U.S. patent application number 11/527969 was filed with the patent office on 2007-01-25 for immunoglobulin variants.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Paula M. Jardieu, Leonard G. Presta.
Application Number | 20070020258 11/527969 |
Document ID | / |
Family ID | 27410640 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070020258 |
Kind Code |
A1 |
Jardieu; Paula M. ; et
al. |
January 25, 2007 |
Immunoglobulin variants
Abstract
The present invention describes IgE antagonists (including
variant anti-IgE antibodies) and their use in diagnosis, therapy or
prophylaxis of allergic and other IgE-mediated disorders, including
asthma, food allergies, hypersensitivity and anaphylactic
reactions.
Inventors: |
Jardieu; Paula M.;
(Berkeley, CA) ; Presta; Leonard G.; (San
Francisco, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
|
Family ID: |
27410640 |
Appl. No.: |
11/527969 |
Filed: |
September 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10968237 |
Oct 19, 2004 |
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11527969 |
Sep 26, 2006 |
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09925179 |
Aug 8, 2001 |
6914129 |
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10968237 |
Oct 19, 2004 |
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08466163 |
Jun 6, 1995 |
6329509 |
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09925179 |
Aug 8, 2001 |
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08405617 |
Mar 15, 1995 |
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08466163 |
Jun 6, 1995 |
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08185899 |
Jan 26, 1994 |
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PCT/US92/06860 |
Aug 14, 1992 |
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08405617 |
Mar 15, 1995 |
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07879495 |
May 7, 1992 |
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08185899 |
Jan 26, 1994 |
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07744768 |
Aug 14, 1991 |
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08185899 |
Jan 26, 1994 |
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Current U.S.
Class: |
424/133.1 ;
530/388.1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 16/32 20130101; C07K 16/468 20130101; C07K 16/4291 20130101;
C07K 16/18 20130101; C07K 2319/00 20130101; C07K 2317/52 20130101;
A61K 38/00 20130101; C07K 2317/565 20130101; C07K 2317/31 20130101;
C07K 16/00 20130101 |
Class at
Publication: |
424/133.1 ;
530/388.1 |
International
Class: |
A61K 39/395 20070101
A61K039/395; C07K 16/42 20070101 C07K016/42 |
Claims
1. A polypeptide which is capable of binding to one of FCEL or FCEH
but which is substantially incapable of binding to the other of
FCEL or FCEH.
2. The polypeptide of claim 1 which comprises amino acid sequence
which is substantially homologous to an Fc.epsilon.3-Fc.epsilon.4
sequence.
3. The polypeptide of claim 2 which comprises amino acid sequence
greater than about 80% homologous with an Fc.epsilon.3-Fc.epsilon.4
sequence and which contains at least about 50 residues.
4. The polypeptide of claim 1 which is an immunoglobulin.
5. The immunoglobulin of claim 4 which is capable of binding to
FCEL but which is substantially incapable of binding to FCEH.
6. The immunoglobulin of claim 5 which is an IgE analogue having a
variant amino acid sequence within about residues 420 to 428,
inclusive.
7. The immunoglobulin of claim 5 which is an IgE analogue having a
variant amino acid sequence within about residues 446 to 453,
inclusive.
8. The immunoglobulin of claim 6 further comprising IgE residues
about from 373-390 and wherein the variant amino acid sequence is a
deletion of one of residues 423-428.
9. The immunoglobulin of claim 4 which further comprises a
cytotoxic polypeptide, an enzyme, a diagnostic label, or an
immunoglobulin variable domain capable of binding a predetermined
antigen.
10. The immunoglobulin of claim 5 which is an IgE analogue having a
variant amino acid sequence within about residues 420-428,
inclusive, and within about residues 446 to 453, inclusive.
11. The immunoglobulin of claim 10 which is capable of binding
complement.
12. The immunoglobulin of claim 9 wherein the antigen is CD8 or
CD3.
13. The immunoglobulin of claim 9 wherein the antigen is a lymphoid
cell surface antigen.
14. An immunoglobulin of claim 9 which comprises an IgG, IgA, IgD
or IgM sequence.
15. A method for treating an allergic disorder which comprises
administering to a patient susceptible to an allergy a
therapeutically effective amount of an FCEL or FCEH specific
polypeptide, provided that the FCEH-specific polypeptide is
incapable of crosslinking FCEH and inducing histamine release.
