U.S. patent application number 11/840665 was filed with the patent office on 2009-03-05 for gamma-1 and gamma-3 anti-human cd23 monoclonal antibodies and use thereof as therapeutics.
This patent application is currently assigned to BIOGEN IDEC INC.. Invention is credited to William S. Kloetzer, Takehiko Nakamura, Mitchell E. Reff.
Application Number | 20090061511 11/840665 |
Document ID | / |
Family ID | 26692224 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090061511 |
Kind Code |
A1 |
Reff; Mitchell E. ; et
al. |
March 5, 2009 |
GAMMA-1 AND GAMMA-3 ANTI-HUMAN CD23 MONOCLONAL ANTIBODIES AND USE
THEREOF AS THERAPEUTICS
Abstract
Monoclonal antibodies which specifically bind human CD23, the
low affinity receptor for IgE (FceRII/CD23), and contain either a
human gamma-1 or human gamma-3 constant domain, are disclosed. The
antibodies are useful for modulating or inhibiting induced IgE
expression. Accordingly, they have practical utility in the
treatment or prophylaxis of disease conditions wherein inhibition
of induced IgE production is therapeutically desirable, including
allergic conditions, autoimmune diseases and inflammatory
diseases.
Inventors: |
Reff; Mitchell E.; (San
Diego, CA) ; Kloetzer; William S.; (Carlsbad, CA)
; Nakamura; Takehiko; (Tokyo, JP) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
BIOGEN IDEC INC.
Cambridge
MA
|
Family ID: |
26692224 |
Appl. No.: |
11/840665 |
Filed: |
August 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10951140 |
Sep 28, 2004 |
7332163 |
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11840665 |
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09019441 |
Feb 5, 1998 |
6893636 |
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10951140 |
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08803085 |
Feb 20, 1997 |
6011138 |
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09019441 |
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Current U.S.
Class: |
435/328 ;
435/243; 435/252.1; 435/255.1; 435/334; 435/410 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 37/08 20180101; C07K 2317/92 20130101; C07K 2317/52 20130101;
C07K 2317/76 20130101; C07K 16/2851 20130101; C07K 2317/54
20130101; C07K 2317/56 20130101; A61P 29/00 20180101; A61P 37/06
20180101; A61P 37/00 20180101; C07K 2317/24 20130101; C07K 2317/21
20130101; C07K 2317/565 20130101; A61P 43/00 20180101; A61P 37/02
20180101; C07K 2319/00 20130101 |
Class at
Publication: |
435/328 ;
435/334; 435/410; 435/243; 435/252.1; 435/255.1 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C12N 5/04 20060101 C12N005/04; C12N 5/08 20060101
C12N005/08; C12N 1/20 20060101 C12N001/20; C12N 1/16 20060101
C12N001/16 |
Claims
1-39. (canceled)
40. A cell that expresses an anti-human CD23 antibody, wherein the
antibody (a) binds human CD23, (b) comprises a human gamma-1
constant region, and (c) inhibits IL-4 induced IgE expression by
B-cells in vitro to a greater extent than the anti-human CD23
monoclonal antibody which lacks a human gamma-1 constant
region.
41. The cell of claim 40, wherein the anti-human CD23 antibody
comprises a primate antigen binding portion.
42. The cell of claim 40, wherein the anti-human CD23 antibody is a
human gamma-1 monoclonal antibody.
43. The cell of claim 40, wherein the anti-human CD23 antibody
comprises a rodent antigen binding portion.
44. The cell of claim 40, wherein the anti-human CD23 antibody is a
humanized antibody.
45. The cell of claim 40, wherein the anti-human CD23 antibody
comprises a CD23 binding affinity ranging from 0.01 nM to 1000
nM.
46. The cell of claim 45, wherein the anti-human CD23 antibody
comprises a CD23 binding affinity ranging from 5 nM to 1000 nM.
47. The cell of claim 46, wherein the anti-human CD23 antibody
comprises a CD23 binding affinity ranging from 100 nM to 1000
nM.
48. The cell of claim 40, wherein the anti-human CD23 antibody
comprises light chain and heavy chain variable domains with
sequences SEQ ID NO: 6 and SEQ ID NO: 8, respectively.
49. The cell of claim 40, wherein the anti-human CD23 antibody
comprises light chain and heavy chain variable domains with
sequences SEQ ID NO:2 and SEQ ID NO:4, respectively.
50. The cell of claim 40, wherein the anti-human CD23 antibody is
capable of inhibiting the binding to CD23 of a monoclonal antibody
which comprises light chain and heavy chain variable domains with
sequences SEQ ID NO: 6 and SEQ ID NO: 8, respectively.
51. The cell of claim 40, wherein the anti-human CD23 antibody is
capable of inhibiting the binding to CD23 of a monoclonal antibody
which comprises light chain and heavy chain variable domains with
sequences SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
52. The cell of claim 48, wherein the anti-human CD23 antibody
comprises a heavy chain variable domain with the sequence set forth
in SEQ ID NO: 8, except that the asparagine corresponding to
position 78 is replaced with lysine.
53. The cell of claim 40, wherein the cell is selected from the
group consisting of mammalian cells, insect cells, plant cells,
yeast, and bacterial cells.
54. The cell of claim 53, wherein the mammalian cell is a Chinese
Hamster Ovary cell.
55. The cell of claim 53, wherein the mammalian cell is a mouse
myeloma cell.
56. The cell of claim 53, wherein the mammalian cell is a rat
myeloma cell.
57. The cell of claim 53, wherein the mammalian cell is a human
cell.
58. The cell of claim 40, wherein the mammalian cell is a
hybridoma.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/803,085, filed Feb. 20, 1997.
FIELD OF THE INVENTION
[0002] The present invention relates to monoclonal antibodies which
specifically bind human CD23, the low affinity receptor for IgE
(FceRII/CD23), and contain either a human gamma-1 or human gamma-3
constant domain, and their usage as therapeutic agents.
BACKGROUND OF THE INVENTION
[0003] IgE is a member of the immunoglobulin family that mediates
allergic responses such as asthma, food allergies, type 1
hypersensitivity and the familiar sinus inflammation allergic
rhinitis and conjunctivitis, and as a result, causes widespread
suffering throughout the general population. IgE is secreted by,
and expressed on the surface of, B-cells. IgE synthesized by
B-cells can be anchored in the B-cell membrane by a short
transmembrane domain linked to the mature IgE sequence. Membrane
and secreted versions of IgE are formed in the same cell by
differential splicing of the IgE RNA transcript.
[0004] IgE also can be bound to B-cells (and T cells, monocytes,
Langerhans cells, follicular dendritic cells, natural killer cells,
eosinophils and platelets) through its Fc region to a low affinity
IgE receptor (FceRII, hereafter "FCEL", and to mast cells and
basophils through its Fc region to a high affinity IgE receptor
FceRI, (hereinafter "FCEH"). The low affinity IgE receptor is
generally referred to in the literature as CD23.
[0005] Upon exposure of a mammal to an allergen, antigen presenting
cells process the antigen for presentation to helper T cells. These
helper T cells secrete cytokines such as IL-4 which assist B-cells
to undergo clonal amplification and secrete more allergen-specific
IgE. This newly synthesized IgE in turn is released into the
circulation where it binds to mast cells and basophils through the
high affinity receptor on their cell surface. Such mast cells and
basophils are thereby sensitized to the specific allergen. The next
exposure to the same allergen causes binding to specific IgE on the
surface of mast cells, and basophils, thereby cross-linking the
FceRI on these cells and thus activating their release of histamine
and other factors which are responsible for clinical
hypersensitivity and anaphylaxis.
[0006] The art has reported antibodies capable of binding to FCEL
(CD23)-bound IgE but not IgE bound to FCEH (see, for example, WO
89/00138 and U.S. Pat. No. 4,940,782). These antibodies are
disclosed to be clinically advantageous because they bind to IgE
which is bound to the low affinity receptor (FCEL) or to
circulating IgE's, but do not bind to IgE bound to the high
affinity receptor (FCEH). Therefore, these antibodies will not
activate mast cells or basophils.
[0007] Moreover, anti-CD23 antibodies have been reported to have
potential as therapeutics, e.g., for the treatment of allergic
disorders, inflammatory diseases, and autoimmune diseases. For
example, Bonnefoy et al., WO 9612741, report that ligands which
bind CD23, e.g., monoclonal antibodies, are useful in the treatment
or prophylaxis of inflammatory, autoimmune and allergic
diseases.
[0008] The usage of monoclonal antibodies to CD23, as both IgE
agonists and antagonists has been reported. IgE antagonists have
been reported to have potential utility in treatment of conditions
or diseases wherein IgE suppression is therapeutically desirable,
e.g., allergic conditions such as allergic rhinitis and
conjuntivitis, atopic dermatitis and asthma. For example, Bonnefoy
et al., WO 8707302 (1987), report monoclonal antibodies to human
CD23, which are assertedly useful for assaying the presence of IgE
receptors on cell types and as therapeutics in diseases wherein
modulation of IgE is therapeutically desirable.
[0009] In part because of their potential as therapeutics and
diagnostics, many groups have reported the generation of monoclonal
antibodies to CD23. See, e.g., Rector et al., Immunol., 55:481-488
(1985); Suemura et al., J. Immunol., 137:1214-1220 (1986); Noro et
al., J. Immunol., 137:1258-1263 (1986); Bonnefoy et al., J.
Immunol., 138:2170-2178 (1987); Flores-Romo et al., Science,
261:1038-1046 (1993); Sherr et al.; J. Immunol., 142:481-489
(1989); and Pene et al., Proc. Natl. Acad. Sci., USA, 85:6880-6884
(1988). Moreover, as discussed supra, the usage of such antibodies
specifically to inhibit IgE production in systems where IgE
synthesis is cytokine (IL-4) induced has also been reported.
(Flores-Romo et al (Id.); Sherr et al. (Id.); Bonnefoy et al. (WO
8707302); Bonnefoy et al. (WO 8707302); Bonnefoy et al. (WO
9612741)); Bonnefoy et al., Eur. J. Immunol 20:139-144 (1990);
Sarfati et al., J. Immunol 141:2195-2199 (1988) and Wakai et al.,
Hybridoma 12:25-43 (1993). Also, Flores-Romo et al. (Id.) disclose
that Fabs prepared from anti-CD23 antibodies inhibit
antigen-specific induced IgE responses in vivo in the rat. However,
notwithstanding what has been reported, the mechanism by which
anti-CD23 antibodies modulate IgE expression and in particular, the
manner by which they block IL-4 induced IgE production remains
unclear.
[0010] It has been suggested that anti-CD23 antibodies inhibit IgE
production by signaling through CD23 present on the surface of IgE
secreting B cells. It has been proposed that the function of CD23,
which is upregulated on IgE secreting B cells, is feedback
inhibition of IgE production (Yu, et al. Nature 369, 753-756
(1994)). This has been theorized because mice in which the CD23
gene has been removed have increased and sustained IgE production
compared to controls (Yu, et al.). In addition, it has been
reported that binding to CD23 by IgE complexes or by a monoclonal
antibody to anti-CD23 suppresses ongoing IgE synthesis by a
lymphoblastoid cell line that constitutively secretes IgE (Sherr et
al. (Id.)). It appears that this is due to down regulation of the
messenger RNA for the secreted IgE heavy chain in this cell (Saxon
et al., J. Immunol., 147:4000-4006 (1991)). However, the exact
mechanism by which IgE expression is inhibited has yet to be
explained in systems in which IgE secretion is IL-4 induced.
[0011] It has also been reported that crosslinking of Fc gamma RII
with surface Ig (B cell receptor) on B cells leads to down
regulation of Ig expression. (D'Ambrosia et al., Science,
268:293-297 (1995). A similar mechanism can be proposed for B cells
secreting IgE which also have cell surface CD23 and Fc gamma RII.
An anti-human CD23 antibody bound to a cell by antigen (CD23) and
also bound to Fc gamma RII through Fc interactions could transmit a
signal to suppress IgE secretion through Fc gamma RII.
[0012] Mechanisms involved in IgE inhibition by anti-CD23
antibodies have been proposed that include blocking interactions
other than the interaction between membrane CD23 and IgE. Related
to this, CD23, which is a member of the C-type lectin family, has
been shown to interact with several other ligands such as CD21,
CD11b and CD11c present on a variety of cell types including T
cells and monocytes. In this context CD23 can be envisioned as a
cellular adhesion molecule.
[0013] Therefore, it has been proposed that the CD21-CD23
interaction may be involved in antigen presentation and subsequent
IgE production. Models suggest CD21 on B cells sending an
activation signal for IgE production after binding to CD23 on
activated T cells present primarily in atopic individuals.
(Leconant et al., Immunol., 88:35-39 (1996); and Bonnefoy et al.,
Int. Amer. Allergy Immunol., 107:40-42 (1995).) Blocking this
interaction with an anti-CD23 could block induced IgE production.
(Aubry et al., Nature, 358:505-507 (1992) and Immunol., 5:944-949
(1993); Grosjean et al. (1992); Bonnefoy et al., Curr. Opin. Eur.
J. Immunol., 24:2982-2988 (1994); Henchoz-Lecoanet et al.,
Immunol., 88:35-39 (1996) Nambu et al., Immunol. Lett., 44:163-167
(1995); Bonnefoy et al., Int. Amer. Allergy Immunol., 107:40-42
(1995).) It is also possible that antigen presentation is
upregulated by CD23 on antigen presenting B cells binding to CD21
on T cells.
[0014] Yet another mechanism which would potentially explain the
effects of CD23 on IgE production involves soluble forms of CD23.
It has been reported that CD23 is cleaved from the cell surface
releasing several different forms of soluble CD23 or IgE binding
factors. (Sarfati et al., Immunol., 53:197-205 (1984).) Soluble
CD23 is a cytokine, with one of its reported activities being the
augmentation of IL-4 induced IgE production from B cells. (Pene et
al., J. Cell Biochem., 39:253-269 (1989); Pene et al., Eur. J.
Immunol., 18:929-935 (1988); Sarfati et al., J. Immunol.,
141:2195-2197 (1988); Sarfati et al. (1984) (Id.); (Saxon et al.,
J. Clin. Immunol. Allergy, 86 (3 pt 1) 333-344 (1990). Also,
certain forms of soluble CD23 have been reported to inhibit IgE
production (Sarfati et al., Immunol., 76:662-667 (1992)).
Accordingly, anti-CD23 antibodies potentially may block IgE
production by 1) inhibiting the IgE augmenting effects of soluble
CD23 and/or 2) blocking the proteolytic release of soluble CD23
from the cell surface.
[0015] Thus, based on the foregoing, it is clear that there is
significant complexity and uncertainty in the art with respect to
the functions of more specifically CD23 and effects on IgE
production, and further with respect to the means by which ligands
specific thereto affect IgE production.
OBJECTS OF THE INVENTION
[0016] Thus, it is an object of the invention to produce novel
ligands (antibodies) specific to CD23 and to use such antibodies to
elucidate the mechanism by which anti-CD23 antibodies modulate IgE
expression.
[0017] It is another object of the invention to produce novel
ligands (antibodies) which bind CD23, in particular human CD23,
having improved ability to inhibit induced IgE expression.
[0018] It is a more specific object of the invention to produce
anti-human CD23 antibodies containing human gamma-1 or human
gamma-3 constant domains.
[0019] It is another object of the invention to produce multivalent
anti-human CD23 antibodies which may be more effective by virtue of
their enhanced potential for cross linking CD23 and Fc
receptors.
[0020] It is another object of the invention to provide
pharmaceutical compositions containing anti-human CD23 monoclonal
antibodies comprising human gamma-1 or gamma-3 constant domains
which are capable of inhibiting induced IgE production.
[0021] It is another object of the invention to use an anti-human
CD23 monoclonal antibody comprising a human gamma-1 or human
gamma-3 constant domain for treatment or prophylaxis of disease
conditions wherein inhibition of induced IgE production is
therapeutically desirable.
[0022] More specifically, it is an object of the invention to treat
or prevent allergic conditions, autoimmune diseases and
inflammatory diseases using an anti-human CD23 monoclonal antibody
comprising either a human gamma-1 or human gamma-3 constant
domain.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 compares the in vitro IgE inhibitory activity of a
murine anti-human CD23 monoclonal antibody (MHM6), to five primate
anti-human CD23 monoclonal antibodies (5E8, 6G5, 2C8, B3B11, and
3G12);
[0024] FIG. 2 shows that primate monoclonal antibodies 5E8 and 6G5
bind an epitope on human CD23 that is distinct from commercially
available murine anti-human CD23 monoclonal antibody MHM6 (middle
panel, FIG. 2) and compete with each other (lower panel, FIG. 2).
Primate anti-human CD23 monoclonal antibodies 2C8 and, B3B11
compete with MHM6 top panel, FIG. 2).
[0025] FIG. 3 compares the in vitro IgE inhibitory activity of a
particular primate anti-human CD23 monoclonal antibody 5E8 to four
different PRIMATIZED.RTM. versions of said primate monoclonal
antibody, the sequences of which are described below.
[0026] p5E8G4P- This PRIMATIZED.RTM. antibody contains the
following sequences:
[0027] Human kappa light chain constant region and a human gamma 4
constant region which contains a P mutation (Angal et al., Mol.
Immunol., 30:105-108 (1993));
[0028] p5E8G4PN- This PRIMATIZED.RTM. antibody contains the human
kappa light chain constant region and a human gamma 4 constant
region having a P mutation (Angal et al. Mol. Immunol., 30:105-108
(1993)). This antibody also contains a mutation in the heavy chain
variable region which changes an asparagine residue (potential
carbohydrate attachment site) to a lysine;
[0029] p5E8G1 This PRIMATIZED.RTM. antibody contains the human
kappa light chain constant region and a human gamma 1 constant
region;
[0030] p5E8G1N- This PRIMATIZED.RTM. antibody contains the human
kappa light chain constant region and human gamma 1 constant
region. This antibody also contains a mutation in the heavy chain
variable region which changes an asparagine residue (carbohydrate
attachment site) to a lysine;
[0031] FIG. 4 contains a table which compares the apparent Kd in nM
of the antibodies identified in FIG. 3 and summarizes their IgE
suppressive activity.
[0032] FIG. 5 compares the in vitro IgE inhibitory activity of a
particular primate anti-human CD23 monoclonal antibody, 6G5, to two
different PRIMATIZED.RTM. versions of 6G5 which are described
below:
[0033] p6G5G1 This PRIMATIZED.RTM. antibody contains the human
lambda light chain constant region and the human gamma 1 constant
region;
[0034] p6G5G4P This PRIMATIZED.RTM. antibody contains the human
lambda light chain constant region and the human gamma 4 constant
region with a P mutation (Angal et al., Mol. Immunol., 30:105-108
(1993));
[0035] FIG. 6 compares the in vitro IgE inhibitory activity of
primate anti-human CD23 monoclonal antibody 2C8 to F(ab').sub.2
derived from 2C8;
[0036] FIG. 7 shows that the F(ab').sub.2 derived from 2C8
antagonizes the suppression of in vitro IgE activity of primate
anti-human CD23 monoclonal antibody 2C8.
[0037] FIG. 8 shows the in vivo IgE inhibitory activity of a
particular primate anti-human CD23 monoclonal antibody, 5E8, in a
SCID animal model;
[0038] FIG. 9 compares the in vivo inhibitory activity of primate
anti-human 6G5 and a PRIMATIZED.RTM. version thereof p6GSG4P.
