9.+-.2%,
p<0.0001). The effect of Siglec-8 crosslinking was time
dependent, with the levels of eosinophil death increasing to more
than 90% by 48-72 h of culture.
[0140] Eosinophil death induced by Siglec-8 crosslinking, as
assessed by dye exclusion, was already approximately 70% after only
24 h of culture. To further explore the effects of Siglec-8
crosslinking on eosinophil death and to more carefully examine the
kinetics, annexin-V staining was used to distinguish apoptosis from
necrosis at various time points. FIG. 5 demonstrates a significant
increase in annexin-V+ eosinophils as early as 4 h of culture with
Siglec-8 crosslinking (15.+-.7%) compared to CD44 control
crosslinking (5.+-.3%) or medium control (3.+-.1%), indicating a
rapid apoptotic effect (n=6). FIG. 4 also demonstrates that the
Siglec-8 effect became even more pronounced by 24-72 h of culture,
especially when compared to effects of the survival promoting
cytokine, IL-5. For an additional assessment of apoptosis induced
by Siglec-8 crosslinking, light microscopic examination of
eosinophils was performed. After 24 h of culture, Siglec-8
crosslinking on eosinophils resulted in changes in morphology
characterized by reduced cell volume, loss of cytoplasmic content,
and condensation of nuclei typical of apoptosis. This was rarely
seen in cells cultured with 1L-5 or control CD44 crosslinking. As
an average from four experiments, the percentage of eosinophils
displaying morphological characteristics of apoptosis with Siglec-8
crosslinking was 43.+-.15% compared to 10.+-.2% and 10.+-.1% with
medium or CD44 Ab crosslinking, respectively. In parallel
experiments, we also studied DNA fragmentation in permeabilized
eosinophils, using PI staining in fixed cells. Siglec-8
crosslinking for 24 h increased DNA fragmentation (48% hypodiploid
DNA staining), compared to 18% and 21% with medium alone, or CD44
crosslinking, respectively. These data provide multiple lines of
evidence demonstrating apoptosis induced by Siglec-8
crosslinking.
[0141] IL-5 and GM-CSF are potent and specific anti-apoptotic
cytokines for eosinophils, and their expression is increased at
sites of allergic inflammation in the airways. When eosinophils are
cultured in the absence of survival-promoting cytokines, they
rapidly undergo apoptosis; in the presence of these cytokines,
their survival can be maintained for weeks. Therefore, we examined
the effect of IL-5 and GM-CSF on Siglec-8 crosslinking-induced
eosinophil apoptosis. When IL-5 (1 ng/ml) was added simultaneously
with Siglec-8 crosslinking antibodies at the beginning of the
culture, the cytokine could not override the Siglec-8
crosslinking-induced cell death. In fact, at 48 h, the level of
eosinophil apoptosis induced by Siglec-8 crosslinking in the
presence of 1 ng/ml of IL-5 appeared somewhat higher compared to
Siglec-8 crosslinking alone (FIG. 6a, n=4). Similar results were
obtained using 10 ng/ml of IL-5 or GM-CSF (data not shown). To
explore this further, we added higher concentrations of IL-5 or
GM-CSF (30 ng/ml) simultaneously with Siglec-8 crosslinking Abs to
the initial eosinophil cultures. Both IL-5 and GM-CSF significantly
enhanced Siglec-8-induced apoptosis. The percentage of apoptosis
increased from 53.+-.5% to 74.+-.3% and 76.+-.3% with IL-5 or
GM-CSF, respectively (n=4). To determine whether these cytokines
were enhancing eosinophil sensitivity to undergo apoptosis induced
by Siglec-8 ligation, experiments were performed in which cells
were cultured with saturating to subsaturating concentrations of
Siglec-8 mAb in the presence or absence of IL-5, GM-CSF (30 ng/ml)
or secondary Ab. Addition of IL-5 or GM-CSF markedly enhanced
eosinophil apoptosis even when sub-saturating concentrations of
Siglec-8 mAb (0.25 mg/ml) were used (FIG. 6b). These data suggest
that the presence of 1L-5 or GM-CSF rendered eosinophils more
sensitive to Siglec-8 crosslinking effects with respect to its
apoptotic effect. Therefore, one additional set of experiments was
performed to determine whether eosinophil priming with these
cytokines, prior to addition of Siglec-8 crosslinking antibodies,
enhanced the pro-apoptotic effect. Eosinophils were preincubated
with IL-5 or GM-CSF for 24 h, then mAbs were added and cells were
cultured for an additional 24 h, after which apoptosis was
analyzed. Remarkably, cytokine priming (4-30 ng/ml) led to a
profound pro-apoptotic response in the presence of Siglec-8 mAb
alone (FIG. 7 and data not shown), a response not seen in the
absence of cytokines. Note that the percentage of apoptosis in the
presence of IL-5 alone or CD44 control Ab alone was 14.+-.3%, and
15.+-.5%, respectively (FIG. 7, n=2). Levels of apoptosis with 2E2
mAb alone (69.+-.4%) in IL-5-primed eosinophils were similar to
those seen in unprimed eosinophils exposed to both 2E2 and
secondary Ab (72.+-.5%). Similar results were obtained using GM-CSF
(FIG. 7).