16. A polypeptide capable of binding to FCEL and having a human IgE
beta strand D sequence which is substantially incapable of binding
to FCEH, said polypeptide containing no more than about 40
residues.
17. The polypeptide of claim 16 having no more than about 30
residues.
18. The polypeptide of claim 17 wherein a residue within the beta
strand D domain has been deleted or substituted, or another residue
inserted within the beta strand D domain.
19. A polypeptide capable of binding to FCEH, containing a beta
strand D sequence of IgE, and having no more than 19 residues.
20. The polypeptide of claim 1 which is capable of binding to FCEH
but not FCEL and comprises IgE sequence selected from about
residues 420 to about 442.
21. The polypeptide of claim 19 which comprises the IgE amino acid
sequence of residues K423-R428.
22. The polypeptide of claim 1 which comprises less than about 20
residues and which is conformationally constrained.
23. The polypeptide of claim 1 which binds FCEL with at least about
75% of the affinity of native IgE and binds FCEH with no greater
than about 10% of the affinity of native IgE.
24. The immunoglobulin of claim 4 which comprises an IgE
complementarity determining region.
25. The immunoglobulin of claim 4 which is capable of binding to
FCEH but which is substantially incapable of binding to FCEL.
26. The immunoglobulin of claim 25 which is an IgE analogue having
a variant amino acid sequence within about residues 373 to 390,
inclusive or residues 446 to 453, inclusive.
27. The immunoglobulin of claim 25 which is an IgE analogue having
a variant amino acid sequence within about residues 382 to 390,
inclusive or residues 446 to 453, inclusive.
28. The immunoglobulin of claim 27 which further comprises a
FCEH-binding loop EF and beta strand D domain.
29. The immunoglobulin of claim 24 which further comprises an
immunoglobulin variable domain capable of binding a predetermined
antigen, an enzyme or a diagnostic label.
30. The immunoglobulin of claim 29 wherein the antigen is CD8 or
CD3.
31. The immunoglobulin of claim 29 wherein the antigen is a
lymphoid cell surface antigen.
32. The immunoglobulin of claim 25 which comprises an IgG, IgA, IgD
or IgM sequence.
33. The immunoglobulin of claim 25 which binds FCEH with at least
about 75% of the affinity of native IgE, and binds FCEL with no
greater than about 10% of the efficiency of native IgE.
34. A polypeptide capable of binding to FCEL and comprising a FCEL
binding domain of the human loop AB-beta strand B of IgE, said
polypeptide having no more than about 25 residues.
35. The polypeptide of claim 34 which is human.
36. The polypeptide of claim 34 having no more than about 10
residues.
37. The polypeptide of claim 34 which is not A358-T389 or
R383-I388.
38. The polypeptide of claim 34 wherein beta strand D is
deleted.
39. The polypeptide of claim 37 wherein the amino acid sequence
comprises the IgE sequence I382-T389.
40. An antibody which is capable of binding to FCEL-bound IgE but
is substantially incapable of binding to FCEH-bound IgE, comprising
a human Kabat CDR domain into which has been substituted an
analogous residue from a Kabat CDR domain of MAE1, MAE13, MAE15,
MAE17.
41. The antibody of claim 40 wherein the residue is from the MAE11,
MAE13 or MAE15 Kabat VH1 CDR domain.
42. The antibody of claim 40 wherein the substituted amino acid
sequence comprises from 1 to about 7 residues from a MAE11, MAE13
or MAE15 Kabat CDR domain
43. The antibody of claim 40 wherein the substituted residue is
from the MAE11, MAE13 or MAE15 Kabat VH1, VH2, VH3, VL1, VL2 and
VL3 domains.
44. The antibody of claim 40 which comprises non-CDR sequence from
a Kabat human consensus antibody.
45. The antibody of claim 44 wherein the consensus antibody is
Kabat subgroup III for heavy chain and kappa subgroup I for light
chain.
46. The antibody of claim 40 further comprising a residue
substituted from a MAE11, MAE13, MAE15 or MAE17 framework or VH-VL
interface domain into the analogous residue of the human
antibody.
47. The antibody of claim 40 wherein the residue is from the heavy
chain framework.