[0039] FIG. 10 shows the in vivo IgE inhibitory activity of the
primate anti-human CD23 monoclonal antibody 6G5 and a
PRIMATIZED.RTM. version thereof, p6G5G1.
DEFINITION OF TERMS USED IN THIS APPLICATION
Chimeric Antibody
[0040] A recombinant antibody containing regions from two different
antibodies, usually different species antibodies, most typically
rodent variable sequences and human constant domain sequences.
Anti-Human CD23 Gamma 1 Antibody
[0041] An antibody that specifically binds human CD23 which
contains a human gamma 1 constant region or fragment or
modification thereof which inhibits induced IgE production. This
includes, in particular, antibodies containing rodent or primate
variable domains or antigen binding portions, humanized,
PRIMATIZED.RTM., and human anti-human CD23 monoclonal antibodies
which comprise a human gamma 1 constant domain, fragment, or
modification thereof, which inhibit induced IgE production in
vitro.
Anti-Human CD23 Gamma 3 Antibody
[0042] An antibody that specifically binds human CD23 which
contains a human gamma 3 constant region or fragment or
modification thereof which inhibits induced IgE production. This
includes, in particular, antibodies containing rodent or primate
variable domains or antigen binding portions, humanized,
PRIMATIZED.RTM., and human anti-human CD23 monoclonal antibodies
which comprise a human gamma 3 constant domain, fragment, or
modification thereof, which inhibit induced IgE production in
vitro.
Modifications of Antibody Constant Domains
[0043] Antibodies according to the present invention containing
mutations, substitutions or deletions of the constant region, that
may create a desired change in the level of efficiency, i.e. in FcR
binding, without changing the basic effector functions mediated by
the constant region.
PRIMATIZED.RTM. Antibody
[0044] A recombinant antibody containing primate variable sequences
or antigen binding portions, and human constant domain
sequences.
Humanized Antibody:
[0045] A recombinant antibody containing a non-human variable
region or antigen binding portion which has been modified to more
closely mimic a human antibody variable region and thereby
eliminate or minimize potential immunogenicity if administered to
humans without sacrificing the specificity or affinity of the
immunoglobulin. There are several known methods of humanization,
including, "veneering" which comprises select modification of
surface residues, framework replacement, (CDR grafting) and
molecular modeling.
Gamma 1 Constant Domain:
[0046] A particular type of constant domain sequence which confers
upon an antibody specific effector activities. In the present
application, gamma 1 constant domain refers to a human gamma 1
constant domain, fragment or modification thereof, which retains
gamma 1 effector functions in combination with anti-CD23 variable
domain sequences or antigen binding portions. Modifications include
human gamma-1 constant domains which comprise the deletion,
substitution or addition of one or more amino acid residues. This
effector function is manifested by the ability of an antibody
containing such a constant domain to inhibit induced IgE
production.
Gamma 3 Constant Domain:
[0047] A particular type of constant domain sequence which confers
upon an antibody specific effector activities. In the present
application, gamma 3 constant domain refers to a human gamma 3
constant domain, fragment or modification thereof, which retains
gamma 3 effector functions in combination with anti-CD23 variable
domain sequences or antigen binding portions. Modifications include
human gamma-3 constant domains which comprise the deletion,
substitution or addition of one or more amino acid residues. This
effector function is manifested by the ability of an antibody
containing such a constant domain to inhibit induced IgE
production.
CD23:
[0048] This refers to the low affinity receptor for IgE,
FceRII/CD23.
Anti-CD23 Antibody:
[0049] An antibody that specifically binds CD23, preferably human
CD23.
DETAILED DESCRIPTION OF THE INVENTION
[0050] As discussed supra, while many groups have previously
reported the production of anti-CD23 antibodies and the use thereof
as antagonists and agonists for modulating IgE production, the
exact mechanism by which such antibodies modulate IgE expression in
systems where IL-4 induces IgE production remains unclear. Thus, it
would be beneficial if the means by which such antibodies modulate
IgE expression were elucidated, or at least better explained, as
such information would be potentially useful in designing
therapeutics for treatment of diseases wherein modulation of IgE
production is therapeutically desirable. In particular, it would be
beneficial if improved antibodies specific to CD23 were obtained
having improved capacity to inhibit induced IgE production, as
enhanced IgE levels are believed to be involved in numerous disease
processes, e.g., allergic conditions, inflammatory conditions and
autoimmune diseases. Such diseases include by way of example,
atopic dermatitis, eczema, allergic rhinitis and conjuntivitis,
Job's syndrome, and asthma.
[0051] Toward that end, the present inventors have surprisingly
discovered that anti-human CD23 monoclonal antibodies which contain
human gamma-1 constant domains inhibit IgE production in systems
where IgE production is induced by IL-4 significantly better than
CD23 monoclonal antibodies of other effector types, e.g., those
comprising human gamma-4 constant domains or CD23 monoclonal
antibodies or antibody fragments lacking effector functions
altogether. Because human gamma-3 constant domains have been shown
to mediate the same effector functions as human gamma-1, anti-human
CD23 monoclonal antibodies containing human gamma-3 constant
domains are also included.
[0052] There are currently five defined effector functions for the
IgG (gamma) class of antibodies. Two of these functions, complement
activation and Fc.gamma.RN interaction, are not found in the in
vitro assays described in the present invention, and are therefore
not likely to be involved in the molecular mechanism. Three other
Fc.gamma.R receptors have been identified which interact with the
IgG class of antibodies: Fc.gamma.RI, Fc.gamma.RII (of which there
are at least six different proteins) and Fc.gamma.RIII (with at
least two different proteins). All three of these receptors
interact with both IgG1 and IgG3.
[0053] Fc.gamma.RI is the only one of the three having an
appreciable affinity for IgG. It binds both monomeric gamma-1 and
gamma-3 with a K.sub.a of about 5.times.10.sup.8 M.sup.-1. However,
its affinity for human gamma-4 is about 10-fold less, and it does
not bind human gamma-2 at all (Fries et al., 1982, J. Immunol. 129:
1041-1049; Kurlander and Batker, 1982, J. Clin. Invest. 69: 1-8;
Woof, 1986, G. Mol. Immunol. 21: 523-527; see also Burton and Woof,
1992, Human Antibody Effector Function, Adv. Immunol. 51:
1-84).
[0054] Although the affinity of human Fc.gamma.RII and
Fc.gamma.RIII for human IgG is generally very low
(K.sub.a<10.sup.7 M.sup.-1), affinity for human IgG1 and human
IgG3 increases significantly (K.sub.a=2 to 5.times.10.sup.7
M.sup.-1) when they are bound to antigen (Karas et al.; 1982, Blood
60: 1277-1282). The affinity of human Fc.gamma.RII for human IgG2
bound to antigen has given conflicting results. Human Fc.gamma.RIII
does not bind to human IgG2. Human Fc.gamma.RII and human
Fc.gamma.RIII do not bind to human IgG4 (Van de Winkel and
Anderson, 1991, J. Leuk. Biol. 49: 511-524; Huizing a et al., 1989,
J. Immunol. 142: 2359-2364).
[0055] While Fc mediated effector functions are sometimes
significant to the therapeutic activity of antibodies, this
discovery was surprising in the case of anti-CD23 antibodies
because the role of effector function in the IgE inhibitory
activity of anti-CD23 antibodies had not been previously reported.
In fact, previous evidence had suggested that antibody effector
function was not significant to the ability of anti-CD23 antibodies
to inhibit induced IgE production. For example, Flores-Romo et al.,
Science, 261:1038-1041 (1993) had reported that Fabs prepared from
a polyclonal anti-CD23 antibody inhibited an in vivo induced IgE
antigen-specific response.
[0056] The discovery that effector functions mediated through the
constant region of the anti-CD23 antibodies are apparently involved
was made after the present inventors isolated various primate
antibodies specific to CD23 having anti-IgE inhibiting activity and
compared these antibodies to PRIMATIZED.RTM. versions with respect
to their ability to inhibit IL-4 induced IgE production in vitro
and in vivo. Antibodies constructed with a human gamma-4 constant
region failed to inhibit IgE antigen-specific responses in vitro,
whereas antibodies containing a human gamma-1 constant region were
successful.
[0057] Because one (or more) of the three classes of Fc.gamma.R
receptors, Fc.gamma.RI, Fc.gamma.RII or Fc.gamma.RIII, is likely
involved in the specific effector function mediated through the
gamma-1 domain, and because these classes of receptors also bind to
antibodies containing gamma-3 domains, it is logical that anti-CD23
antibodies containing gamma-3 domains will also be successful at
inhibiting IgE-antigen specific-response in vitro.
[0058] More specifically, and as described in greater detail infra,
five primate monoclonal antibodies which specifically bound both
cellular and soluble CD23 were isolated from an Old World monkey
(macaque) according to the methodology which is disclosed in
commonly assigned application Ser. No. 08/379,072 (now allowed),
which application is incorporated by reference in its entirety
herein. This application described in detail a means for producing
monoclonal antibodies to desired antigens, desirable human
antigens, in Old World monkeys and their advantages in relation to
antibodies of other species as therapeutics, for example reduced or
potentially lack of immunogenicity in humans because of the
phylogenetic closeness of humans and Old World monkeys. In fact,
because of the phylogenetic closeness of these species, it is
difficult to distinguish Old World monkey immunoglobulins from
human immunoglobulins by sequence comparison.
[0059] Four of these five primate monoclonal anti-human CD23
antibodies were demonstrated to be capable of inhibiting IL-4
induced IgE production in an in vitro B cell assay described in
detail infra and the most potent was also shown to inhibit IL-4
induced IgE in a SCID mouse animal model (also described in detail
infra). Based on this IgE inhibitory activity, and expected low
immunogenicity in humans, such antibodies are potentially suitable
as therapeutics for treating diseases wherein inhibition of IgE
production is therapeutically desirable.
[0060] However, in order to further reduce immunogenicity, it was
elected to PRIMATIZE.RTM. two primate monoclonal antibodies (a type
of chimerization of antibodies) according to the methodology which
is also described in U.S. Ser. No. 08/379,072 (now allowed),
incorporated by reference herein. PRIMATIZATION.RTM. essentially
refers to the production of recombinant antibodies developed by
IDEC Pharmaceuticals Corporation which comprise primate variable
regions and human constant regions. Primatization of the two
primate anti-human CD23 monoclonal (5E8 and 6G5) antibodies having
potent IgE inhibiting activity was effected in order to eliminate
any potential immunogenicity attributable to the primate constant
domains in humans.
[0061] Again, because of the inventors' initial expectation from
published literature that Fc effector function was not necessary
for induced IgE inhibition, human gamma 4 versions of these
particular antibodies were initially produced. However, quite
surprisingly, it was found that the gamma-4 versions produced from
both of these primate monoclonal antibodies were ineffective, i.e.,
they required significantly higher concentrations of
PRIMATIZED.RTM. gamma 4 antibody than the primate antibody to
inhibit IL-4 induced IgE production in in vitro assays.
[0062] Moreover, even more surprising was the discovery that when
the same two primate antibodies were then converted to human
gamma-1 versions (by substitution of the primate constant domains
with human gamma-1 constant domains), that these gamma-1 antibodies
very effectively inhibited induced IgE production in vitro. Thus,
our results suggested that Fc effector function is apparently
significant to the ability of anti-human CD23 antibodies to inhibit
induced IgE production. This hypothesis was confirmed when a third
primate anti-human CD23 monoclonal, i.e., the 2C8 antibody, which
was shown by us to inhibit IgE production in vitro, was converted
to a F(ab').sub.2, which was found to be substantially incapable of
inhibiting induced IgE production in vitro. In fact, this
F(ab').sub.2 was found to antagonize the suppressive effects on
induced IgE blocking activity of the primate anti-human CD23
monoclonal antibody 2C8.
[0063] In addition, it was found that removing a glycosylation site
in the heavy chain variable region of one of the antibodies (5E8)
had no effect on binding of the antibody to CD23 (as evidenced by
obtained Kd values), or on induced IgE inhibition. Thus, the
differences in IgE inhibition were shown to apparently not involve
glycosylation differences.
[0064] The PRIMATIZED.RTM. gamma 1 version of primate 6G5 was found
to inhibit induced IgE expression in SCID mice while the same
concentration of either the primate 6G5 or the PRIMATIZED.RTM.
p6G5F4p did not inhibit induced IgE expression. Therefore, an
antibody containing human gamma-1 constant domains was found to be
even more effective in an in vivo animal model then the primate
monoclonal antibody. Furthermore, the inventors anticipate that
anti-CD23 antibodies containing human gamma-3 constant domains will
be just as effective as those having gamma-1 constant domains,
because gamma-1 and gamma-3 constant domains have affinity for the
same classes of Fc receptors.
[0065] Accordingly, based on these results, it has been
surprisingly discovered that an active Fc region, in particular
that of human gamma-1 or human gamma-3, is significantly involved
in the mechanism of IL-4 induced IgE inhibition by anti-human CD23
monoclonal antibodies. This discovery is quite unexpected
especially based on earlier reports that Fabs derived from
polyclonal anti-CD23 antibodies were capable of inhibiting induced
IgE production, and also based on the various theories as to how
CD23 affects induced IgE expression.
[0066] Accordingly, the present invention relates to anti-human
CD23 antibodies containing human gamma-1 or gamma-3 constant
domains and their use as therapeutics based on their ability to
effectively inhibit IgE expression.
[0067] The skilled artisan can prepare anti-human CD23 antibodies
containing either human gamma-1 or gamma-3 constant domains by
methods which are well known in the art for the manufacture of
chimeric antibodies. Essentially, such methods comprise producing
anti-human CD23 antibodies in a desired host or in vitro, cloning a
hybridoma or cell line which produces an anti-human CD23 monoclonal
antibody exhibiting desirable characteristics, e.g., adequate CD23
binding affinity, cloning the nucleic acid sequences which encode
such antibody from said hybridoma or cell line, e.g. by polymerase
chain reaction using suitable primers, isolating the variable
domains contained therein, recombining such variable domains with
human gamma-1 or gamma-3 constant domains and the appropriate human
light chain constant domain, and expressing the resultant nucleic
acid sequence encoding a chimeric anti-human CD23 gamma-1 or
gamma-3 immunoglobulin in a suitable expression system. Preferably,
the anti-human CD23 antibodies of the invention will have apparent
CD23 binding affinities ranging from 0.1 nM to 1000 nM, more
preferably at least 50 nM, and most preferably at least 5 nM.
[0068] Host cells suitable for expression of recombinant
immunoglobulins are well known in the art. For example, recombinant
antibodies may be expressed in Chinese hamster ovary (CHO) cells,
DG44 or DUXB11; or CHO cells CHO K-1; mouse myeloma cells SP2/0 or
X63-Ag8.653 or NSO; rat myeloma cells YB2/0; baby hamster kidney
cells, BHK; human embryonic kidney line, 293; monkey kidney cells,
CV1; human lung fibroblasts, WI38; human cervical carcinoma cells,
HELA; insect cells, plant cells, yeast or in bacteria. Further,
vectors suitable for expression of immunoglobulins are also well
known in the art and are commercially available.
[0069] A particularly preferred vector system is the
translationally impaired vector system disclosed in U.S. Ser. No.
08/147,696 (now allowed), which comprises a (neo) containing an
intron into which a desired heterologous DNA is inserted. This
vector system has been found to provide for very high yields of
recombinant proteins, e.g., immunoglobulins. However, the subject
anti-CD23 antibodies may be produced in any vector system which is
suitable for expression of functional immunoglobulins.
[0070] Also, the present invention embraces human monoclonal
antibodies of the gamma-1 or gamma-3 types which are specific to
human CD23. Methods for isolation of human monoclonal antibodies
are also well known in the art and include in vitro methods, e.g.,
in vitro immunization of human B cells in tissue culture, and in
vivo methods, e.g. synthesis of human monoclonal antibodies in SCID
mice. A preferred means of producing human monoclonal antibodies in
SCID mice which combines in vitro priming of human spleen cells
which are then introduced into SCID mice is disclosed in U.S. Ser.
No. 08/488,376 (incorporated by reference in its entirety herein).
This method is advantageous as it provides for the reproducible
recovery of monoclonal antibodies having high affinity against a
desired antigen, e.g., a human antigen.
[0071] Also, the present invention embraces human monoclonal
antibodies which compete with the primate anti-human CD23
monoclonal antibodies 5E8 and 6G5 for binding to CD23.
EXAMPLE 1
Production of Primate Anti-CD23 Antibodies
[0072] Five primate monoclonal antibodies specific to CD23 were
isolated from macaques substantially according to the methodology
disclosed in Ser. No. 08/379,072, which has been incorporated by
reference herein. The exact techniques utilized are described in
detail below.
Methodology for Isolation and Characterization of Anti-Human CD23
Monoclonal Antibodies
[0073] Purification of the Immunogen sCD23 from 8866 Cells
[0074] During purification, soluble CD23 (sCD23) was quantified by
a three-step ELISA using a murine anti-CD23 antibody (Binding Site;
catalog #MC112) as a capture. The antigen was partially purified
from cultures of 8866 cells maintained in suspension bioreactors
using RPMI 1640 (JRH Biosciences; catalog #56-509) supplemented
with 10% fetal bovine serum (JRH Biosciences) and 4 mM glutamine
(JRH Biosciences; catalog #90114) at 37.degree. C. Carbon dioxide
was used to maintain pH 7.1. After removing cells by 0.45 .mu.m
filtration, phenylmethyl sulfonyl fluoride (final concentration 0.2
mM, Sigman Chemical Co.; catalog #P-7626) and
ethylenediaminetetraacetic acid (final concentration 3 mM, Sigma
Chemical Co.; catalog #EDS) were added to the supernate and the
solution stored at 2-8.degree. C. The cell-free supernate was
concentrated approximately 15 to 20-fold using a hollow-fiber
ultrafiltration cartridge (A/T Technology; catalog #UFP-10-C-9A;
10,000 d MWCO) or tangential flow ultrafiltration cartridge
(Filtron Corporation; 10,000 d MWCO) at ambient temperature. The
concentrated supernate was sterile filtered and stored at
-70.degree. C. Thawed concentrates were de-lipidated by adding SM-2
BioBeads (BioRad Industries; catalog #152-3920) at 5 g/L and
stirring overnight at 2-8.degree. C. The resin was removed by
filtration and the solution stored at 2-8.degree. C. For some
preparations of sCD23, concentrates were fractionated using
ammonium sulfate (35-70% (w/v); Fisher; catalog #A702-3) before or
after de-lipidation.
[0075] The de-lipidated solution was subsequently purified using
affinity chromatography at 2-8.degree. C. The affinity matrix was
prepared by covalently linking a murine anti-CD23 monoclonal
antibody (BU38) to Sepharose using CNBr-activated Sepharose 4B
(Sigma Chemical Co.; catalog #C-9142). The BU38 antibody was
purified to >90% homogeneity from ascites (Binding Site; catalog
#CUS830) using Protein A chromatography. The delipidated solution
was applied to the affinity column (1.5.times.5 cm) equilibrated
with 1.times.PBS (Gibco BRL; catalog #70013-0.32), pH 7.2 and the
column washed with 1.times.PBS, pH 7.2, containing 0.05% NP40
(Sigma Chemical Co.) to remove non-bound protein. Soluble CD23 was
eluted using 3.5 M MgCl.sub.2 (Fisher; catalog #M33-500). Fractions
containing sCD23 were combined and dialyzed (Baxter Spectra/Por;
catalog #D1615-1) against 1.times.PBS, pH 7.2 at 2-8.degree. C.
After dialysis, the protein solution was concentrated by
centrifugation using Centriprep 10 spin filters (Amicon
Corporation; MWCO 10,000 d) and preparations stored at -70.degree.