[0142] Crosslinking of Siglec-8 on eosinophils isolated after
allergen challenge of the lower airways by bronchoalveolar lavage
using the instant monoclonal antibodies caused apoptosis of those
eosinophils, both in the presence and absence of a secondary,
crosslinking antibody (FIG. 8).
[0143] The functional consequences of Siglec-8 crosslinking in
human basophils and mast cells was also explored. Culture of human
basophils under crosslinking conditions resulted in reductions in
total cellular histamine content as well as IgE-dependent histamine
release responses. Although apoptosis was not specifically studied
in this preliminary experiment, the most likely explanation for the
results observed is induction, by Siglec-8 crosslinking, of
apoptosis and subsequent necrosis during culture, as well as
reduced IgE-dependent releasability. In separate experiments, HMC-1
cells subjected to Siglec-8 crosslinking for 72 hours displayed
enhanced apoptosis as determined by annexin-V staining (47%
apoptosis compared to 26% apoptosis under control conditions, means
of n=2). These data suggest that crosslinking of Siglec-8 may have
profound consequences on mast cell and basophil function and
survival.
Sequence CWU 1
1
14 1 360 DNA Homo sapien 1 caggttcagc taaaggagtc aggacctggc
ctggtggcgc cctcacagag cctgtccatc 60 acttgcactg tctctgggtt
ttcattaacc atctatggtg cacactgggt tcgccagcct 120 ccaggaaagg
gtctggagtg gctgggagta atatgggctg gtggaagcac aaattataat 180
tcggctctca tgtccagact gagcatcagc aaagacaact ccaagagcca agttttctta
240 aaaataaaca gtctgcaaac tgatgacaca gccctgtact actgtgccag
agacggtagt 300 agcccctatt actattctat ggaatactgg ggtcaaggaa
cctcagtcac cgtctcctca 360 2 119 PRT Homo sapien 2 Gln Val Gln Leu
Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Tyr Gly 20 25 30
Ala His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly 35
40 45 Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
Ser 50 55 60 Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu Lys 65 70 75 80 Ile Asn Ser Leu Gln Thr Asp Asp Thr Ala Leu
Tyr Tyr Cys Ala Arg 85 90 95 Asp Gly Ser Ser Pro Tyr Tyr Tyr Ser
Met Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser
115 3 321 DNA Homo sapien 3 gagataatcc tgacccagtc tccagcaatc
atgtctgcat ctccagggga gaaggtctcc 60 ataacctgca gtgccacctc
aagtgtaagt tacatgcact ggttccagca gaagccaggc 120 acttctccca
aactctggat ttatagcaca tccaacctgg cttctggagt ccctgttcgc 180
ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa
240 gatgctgcca cttattactg ccagcaaagg agtagttacc cattcacgtt
cggctcgggg 300 acaaagttgg aaataaaacg g 321 4 107 PRT Homo sapien 4
Glu Ile Ile Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Ser Ile Thr Cys Ser Ala Thr Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu
Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Val
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Arg Ser Ser Tyr Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 5 5 PRT Homo sapien 5 Ile Tyr Gly
Ala His 1 5 6 16 PRT Homo sapien 6 Val Ile Trp Ala Gly Gly Ser Thr
Asn Tyr Asn Ser Ala Leu Met Ser 1 5 10 15 7 12 PRT Homo sapien 7
Asp Gly Ser Ser Pro Tyr Tyr Tyr Ser Met Glu Tyr 1 5 10 8 10 PRT
Homo sapien 8 Ser Ala Thr Ser Ser Val Ser Tyr Met His 1 5 10 9 7
PRT Homo sapien 9 Ser Thr Ser Asn Leu Ala Ser 1 5 10 9 PRT Homo
sapien 10 Gln Gln Arg Ser Ser Tyr Pro Phe Thr 1 5 11 19 DNA Homo
sapien 11 ctgcaggaag aaatcggca 19 12 19 DNA Homo sapien 12
atgctcggtg tggagaagc 19 13 19 DNA Homo sapien 13 ctgcaggaag
aaatcggca 19 14 21 DNA Homo sapien 14 tgtgattcct caaacaggcc t
21
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