48. The antibody of claim 47 wherein the residue is VH78, VH60 or
VH61.
49. An antibody which is capable of binding to FCEL-bound IgE but
is substantially incapable of binding to FCEH-bound IgE, comprising
the heavy and light chain sequences of humae11ver. 1, 2, 3, 4, 5,
6, 7, 7a, 8, 8a, 8b or 9.
50. The antibody of claim 48 which is humae11ver. 9.
51. A bispecific antibody which is capable of binding to FCEL-bound
IgE but is substantially incapable of binding to FCEH-bound
IgE.
52. An antibody which is (a) monovalent for FCEL-bound IgE but is
substantially incapable of binding to FCEH-bound IgE and (b) is
capable of an immunoglobulin effector function and comprises an Fc
domain containing at least two heavy chains.
53. An antibody which is capable of binding to FCEL-bound IgE but
is substantially incapable of binding to FCEH-bound IgE, comprising
a human consensus heavy chain and light chain sequence.
54. The antibody of claim 52 wherein the consensus heavy chain is
Kabat subgroup III and the consensus light chain is Kabat kappa
subgroup I.
55. An antibody which is capable of binding to FCEL-bound IgE but
is substantially incapable of binding to FCEH-bound IgE, comprising
a human heavy chain and light chain sequence, and which has an IgE
affinity which is substantially the same as or greater than that of
MAE11 for IgE.
56. The antibody of claim 54 wherein the affinity for IgE is about
0.1 to 100 times greater than that of MAE11 for IgE.
57. The antibody of claim 54 wherein the human heavy chain or light
chain sequence comprises a residue substituted from MAE11, MAE13 or
MAE15.
Description
RELATION BACK AND PRIORITY INFORMATION
[0001] This is a continuation filed under 37 C.F.R. .sctn.
1.53(b)(1) of U.S. Ser. No. 10/968,237, filed Oct. 19, 2004, now
pending, which is a continuation of U.S. Ser. No. 09/925,179, filed
on Aug. 8, 2001, now U.S. Pat. No. 6,914,129, which is a
continuation of U.S. Ser. No. 08/466,163, filed on Jun. 6, 1995,
now U.S. Pat. No. 6,329,509, which is a divisional of U.S. Ser. No.
08/405,617, filed on Mar. 15, 1995, now abandoned, which is a
continuation of U.S. Ser. No. 08/185,899, filed on Jan. 26, 1994,
now abandoned, which is a 35 U.S.C. .sctn.371 of PCT/US92/06860
filed Aug. 14, 1992, now inactive, which is a Continuation-in-Part
of U.S. Ser. No. 07/879,495 filed May 7, 1992, now abandoned, which
is a Continuation-in-Part of U.S. Ser. No. 07/744,768, filed on
Aug. 14, 1991, now abandoned; all of which are incorporated by
reference and to which application priority is claimed under 35
U.S.C. .sctn. 120.
BACKGROUND OF THE INVENTION
[0002] This invention relates to amino acid sequence variant
anti-IgE antibodies and to polypeptides containing IgE sequences,
especially IgE antagonists and to polypeptides capable of
differential binding to Fc.epsilon.RI and Fc.epsilon.RII.
[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 suffered on a
widespread basis. IgE is secreted by, and expressed on the surface
of, B-cells. IgE synthesized by B-cells is anchored in the B-cell
membrane by a transmembrane domain linked to the mature IgE
sequence by a short membrane binding region. IgE also is bound to
B-cells (and monocytes, eosinophils and platelets) through its Fc
region to a low affinity IgE receptor (Fc.epsilon.RII, hereafter
"FCEL"). Upon exposure of a mammal to an allergen, B-cells are
clonally amplified which synthesize IgE that binds the allergen.
This IgE in turn is released into the circulation by the B-cells
where it is bound by B-cells (through the FCEL) and by mast cells
and basophils through the so-called high affinity receptor
(Fc.epsilon.RI, hereinafter "FCEH") found on the surface of the
mast cells and basophils. Such mast cells and basophils are thereby
sensitized for allergen. The next exposure to the allergen
cross-links the Fc.epsilon.RI on these cells and thus activates
their release of histamine and other factors which are responsible
for clinical hypersensitivity and anaphylaxis.