C. The purity of sCD23 was estimated to be >70% using SDS-PAGE
analysis (4-20% pre-cast gels, Novex Corporation) and Coomasie
staining.
Immunization of Primates and Isolation of Immune Cells
[0076] Cynomolgus monkeys (White Sands Research Center, Alamogordo,
N. Mex.) were immunized with soluble CD23 which had been purified
from the supernatant of human RPMI 8866 cells (B cell lymphoma,
Hassner and Saxon, J. Immunol., 132:2844 (1984)). Each monkey was
immunized every third week with 200 .mu.g soluble CD23 in 500 .mu.l
PBS mixed with 167 .mu.l Temuritide (adjuvant peptide) (Sigma, St.
Louis, Mo., Catalog #A-9519) and 333 .mu.l 3.times. PROVAX.RTM.
(IDEC Pharmaceuticals Corporation). Immunization was effected
intradermally, intraperitoneally, intramuscularly and
subcutaneously. The titer of anti-CD23 antibodies in the serum of
the monkeys was measured by ELISA on 8866 cells and compared to a
pre-bleed from the same monkeys.
[0077] Monkey PRO978, with a serum titer of fifty thousand was
sacrificed, and the spleen and lymph nodes were surgically removed,
and shipped on ice to IDEC pharmaceuticals, submerged in sterile
RPMI-1640 (Gibco BRL, Gaithersburg, Md., Catalog #21870-050)
supplemented with 10% fetal calf serum, 2 mM L-glutamine, 2 mM
sodium pyruvate and 50 .mu.g/ml gentamicin. Immediately upon
arrival the spleen was homogenized by squeezing it through a wire
mesh with a glass pistil. Red blood cells were lysed in an ammonium
chloride based hypotonic buffer and the remaining lymphocytes
collected and washed in RPMI-1640 at least three times. Lymph nodes
were homogenized similarly into a single cell suspension, collected
and washed at least three times in RPMI-1640.
Production of Hybridomas
[0078] After the last wash, the cells were counted, and the primate
cells obtained above were then somatically fused to the mouse-human
heterohybridoma cell line H6K6/B5 (Carroll et al., J. Immunol.
Methods, 89:61 (1986)) using standard techniques (Boerner et al.,
J. Immunol., 147:86) (1991)) and plated into 96 well dishes (175
dishes or 14,700 wells for the spleen, and 17 dishes or 1386 wells
for the lymph nodes) at 300,000 cells per well.
[0079] This procedure involved the mixing of lymphocytes and the
above-identified fusion partner, at a 2:1 ratio, which cells were
slowly resuspended into 50% PEG 1500 (Sigma, Catalog #P5402) for 1
minute. These cells were then allowed to rest for 1 minute and then
slowly further resuspended in excess RPMI-1640. Afterward, the
cells were again allowed to rest, this time for 15 minutes before a
light spin at 250.times.g. The cells were then resuspended in
RMPI-1640 growth media, which was supplemented with 20% Fetal Calf
Serum, 2 mM L-Glutamine, Sodium Pyruvate, Non-Essential Amino Acids
and 50 .mu.g/ml Gentamicin, containing 100 .mu.M Hypoxanthine, 16
.mu.M Thymidine (BoehringerMannheim, Germany, # 623091) and 5.8
.mu.M Azaserine (Sigma, Catalog #A 1164) (HTA). HTA is a selection
agent which provides the survival of successfully fused cells
(primate lymphocyte fused with heterohybridoma fusion partner).
[0080] Approximately 65% of the wells showed growth (10,500 wells).
These wells were then screened for the presence of anti-human CD23
antibody by a three step cell ELISA.
ELISA Procedure
[0081] The first step of the ELISA comprised the transferal of
fifty microliters of supernatant from each well to ninety-six well
plates which had previously been coated with 10.sup.5 8866 cells
(CD23 positive cell line) per well. These plates were made by first
coating the plates with 50 .mu.l of aqueous solution containing
twenty .mu.g/ml Poly L-Lysine (Sigma Catalog #P1399, MW
150,000-300,000) for thirty minutes at room temperature. The
remaining solution was removed ("flicked out") and the plates left
to dry. Once dry, fifty .mu.l of 8866 cells in PBS were transferred
and spun at 600 g for five minutes. The 8866 cells were covalently
bound to the plate by adding fifty .mu.l 0.5% glutaraldehyde (Sigma
Catalog #G6257) in phosphate buffered saline (PBS) for 15 minutes.
The glutaraldehyde was removed (flicked out) and the plates blocked
with one hundred fifty .mu.l 100 mM glycine (Sigma Catalog #G-2879)
in 0.1% BSA-PBS. After the addition of supernatants, the plates
were incubated at 37.degree. C. for one to two hours and washed
seven to nine times with tap water, and a goat anti-human IgG
antibody coupled to horse radish peroxidase (HRPO) (Southern
Biotech, Birmingham, Ala., Catalog #2040-05) diluted 1:2000 into 1%
dry skimmed milk (Vons) in PBS --0.05% Tween 20 (Sigma, Catalog
#P1379) was added. The plates were incubated for forty-five minutes
at 37.degree. C., and again washed seven to nine times in tap
water. The presence of the HRPO was detected by a color development
after the addition of a TMB reagent (Kirkegaard & Perry,
Gaithersburg, Md., Catalog #50-76-02 and 50-65-02), 100 .mu.l/well.
The reaction was stopped by adding twenty-five .mu.l 4N
H.sub.2SO.sub.4. Optical density (OD) was measured at 470 nM on a
spectrophotometer (Titertek Multiscan). The OD values greater than
two times the background were scored as positive.
[0082] The second step in the ELISA was effected to confirm that
the supernatants which had been scored positive in the first ELISA
reacted to CD23 and not to some irrelevant antigen. This was
effected by testing the supernatants on SupT1 cells (ERC
BioServices Corporation, Rockville, Md., Catalog #100), a CD23
negative human cell line, using the same ELISA procedure.
Supernatants that scored similarly in both tests were discarded.
These results indicated that fifty-six of the 10,500 wells with
growth showed the presence of a primate monoclonal antibody that
bound to 8866 cells in two separate screenings at different times
and did not bind to SupT1 cells.
[0083] The third step of the ELISA was conducted to determine
whether the supernatants identified according to the first two
ELISA steps, reacted with soluble CD23. In this third ELISA, 96
well plates were coated at 4.degree. C. overnight with 2 .mu.g/ml
BG-6 (Biosource International, Camarillo, Calif., Catalog
#CT-CD23-CF), a mouse monoclonal antibody that binds to soluble
CD23 but does not block CD23-IgE binding, contained in a 50 mM
bicarbonate buffer, pH 9.3. After removing the coating buffer,
fifty .mu.l of semi-purified soluble CD23 at a predetermined
dilution in PBS were added to the plate and incubated for two hours
at room temperature. After washing the plate with tap water seven
to nine times, 50 .mu.l supernatants from selected wells were
added. After washing the plate in tap water seven to nine times, 50
.mu.l rabbit anti-human IgG (mouse adsorbed)-HRPO (Southern
Biotech, Catalog #6145-05) diluted 1:4000 in 1% dry skimmed milk in
PBS with 0.05% Tween 20 were added, incubated for two hours at
37.degree. C., washed seven to nine times in tap water and
developed with TMB as described above. Wells with OD's greater than
two times the background were again scored as positive.
[0084] Twenty-one of the fifty-six wells that showed binding to
8866 cells also bound to sCD23 in the ELISA. These wells were
expanded and subcloned at least twice by plating out cells at one
cell per three wells. After approximately three months, five stable
hybridomas producing primate monoclonal antibodies to CD23 were
obtained.
Antibody Purification by Protein A Methods
[0085] Essentially, antibodies are purified by centrifugation of
the culture supernatant to remove cells and debris. The resultant
centrifuged samples are then filtered through a 0.2 .mu.m filter. A
protein A sepharose Fast flow column is then prepared and
equilibrated using PBS (pH 7.4). The supernatant is then loaded on
the column at an appropriate flow rate (2 ml/min). After loading,
the wash column is washed with 10 column volume of PBS (pH 7.4).
The antibody is then eluted from the column with elution buffer
(0.2 M acetic acid, 0.1 M glycine pH 3.5) at 1 ml/min flow rate.
One milliliter fractions/tube (2.0 M Tris-Hcl pH 10.0) including
100 .mu.l of Tris, are then collected. Afterward, spectrophotometer
readings are taken at 280 nm. The resultant fractions with high
absorbance at 280 nm containing the antibody are then collected and
dialyzed against PBS overnight. The product is then sterilized by
filtration through 0.22 .mu.m membrane and stored at -20.degree.
C.
[0086] Four of these five primate anti-human CD23 monoclonal
antibodies (1H6, 2C8, 5E8 and 6G5) were demonstrated to inhibit IgE
production in an in vitro assay which measures IgE production by
IL-4-hydrocortisone induced peripheral blood mononuclear cell
(PBMC) cultures. These results are shown in FIG. 1. The assay
conditions are described below. The fifth primate monoclonal
anti-human CD23 antibody B3B11 was inactive in this assay.
IL-4 Stimulated IgE Production by Peripheral Blood Mononuclear
Cells
[0087] As discussed supra, the subject primate antibodies and
PRIMATIZED.RTM. forms thereof were assessed for their ability to
inhibit IgE production in an in vitro assay which measured the
effect of such antibodies on IgE production by IL-4 stimulated
peripheral blood mononuclear cells.
Materials for In Vitro IL-4 IgE Assay
[0088] Forty-eight well flat bottom cluster plates (Costar Catalog
#3548) (1.5 million PBMCs per ml per well (48 well plate))
[0089] Human recombinant IL-4 (Genzyme Catalog #2181-01; 10 .mu.g
(2.5.times.10.sup.7 units).
[0090] anti-CD23 Mabs: [0091] murine Mab (MHM6; DAKO. Catalog
#M763) [0092] primate Mabs (no preservatives) [0093]
PRIMATIZED.RTM. (no preservatives)
[0094] HB101 basal medium: (Irvine Scientific Catalog #T000)
[0095] HB101 supplement: (Irvine Scientific Catalog #T151)
[0096] Fetal Bovine Serum: (FBS; Bio-Whittaker Catalog
#14-501F)
[0097] dimethylsulfoxide: (DMSO; Fisher Scientific Catalog
#D128-500)
[0098] hydrocortisone: (Sigma Catalog #H-0888)
[0099] puromycin: (Sigma Catalog #P-7255)
[0100] cyclohexamide: (Sigma Catalog #C-7698)
[0101] Histopaque.RTM.: (Sigma Catalog #H-8889)
[0102] Hank's Buffered Salt Solution: (HBSS; Irvine Scientific
Catalog #9232)
[0103] 1% FBS in HBSS
[0104] concentrated Dulbecco's phosphate buffered saline
(10.times.DPBS; Bio-Whittaker, Catalog #17-517Q)
[0105] Bath Clear Microbicide (Fisher, Catalog #13-641-334) in
DPBS
Solutions:
[0106] puromycin solution: 40 .mu.g/ml in HB101 growth medium
[0107] cyclohexamide solution: 200 .mu.g/ml in HB101 growth
medium
[0108] hydrocortisone solution: 0.1 M solution in DMSO
[0109] anti-CD23 murine Mabs were extensively dialyzed to remove
preservatives
[0110] HB101 growth medium
TABLE-US-00001 HB101 basal medium 500 ml HB101 supplement in 10 ml
5 ml sterile filtered distilled H.sub.2O FBS 10 ml hydrocortisone
solution 0.25 ml (final conc. 5 .mu.M)
In Vitro Assay Procedure
[0111] Buffy coat cells 1:4 are diluted using HBSS at room
temperature. These cells are derived from whole blood after an
overnight incubation at room temperature to resolve and separate
the plasma components, clotted platelets and fibrin, and buffy coat
cells.
[0112] Thirty microliters of diluted buffy coat are then overlayed
onto fifteen microliters of Histopaque in fifty ml conical tubes.
These tubes are then centrifuged for twenty minutes at 1700 rpm at
room temperature without brakes (IEC 216 swinging bucket rotor).
The white PBMC layer is then collected using a sterile pipette,
taking care not to disturb the other layers. The PBMCs (peripheral
blood mononuclear cells) are the buffy coat cells which have been
sedimented by centrifugation partially through a HISTOPAQUE.RTM.
density gradient to form a distinctly visible white layer of cells.
These cells are collected with a pipette, rinsed with HBSS, and
then counted using a hemocytometer. Typically, 300 to 600 million
PBMCs can be recovered from a single 450 ml buffy coat package.
[0113] The collected PBMCs are then washed three times in 1%
FBS/HBSS. The washed cells are collected by centrifugation for
seven minutes at 1300 rpm at 7.degree. C.
[0114] The number of cells collected is then determined using a
hemocytometer. The cell concentration is adjusted to about three
million cells per milliliter of HB101 growth medium.
[0115] Approximately about 1.5 million cells (0.5 ml) are then
added to each well of a 48 well plate. In general, five replicate
samples are prepared for each experiment. The perimeter wells of
each plate are not used for cell samples. Accordingly, these wells
are filled, e.g., using 0.5 ml of 0.05% BathClear/DPBS.
[0116] 0.5 ml HB101 growth medium containing desired amounts of
IL-4 and Mab is then added to the wells. The IL-4 used is
recombinant DNA-generated human interleukin 4. The Mab used in the
assay is a murine, primate or PRIMATIZED.RTM. antibody. Typically,
IL-4 is added at a final concentrate of 100 U/ml and Mab is added
at a final concentrate ranging from 0.01 to 3 .mu.g/ml.
[0117] The cells are then incubated for nine to eleven days at
37.degree. C. in a moist incubator set at 5% CO.sub.2. After
incubation, the supernatant fluids are collected and the IgE
content is measured.
IgE ELISA
[0118] The following list identifies materials and solutions used
in the IgE ELISAs.
Materials and Solutions Needed for IgE ELISA
[0119] sulfuric acid, 4 M coating buffer: 10 mM sodium bicarbonate
buffer, pH 9.6 concentrated phosphate buffered saline
(10.times.PBS) stock solution:
TABLE-US-00002 NaH.sub.2PO.sub.4 26.6 gm Na.sub.2HPO.sub.4 289 gm
NaCl 1064 gm distilled H.sub.2O 10 L
blocking buffer: 10% FBS/PBS dilution buffer: 1% BSA/0.05% Tween
20/PBS washing buffer: 0.05% Tween 20/PBS goat anti-human IgE
(epsilon chain-specific), unlabeled: (Tago Catalog #4104) human IgE
standard: (The Binding Site Catalog #BP094) goat anti-human IgE,
HRP-labeled: (Tago Catalog #AHI 0504) TMB peroxidase substrate:
(KPL Catalog #50-76-02) peroxidase solution B: (KPL Catalog
#50-65-02) working substrate solution: mix substrate and Solution B
at 1:1 ratio Immulon II microtiter plates (Dynatech Labs Catalog
#011-010-3455)
IgE ELISA Procedure
[0120] Each well of a microtiter plate is coated using 100 .mu.l of
a coating buffer containing 2 .mu.g/ml goat anti-human IgE.
[0121] The coated plate is then incubated overnight at 4.degree.
C.
[0122] After incubation, each well in the plate is then washed
three times with 200 .mu.l of Tween 20/PBS. After washing, the
non-specific binding sites are blocked with 200 .mu.l blocking
buffer/well for 1 hour at 37.degree. C.
[0123] One hundred .mu.l of samples or standards are then added to
each well; which wells are then incubated over-night at 4.degree.
C. After incubation, the samples are tested with or without
dilution. A standard concentration curve is prepared for each plate
using several dilutions of IgE ranging from 0.1 to 50 ng/ml.
[0124] After overnight incubation, each plate is washed five times
with Tween 20/PBS.
[0125] One hundred .mu.l of horseradish peroxidase (HRP) labeled
goat anti-human IgE diluted 1:10,000 in dilution buffer is then
added to each drained well. The plate is then incubated for 4 hours
at 37.degree. C.
[0126] The plates are then washed 5 times with Tween 20/PBS and 3
times with water.
[0127] One hundred .mu.l of 3,3',5,5'-tetramethylbenzidine working
substrate solution is then added to each well. The plate is then
incubated for twenty-five minutes in the dark at room temperature.
After incubation the developing reaction is stopped by the addition
of fifty p1 of 4 M sulfuric acid.
[0128] The absorbency is then read concurrently at 450 and 540 nm.
The 540 nm absorbency values are subtracted as background.
Assay for Kd Measurement of Primate Monoclonal Anti-Human CD23
Antibodies
Scatchard Analysis Procedure
1. Radiolabeling Procedure
[0129] IODO-BEADS are washed with 100 mN Phosphate Buffer, pH 7.4
twice using 1 mL of buffer per 2 beads. The beads are then dried on
filter paper.
[0130] The two beads are then added to 100 .mu.l .sup.125I
solution, containing about 1 mCi of I, diluted with 200 .mu.l of
the phosphate buffer, and left at room temperature for 5
minutes.
[0131] The antibody (50 .mu.gs) is added to the preloaded beads.
The reaction time for maximal incorporation of radioactivity is 6
minutes.
[0132] The reaction is stopped by removing the radiolabeled
antibody from the reaction vessel.
[0133] Gel filtration is then performed to remove excess .sup.125I
or unincorporated .sup.125I from the radiolabeled antibody
solution. This is effected by passing the radiolabeled antibody
over a column made up of 1.5 mL Sephadex-G25, 1.5 mL DEAE
Sephadex-A25 and 0.5 mL Amberlite. The radiolabeled antibody is
eluted off in a total volume of 5 mL at a concentration of about 10
.mu.g/mL. (Elution Buffer: 1.times.PBS containing 10% Gelatin, 2%
Sodium Azide and 1% BSA).
2. Optimization Assay (Direct Binding Study)
[0134] The specific activity of the 10 .mu.g/mL radiolabeled
solution is determined by taking a 1 .mu.l sample and running the
sample on a gamma counter.
EXAMPLE
[0135] 1.times.10.sup.5 cpm/.mu.l.times.1000 .mu.l/10 .mu.g
antibody [0136] 1.times.10.sup.5 cpm/.mu.g antibody [0137]
1.times.0.sup.4 cpm/ng antibody [0138] Molecular wt. of
antibody=75,000 ng/nmole
[0139] Specific Activity: [0140] 1.times.0.sup.4
cpm/ng.times.75,000 ng/nmole=7.5.times.0.sup.8 cpm/nmole
[0141] The antigen-coated plate is blocked (to eliminate
non-specific binding, e.g., with mB7.1-CHO) and the background
plate (i.e., Untransfected-CHO) for one hour at room
temperature--with 200 .mu.l/well of blocking buffer (Blocking
Buffer: 1.times.PBS containing 10% Gelatin, 2% Sodium Azide, 1% BSA
and 10% FBS).
[0142] The plate(s) are then washed, typically ten times by hand
with tap water.
[0143] The 10 .mu.g/mL radiolabeled antibody (50 .mu.ls) is then
titrated by two-fold serial dilutions across the plate(s) using a
multichannel pipette. Incubate for one hour at room
temperature.
[0144] The plate(s) are again washed about 6-7 times with 200
.mu.l/well of wash buffer (Wash Buffer:--1.times.PBS containing 10%
Gelatin and 2% Sodium Azide).
[0145] The radio activity counts in each well are then determined
by running the wells on a gamma counter.