[0004] The art has reported antibodies capable of binding to
FCEL-bound IgE but not IgE located on 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
found on B-cells or circulating free in the body, but do not bind
to FCEH and thus will not activate mast cells or basophils. In
addition, various amino acid sequence variants of immunoglobulins
are known, e.g., "chimeric" and "humanized" antibodies (see, for
example, U.S. Pat. No. 4,816,567; WO 91/09968; EP 452,508; and WO
91/16927). Humanized antibodies are immunoglobulins, immunoglobulin
chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or
other antigen-binding subsequences of antibodies) which contain
minimal sequence derived from non-human immunoglobulin. For the
most part, humanized antibodies are human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibody may comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine
and optimize antibody performance as will be more further described
infra. Also known per se are monovalent and bispecific
antibodies.
[0005] It is generally understood that FCEH, like FCEL, binds to
recognition site(s) in the IgE constant (Fc) domain. The IgE
recognition site(s) for the two receptors are poorly defined,
despite considerable effort in the past directed to the
problem.
[0006] Over the past decade several studies have been undertaken to
determine which portion of the IgE molecule is involved in binding
to Fc.epsilon.RI and Fc.epsilon.RII. Essentially three approaches
have been tried. First, peptides corresponding to specific portions
of IgE sequence have been used as either competitive inhibitors of
IgE-receptor binding (Burt et al., Eur. J. Immun, 17:437-440
[1987]; Helm et al., Nature, 331:180-183 [1988]; Helm et al., Proc.
Natl. Acad. Sci., 86:9465-9469 [1989]; Vercelli et al., Nature,
338:649-651 [1989]; Nio et al., Peptide Chemistry, 203-208 [1990])
or to elicit anti-IgE antibodies which would block IgE-receptor
interaction (Burt et al., Molec. Immun. 24:379-389 [1987];
Robertson et al., Molec. Immun., 25:103-113 [1988]; Baniyash et
al., Molec. Immun. 25:705-711 [1988]). The most effective
competitive peptide was a sequence that was 1000-fold less active
than IgE (Burt et al., Eur. J. Immun., 17:437-440 [1987]).
[0007] Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 (1989)
found that a peptide corresponding to IgE residues 329-409 blocked
in vivo sensitization of human basophil granulocytes with human IgE
antibodies. Further studies indicated that residues 395-409 were
not essential for binding of the 329-409 peptide to Fc.epsilon.RI
(Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 [1989]). Note
that the IgE sequence variants described below had the sequence of
Padlan et al., Mol. Immun., 23:1063 (1986), but that the
immunoglobulin residue numbers used herein are those of Kabat et
al., Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. 1987).
[0008] Vercelli et al., Nature, 338:649-651 (1989) used recombinant
IgE peptides as well as anti-Fc.epsilon. monoclonal antibodies to
investigate the B-cell (Fc.epsilon.RII) binding site of human IgE.
They concluded that the Fc.epsilon.RII binding site is in
Fc.epsilon.3 near K399-V402.
[0009] Burt et al., Eur. J. Immun., 17:437-440 (1987) investigated
seven peptides for competition against rat IgE in binding to rat
mast cells. Their most active peptide, p129, was 1000-fold less
active than IgE. p129 corresponds to human sequence 439-453 which
includes loop EF. Another of their peptides, p130, corresponding to
residues 396-419 in the Fc.epsilon.3 domain, had no activity.
[0010] Robertson et al., Molec. Immun., 25:103-113 (1988) assessed
IgE binding by sequence-directed antibodies induced by several
synthetic peptides. They concluded that the sequence defined by
their .epsilon.-peptide-4 (corresponding to residues 446-460), was
not significantly involved in receptor binding while the sequence
defined by their .epsilon.-peptide-3 (corresponding to residues
387-401), was likely to be proximal to the IgE-receptor recognition
site.
[0011] Nio et al., Peptide Chemistry, 203-208 (1990) evaluated
numerous peptides with respect to their ability to inhibit
histamine release by human basophils in vitro. Only one peptide
(peptide 2, Table 1), exhibited specific inhibition; this peptide
encompassed residues 376-388. However, a larger peptide which
incorporated this sequence (peptide 3, Table 1), had no inhibitory
activity.