[0146] The optimal radiclabeled antibody concentration is the
concentration in which the difference between the specific counts
and background counts is at a maximum.
3. Scatchard Analysis of Competition Assay
[0147] The 10 .mu.g/mL radiolabeled solution is diluted to the
optimal concentration determined in the Direct Binding
experiment.
[0148] The antigen-coated plate and the background plate are
blocked for one hour at room temperature with 200 .mu.l/well of
blocking buffer.
[0149] The plate(s) are then washed, e.g., about 10 times, by hand
with tap water.
[0150] The "cold" (no radiolabel) antibody is then titrated by
two-fold dilutions in a separate U-bottom microtitre plate. The
starting concentration of the "cold" antibody should be at least
100 times greater than that of the optimal radiolabeled antibody
concentration.
EXAMPLE
[0151] Optimal Radiolabeled Conc.: 0.5 .mu.g/mL [0152] "Cold"
Antibody Conc.: 100 .mu.g/mL (Note: 1:2 titration in the first well
will adjust the "cold" antibody concentration to 50 .mu.g/mL.)
[0153] Fifty .mu.l/well of optimal radiolabeled antibody are then
added to the wells containing "cold" antibody.
[0154] One hundred .mu.l/well of the mixed solution are then
transferred to the corresponding wells of the antigen-coated plate,
and incubated for one hour at room temperature.
[0155] Also, it is desirable also that the following controls be
effected:
[0156] a) Direct binding of radiolabeled antibody to antigen-coated
plate (5 wells),
[0157] b) Direct binding of radiolabeled antibody to background
plate (5 wells).
[0158] After incubation, the plate(s) are washed, e.g., about 6-7
times, with 200 .mu.l/well of wash buffer.
[0159] The radio activity counts in each well are then determined
by running the wells on a gamma counter.
[0160] These calculations are determined by calculating the
specific counts in each well tested by subtracting the background
counts from the counts bound to the antigen-coated plate.
4. Calculations for Scatchard Analysis
[0161] The Molar Concentration of Bound antibody [B] can then be
determined as follows:
[0162] Example: At 50 .mu.g/mL "cold antibody"
Specific counts bound: 4382 cpm Counts bound in the presence of 50
.mu.g/mL "cold" ab: 215 cpm Difference: 4382 cpm-215 cpm=4167
cpm
Specific Activity ( radiolabeled ab ) : 5.54 .times. 10 9 cpm /
nmole ##EQU00001## 4167 cpm / 5.54 .times. 10 9 cpm / nmole = 7.52
.times. 10 - 7 nmole ##EQU00001.2## 7.53 .times. 10 - 7 nmole /
0.05 mL ( sample vol . ) = 1.50 .times. 10 - 5 nmole / mL = 1.50
.times. 10 - 8 mole / mL [ B ] = 1.50 .times. 10 - 11 mole / mL ( M
) ##EQU00001.3##
Total Molar Concentration [T] is determined as follows:
50 g / mL .times. 1 mole / 75 , 000 g = 6.67 .times. 10 - 4 mole /
mL = 6.67 .times. 10 - 7 mmole / mL ( M ) [ T ] = 66667 .times. 10
- 11 mmole / mL ( M ) ##EQU00002##
Free antibody [F] is determined as follows:
Free Molar Conc . = Total minus Bound ##EQU00003## [ F ] = ( 66667
.times. 10 - 11 ) - ( 1.50 .times. 10 - 11 ) = 66665.5 .times. 10 -
11 mmole / mL ( M ) ##EQU00003.2##
Calculate B/F.
[0163] Plot B versus B/F on Cricket Graph software.
Activity and Affinity of Anti-Human CD23 Antibodies According to
the Invention
[0164] Four of the five isolated primate anti-human CD23 monoclonal
antibodies (B3B11, 2C8, 5E8 and 6G5) were found to inhibit IgE
production in the above-identified in vitro assay which measures
IgE production by IL-4-hydrocortisone induced peripheral blood
mononuclear cell (PBMC) cultures. These results are shown in FIG.
1. The fifth primate monoclonal anti-human CD23 antibody 3G12 was
inactive in this assay.
[0165] Two of the four primate monoclonal anti-human CD23
antibodies (B3B11 and 2C8) found to be active in this in available
mouse anti-human CD23 antibody MHM6 (CAKO A/S, Glostrup, Denmark
Catalog #M763). (FIG. 2, top panel.) However, in repeated assays
these antibodies were not as potent IgE inhibitors as MHM6 (data
not shown). By contrast, the other primate anti-CD23 monoclonal
antibodies (5E8 and 6G5) were found to compete with each other and
did not complete with MHM6. (FIG. 2, middle and bottom panels.)
Moreover, the primate anti-human CD23 monoclonal antibody 5E8 was
found to be a potent inhibitor of IL-4 induced IgE in the in vitro
assay. (See FIGS. 1 and 3.)
Modified Hu-SCID-Mouse Model for Human IgE Synthesis and Measuring
the Inhibition of IL-4 Induced IgE Production by Anti-CD23
Antibodies In Vivo
[0166] A modified hu-PBMC-SCID mouse model was also developed to
detect the effect of the subject antibodies on induced human IgE
production in vivo. PBMCs obtained from two donors were cultured
with IL-4 in vitro for two days. PBMCs were pooled and used to
reconstitute groups of C.B.-17 SCID mice with and without
antibodies. Mice were bled on day 14, 21, 28 and 35 and serum IgG
and IgE levels were determined by ELISA. This in vivo model was
used to assay primate and two different versions of PRIMATIZED.RTM.
antibodies to CD23 for their ability to inhibit the production of
IgE.
[0167] A modified SCID mouse model was used because it is known
that severe combined immunodeficiency scid/scid (SCID) mice,
C.B.-17 (Bosma et al., Nature, 301:527 (1983)) reconstituted with
human peripheral blood mononuclear cells (hu-PBMC-SCID) can produce
significant quantities of human immunoglobulins (Ig) (Moiser et
al., Nature, 335:256 (1988); Moiser et al., J. Clin. Immunol.,
10:185 (1990); Abedi et al., J. Immunol., 22:823 (1992); and
Mazingue et al., Eur. J. Immunol., 21:1763 (1991).) The predominant
isotype of human immunoglobulin (Ig) produced in hu-PBMC-SCID mice
is IgG. Generally, IgM, IgA and IgE isotypes are found in very low
or non-detectable levels except in cases where PBMC is obtained
from donors with certain autoimmune or allergic disease conditions.
It has also been reported that manipulation of hu-PBMC SCID mouse
model with certain cytokines may be provided for the generation of
significant levels of non-IgG isotypes, including IgE (Kilchherr et
al., Cellular Immunology, 151:241 (1993); Spiegelberg et al., J.
Clin. Investigation, 93:711 (1994); and Carballido et al., J.
Immunol., 155:4162 (1995)). The hu-PBMC-SCIDs, has been also used
to generate antigen specific Ig provided the donor has been primed
for the antigen in vivo.
[0168] Therefore, the aim of the present inventors was focused on
establishing a suitable human IgE producing hu-PBMC-SCID mouse
model that could be used to test the efficacy of therapeutic for
treatment of IgE related diseases such as allergic disorders,
including the subject anti-CD23 antibodies.
Materials and Methods:
[0169] The following materials and methods were used in the
hu-PBMC-SCID mouse model described below.
[0170] SCID mice: C.B-17 scid/scid immunodeficient mice were
obtained from Taconic (C.B.-17/IcrTac-scidfDF) and maintained in
IDEC Pharmaceuticals' animal facility. Mice were housed in
sterilized microbarrier units with sterilized bedding. Animal
studies were performed in accordance with the "Guide for the Care
and Use of Laboratory Animals" specified by the Committee on Care
of Laboratory Animal Resources Commission on Life Science-National
Research Council (Guide for the Care and Use of Laboratory Animals,
DHHS Publ. No. (NIH) 86-23, Bethesda, Md., NIH, 1985).
[0171] Human PBMC: PBMCs were isolated from buffy coats obtained
from a blood bank by centrifugation through Ficoll-Hypaque
(Histopaque-1077) as recommended by the manufacturer (Sigma
Diagnostics Catalog #1077-1). Lymphocyte preparation at the
interface of the gradient were harvested and washed three times in
Hanks Balanced Salt Solution (HBSS) (Bio-Whittaker Catalog
#10-527F). For each experiment PBMCs were obtained from two
separate donors and cultured separately in vitro. PBMCs were
resuspended at 1-3.times.10.sup.6 cells/ml concentration in
HB-Basal medium plus 1% HB101 lyophilized supplement (Irvine
Scientific Catalog #T000 & T151) containing 5% FCS plus 1000
IU/ml of IL-4 (Genzyme, Inc. Catalog #2181-01) and incubated for 48
hours at 37.degree. C. with 5% CO.sub.2. After incubation, the
cells from different buffy coats were harvested, pooled and used to
reconstitute SCID mice.
In Vivo Assay Conditions
[0172] Groups of mice (four to five per group) were injected with
fifty-sixty.times.10.sup.6 lymphocytes in 200-300 .mu.l volume of
HBSS intraperitoneally (i.p.) on day zero. For the groups that
received anti-CD23 antibody, on day zero, PBMCs were mixed with
anti-CD23 antibody (200 to 400 .mu.g/mouse) before i.p., injection
and the second injection was given on day seven. All mice received
5000 IU per mouse of IL-4 i.p., between day zero to day five. A
group which was not injected with antibody served as the control
group. Mice were bled from a retro-orbital vein and the serum was
analyzed for IgG and IgE on days fourteen, twenty-one, twenty-eight
and thirty-five by ELISA.
[0173] FIG. 8 shows that the primate anti-human CD23 monoclonal
antibody 5E8 is effective in inhibiting IL-4 induced IgE production
in vivo in the SCID mouse model.
Cloning and Expression of PRIMATIZED.RTM. Anti-Human CD23
Monoclonal Antibodies
[0174] In order to clone primate immunoglobulin variable domains,
Poly A+ RNA was separately isolated from approximately
2.times.10.sup.6 cells from the primate heterohybridomas secreting
the anti-human CD23 monoclonal antibodies 6G5 and 5E8 by using the
Micro-FastTrack mRNA isolation Kit (Invitrogen Catalog #K1520-02)
according to methods set forth by the manufacturer.
[0175] The first strand of cDNA was synthesized from the poly A+
RNA by using the cDNA Cycle Kit (Invitrogen Catalog #L1310-01)
according to conventional methods.
[0176] The light and heavy chain variable regions of 6G5 and 5E8
were then isolated by PCR from cDNA using PCR primers that were
selected based upon different consensus families of human
immunoglobulins. 5' primers were selected which corresponded to the
beginning of the leader sequences of the light and heavy variable
region and 3' primers were selected which corresponded to the J
region (The specific primers used to PCR amplify the lambda light
chain variable domain of 6G5, the kappa light chain variable
domains of 5E8, and the heavy chain variable domains of 6G5 and 5E8
are set forth in Tables 1-3). PCR was performed according to
standard methods (30 cycles with 1 minute at 94.degree. C., 1.5
minutes at 54.degree. C. and 2 minutes at 72.degree. C. in a Hot
start 100 tube (Gibco BRL Catalog #10332-013). PCR was set up in 50
.mu.l reactions containing 5 .mu.l out of 80 .mu.l cDNA (from
2.times.10.sup.6 cells) as a template, 2 .mu.l of 5 nM DNTP, 1
.mu.l of Taq polymerase, 5 .mu.l of Taq polymerase buffer, 2 .mu.l
of the 5' primer (25 pmoles/.mu.l), 2 .mu.l of the 3' primer (25
pmoles/.mu.l), and 36 .mu.l of water. (Taq polymerase and buffer
were obtained from Stratagene Catalog #600131, DNTP from Boehringer
Mannheim Catalog #1581295.)
A) Construction of the Plasmids N5LG1+6G5 and N5LG4P+6G5
1) Cloning the Light Chain Variable Domain of Primate Monoclonal
Anti-Human CD23 Antibody 6G5 by PCR
[0177] The first PCR amplification of the light chain variable
region from the cDNA of primate monoclonal antibody 6G5 showed
bands which were consistent in situ with the lambda light chain
variable region. These bands appeared in all reactions using the
three different early leader sequence primers. (See Tables 1-3.)
However, the PCR product obtained using primer 745 (Family 2) was
considered more specific because of the relatively greater
intensity of the PCR product band.
[0178] This PCR product was isolated using a Qiaquick Gel
Extraction Kit (Qiagen Catalog #28704). The purified PCR fragment
was digested with Bgl II and Avr II restriction endonucleases, and
ligated into the mammalian expression vector N5LG1 which was
digested with the same restriction endonucleases. Twenty
microliters of the ligation mixture containing the purified PCR
product from one fifty microliter PCR reaction, 100 mg N5LG1
vector, two microliters of 10.times. ligation buffer (NEE Catalog
#202S) and two microliters of T4 ligase (NEB Catalog #202S), were
then incubated at 14.degree. C. overnight.
[0179] The mammalian expression vector N5LG1 contains genetic
sequences (e.g., regulatory sequences, coding sequences) which
provide for the expression of four separate proteins in a mammalian
cell. They are:
[0180] (i) a partial immunoglobulin light chain with the human
lambda light chain constant region and unique restriction
endonuclease sites for inserting light chain variable domains;
[0181] (ii) a partial immunoglobulin heavy chain with the and human
gamma 1 chain constant region coding sequences and unique
restriction endonuclease sites for inserting heavy chain variable
domains;
[0182] (iii) a neomycin phosphotransferase gene used to select for
cells that have incorporated the plasmid and are resistant to the
antibiotic Geneticin (Gibco BRL Catalog #10131-1209); and
[0183] (iv) a murine dihydrofolate reductase gene (DHFR) which
provides for the selection and genomic amplification when cells are
cultured in the presence of methotrexate (MTX, Sigma Catalog
#A-6770) (Reff et al., Blood, 83:433-445 (1994).
[0184] After ligation, the mixture was digested using Pme I
restriction endonuclease, which digests the parent N5LG1 plasmid,
but not the NSLGl plasmid which has been ligated to the light
variable domain of 6G5. After digestion, the mixture was
transformed into Epicurian coli.RTM. XL1-Blue competent cells
(Stratagene Catalog #200249) as follows.
[0185] One hundred microliters of competent cells were mixed with
10 .mu.l of the above ligation mixture, set on ice for 30 minutes,
then heated at 45.degree. C. for 30 seconds. This mixture was
placed on ice for 2 minutes, and 900 .mu.l of SOC, prewarmed to
room temperature, was then added. (SOC is LB broth Gibco BRL
Catalog #10855-013, plus 0.02 M MgCl.sub.2, 0.02 M MgSO.sub.4 and
0.02 M D-glucose.) After incubation at 37.degree. C. for an hour,
the mixture was centrifuged at 4000 g for a minute, and 800 .mu.l
of supernatant discarded. The rest of the mixture was plated onto a
LB agar (Gibco BRL Catalog #12945-044) dish containing 50 .mu.g/ml
ampicillin (Amp, Gibco BRL Catalog #13075-015). Plasmid DNA was
isolated from individual colonies of E. coli that grew on the Amp
plate by using the Wizard.RTM. Miniprep DNA purification system
(Promega Catalog #A7510).
[0186] The isolated plasmid DNA was then characterized by digestion
with Bgl II and Avr II followed by agarose gel electrophoresis. An
ethidium stained DNA band of 400 bp was indicative of a potential
successful cloning of a light chain variable domain.
[0187] To confirm this was an immunoglobulin light chain variable
domain, sequencing was done using the Sequenase 7-Deaza-dGTP DNA
Sequencing Kit (USB catalog #70990) with sequencing primers 607 and
GE 108. (See Sequencing Primers in Table 4.)
[0188] A second independent PCR amplification of the light chain
from cDNA of primate monoclonal antibody 6G5 was effected using a
5' primer early leader sequence of lambda light chain family 2
(primer 745) and the 3' J region primer 926. (See Primers for PCR
of the lambda light chain variable domain of 6G5 in Tables 1-3.)
The isolated PCR product (see technique above) was cloned into TA
vector by using the Original TA Cloning (Kit (Invitrogen Catalog
#K2000-01). The isolated miniprep DNA (see technique above) was
examined under agarose gel electrophoresis after digestion with
EcoR I restriction endonuclease. The resultant PCR product
comprised in the TA vector was then sequenced (as described
previously) using Sp6 and M13 (-40) forward primers (See Sequencing
primers in Table 4). The resultant light chain sequence was
identical to that of light chain from the first PCR. This entire
sequence of the light chain variable domain of primate monoclonal
anti-human CD23 antibody 6G5 is presented below.
TABLE-US-00003 Light chain variable region of primate monoclonal
antibody anti-human CD23 6G5 Leader Met Ala Trp Thr Leu Leu Leu Val
Thr Leu Leu Thr Gln Gly Thr ATG GCC TGG ACT CTG CTC CTC GTC ACC CTC
CTC ACT CAG GGC ACA -1 Gly Ser Trp Ala GGA TCC TGG GCT Mature
Protein (Numbering is Kabat) Framework 1 1 9 11 Gln Ser Ala Pro Thr
Gln Pro Pro Ser Val Ser Gly Ser Pro Gly CAG TCT GCC CCG ACT CAG CCT
CCC TCT GTG TCT GGG TCT CCT GGA 20 23 Gln Ser Val Thr Ile Ser Cys
CAG TCG GTC ACC ATC TCC TGC CDR 1 24 27 27A 27B 27C 28 34 Thr Gly
Thr Ser Asp Asp Val Gly Gly Tyr Asn Tyr Val Ser ACT GGA ACC AGC GAT
GAC GTT GGT GGT TAT AAC TAT GTC TCC Framework 2 35 40 49 Trp Tyr
Gln His His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr TGG TAC CAA CAC
CAC CCA GGC AAA GCC CCC AAA CTC ATG ATT TAT CDR2 50 56 Asp VaT Ala
Lys Arg Ala Ser GAT GTC GCT AAG CGG GCC TCA Framework 3 57 60 70
Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala GGG GTC
TCT OAT COC TTC TCT GGC TCC AAG TCT GGC AAC ACG GCC 80 Ser Leu Thr
Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr TCC CTG ACC ATC TCT
GGG CTC CAG GCT GAG GAC GAG GCT GAT TAT 88 Tyr Cys TAC TGT CDR 3 89
90 95 95A 96 97 Cys Ser Tyr Thr Thr Ser Ser Thr Leu Leu TGT TCA TAT
ACA ACC AGT AGC ACT TTG TTA Framework 4 98 100 106 106A 107 Phe Gly
Arg Gly Thr Arg Leu Thr Val Leu Gly TTC GGA AGA GGG ACC CGG TTG ACC
GTC CTA GGT
2) Cloning the Heavy Chain Variable Domain of Primate Monoclonal
Anti-Human CD23 Antibody 6G5 by PCR
[0189] The first PCR amplification of the heavy chain variable
domain from cDNA of primate monoclonal antibody 6G5 was performed
by using the set of early leader sequence primers described supra
and the 3' J region primer GE244. These primers are in Tables 1-3
infra. This reaction resulted in a 350 base PCR product. This 350
base product (purified as described supra), was digested with Nhe I
and Sal I, and ligated into N5LG1 and digested with the same
endonucleases in the first PCR amplification. The resultant
ligation mixture was transformed into host cells using the same
techniques for cloning the light chain. Plasmid N5LG1 containing
the 350 base PCR product was then isolated and sequenced (using
sequencing primers 266 and 268). (These Sequencing primers are set
forth in Table 4.)
[0190] Sequencing revealed that the PCR product contained only part
of the heavy variable domain and comprised a deletion in its amino
terminus (Sequence began at framework 2, codon 36).
[0191] A second independent PCR reaction was conducted to amplify
and isolate the heavy chain variable domain of primate monoclonal
antibody 6G5 using a 5' early leader sequence primer for family 1
(MB1503) and a 3`J` region primer GE244. (These primers are also
contained in Tables 1-3.) The resultant PCR product was then cloned
into the N5LG1 using the same techniques described supra. Its
sequence was found to be identical to the first PCR product.
[0192] Therefore, in order to clone the whole heavy variable domain
of 6G5 including the missing 5' terminus a new longer 3' primer
(MB1533) which included the CDR3 and framework 4 regions of the 6G5
heavy variable chain was then used in a third independent PCR
reaction with the family 1 5' primer (MB1503). (These primers are
also contained in Tables 1-3.)
[0193] After PCR, a larger 420 base PCR product was observed on the
agarose gel. This PCR product was isolated as described previously,
and cloned into a TA vector. The resultant PCR product contained in
the TA this DNA contained the whole heavy variable domain and that
the 3' part was identical to that of previously cloned partial
heavy chain variable domain from the first two PCR reactions.
[0194] A fourth independent PCR was performed using the same
primers as the third PCR amplification. This resulted in a PCR
product which was isolated and cloned into the TA vector as
described previously. The sequence of the fourth independent PCR
product was found to be identical to that obtained in the third PCR
amplification. This sequence, which comprises the heavy chain
variable domain of primate monoclonal anti-human CD23 antibody 6G5,
is presented below.
TABLE-US-00004 Heavy chain variable region of primate monoclonal
antibody anti-human CD23 6G5 Leader Met Lys His Leu Trp Phe Phe Leu
Leu Leu Val Ala Ala Pro Arg ATG AAA CAC CTG TGG TTC TTC CTC CTC CTG
GTG GCA GCT CCC AGA -1 Trp Val Leu Ser TGG GTC CTG TCC Mature
Protein (Numbering is Kabat) Framework 1 1 10 Gln Leu Gln Leu Gln
Glu Ser Gly Pro Gly Val Val Lys Pro Ser CAG CTG CAG CTG CAG GAG TCG
GGC CCA GGA GTG GTG AAG CCT TCG 20 30 Glu Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly Gly Ser Val Ser GAG ACC CTG TCC CTC ACC TGC GCT GTC
TCT GGT GGC TCT GTC AGC CDR 1 31 35 35a Ser Ser Asn Trp Trp Thr AGT
AGT AAC TGG TGG ACC Framework 2 36 40 49 Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile Gly TGG ATC CGC CAG CCC CCA GGG AAG GGA
CTG GAG TGG ATT GGA CDR2 50 52 52A 53 60 Arg Ile Ser Gly Ser Gly
Gly Ala Thr ASn Tyr Asn Pro Ser Leu CGT ATC TCT GGT AGT GGT GGG GCC
ACC AAC TAC AAC CCG TCC CTC 65 Lys Ser AAG AGT Framework 3 66 70 80
Arg Val Ile Ile Ser Gln Asp Thr Ser Lys Asn Gln Phe Ser Leu CGA GTC
ATC ATT TCA CAA GAC ACG TCC AAG AAC CAG TTC TCC CTG 82 82a 82b 82c
83 90 Asn Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
AAC CTG AAC TCT GTG ACC GCC GCG GAC ACG GCC CTG TAT TAC TGT 94 Ala
Arg GCC AGA CDR 3 95 100 100a 100b 100c 100d 101 102 Asp Trp Ala
Gln Ile Ala Gly Thr Thr Leu Gly Phe GAT TGG GCC CAA ATA GCT GGA ACA
ACG CTA GGC TTC Framework 4 103 110 113 Trp Gly Gln Gly Val Leu Val
Thr Val Ser Ser TGG GGC CAG GGA GTC CTG GTC ACC GTC TCC TCA
3) Construction of Mammalian Expression Vectors
[0195] In order to insert the cloned heavy chain variable domains
of 6G5 into a mammalian expression vector, the heavy chain variable
domain in the TA vector (obtained in the 3rd independent PCR) was
digested with Nhe I and Sal I and cloned into the N5LG1 vector
which was digested with the same restriction enzymes and which
vector already contains the light chain variable domain. The
resultant mammalian expression vector was named N5LG1+6G5.
[0196] To construct the N5LG4P+6G5 vector, both the light and heavy
chain variable domains were isolated from N5LG1+6G5 by digestion of
Bgl II and Avr II, and Nhe I and Sal I respectively. The mammalian
expression vector N5LG4P vector is identical to the N5LG1 vector
described above, except the human gamma 1 was replaced with a human
gamma 4 constant region containing a mutation of a serine to a
proline in the hinge region to increase stability of the
immunoglobulin and improve pharmacokinetics in vivo ("P" mutation).
The light chain variable domain was cloned in the plasmid first and
the heavy chain variable domain was cloned into the vector
containing the light chain variable domain using techniques
previously described. This mammalian expression vector was named
N5LG4P+6G5.
B. Construction of the Plasmids N5KG4P+5E8, N5KG1+5E8,
N5KG4P+5E8N-, and N5KG1+5E8N-
1. Cloning the Light Chain Variable Domain of Primate Monoclonal
Anti-Human CD23 Antibody 5E8 by PCR
[0197] The first PCR reaction of the light chain variable domain
from 5E8 cDNA was carried out using a set of kappa early leader
sequence primers and the 3' J region primer GE204. (See primers for
PCR of the kappa light chain variable domain of 5E8 in Tables 1-3).
A 420 base PCR product was obtained. The isolated 420 base PCR
product was digested with Bgl II and BsiW I restriction
endonucleases, cloned into the mammalian expression vector N5KG4P
and sequenced using GE108 and 377 primers (which are contained in
Table 4). The mammalian expression vector N5KG4P is identical to
the vector N5LG4P except it contains the human kappa light chain
constraint region is place of the human lambda light chain constant
region. Sequencing of this 420 polynucleotide DNA revealed that it
contains the entire kappa light chain variable domain.
[0198] A second independent PCR of the light chain variable region
was performed using the 5'-family 1 primer GE201 and the 3' primer
GE204. (See primers for PCR of the kappa light chain variable
domain of 5E8 in Tables 1-3). The isolated PCR product was cloned
into the TA vector (using methods previously described) and
sequenced using Sp6 and T7 promoter primers. Sequencing revealed
that this PCR product was identical to that obtained from the first
PCR. The entire sequence of the light chain variable domain of
primate monoclonal anti-human CD23 antibody 5E8 is presented
below.
TABLE-US-00005 Light chain variable region of primate monoclonal
antibody anti-human CD23 5E8 Leader Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu ATG GAC ATG AGG GTC CCC GCT GAG CTC CTG
GGG CTC CTT CTG CTC -1 Trp Leu Pro Gly Ala Arg Cys TGG CTC CCA GGT
GCC AGA TGT Mature Protein (Numbering is Kabat) Framework 1 1 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GAG ATC
GAG ATG ACC GAG TCT CCA TCT TCC CTG TCT GCA TCT GTA 20 23 Gly Asp
Arg Val Thr Ile Thr Cys GGG GAG AGA GTC ACC ATG ACT TGC CDR 1 24 30
34 Arg Ala Ser Gln Asp Ile Arg Tyr Tyr Leu Asn AGG GCA AGT CAG GAC
ATT AGG TAT TAT TTA AAT Framework 2 35 40 49 Trp Tyr Gln Gln Lys
Pro Gly Lys Aia Pro Lys Leu Leu Ile Tyr TGG TAT GAG GAG AAA CCA GGA
AAA GCT CCT AAG GTG GTG ATG TAT CDR2 50 56 Val Ala Ser Ser Leu Gln
Ser GTT GCA TCC AGT TTG GAA AGT Framework 3 57 60 70 Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe GGG GTG GGA TGA AGG
TTC AGC GGC AGT GGA TCT GGG ACA GAG TTC 80 Thr LeU Thr Val Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr ACT CTC ACC GTC AGC AGC CTG CAG
CCT GAA GAT TTT GCG ACT TAT 88 Tyr Cys TAC TGT CDR 3 89 90 97 Leu
Gln Val Tyr Ser Thr Pro Arg Thr CTA CAG GTT TAT AGT ACC CCT CGG ACG
Framework 4 98 100 107 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys TTC
GGC CAA GGG ACC AAG GTG GAA ATC AAA
2) Cloning the Heavy Chain Variable Domain of Primate Monoclonal
Anti-Human CD23 Antibody 5E8 by PCR
[0199] The first PCR of the heavy chain variable domain of 5E8 was
performed using a set of 5' early leader heavy chain sequence
primers and the 3' primer GE210. (See primers for PCR of the heavy
chain variable domain of 6G5 and 5E8 in Table 1). A 420 base PCR
product appeared in the family 3 primer reaction. The PCR product
was purified and then digested with Nhe I and Sal I and cloned into
the mammalian expression vector N5KG4P vector (as described
previously). The PCR product was sequenced using the 268 and 928
primers. (See sequencing primers in Table 4.)
[0200] A second independent PCR of the heavy chain variable domain
of 5E8 was performed using the family 3 5' primer GE207 and the 3'
primer GE210. (See primers for PCR of the heavy chain variable
domain of 6G5 and 5E8 in Tables 1-3). The isolated PCR product was
cloned into a TA vector using the same techniques previously
described and sequenced by using Sp6 and T7 primers. Sequencing
revealed that the TAC at codon 91 had been changed into TGC.
[0201] In order to determine the appropriate codon at 91, a third
independent PCR was performed using the same primers as the second
PCR (see above). The PCR product was again cloned into a TA vector
and sequenced using Sp6 and T7 primers. The sequence was found to
be identical to the heavy chain variable sequence obtained in the
first PCR. Therefore, the TGC at position 91 in the second
independent PCR product is apparently the result of an error
introduced during PCR. This entire sequence of the heavy chain
variable domain of primate monoclonal anti-human CD23 antibody 5E8
is presented below.
TABLE-US-00006 Heavy chain variable region of primate monoclonal
antibody anti-human CD23 5E8 Leader Met Glu Phe Gly Leu Ser Trp Val
Phe Leu Val Pro Leu Leu Lys ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT
GTT CCT CTT TTG AAA -1 Gly Val Gln Cys GGT GTC CAG TGT Mature
Protein (Numbering is Kabat) Framework 1 1 10 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Ala Lys Pro Gly GAG GTG CAG CTG GTG GAG TCT
GGG GGC GGC TTG GCA AAG OCT GGG 20 30 Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Arg Phe Thr GGG TCC CTG AGA CTC TCC TGC GCA GCC
TCC GGG TTC AGG TTC ACC CDR 1 31 35 35a 35b Phe Asn Asn Tyr Tyr Met
Asp TTC AAT AAC TAC TAC ATG GAC Framework 2 36 40 49 Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Val Ser TGG GTC CGC CAG GCT CCA
GGG CAG GGG CTG GAG TGG GTC TCA CDR2 50 52 52A 53 60 Arg Ile Ser
Ser Ser Gly Asp Pro Thr Trp Tyr Ala Asp Ser Val CGT ATT AGT AGT AGT
GGT GAT CCC ACA TGG TAC GCA GAC TCC GTG 65 Lys Gly AAG GGC
Framework 3 66 70 80 Arg Phe Thr Ile Ser Arg Glu Asn Ala Asn Asn
Thr Leu Phe Leu AGA TTC ACC ATC TCC AGA GAG AAC GCC AAC AAC ACA CTG
TTT CTT 82 82a 82b 82c 83 90 Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys CAA ATG AAC AGC CTG AGA GCT GAG GAC ACG GCT
GTC TAT TAC TGT 94 Ala Ser GCG AGC CDR 3 95 100 101 Leu Thr Thr Gly
Ser Asp Ser TTG ACT ACA GGG TCT GAC TCC Framework 4 103 110 113 Trp
Gly Gln Gly Val Leu Val Thr Val Ser Ser TGG GGC CAG GGA GTC CTG GTC
ACC GTC TCC TCA
3) Construction of Mammalian Expression Vectors
[0202] The heavy variable domain in N5KG4P was digested with Nhe I
and Sal I, purified, and cloned into N5KG4P which contains the
light chain variable domain of 5E8. This plasmid was then digested
with the restriction endonucleases as previously described. This
resulted in a vector containing both the light and heavy variable
domain of 5E8. This vector was named N5KG4P+5E8. The heavy and
light variable domains of N5KG4P+5E8 were then both inserted into
the mammalian expression vector N5KG1 to create the N5KG1+5E8
vector.
4) Alteration of an Amino Acid in the Heavy Chain Variable Region
of Primate Monoclonal Antibody 5E8 by Site Specific Mutagenesis and
Construction of Mammalian Expression Vectors
[0203] Based upon the sequence of 5E8 heavy variable domain, there
is a potential glycosylation site of the immunoglobulin at
asparagine codon 75. This potential glycosylation site corresponds
to a conserved asparagine-linked glycosylation motif having the
following tripeptide sequence: (Asp)--(Any amino acid except
proline)--(Serine or threonine). Therefore, a glycosylation mutant
of 5E8, which would be unable to be glycosylated at this position
because of modification of this glycosylation motif, was generated
by replacing the asparagine codon 75 with a lysine (which is found
in many human immunoglobulins at this position). Site specific
mutagenesis was effected by the following methods.
[0204] A first PCR was done using N5KG4P+5E8 as a template and a 3'
primer (corresponding to codon 71 to 79) and which contains a
mutation at codon 75 (AAC changed to AAG, Primer MB1654, and a 5'
primer at the beginning of the leader sequence (Primer MB1650).
(See PCR Primers Used for the Generation of a Glycosylation Mutant
of the Heavy Chain Variable Region 5E8 set forth in Table 5).
[0205] A second PCR was performed on the same template by using a
5' primer (corresponding to codon 71 to 79) containing the same
mutation (Primer MB1653) and a 3' primer from the end of framework
4 (Primer MB1651) (See PCR Primers Used for the Generation of a
Glycosylation Mutant of the Heavy Chain Variable Region of 5E8 in
Table 5.)
[0206] These two PCR products were isolated and mixed in equal
molar ratios. A third independent PCR was then carried out by using
the mixture of the first and second PCR products as a template with
a 5' primer used in the first PCR (MB1650) and a 3' primer used in
the second PCR (MB 1651). (See PCR Primers Used for the Generation
of a Glycosylation Mutant of the Heavy Chain Variable Region in
Table 5.) The PCR product obtained in third PCR was found to
contain the heavy variable domain coding region of 5E8 wherein the
asparagine 75 had been changed to lysine.
[0207] The third PCR product was purified, digested with
restriction endonucleases Sal I and Nhe I, and cloned into the
N5KG4P containing the light variable domain only. The PCR product
was sequenced to confirm that it comprised the mutant heavy
variable domain. This mammalian expression plasmid was named
NSKG4P+5E8N-.
[0208] To construct of N5KG1+5E8N-, both light and heavy variable
domains from N5KG4P-5E8N- were digested with Bgl II and BsiW I, and
Nhe I and Sal I respectively. The light variable domain was cloned
first and then the heavy variable domain was inserted into the
mammalian expression plasmid N5KG1, to create the plasmid
N5KG1+5E8N-.
TABLE-US-00007 TABLE 1 Primers for PCR of the kappa light chain
variable domain of 5E8 NAME FAMILY Light chain Vk-early leader 5'
(Bgl II) -22 -21 -20 -19 -18 -17 -16 -15 -14 GE201 5' AT CAC AGA
TCT CTC ACC ATG GAC ATG AGG GTC CCC GCT CAG 3' 1 GE200 5' AT CAC
AGA TCT CTC ACC ATG AGG CTC CCT GCT CAG 3' 2 GE202 5' AT CAC AGA
TCT CTC ACC ATG GAA (A/G)CC CCA GC(T/G) CAG 3' 3 GE203 5' AT CAC
AGA TCT CTC ACC ATG GTG TTG CAG ACC CAG GTC 3' 4 Light chain Vk-3'
primer (BsiW I) 113 112 111 110 109 108 107 106 105 104 103 GE204
5' GG TGC AGC CAC CGT AGC TTT GAT (C/T)TC CA(G/C) CTT 3'
TABLE-US-00008 TABLE 2 Primers for PCR of the lambda light chain
variable domain of 6G5 NAME FAMILY Light chain V1-early leader 5'
(Bgl II) -20 -19 -18 -17 -16 -15 744 5' AT CAC AGA TCT CTC ACC ATG
(G/A)CC TG(G/C) TCC CCT CT 3' 1 745 5' AT CAC AGA TCT CTC ACC ATG
GCC TGG (A/G)CT C(T/C)G CT 3' 2 910 5' AT CAC AGA TCT CTC ACC ATG
GC(A/C) TGG A(T/C)C CCT CTC 3' 3 Light chain V1-3' primer (Avr II)
110 109 108 107 106 105 104 926 5' (AC)10 CTT GGG CTG ACC TAG GAC
GGT 3'
TABLE-US-00009 TABLE 3 Primers for PCR of the heavy chain variable
domains from 6G5 and 5E8 NAME Family Heavy chain-early leaders 5'
(Sal I) -20 -19 -18 -17 -16 -15 MB1503 5' GCG ACT AAG TCG ACC ATG
GAC TGG ACC TGG 3' 1 MB1502 5' GCG ACT AAG TCG ACC ATG AAA CAC CTG
TGG 3' 2, 4 GE207 5' GCG ACT AAG TCG ACC ATG GAG TTT GGG CTG AGC 3'
3 GE208 5' GCG ACT AAG TCG ACC ATG GGG TCA ACC GCC ATC 3' 5 GE209
5' GCG ACT AAG TCG ACC ATG TCT GTC TCC TTC CTC 3' 6 Heavy chain-3'
Primer (Nhe I) 120 119 118 117 116 115 114 113 112 111 110 GE244 5'
GC CAG GGG GAA GAC CGA TGG GCC CTT GGT GCT AGC TGA GGA GAC GG 3'
GE210 5' GA TGG GCC CTT GGT GCT AGC TGA GGA GAC GG 3' MB1533 5' GGT
GCT AGC TGA GGA GAC CGT 109 108 107 106 105 104 103 101 100 99 GAC
CAG GAC TCC CTG GCC CCA GAA GCC TAG 3'
TABLE-US-00010 TABLE 4 Sequencing Primers Sp6 primer 5' AT TTA GGT
GAC ACT ATA 3' M13 (-40) 5' GTT TTC CCA GTC ACG A 3' Forward Primer
T7 Promoter 5' AT ATA CGA CTC ACT ATA GGG 3' Primer GE 108 Primer
5' CCG TCA GAT CGC CTG GAG ACG CCA 3' 377 Primer 5' GCA GTT CCA GAT
TTC AAC TG 3' 607 PRIMER 5' CCA GGC CAC TGT CAC GGC TTC 3' 266
PRIMER 5' CAG AGC TGG GTA CGT CCT CA 3' 268 PRIMER 5' GCC CCC AGA
GGT GCT CTT GG 3' 876 PRIMER 5' ACA CAG ACC CGT CGA CAT GG 3' 928
PRIMER 5' GCT CTC GGA GGT GCT CCT GG 3'
TABLE-US-00011 TABLE 5 PCR Primers Used for the Generation of a
Glycosylation Mutant of the Heavy Chain Variable Region of 5E8 Sal
I -20 -19 -18 -17 -16 MB1650 5' ACA GAC CCG TCG ACC ATG GAG TTT GGG
CTG 3' Nhe I 118 117 116 115 114 113 112 111 110 MB1651 5' CCC CTT
GGT GCT AGC TGA GGA GAC GGT 3' 71 72 73 74 75 76 77 78 79 MB1653 5'
AGA GAG AAC GCC AAG AAC ACA CTG TTT 3' 79 78 77 76 75 74 73 72 71
MB1654 5' AAA CAG TGT GTT CTT GGC GTT CTC TCT 3'
C) Construction of Gamma-3 Vectors
[0209] Numerous methods exist for conversion of murine antibodies
to chimeras in which the heavy and light chain constant regions are
substituted with human versions or in which all but the CDRs
(complementary determining regions) are substituted with their
human equivalents. (See Biochem. J. 281:317, 1992; Proc. Nat. Acad.