[0012] Second, mutations in IgE have been partially explored.
Schwarzbaum et al., Eur. J. Immun., 19:1015-1023 [1989] (supra)
found that a point mutant P404H (P442H by the numbering system used
herein) had 2-fold reduced affinity for Fc.epsilon.RI on rat
basophilic leukemia (RBL) cells, but the interpretation of this
finding is controversial (Weetall et al., J. Immunol.,
145:3849-3854 [1990]).
[0013] Third, chimeric molecules have been constructed. Human IgE
does not bind to the murine receptor (Kulczycki Jr., et al., J.
Exp. Med., 139:600-616 [1974]) while rodent IgE binds to the human
receptor with a reduced affinity (Conrad, et al., J. Immun.,
130:327-333 [1983]); human IgG1 does not bind to IgE receptors
(Weetall et al., J. Immun., 145:3849-3854 [1990]). Based on these
observations, several groups have constructed human-murine chimeras
or human IgE-IgG chimeras. Weetall et al., J. Immun., 145:3849-3854
(1990) made a series of human IgG1-murine IgE chimeras and
concluded that the Fc.epsilon.2 and Fc.epsilon.3 domains are
involved in binding murine Fc.epsilon.RI while the Fc.epsilon.4
domain is unlikely to be involved in binding to murine
Fc.epsilon.RI (but may possibly be involved in binding to
Fc.epsilon.RII). However, these conclusions are uncertain since
they rest primarily on lack of binding by chimeras and three of
five chimeras lacked some interchain disulfide bonds.
[0014] Nissim et al., EMBO J., 10:101-107 (1991) constructed a
series of human-murine IgE chimeras and measured binding to RBL
cells and concluded that the portion of IgE which binds with high
affinity to the specialized Fc.epsilon. receptor on RBL cells could
be assigned to Fc.epsilon.3.
[0015] The results reported by these authors (e.g., Helm et al. and
Burt et al.) are inconsistent. Further, in the case of anti-IgE
antibodies it is difficult to eliminate the possibility of
nonspecific blocking due to steric hindrance (Schwarzbaum et al.,
Eur. J. Immun., 19:1015-1023 [1989]). It is apparent that
considerable confusion exists in the art as to the domains of IgE
Fc which are involved in the binding of IgE to FCEH or in the
maintenance of IgE conformation responsible for IgE binding to
FCEH.
[0016] It is an object of this invention to identify polypeptides
capable of differential binding to FCEL and FCEH.
[0017] It is an object herein to determine an IgE domain which is
implicated in FCEH receptor binding, but which is not involved in
FCEL receptor binding, and vice-versa.
[0018] It is another object herein to identify antagonists which
are capable of inhibiting allergic responses, including antagonists
that neutralize the FCEH or FCEL receptor-binding domains of
Fc.epsilon., immunoglobulin analogues that bind FCEL but do not
bind FCEH, or that bind FCEH but not FCEL and humanized anti-huIgE
antibodies that bind to FCEL-bound IgE but not to FCEH-bound IgE or
which bind to IgE but do not induce histamine release or
degranulation of mast cells.
[0019] It is another object to provide novel polypeptides for use
in the assay of Fc.epsilon. receptors and for use as immunogens or
for selecting anti-IgE antibodies.
SUMMARY OF THE INVENTION
[0020] We have identified domains and specific residues of IgE
which play an important role in binding IgE to its FCEL and FCEH
receptors, and based on this information we have designed
polypeptides which remain capable of substantially binding to only
one of these two receptors while being substantially incapable of
binding to the other of the receptors. These polypeptides are
referred to as differential binding polypeptides. In particular,
differential binding polypeptides that bind FCEL comprise IgE
sequences in which one or more residues in loop EF or the
.beta.-strand D domain are varied, while FCEH-binding polypeptides
comprise IgE sequences in which loop AB and/or .beta.-strand B
sequences are varied. Conversely, included herein are certain
polypeptides comprising functional IgE loop EF and .beta.-strand D
domains but loop AB and/or .beta. strand B domains having reduced
functionality compared to wild-type, which bind differentially to
FCEH, and polypeptides comprising functional loop AB and
.beta.-strand B domains but .beta.-strand D and/or loop EF domains
having reduced functionality compared to wild-type, which bind
differentially to FCEL.