Sci. USA 86:10029, 1989; Methods Enzymol. 178:515, 1989; Cancer
Res. 51:181, 1991; Biotechniques 7:360, 1989; J. Immunol. 143:3589,
1989; Int. J. Cancer 44:424, 1989; Proc. Nat. Acad. Sci. USA
86:3833, 1989).
[0210] Further, the previous sections specifically demonstrate that
construction of vectors for expression of PRIMATIZED.RTM. gamma-1
and gamma-4 antibodies may be accomplished by amplifying the DNA
encoding the light and heavy chain variable regions using PCR, and
cloning these regions into an expression vector or vectors encoding
the appropriate human constant region domains. Thus, it should be
clear that similar constructs may be made which encode other human
constant region domains, so long as appropriate mammalian
expression vectors are available which also contain cloning sites
which allow insertion of the amplified variable region DNA from the
primate antibodies.
[0211] It should also be apparent that the PCR primers used to
amplify the variable region DNA may be designed to accommodate
various restriction cloning sites depending on the vector used.
[0212] Vectors encoding the constant region domain from human IgG3
are available in the art, and may be used for constructing the
PRIMATIZED.RTM. antibodies of the present invention. For instance,
Co et al. have used separate vectors expressing human light and
heavy chain constant regions for constructing chimeric and
humanized antibodies, and provide heavy chain vectors for both
human gamma-1 and gamma-3. For cloning the variable domain regions
of the antibody of interest into these vectors, an XbaI restriction
site is conveniently located just before the constant regions in
the J.sub.k4 and the J.sub.H3 intronic segments for the light and
heavy chains, respectively (Co et al., 1996, Cancer Res. 56:
1118-1125; Co et al., 1992, J. Immunol. 148: 1149-1154).
[0213] Thus, by designing PCR primers which incorporate XbaI
restriction sites, or sites which create the same single stranded
overhangs as the XbaI restriction enzyme (i.e. NheI, SpeI), the
variable regions from a primate antibody may be associated with the
human gamma-3 constant region domain using the vectors described in
Co et al. Alternatively, linkers or adaptors may be used to
facilitate cloning.
[0214] It is also possible to construct a cDNA version of the human
gamma-3 constant region using DNA synthesis techniques and insert
it into the N5KG1 vectors described herein in place of the human
gamma-1 constant region between the NheI and BamHI sites. Depending
on which restriction sites are most convenient for cloning the
particular variable region of interest, there are also other
gamma-3 vectors which have been described in the art, and any of
these may be used to construct and express the gamma-3 antibodies
of the present invention. (See, for example, Parren et al., 1992,
J. Immunol. 148(3): 695-701; Steplewski et al., 1988, Proc. Natl.
Acad. Sci. USA 85: 4852-4856; and U.S. Pat. No. 4,975,369, herein
incorporated by referenced).
D) Variations of PRIMATIZED.RTM. Antibodies
[0215] The present invention also encompasses PRIMATIZED.RTM.
antibodies containing mutations, substitutions or deletions of the
constant region. Such mutations may be constructed using
site-directed mutagenesis techniques as described above for the
glycosylation mutant, which are well-known techniques for those of
skill in the art.
[0216] The mutated antibodies of the present invention are designed
with the purpose of creating a desired change in the level of
therapeutic efficiency, i.e. in FcR binding. However, it should be
understood that any mutation in the constant region of the
antibodies should not alter the basic effector functions as
mediated by the gamma-1 or gamma-3 constant region domains. It may
also be possible to alter other subclasses of gamma antibodies by
mutagenesis such that they have similar effector functions as an
antibody containing a gamma-1 or a gamma-3 domain. Such antibodies
are also encompassed by the present invention.
[0217] For example, the regions of the IgG constant region involved
in FcR binding and interaction with the C1q complement component
have been characterized, and mutations have been identified which
either increase or decrease binding affinity. As disclosed in U.S.
Pat. No. 5,648,260, herein incorporated by reference, changing Leu
235 to Glu in the human IgG3 constant region destroys the
interaction of the mutant for the human Fc gamma R1 receptor.
Furthermore, mutations on adjacent or close sites in the hinge link
region (i.e. replacing residues 234, 236 or 237 by Ala) indicate
that alterations in residues 234, 235, 236 and 237 at least affect
affinity for the Fc gamma R1 receptor.
[0218] Similarly, in U.S. Pat. No. 5,348,876, also incorporated by
reference, it was shown that by decreasing the length of the hinge
region of the human IgG3 constant region and altering the amino
acid sequence of this region, C1q binding and thus complement
mediated cytolysis were further improved. In fact, the variants
bound more C1q than wild type IgG3 and much more than IgG1.
[0219] Thus, it is clear from the art that mutations may be used to
create therapeutic antibodies with decreased affinities for human
Fc receptors, perhaps for the treatment of more mild medical
conditions. In addition, mutations may be identified which increase
effector function activity, thereby leading to stronger versions
for therapeutic use. It should be recognized that manipulations of
gene sequence to create alterations in drug efficiency are
envisioned, and well within the spirit and scope of the present
invention.
E) Expression of PRIMATIZED.RTM. Antibodies in Chinese Hamster
Ovary Cells
[0220] In order to isolate the PRIMATIZED.RTM. antibodies, the
vectors encoding the antibodies were expressed in Chinese Hamster
Ovary cells using the following protocol.
[0221] A large scale plasmid DNA was purified using the WIZARD.RTM.
Maxipreps DNA Purification System (Promega Catalog #A7421). The
purified DNA was digested with Ssp I and BspLU11 I, precipitated
with ethanol once, and resuspended in sterile TE.
[0222] Purified, endonuclease restricted plasmid DNA was then
introduced into Chinese hamster ovary (CHO) dihydrofolate reductase
minus DG44 cells using electroporation. The electroporation
technique used is described below.
[0223] Approximately 1.6.times.10.sup.8 CHO cells were spun in an
appropriate size sterile Corning tube for one minute at 1000 RPM.
The media was removed and the cells were washed in fifteen
milliliters of sterile ice cold SBS (sterile sucrose buffered
solution is 272 mM sucrose, 7 mM sodium phosphate pH 7.4, 1 mM
MgCl.sub.2) and spun for 5 minutes at 1000 RPM. The SBS was removed
and cells were suspended using fresh ice cold sterile SBS at a cell
concentration of 1.times.10.sup.7 cells were per ml and left on ice
for 15 minutes. The BTX 600 electroporator was turned on and preset
at 230 volts, with the maximum voltage knobs being set at 500
volts/capacitance & resistance. The capacitance was set at 400
microfaradays and the resistance was set at 13 ohms (setting
R1).
[0224] Plasmid DNA (4 .mu.g DNA or 2 .mu.g DNA) and 0.4 ml of cells
(4.times.10.sup.6 cells) were then placed in BTX 0.4 ml cuvettes
(BTX Catalog #620). The cells were shocked by placing the cuvette
into the BTX 600 stand and pressing the automatic charge &
pulse button. Approximately 20 separate electroporations were
performed with each mammalian expression plasmid.
[0225] After shocking, the cuvettes were left at ambient
temperature for fifteen minutes. The cells and DNA were from each
cuvette were resuspended in 20 ml of CHO-SSFMII containing no
hypoxanthine or thymidine (Gibco BRL Catalog #31033-012) to which
HT supplement (100.times. supplement is 10 mM sodium hypoxanthine,
1.6 mM thymidine Gibco BRL Catalog #11067-014) had been added. The
cells from a single electroporated cuvette were then plated into a
96 well plates (200 .mu.l/well) and placed into a 37.degree. C.
CO.sub.2 incubator. Selection was started two or three days later
by changing the media to the above media with the addition of 400
mg/ml of Geneticin.RTM. (G418, Gibco BRL Catalog #10131-019). The
cells were grown at 37.degree. C. and the cell media were changed
every 3-5 days. After sixteen days G418 resistant clones appeared
in the wells and the supernatant was assayed for antibody
expression by ELISA. The highest expressing clones were then
expanded individually. Monoclonal antibodies were purified as
described below.
Immunoglobulin ELISA
[0226] Plates (Immulon 2, Dynatech Laboratories, Inc. Catalog
#011-010-3455) are coated overnight at 4.degree. C. with 200 ng
unlabeled goat anti-human IgG antibody at 100 .mu.l/well. This is
effected using twenty milliliters of unlabeled goat anti-human
IgG/10 mls Coating Buffer/plate (Boehringer Mannheim Ab Catalog
#605 400). (1:500 dilution of .sup..about.1 mg/ml stock.) The
coating buffer is then removed from the plates and dried using a
paper towel. One hundred microliters of a dilution buffer/well is
then added.
[0227] Antibody solutions and standards (100 ng/ml-2.5 ng/ml) are
then added in duplicate at 100 .mu.l/well directly to the 100 ml
dilution buffer. The antibody solutions and standards are contained
in dilution buffer. The resultant solutions are then incubated for
at least 1 hour at 37.degree. C.
[0228] After incubation, the contents of each plate are removed and
the plates are washed with tap water five times. The plates are
then dried on a paper towel.
[0229] After drying of the plates, a second antibody is then added
at 100 .mu.l/well. This second antibody is either goat anti-human
Kappa-HRPO: added at 1/10,000 dilution or 1 .mu.l Ab/10 mls
dilution buffer/plate, available from Southern Biotechnology
Associates, Inc. Catalog #2060-05 or a goat anti-human Lambda-HRPO;
used at 1/20,000 dilution or 1 .mu.l Ab/20 mls dilution buffer/2
plates (available from Southern Biotechnology Associates, Inc.
Catalog #2070-05).
[0230] The antibody and contents of the plate are allowed to
incubate for one hour at 37.degree. C. After incubation the
contents of each plate are removed. The plates are again washed
five time with tap water, and the washed plates are dried. To the
dried plates is then added HRPO substrate (TMB Microwell--two
component) in an amount of 100 .mu.l/well. (Five milliliters of TMB
Peroxidase Substrate+five milliliters of Peroxidase Solution
B/plate (Kirdgaard and Perry Labs, TMB Microwell two component
reagents Catalog #50-76-00).
[0231] The reaction is stopped by the addition of one hundred
microliters of 2M H.sub.2SO.sub.4 to each well when the weakest
standard (2.5 ng/ml) is visible over background. The optical
density of wells in plates is then read using a plate reader, e.g.,
Molecular Devices Emax precision microplate reader set at
wavelength: OD 450 and OPT2 (OD 540).
ELISA BUFFERS
Coating Buffer
[0232] Sodium Carbonate 0.8 gram/liter
[0233] Sodium Bicarbonate 1.55 gram/liter
[0234] Adjust pH to 9.5 with .sup..about.1 ml 1N HCl
Dilution Buffer
[0235] 0.5% Nonfat Dry Milk in PBS plus 0.01% Thimerosal (5 gm/L)
(100 mg/L)
[0236] Examples of ELISA values obtained using the above-described
assay are set forth below.
TABLE-US-00012 Standard OD 450 OD450 Average 100 ng/ml 0.805 0.876
0.841 50 ng/ml 0.395 0.472 0.434 25 ng/ml 0.213 0.252 0.233 10
ng/ml 0.089 0.105 0.097 5 ng/ml 0.054 0.055 0.055 2.5 ng/ml 0.031
0.035 0.033 0 ng/ml 0.004 0.006 0.005
Standards in Dilution Buffer
[0237] Appropriate dilution of stock AB (sterile filtered in normal
saline, protein determination by OD) to give 1 mg/ml
EXAMPLE
[0238] Chimeric monkey/human anti-CD4 (CE9.1) is 4.18 mg/ml
[0239] 24 .mu.l of above into 76 .mu.l Dilution Buffer is 1
mg/ml
[0240] 50 .mu.l Stock Ab (1 mg/ml) into 450 .mu.l Dilution Buffer
(D.B.) is 100 .mu.g/ml
[0241] 50 .mu.l of above mixture into 450 .mu.l D.B. is 10
.mu.g/ml
[0242] 200 .mu.l of above mixture into 1.8 mls D.B. is 1
.mu.g/ml
[0243] 1 ml of above mixture into 9 mls D.B. is 100 ng/ml *
[0244] 5 ml of above mixture into 5 ml D.B. is 50 ng/ml *
[0245] 5 ml of above mixture into 5 ml D.B. is 25 ng/ml *
[0246] 4 ml of above mixture into 6 ml D.B. is 10 ng/ml *
[0247] 5 ml of above mixture into 5 ml D.B. is 5 ng/ml *
[0248] 5 ml of above mixture into 5 ml D.B. is 2.5 ng/ml *
[0249] * Standards used in the ELISA
Antibody Purification by Protein A
Procedure
[0250] The culture supernatant is centrifuged to remove cells and
debris. The centrifuge is then filtered through a 0.2 .mu.m filter.
A protein A sepharose Fast flow column (recombinant protein A
Sepharose Fast floe) (Pharmacia Biotech Catalog #71-5000-09) is
then prepared and equilibrated using PBS (pH 7.4).
[0251] The supernatant is loaded on the column at an appropriate
flow rate (e.g., 2 ml/min). After loading, the column is washed
with 10 column volume of PBS (pH 7.4). The antibody is eluted from
the column using an elution buffer (0.2M acetic acid, 0.1 M glycine
pH 3.5) at 1 ml/min flow rate. One milliliter fractions/tube
including 100 .mu.l of Tris are then collected. A spectrophotometer
absorbance reading is then taken at 280 nm. The antibody fractions
are then collected and dialyzed against PBS (pH 7.9) overnight. The
dialysate is then sterilized by filtration through a 0.22 .mu.m
membrane and stored at -20.degree. C.
Assay Results
[0252] The PRIMATIZED.RTM. human gamma-4 anti-human CD23
anti-bodies which are described supra, were purified and assayed
for induced IgE inhibitory activity in vitro. These results are
contained in FIGS. 3 and 5. This was effected using the in vitro
IL-4 IgE assay described supra.
[0253] These assay results surprisingly indicated that both human
gamma-4 anti-human CD23 antibodies were not as active as the
corresponding primate anti-human CD23 antibodies, i.e., they did
not significantly inhibit induced IgE production in vitro.
[0254] However, because primate 5E8 and p5E8G4P have a potential
asparagine linked glycosylation site in the heavy chain variable
region, the effects of glycosylation at this site were
investigated. (It was found that both these antibodies contain
N-linked oligosaccharides at this site. (Data not shown.))
[0255] Therefore, in order to prevent glycosylation, the asparagine
in the glycosylation site was changed to a lysine in order to
eliminate carbohydrate addition. This mutated antibody was named
p5E8G4PN-. Assay results demonstrated that this antibody behaved
identically to p5E8G4P in the IL-4 IgE assay (see FIG. 3) and also
exhibited an identical apparent affinity Kd for human CD23. (See
FIG. 4.) Therefore, these results indicated that the difference in
IgE inhibition observed from the 5E8 gamma 4 PRIMATIZED.RTM.
antibody in comparison to primate 5E8 antibody was not attributable
to glycosylation differences.
[0256] The three primate antibodies (p5E8G4P, p5E8G4PN-, and
pGG5G4P) were then expressed as human gamma-1 versions using
substantially the same methodology. All three human gamma-1
anti-human CD23 antibodies, respectively designated p5E8G1,
p5E8G1N- and pGG5G1, were found to be active in the in vitro
IL-4/IgE assay (FIGS. 3 and 5).
[0257] p5E8G1 was found to be statistically more suppressive than
p5E8G4P at a concentration of 0.3 .mu.g/ml (P[T,t] one tail+0.0055)
and at 3 .mu.g/ml (p[T<t] one tail+0.0019). In addition,
p5E8GlN- is statistically more suppressive than p5E8G4PN- at both
0.3 .mu.g/ml (p[T<t] one tail+0.0392) and at 3 .mu.g/ml
(p[T<t] one tail+0.0310) (FIG. 3).
[0258] Similarly, p6GSGl completely inhibited induced IgE
production at 3 mg/ml, while p6G5G4P did not. (These results are in
FIG. 5).
[0259] Thus, these results suggested that an active Fc region, in
particular that of human gamma-1, is significant for induced IgE
inhibition by anti-human CD23 antibodies. These results also
suggest that human anti-CD23 antibodies containing human gamma-3
constant regions will be significant for induced IgE inhibition
because a gamma-3 constant region binds to the same Fc.gamma.R
receptors that gamma-1 binds to, and will likely mediate the same
result.
EXAMPLE 2
[0260] To confirm our hypothesis as to the involvement of the Fc
effector portion in IgE inhibition of anti-human CD23 antibodies, a
third primate antibody, designated 2C8, also shown to inhibit IgE
in in vitro) was converted to a F(ab').sub.2. IgE inhibitory
activity was determined using the same IL-4/IgE assay described
previously.
Materials
[0261] The following materials were used in this example.
[0262] ImmunoPure F(ab').sub.2 Preparation Kit (Pierce Catalog
#44888)
[0263] digestion buffer: 20 mM sodium acetate buffer, pH 4.5
[0264] 0.1 M citric acid, pH 3.0 (adjust pH with NaOH)
[0265] 0.1% sodium azide in water
[0266] dialysis tubing; 50,000 MW cutoff (Spectra Por Catalog #132
128)
[0267] shaking water bath capable of maintaining 37.degree. C.
[0268] polystyrene culture tubes, 17.times.100 mm (Fisher Catalog
#14-956-6B)
[0269] BCA protein assay (Pierce Catalog #23224)
[0270] Centricon-50 concentrators (Amicon Catalog #4225).
Equilibration of Immobilized Pepsin
[0271] 0.25 milliliters of the 50% slurry of Immobilized Pepsin is
added to a 17.times.100 mm test tube (0.125 ml of gel). Four
milliliters of digestion buffer are then added. The pepsin is then
separated from the buffer using the serum separator. The buffer is
then discarded and the wash procedure repeated using another four
milliliters of buffer. The immobilized pepsin is then resuspended
in 0.5 ml of digestion buffer.
Preparation of Immobilized Protein a Column
[0272] Protein A AffinityPak.RTM. columns and ImmunoPure Binding
and Elution buffers are brought to room temperature.
Preparation of 2C8 F(ab').sub.2 Fragments
[0273] 2C8 F(ab').sub.2 fragments are prepared by methods well
known in the antibody art. The inventors elected to use a
commercially available kit, ImmunoPure F(ab').sub.2 Preparation Kit
(Pierce Catalog #44888), using the manufacturer's protocols.
[0274] Ten milligrams of lyophilized 2C8 antibody were dissolved in
one milliliter of a digestion buffer (20 mM sodium acetate buffer,
pH 4.5). One milliliter of the antibody containing sample was than
added to a tube containing immobilized pepsin.
[0275] The antibody and immobilized pepsin were then incubated for
four hours in a high speed shaker water bath at 37.degree. C. (at
high speed), taking care to maintain the mixing constant during the
incubation.