[0021] The differential binding polypeptides of this invention are
sufficiently homologous with the amino acid sequence of an IgE
heavy chain that they retain the capability to bind FCEL or FCEH,
but are varied such that they exhibit reduced ability to bind to
one of the two receptors as compared to native IgE. In various
embodiments, the polypeptides of this invention additionally
comprise cytotoxic polypeptides, detectable labels,
conformation-restraining groups and/or amino acid sequences which
are heterologous to IgE, e.g., sequences from receptors or
immunoglobulins as further described below. In other embodiments,
the differential binding polypeptides comprise IgE sequences in
addition to the above-mentioned receptor binding domains, e.g., at
least one variable domain capable of binding a predetermined
antigen. In another embodiment, the differential binding
polypeptide is an IgE variant which is monovalent for a
predetermined antigen. In a still further embodiment, the
differential binding polypeptide comprises an inactive IgE variable
domain, i.e., one which is incapable of binding to any antigen, or
which is devoid of a variable domain or functional CDR.
[0022] The differential binding polypeptides of this invention are
useful in diagnostic procedures for IgE receptors or in the therapy
of IgE-mediated disorders such as allergies. They also are useful
in preparing antibodies capable of binding regions of IgE that
participate in receptor binding.
[0023] In an embodiment of this invention, variant anti-IgE
antibodies are provided for use in diagnosis or for the therapy or
prophylaxis of allergic and other IgE-mediated disorders. In
particular embodiments of this invention anti-IgE variant
antibodies are provided in which one or more human (recipient)
light chain residues 4, 13, 19, 24, 29, 30, 33, 55, 57, 58, 78, 93,
94, or 104, or heavy chain residues 24, 37, 48, 49, 54, 57, 60, 61,
63, 65, 67, 69, 78, 82, 97 or 100 have been modified, preferably by
substitution with the residue found in the corresponding position
in the donor (generally murine) antibody. In preferred embodiments,
the selected residues are light chain 13, 19, 58, 78, or 104, or
heavy chain residues 48, 49, 60, 61, 63, 67, 69, 82 or 82c, and
most preferably are heavy chain residues 60, 61 or light chain
residue 78.
[0024] In other embodiments we provide antibodies which are capable
of binding FCEL-bound IgE but which are substantially incapable of
binding FCEH-bound IgE or inducing histamine release from mast
cells or basophils, comprising a human Kabat CDR domain into which
has been substituted a positionally analogous residue from a Kabat
CDR domain of the murine anti-huIgE antibodies MAE11, MAE13, MAE15
or MAE17. Also provided herein are bispecific antibodies and
IgE-monovalent antibodies; and humanized antibodies exhibiting an
affinity for IgE which ranges from about 0.1 to 100 times that of
MAE11.
BRIEF DESCRIPTION OF THE FIGURE
[0025] FIG. 1 depicts the sequence of human IgE Fc.epsilon.2 and
Fc.epsilon.3 (SEQ ID NO: 1). This particular sequence is from
Padlan et al., Molec. Immun., 23:1063-1075 (1986). Residues are
numbered according to Kabat (supra). "X" residues are included to
align the Padlan IgE sequence with the Kabat numbering scheme.
Sequences which were altered in preparing various IgE mutants are
underlined; bold numbers below the lines denote the mutant number.
.beta.-strand residues are overlined; loop residues are defined by
all residues intervening between two .beta.-strands.
[0026] FIG. 2 depicts light and heavy chain sequences for MAE11
(SEQ ID NOS: 2 and 3), MAE13 (SEQ ID NOS: 4 and 5) and MAE15 (SEQ
ID NOS: 6 and 7).
[0027] FIG. 3 depicts heavy and light chain sequences for HuMae11V1
(SEQ ID NOS: 8 and 9).
[0028] FIGS. 4a and 4b depicts the percent inhibition of IgE
binding to FCEL and FCEH receptors, respectively, by murine
monoclonal antibody Mae11 as well as 3 humanized variants (v1, v8
and v9).