[0276] The resultant solubilized F(ab').sub.2 and Fc fragments and
the undigested IgGs were then recovered from the immobilized pepsin
gel using a serum separator. The crude digest is then decanted into
a clean tube.
[0277] In order to enhance recovery of F(ab').sub.2 fragments, the
immobilized pepsin desirably is then washed with 1.5 of milliliters
of the ImmunoPure IgG binding buffer. The wash is then added to the
crude digest.
[0278] The antibody fragments were then recovered using a protein A
column. This is effected by opening an immobilized protein A
column. Care is taken to avoid air bubbles from being drawn into
the gel. The storage solution (which contains 0.02% sodium azide)
is discarded.
[0279] The immobilized protein A column was then equilibrated using
twelve milliliters of binding buffer (contained in ImmunoPure
Preparation Kit). The column was then transferred to a 17.times.100
nm test tube contained in the kit (labeled "F(ab').sub.2") to
collect eluate.
[0280] Three milliliters of the crude digest was then applied to a
column and are allowed to flow completely into the gel. The use of
AffinityPak.TM. columns is desirable as these columns stop flowing
automatically when the level reaches the top frit.
[0281] The column is then washed using six milliliters of binding
buffer. The eluate which contains F(ab').sub.2 fragments was then
collected. This eluate also contains small Fc fragments that can no
longer bind protein A (which are not bound to the Protein A
column). However, the substantial portion thereof was eliminated by
dialysis.
[0282] Dialysis was effected by taking the F(ab').sub.2 containing
eluate and dialyzing the eluate against pH 7.4 phosphate buffered
saline, using dialysis tubing with a molecular weight cut-off of
50,000 so as to eliminate the small Fc fragment containments
(Spectra Pur. Catalog #132 128).
[0283] This resulted in a F(ab').sub.2 fraction having an optical
density of 280 nm of 0.707 (6 ml). After dialysis and concentration
with Centricon-S0 concentrators (Amicon Catalog #4225), the 2C8
F(ab').sub.2 product was assayed for protein content using a BCA
protein assay (Pierce Catalog #23224). The protein content was
found to be 3.76 mg per milliliter.
[0284] The 2C8 F(ab).sub.2's were assayed for IgE inhibitory
activity and were found to be substantially incapable of inhibiting
IgE production in the same in vitro assays described previously.
These results are contained in FIG. 6. In fact, the F(ab').sub.2
was found to antagonize the suppressive effects of induced IgE on
the monoclonal antibody 2C8. These results are in FIG. 7.
EXAMPLE 3
[0285] The PRIMATIZED.RTM. gamma 1 and gamma 4P versions of primate
monoclonal 6G5 were both evaluated for their effect on inhibition
of induced IgE production in vivo in the SCID mouse model described
previously. p5E8G1N- was found to be as efficient as primate 5E8 in
inhibiting induced IgE. (See FIGS. 8 and 9). While neither primate
6G5 nor the primatized p6G5G4P were effective at inhibiting induced
IgE in vivo, primatized p6G5G1 inhibited induced IgE production.
(See FIGS. 9 and 10.) These results further substantiate our
conclusion that an active Fc region is significant to the ability
of an anti-human CD23 antibody to effectively inhibit induced IgE
production.
Proposed Mechanism
[0286] Although the results of the present invention conclusively
demonstrate that an active Fc region is significant to the ability
of an anti-human CD23 antibody to effectively inhibit induced IgE
production, the molecular mechanism has not been elucidated.
However, several hypotheses may be made.
[0287] Firstly, it may be that the anti-CD23 antibodies of the
present invention function to trigger a similar signal transduction
pathway as is apparently triggered by cross-linking B cell receptor
with anti-BCR gamma-1 antibodies. It has been hypothesized that the
down-regulation of antigen receptor signaling in this case results
from coligation of Fc gamma RII and surface Ig (B cell receptor) on
the same B cell, leading to down regulation of Ig expression.
(D'Ambrosia et al., 1995, Science, 268:293-297; Ono et al., 1997,
Cell 90: 293-301). Indeed, CD23 is upregulated on the surface of
IgE-expressing B cells, so it is possible that coligation of CD23
and the Fc.gamma.RII-B receptor via the gamma-1 constant region
results in a similar signal transduction pathway.
[0288] Alternatively, inhibition of induced IgE expression may be
occurring through cross-linking of surface IgE on B cells at the
same time as the Fc region interacts with an appropriate Fc.gamma.R
receptor on another cell type, i.e. a killer T cell, which may then
"instruct" the B cell to undergo apoptosis, or lead to cell death
in some other manner (i.e. phagocytosis). For instance, U.S. Pat.
No. 5,543,144, herein incorporated by reference, proposes a similar
mechanism for inhibiting IgE-expressing B cells with anti-IgE
antibodies. As discussed therein, antibodies of certain IgG
subclasses, such as mouse IgG2a and human IgG1 and IgG3, can
mediate ADCC (Antibody Dependent Cellular Cytotoxicity) carried out
by certain Fc receptor-bearing phagocytic leukocytes. For example,
OKT3, a mouse IgG2a monoclonal antibody specific for human T cell
surface antigen (which was the first monoclonal antibody product
approved by the FDA for marketing as a therapeutic agent) is used
in patients to provide rapid depletion of T cells in the blood and
to induce an immunosuppressed state (for kidney transplantation).
(Russell, P. S. et. al, Transpl. Proc. 17:39-41 (1985)). OKT3, at a
dosage of 5 mg/day/subject, can completely deplete circulating T
cells. The monoclonal antibodies described in U.S. Pat. No.
5,543,144, especially in the form of mouse gamma 2a antibodies or
human or humanized antibodies bearing human gamma-1 or gamma-3
chains, are proposed to deplete IgE-expressing B cells by the ADCC
mechanism in a similar manner.
[0289] But whatever the actual molecular mechanism for inhibiting
IgE expression using anti-CD23 antibodies may be, it is clear that
the Fc region, and accordingly effector function, of anti-CD23
antibodies are important considerations. As discussed previously,
since complement is not present in the in vitro assays of the
present invention, the phenomenon likely involves one or more
Fc.gamma.R receptors. Once the relevant molecular components are
identified, as well as molecules internal to the cells involved
which are involved in signal transduction pathways, it will be
possible to design other therapeutics which may also be used to
inhibit induced IgE expression.
UTILITY
[0290] Because of their ability to effectively inhibit IgE
production, the subject anti-human CD23 antibodies which comprise
human gamma-1 constant domains, and those that may also be
constructed containing gamma-3, are effective in treating any
disease wherein inhibition of IgE production is therapeutically
desirable. Such diseases include by way of example allergic
diseases, autoimmune diseases and inflammatory disease.
[0291] Specific conditions which are potentially treatable by
administration of the subject anti-CD23 human gamma-1/gamma-3
constant domain containing antibodies include the following:
[0292] Allergic bronchopulmonary aspergillosis; Allergic rhinitis
and conjunctivitis autoimmune hemolytic anemia; Acanthosis
nigricans; Allergic contact dermatitis; Addison's disease; Atopic
dermatitis; Alopecia greata; Alopecia universalis; Amyloidosis;
Anaphylactoid purpura; Anaphylactoid reaction; Aplastic anemia;
Angioedema, hereditary; Angioedema, idiopathic; Ankylosing
spondylitis; Arteritis, cranial; Arteritis, giant cell; Arteritis,
Takayasu's; Arteritis, temporal; Asthma; Ataxia-telangiectasia;
Autoimmune oophoritis; Autoimmune orchitis; Autoimmune
polyendocrine failure; Behcet's disease; Berger's disease;
Buerger's disease; bronchitis; Bullous pemphigus; Candidiasis,
chronic mucocutaneous; Caplan's syndrome; Post-myocardial
infarction syndrome; Post-pericardiotomy syndrome; Carditis; Celiac
sprue; Chagas's disease; Chediak-Higashi syndrome; Churg-Strauss
disease; Cogan's syndrome; Cold agglutinin disease; CREST syndrome;
Crohn's disease; Cryoglobulinemia; Cryptogenic fibrosing
alveolitis; Dermatitis herpetifomis; Dermatomyositis; Diabetes
mellitus; Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid
lupus erythematosus; Eosinophilic fasciitis; Episcleritis; Drythema
elevatum diutinum; Erythema marginatum; Erythema multiforme;
Erythema nodosum; Familial Mediterranean fever; Felty's syndrome;
Fibrosis pulmonary; Glomerulonephritis, anaphylactoid;
Glomerulonephritis, autoimmune; Glomerulonephritis,
post-streptococcal; Glomerulonephritis, post-transplantation;
Glomerulopathy, membranous; Goodpasture's syndrome; Graft-vs.-host
disease; Granulocytopenia, immune-mediated; Granuloma annulare;
Granulomatosis, allergic; Granulomatous myositis; Grave's disease;
Hashimoto's thyroiditis; Hemolytic disease of the newborn;
Hemochromatosis, idiopathic; Henoch-Schoenlein purpura; Hepatitis,
chronic active and chronic progressive; Histiocytosis_X;
Hypereosinophilic syndrome; Idiopathic thrombocytopenic purpura;
Job's syndrome; Juvenile dermatomyositis; Juvenile rheumatoid
arthritis (Juvenile chronic arthritis); Kawasaki's disease;
Keratitis; Keratoconujunctivitis sicca;
Landry-Guillain-Barre-Strohl syndrome; Leprosy, lepromatous;
Loeffler's syndrome; lupus; Lyell's syndrome; Lyme disease;
Lymphomatoid granulomatosis; Mastocytosis, systemic; Mixed
connective tissue disease; Mononeuritis multiplex; Muckle-Wells
syndrome; Mucocutaneous lymph node syndrome; Mucocutaneous lymph
node syndrome; Multicentric reticulohistiocytosis; Multiple
sclerosis; Myasthenia gravis; Mycosis fungoides; Necrotizing
vasculitis, systemic; Nephrotic syndrome; Overlap syndrome;
Panniculitis; Paroxysmal cold hemoglobinuria; Paroxysmal nocturnal
hemoglobinuria; Pemphigoid; Pemphigus; Pemphigus erythematosus;
Pemphigus foliaceus; Pemphigus vulgaris; Pigeon breeder's disease;
Pneumonitis, hypersensitivity; Polyarteritis nodosa; Polymyalgia
rheumatic; Polymyositis; Polyneuritis, idiopathic; Portuguese
familial polyneuropathies; Pre-eclampsia/eclampsia; Primary biliary
cirrhosis; Progressive systemic sclerosis (Scleroderma); Psoriasis;
Psoriatic arthritis; Pulmonary alveolar proteinosis; Pulmonary
fibrosis, Raynaud's phenomenon/syndrome; Reidel's thyroiditis;
Reiter's syndrome, Relapsing polychrondritis; Rheumatic fever;
Rheumatoid arthritis; Sarcoidosis; Scleritis; Sclerosing
cholangitis; Serum sickness; Sezary syndrome; Sjogren's syndrome;
Stevens-Johnson syndrome; Still's disease; Subacute sclerosing
panencephalitis; Sympathetic ophthalmia; Systemic lupus
erythematosus; Transplant rejection; Ulcerative colitis;
Undifferentiated connective tissue disease; Urticaria, chronic;
Urticaria, cold; Uveitis; Vitiligo; Weber-Christian disease;
Wegener's granulomatosis; Wiskott-Aldrich syndrome.
[0293] Of these, the preferred indications treatable or presentable
by administration of anti-CD23 antibodies include allergic rhinitis
and conjunctivitis, atopic dermatitis; eczema; Job's syndrome,
asthma; allergic conditions; and chronic inflammatory diseases and
conditions, including CLL chronic lymphocytic leukemia, typically
characterized by high levels of membrane CD23 and high circulating
levels of sCD23 (Sarfart et al., Blood 71: 94-98 (1988)).
[0294] The amount of antibody useful to produce a therapeutic
effect can be determined by standard techniques well known to those
of ordinary skill in the art. The antibodies will generally be
provided by standard technique within a pharmaceutically acceptable
buffer, and may be administered by any desired route. Because of
the efficacy of the presently claimed anti-bodies and their
tolerance by humans it is possible to administer these antibodies
repetitively in order to combat various diseases or disease states
within a human.
[0295] One skilled in the art would be able, by routine
experimentation, to determine what an effective, non-toxic amount
of antibody would be for the purpose of effecting allergic diseases
and inflammatory conditions. Generally, however, an effective
dosage will be in the range of about 0.05 to 100 milligrams per
kilogram body weight per day.
[0296] The antibodies of the invention may be administered to a
human or other animal in accordance with the aforementioned methods
of treatment in an amount sufficient to produce such effect to a
therapeutic or prophylactic degree. Such antibodies of the
invention can be administered to such human or other animal in a
conventional dosage form prepared by combining the antibody of the
invention with a conventional pharmaceutically acceptable carrier
or diluent according to known techniques. It will be recognized by
one of skill in the art that the form and character of the
pharmaceutically acceptable carrier or diluent is dictated by the
amount of active ingredient with which it is to be combined, the
route of administration and other well-known variables.
[0297] The route of administration of the antibody of the invention
may be oral, parenteral, by inhalation or topical. The term
parenteral as used herein includes intravenous, intramuscular,
subcutaneous, rectal, vaginal or intraperitoneal administration.
The intravenous form of parenteral administration is generally
preferred.
[0298] The daily parenteral and oral dosage regimens for employing
compounds of the invention to prophylactically or therapeutically
induce immunosuppression will generally be in the range of about
0.05 to 100, but preferably about 0.5 to 10, milligrams per
kilogram body weight per day.
[0299] The antibody of the invention may also be administered by
inhalation. By "inhalation" is meant intranasal and oral inhalation
administration. Appropriate dosage forms for such administration,
such as an aerosol formulation or a metered dose inhaler, may be
prepared by conventional techniques. The preferred dosage amount of
a compound of the invention to be employed is generally within the
range of about 10 to 100 milligrams.
[0300] The antibody of the invention may also be administered
topically. By topical administration is meant non-systemic
administration and includes the application of an antibody (or
fragment thereof) compound of the invention externally to the
epidermis, to the buccal cavity and instillation of such an
antibody into the ear, eye and nose, and where it does not
significantly enter the blood stream. By systemic administration is
meant oral, intravenous, intraperitoneal and intramuscular
administration. The amount of an antibody required for therapeutic
or prophylactic effect will, of course, vary with the antibody
chosen, the nature and severity of the condition being treated and
the animal undergoing treatment, and is ultimately at the
discretion of the physician. A suitable topical dose of an antibody
of the invention will generally be within the range of about 1 to
100 milligrams per kilogram body weight daily.
Formulations
[0301] While it is possible for an antibody or fragment thereof to
be administered alone, it is preferable to present it as a
pharmaceutical formulation. The active ingredient may comprise, for
topical administration, from 0.001% to 10% w/w, e.g., from 1% to 2%
by weight of the formulation, although it may comprise as much as
10% w/w but preferably not in excess of 5% w/w and more preferably
from 0.1% to 1% w/w of the formulation.
[0302] The topical formulations of the present invention, comprise
an active ingredient together with one or more acceptable
carrier(s) therefor and optionally any other therapeutic
ingredients(s). The carrier(s) must be "acceptable" in the sense of
being compatible with the other ingredients of the formulation and
not deleterious to the recipient thereof.
[0303] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of where treatment is required, such as
liniments, lotions, creams, ointments or pastes, and drops suitable
for administration to the eye, ear or nose.
[0304] Drops according to the present invention may comprise
sterile aqueous or oily solutions or suspensions and may be
prepared by dissolving the active ingredient in a suitable aqueous
solution of a bactericidal and/or fungicidal agent and/or any other
suitable preservative, and preferably including a surface active
agent. The resulting solution may then be clarified by filtration,
transferred to a suitable container which is then sealed and
sterilized by autoclaving or maintaining at 90.degree.-100.degree.
C. for half an hour. Alternatively, the solution may be sterilized
by filtration and transferred to the container by an aseptic
technique. Examples of bactericidal and fungicidal agents suitable
for inclusion in the drops are phenylmercuric nitrate or acetate
(0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate
(0.01%). Suitable solvents for the preparation of an oily solution
include glycerol, diluted alcohol and propylene glycol.
[0305] Lotions according to the present invention include those
suitable for application to the skin or eye. An eye lotion may
comprise a sterile aqueous solution optionally containing a
bactericide and may be prepared by methods similar to those for the
preparation of drops. Lotions or liniments for application to the
skin may also include an agent to hasten drying and to cool the
skin, such as an alcohol or acetone, and/or a moisturizer such as
glycerol or an oil such as castor oil or arachis oil.
[0306] Creams, ointments or pastes according to the present
invention are semi-solid formulations of the active ingredient for
external application. They may be made by mixing the active
ingredient in finely-divided or powdered form, alone or in solution
or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable machinery, with a greasy or non-greasy basis. The basis
may comprise hydrocarbons such as hard, soft or liquid paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural
origin such as almond, corn, arachis, castor or olive oil; wool fat
or its derivatives, or a fatty acid such as stearic or oleic acid
together with an alcohol such as propylene glycol or macrogels. The
formulation may incorporate any suitable surface active agent such
as an anionic, cationic or non-ionic surface active such as
sorbitan esters or polyoxyethylene derivatives thereof. Suspending
agents such as natural gums, cellulose derivatives or inorganic
materials such as silicaceous silicas, and other ingredients such
as lanolin, may also be included.
[0307] It will be recognized by one of skill in the art that the
optimal quantity and spacing of individual dosages of an antibody
or fragment thereof of the invention will be determined by the
nature and extent of the condition being treated, the form, route
and site of administration, and the particular animal being
treated, and that such optimums can be determined by conventional
techniques. It will also be appreciated by one of skill in the art
that the optimal course of treatment, i.e., the number of doses of
an antibody or fragment thereof of the invention given per day for
a defined number of days, can be ascertained by those skilled in
the art using conventional course of treatment determination
tests.
[0308] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following are,
therefore, to be construed as merely illustrative examples and not
a limitation of the scope of the present invention in any way.
Capsule Composition
[0309] A pharmaceutical composition of this invention in the form
of a capsule is prepared by filling a standard two-piece hard
gelatin capsule with 50 mg. of an antibody or fragment thereof of
the invention, in powdered form, 100 mg. of lactose, 32 mg. of talc
and 8 mg. of magnesium stearate.
Injectable Parenteral Composition
[0310] A pharmaceutical composition of this invention in a form
suitable for administration by injection is prepared by stirring
1.5 k by weight of an antibody or fragment thereof of the invention
in 10 k by volume propylene glycol and water. The solution is
sterilized by filtration.
Ointment Composition
[0311] Antibody or fragment thereof of the invention 10 g.
[0312] White soft paraffin to 100.0 g.
[0313] The antibody or fragment thereof of the invention is
dispersed in a small volume of the vehicle to produce a smooth,
homogeneous product. Collapsible metal tubes are then filled with
the dispersion.
Topical Cream Composition
[0314] Antibody or fragment thereof of the invention 1.0 g.
[0315] Polawax GP 200 20.0 g.
[0316] Lanolin Anhydrous 2.0 g.
[0317] White Beeswax 2.5 g.
[0318] Methyl hydroxybenzoate 0.1 g.
[0319] Distilled Water to 100.0 g.
[0320] The polawax, beeswax and lanolin are heated together at
60.degree. C. A solution of methyl hydroxybenzoate is added and
homogenization is achieved using high speed stirring. The
temperature is then allowed to fall to SOOC. The antibody or
fragment thereof of the invention is then added and dispersed
throughout, and the composition is allowed to cool with slow speed
stirring.
Topical Lotion Composition
[0321] Antibody or fragment thereof of the invention 1.0 g.
[0322] Sorbitan Monolaurate 0.6 g. Polysorbate 20 0.6 g.
[0323] Cetostearyl Alcohol 1.2 g. Glycerin 6.0 g.
[0324] Methyl Hydroxybenzoate 0.2 g.
[0325] Purified Water B.P. to 100.00 ml. (B.P.=British
Pharmacopeia)
[0326] The methyl hydroxybenzoate and glycerin are dissolved in 70
ml. of the water at 75.degree. C. The sorbitan monolaurate,
polysorbate 20 and cetostearyl alcohol are melted together at
75.degree. C. and added to the aqueous solution. The resulting
emulsion is homogenized, allowed to cool with continuous stirring
and the antibody or fragment thereof of the invention is added as a
suspension in the remaining water. The whole suspension is stirred
until homogenized.
Eye Drop Composition
[0327] Antibody or fragment thereof of the invention 0.5 g.
[0328] Methyl Hydroxybenzoate 0.01 g.
[0329] Propyl Hydroxybenzoate 0.04 g.
[0330] Purified Water B.P. to 100.00 ml.
[0331] The methyl and propyl hydroxybenzoates are dissolved in 70
ml. purified water at 75.degree. C. and the resulting solution is
allowed to cool. The antibody or fragment thereof of the invention
is then added, and the solution is sterilized by filtration through
a membrane filter (0.022 Am pore size), and packed aseptically into
suitable sterile containers.
Composition for Administration by Inhalation
[0332] For an aerosol container with a capacity of 15-20 ml: mix 10
mg. of an antibody or fragment thereof of the invention with
0.2-0.5 k of a lubricating agent, such as polysorbate 85 or oleic
acid, and disperse such mixture in a propellant, such as freon,
preferably in a combination of (1,2 dichlorotetrafluoroethane) and
difluorochloromethane and put into an appropriate aerosol container
adapted for either intranasal or oral inhalation administration.
Composition for Administration by Inhalation For an aerosol
container with a capacity of 15-20 ml: dissolve 10 mg. of an
antibody or fragment thereof of the invention in ethanol (6-8 ml.),
add 0.1-0.2 k of a lubricating agent, such as polysorbate 85 or
oleic acid; and disperse such in a propellant, such as freon,
preferably in combination of (1-2 dichlorotetrafluoroethane) and
difluorochloromethane, and put into an appropriate aerosol
container adapted for either intranasal or oral inhalation
administration.
Parenteral Administrable Antibody Compositions
[0333] The antibodies and pharmaceutical compositions of the
invention are particularly useful for parenteral administration,
i.e., subcutaneously, intramuscularly or intravenously. The
compositions for parenteral administration will commonly comprise a
solution of an antibody of the invention or a cocktail thereof
dissolved in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be employed, e.g., water,
buffered water, 0.4 k saline (normal saline), 0.3% glycine, and the
like. The use of normal saline is preferred. These solutions are
sterile and generally free of particulate matter. These solutions
may be sterilized by conventional, well-known sterilization
techniques. 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 or fragment thereof of the
invention in such pharmaceutical formulation can vary widely. Such
concentrations will be selected primarily based on fluid volumes,
viscosities, etc., according to the particular mode of
administration selected. Generally suitable intravenous
concentrations range from about one to one hundred milligrams per
milliliter.
[0334] Thus, a pharmaceutical composition of the invention for
intravenous injection could comprise 10 mL normal saline containing
40-50 mg of an anti-human CD23 antibody of the invention. 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., hereby incorporated by reference herein.
[0335] The antibodies of the invention can be lyophilized for
storage and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional immune
globulins and art-known lyophilization and reconstitution
techniques can be employed.
[0336] Depending on the intended result, the pharmaceutical
composition of the invention can be administered for prophylactic
and/or therapeutic treatments. In therapeutic application,
compositions are administered to a patient already suffering from a
disease, in an amount sufficient to cure or at least partially
arrest the disease and its complications. In prophylactic
applications, compositions containing the present antibodies or a
cocktail thereof are administered to a patient not already in a
disease state to enhance the patient's resistance.
[0337] Single or multiple administrations of the pharmaceutical
compositions can be carried out with dose levels and pattern being
selected by the treating physician. In any event, the
pharmaceutical composition of the invention should provide a
quantity of the subject anti-CD23 antibodies sufficient to
effectively treat the patient.
[0338] It should also be noted that the antibodies of this
invention may be used for the design and synthesis of either
peptide or non-peptide compounds (mimetics) which would be useful
in the same therapy as the antibody. See, e.g., Saragovi et al.,
Science, 253:792-795 (1991).
[0339] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without diverting from the scope of the invention. Accordingly, the
invention is not limited by the appended claims.
Sequence CWU 1
1
391390DNAArtificial SequenceDescription of Artificial Sequence
Mature peptide is derived from Old World Monkey (macaque); leader
sequence is a primate leader sequence with primer terminal residues
to facilitate cloning 1atg gcc tgg act ctg ctc ctc gtc acc ctc ctc
act cag ggc aca gga 48Met Ala Trp Thr Leu Leu Leu Val Thr Leu Leu
Thr Gln Gly Thr Gly-15 -10 -5tcc tgg gct cag tct gcc ccg act cag
cct ccc tct gtg tct ggg tct 96Ser Trp Ala Gln Ser Ala Pro Thr Gln
Pro Pro Ser Val Ser Gly Ser-1 1 5 10cct gga cag tcg gtc acc atc tcc
tgc act gga acc agc gat gac gtt 144Pro Gly Gln Ser Val Thr Ile Ser
Cys Thr Gly Thr Ser Asp Asp Val15 20 25ggt ggt tat aac tat gtc tcc
tgg tac caa cac cac cca ggc aaa gcc 192Gly Gly Tyr Asn Tyr Val Ser
Trp Tyr Gln His His Pro Gly Lys Ala30 35 40 45ccc aaa ctc atg att
tat gat gtc gct aag cgg gcc tca ggg gtc tct 240Pro Lys Leu Met Ile
Tyr Asp Val Ala Lys Arg Ala Ser Gly Val Ser50 55 60gat cgc ttc tct
ggc tcc aag tct ggc aac acg gcc tcc ctg acc atc 288Asp Arg Phe Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile65 70 75tct ggg ctc
cag gct gag gac gag gct gat tat tac tgt tgt tca tat 336Ser Gly Leu
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr80 85 90aca acc
agt agc act ttg tta ttc gga aga ggg acc cgg ttg acc gtc 384Thr Thr
Ser Ser Thr Leu Leu Phe Gly Arg Gly Thr Arg Leu Thr Val95 100
105cta ggt 390Leu Gly1102130PRTArtificial SequenceDescription of
Artificial Sequence Mature peptide is derived from Old World Monkey
(macaque); leader sequence is a primate leader sequence with primer
terminal residues to facilitate cloning 2Met Ala Trp Thr Leu Leu
Leu Val Thr Leu Leu Thr Gln Gly Thr Gly-15 -10 -5Ser Trp Ala Gln
Ser Ala Pro Thr Gln Pro Pro Ser Val Ser Gly Ser-1 1 5 10Pro Gly Gln
Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Asp Asp Val15 20 25Gly Gly
Tyr Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys Ala30 35 40
45Pro Lys Leu Met Ile Tyr Asp Val Ala Lys Arg Ala Ser Gly Val Ser50
55 60Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile65 70 75Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys
Ser Tyr80 85 90Thr Thr Ser Ser Thr Leu Leu Phe Gly Arg Gly Thr Arg
Leu Thr Val95 100 105Leu Gly1103423DNAArtificial
SequenceDescription of Artificial Sequence Mature peptide is
derived from Old World Monkey (macaque); leader sequence is a
primate leader sequence with primer terminal residues to facilitate
cloning 3atg aaa cac ctg tgg ttc ttc ctc ctc ctg gtg gca gct ccc
aga tgg 48Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro
Arg Trp-15 -10 -5gtc ctg tcc cag ctg cag ctg cag gag tcg ggc cca
gga gtg gtg aag 96Val Leu Ser Gln Leu Gln Leu Gln Glu Ser Gly Pro
Gly Val Val Lys-1 1 5 10cct tcg gag acc ctg tcc ctc acc tgc gct gtc
tct ggt ggc tct gtc 144Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val
Ser Gly Gly Ser Val15 20 25agc agt agt aac tgg tgg acc tgg atc cgc
cag ccc cca ggg aag gga 192Ser Ser Ser Asn Trp Trp Thr Trp Ile Arg
Gln Pro Pro Gly Lys Gly30 35 40 45ctg gag tgg att gga cgt atc tct
ggt agt ggt ggg gcc acc aac tac 240Leu Glu Trp Ile Gly Arg Ile Ser
Gly Ser Gly Gly Ala Thr Asn Tyr50 55 60aac ccg tcc ctc aag agt cga
gtc atc att tca caa gac acg tcc aag 288Asn Pro Ser Leu Lys Ser Arg
Val Ile Ile Ser Gln Asp Thr Ser Lys65 70 75aac cag ttc tcc ctg aac
ctg aac tct gtg acc gcc gcg gac acg gcc 336Asn Gln Phe Ser Leu Asn
Leu Asn Ser Val Thr Ala Ala Asp Thr Ala80 85 90gtg tat tac tgt gcc
aga gat tgg gcc caa ata gct gga aca acg cta 384Val Tyr Tyr Cys Ala
Arg Asp Trp Ala Gln Ile Ala Gly Thr Thr Leu95 100 105ggc ttc tgg
ggc cag gga gtc ctg gtc acc gtc tcc tca 423Gly Phe Trp Gly Gln Gly
Val Leu Val Thr Val Ser Ser110 115 1204141PRTArtificial
SequenceDescription of Artificial Sequence Mature peptide is
derived from Old World Monkey (macaque); leader sequence is a
primate leader sequence with primer terminal residues to facilitate
cloning 4Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro
Arg Trp-15 -10 -5Val Leu Ser Gln Leu Gln Leu Gln Glu Ser Gly Pro
Gly Val Val Lys-1 1 5 10Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val
Ser Gly Gly Ser Val15 20 25Ser Ser Ser Asn Trp Trp Thr Trp Ile Arg
Gln Pro Pro Gly Lys Gly30 35 40 45Leu Glu Trp Ile Gly Arg Ile Ser
Gly Ser Gly Gly Ala Thr Asn Tyr50 55 60Asn Pro Ser Leu Lys Ser Arg
Val Ile Ile Ser Gln Asp Thr Ser Lys65 70 75Asn Gln Phe Ser Leu Asn
Leu Asn Ser Val Thr Ala Ala Asp Thr Ala80 85 90Val Tyr Tyr Cys Ala
Arg Asp Trp Ala Gln Ile Ala Gly Thr Thr Leu95 100 105Gly Phe Trp
Gly Gln Gly Val Leu Val Thr Val Ser Ser110 115 1205387DNAArtificial
SequenceDescription of Artificial Sequence Mature peptide is
derived from Old World Monkey (macaque); leader sequence is a
primate leader sequence with primer terminal residues to facilitate
cloning 5atg gac atg agg gtc ccc gct cag ctc ctg ggg ctc ctt ctg
ctc tgg 48Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu
Leu Trp-20 -15 -10ctc cca ggt gcc aga tgt gac atc cag atg acc cag
tct cca tct tcc 96Leu Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser-5 -1 1 5 10ctg tct gca tct gta ggg gac aga gtc acc
atc act tgc agg gca agt 144Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser15 20 25cag gac att agg tat tat tta aat tgg
tat cag cag aaa cca gga aaa 192Gln Asp Ile Arg Tyr Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys30 35 40gct cct aag ctc ctg atc tat gtt
gca tcc agt ttg caa agt ggg gtc 240Ala Pro Lys Leu Leu Ile Tyr Val
Ala Ser Ser Leu Gln Ser Gly Val45 50 55cca tca agg ttc agc ggc agt
gga tct ggg aca gag ttc act ctc acc 288Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr60 65 70gtc agc agc ctg cag cct
gaa gat ttt gcg act tat tac tgt cta cag 336Val Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln75 80 85 90gtt tat agt acc
cct cgg acg ttc ggc caa ggg acc aag gtg gaa atc 384Val Tyr Ser Thr
Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile95 100 105aaa
387Lys6129PRTArtificial SequenceDescription of Artificial Sequence
Mature peptide is derived from Old World Monkey (macaque); leader
sequence is a primate leader sequence with primer terminal residues
to facilitate cloning 6Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp-20 -15 -10Leu Pro Gly Ala Arg Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser-5 -1 1 5 10Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser15 20 25Gln Asp Ile Arg Tyr Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys30 35 40Ala Pro Lys Leu Leu
Ile Tyr Val Ala Ser Ser Leu Gln Ser Gly Val45 50 55Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr60 65 70Val Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln75 80 85 90Val
Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile95 100
105Lys7411DNAArtificial SequenceDescription of Artificial Sequence
Mature peptide is derived from Old World Monkey (macaque); leader
sequence is a primate leader sequence with primer terminal residues
to facilitate cloning 7atg gag ttt ggg ctg agc tgg gtt ttc ctt gtt
cct ctt ttg aaa ggt 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val
Pro Leu Leu Lys Gly-15 -10 -5gtc cag tgt gag gtg cag ctg gtg gag
tct ggg ggc ggc ttg gca aag 96Val Gln Cys Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Ala Lys-1 1 5 10cct ggg ggg tcc ctg aga ctc tcc
tgc gca gcc tcc ggg ttc agg ttc 144Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Arg Phe15 20 25acc ttc aat aac tac tac atg
gac tgg gtc cgc cag gct cca ggg cag 192Thr Phe Asn Asn Tyr Tyr Met
Asp Trp Val Arg Gln Ala Pro Gly Gln30 35 40 45ggg ctg gag tgg gtc
tca cgt att agt agt agt ggt gat ccc aca tgg 240Gly Leu Glu Trp Val
Ser Arg Ile Ser Ser Ser Gly Asp Pro Thr Trp50 55 60tac gca gac tcc
gtg aag ggc aga ttc acc atc tcc aga gag aac gcc 288Tyr Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala65 70 75aac aac aca
ctg ttt ctt caa atg aac agc ctg aga gct gag gac acg 336Asn Asn Thr
Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr80 85 90gct gtc
tat tac tgt gcg agc ttg act aca ggg tct gac tcc tgg ggc 384Ala Val
Tyr Tyr Cys Ala Ser Leu Thr Thr Gly Ser Asp Ser Trp Gly95 100
105cag gga gtc ctg gtc acc gtc tcc tca 411Gln Gly Val Leu Val Thr
Val Ser Ser110 1158137PRTArtificial SequenceDescription of
Artificial Sequence Mature peptide is derived from Old World Monkey
(macaque); leader sequence is a primate leader sequence with primer
terminal residues to facilitate cloning 8Met Glu Phe Gly Leu Ser
Trp Val Phe Leu Val Pro Leu Leu Lys Gly-15 -10 -5Val Gln Cys Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Lys-1 1 5 10Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Phe15 20 25Thr Phe
Asn Asn Tyr Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Gln30 35 40
45Gly Leu Glu Trp Val Ser Arg Ile Ser Ser Ser Gly Asp Pro Thr Trp50
55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn
Ala65 70 75Asn Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr80 85 90Ala Val Tyr Tyr Cys Ala Ser Leu Thr Thr Gly Ser Asp
Ser Trp Gly95 100 105Gln Gly Val Leu Val Thr Val Ser Ser110
115941DNAArtificial SequenceDescription of Artificial Sequence
Primer 9atcacagatc tctcaccatg gacatgaggg tccccgctca g
411035DNAArtificial SequenceDescription of Artificial Sequence
Primer 10atcacagatc tctcaccatg aggctccctg ctcag 351135DNAArtificial
SequenceDescription of Artificial Sequence Primer 11atcacagatc
tctcaccatg gaarccccag ckcag 351238DNAArtificial SequenceDescription
of Artificial Sequence Primer 12atcacagatc tctcaccatg gtgttgcaga
cccaggtc 381332DNAArtificial SequenceDescription of Artificial
Sequence Primer 13ggtgcagcca ccgtagcttt gatytccasc tt
321434DNAArtificial SequenceDescription of Artificial Sequence
Primer 14atcacagatc tctcaccatg rcctgstccc ctct 341534DNAArtificial
SequenceDescription of Artificial Sequence Primer 15atcacagatc
tctcaccatg gcctggrctc ygct 341635DNAArtificial SequenceDescription
of Artificial Sequence Primer 16atcacagatc tctcaccatg gcmtggaycc
ctctc 351721DNAArtificial SequenceDescription of Artificial
Sequence Primer 17cttgggctga cctaggacgg t 211830DNAArtificial
SequenceDescription of Artificial Sequence Primer 18gcgactaagt
cgaccatgga ctggacctgg 301930DNAArtificial SequenceDescription of
Artificial Sequence Primer 19gcgactaagt cgaccatgaa acacctgtgg
302033DNAArtificial SequenceDescription of Artificial Sequence
Primer 20gcgactaagt cgaccatgga gtttgggctg agc 332133DNAArtificial
SequenceDescription of Artificial Sequence Primer 21gcgactaagt
cgaccatggg gtcaaccgcc atc 332233DNAArtificial SequenceDescription
of Artificial Sequence Primer 22gcgactaagt cgaccatgtc tgtctccttc
ctc 332346DNAArtificial SequenceDescription of Artificial Sequence
Primer 23gccaggggga agaccgatgg gcccttggtg ctagctgagg agacgg
462431DNAArtificial SequenceDescription of Artificial Sequence
Primer 24gatgggccct tggtgctagc tgaggagacg g 312551DNAArtificial
SequenceDescription of Artificial Sequence Primer 25ggtgctagct
gaggagacgg tgaccaggac tccctggccc cagaagccta g 512617DNAArtificial
SequenceDescription of Artificial Sequence Primer 26atttaggtga
cactata 172716DNAArtificial SequenceDescription of Artificial
Sequence Primer 27gttttcccag tcacga 162820DNAArtificial
SequenceDescription of Artificial Sequence Primer 28atatacgact
cactataggg 202924DNAArtificial SequenceDescription of Artificial
Sequence Primer 29ccgtcagatc gcctggagac gcca 243020DNAArtificial
SequenceDescription of Artificial Sequence Primer 30gcagttccag
atttcaactg 203121DNAArtificial SequenceDescription of Artificial
Sequence Primer 31ccaggccact gtcacggctt c 213220DNAArtificial
SequenceDescription of Artificial Sequence Primer 32cagagctggg
tacgtcctca 203320DNAArtificial SequenceDescription of Artificial
Sequence Primer 33gcccccagag gtgctcttgg 203420DNAArtificial
SequenceDescription of Artificial Sequence Primer 34acacagaccc
gtcgacatgg 203520DNAArtificial SequenceDescription of Artificial
Sequence Primer 35gctctcggag gtgctcctgg 203630DNAArtificial
SequenceDescription of Artificial Sequence Primer 36acagacccgt
cgaccatgga gtttgggctg 303727DNAArtificial SequenceDescription of
Artificial Sequence Primer 37ccccttggtg ctagctgagg agacggt
273827DNAArtificial SequenceDescription of Artificial Sequence
Primer 38agagagaacg ccaagaacac actgttt 273927DNAArtificial
SequenceDescription of Artificial Sequence Primer 39aaacagtgtg
ttcttggcgt tctctct 27
* * * * *