[0029] FIGS. 5a-5c compare the binding of the MAE11, MAE15 and
MAE17 antibodies to various huIgE variants. MAE1 is provided as a
control which binds to both B cells and mast cell-bound IgE. The
mutants scheduled in the boxes in each figure are identified in
Table 11.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The IgE analogue polypeptides of this invention contain an
amino acid sequence which is homologous to that of a naturally
occurring IgE and have the ability to bind specifically or
differentially to FCEL or FCEH but, in varying degree, not to both.
The degree of homology of such polypeptides to wild-type IgE is not
critical since only enough IgE sequence needs to be retained to
enable the IgE to bind differentially or specifically to one of the
two receptors. In general, the polypeptides of this invention will
be IgE Fc analogues and will be about from 80% to 99% homologous
with a polypeptide sequence of a naturally occurring IgE heavy
chain Fc region. Homology is determined by conventional methods in
which all substitutions are considered to be nonhomologous (whether
conservative or nonconservative) and in which the sequences are
aligned to achieve maximal homology.
[0031] It will be understood that the IgE Fc residue numbers
referred to herein are those of Kabat. In applying the residue
teachings of this invention to other IgE Fc domains it will be
necessary to compare the entire candidate sequence with the FIG. 1
sequence in order to align the residues and correlate the residue
numbers. In addition, the identity of certain individual residues
at any given Kabat site number may vary from IgE to IgE due to
interspecies or allelic divergence. When for example it is stated
that substitutions are introduced at residue R383 (human IgE) it
will be understood that this means introducing a substitution at
the same site in IgE even though this same site (in loop AB) may be
located at a different residue number or may be represented in the
parental or starting IgE by a residue which is different than that
described by Kabat. However, for the sake of clarity and simplicity
the residue numbers and identities of the Kabat human IgE heavy
chain sequences will be used herein. Note that some Kabat residues
were deleted in the Padlan sequence, in which case the Kabat
numbering system is preserved by insertion of a spacer residue
designated "X" (See FIG. 1).
[0032] Similarly, the Kabat system is used to designate
immunoglobulin residues used in the preparation of variant, e.g.
humanized, anti-IgE immunoglobulins such as IgG, IgE, IgA or IgD.
In preferred embodiments the recipient human immunoglobulin site is
numbered in accord with Kabat subgroups III (V.sub.H) consensus and
.kappa. subgroup I (V.sub.L) consensus sequences. In order to
determine which donor residues correspond to these Kabat consensus
residues the sequences are maximally aligned, introducing gaps as
necessary, using the variable domain cysteine residues as principal
guideposts. Note that CDRs vary considerably from antibody to
antibody (and by definition will not exhibit homology with the
Kabat consensus sequences). Maximal alignment of framework residues
(particularly the cysteines) frequently will require the insertion
of "spacer" residues in the numbering system, to be used for the
F.sub.v region of the donor antibody. For example, the residue
"29a" referred to infra. This represents an extra residue found in
the murine donor antibody V.sub.H1 CDR for which a counterpart does
not exist in the consensus sequence but whose insertion is needed
to obtain maximal alignment of consensus and donor sequences. In
practice, then, when a humanized antibody (ver. 1) is prepared from
this donor it will contain V.sub.H1 with residue 29a. The
differential binding polypeptides of this invention typically
contain about from 5 to 250 residues which are homologous to an IgE
heavy chain Fc region, but ordinarily will contain about from 10 to
100 such residues. Usually, the IgE Fc3 and Fc4 regions will be
present, with the Fc3 domain providing residues directly involved
in receptor binding with Fc4 being present to ensure conformational
integrity.
[0033] Generally, the IgE is human IgE, although animal IgE such as
rat, murine, equine, bovine, feline or porcine IgE is included. As
noted above, there will be variation in the residue identities and
numbers for these IgEs compared to the FIG. 1 sequence.
[0034] FCEH and FCEL are respectively defined to be the high
affinity IgE receptor (FC.epsilon.RI, Ishizaka et al.,
Immunochemistry, 7:687-702 [1973]) found on mast cells or
basophils, and the low affinity receptor (FC.epsilon.RII, or CD23)
found on cells involved in inflammation such as monocytes,
eosinophils and platelets, as well as B-cells (Capron et al.,
Immun. Today, 7:15-18 [1986]). FCEH and FCEL include alleles and
predetermined amino acid sequence variants thereof which bind IgE.
While FCEH contains several polypeptide chains, the binding of
candidate polypeptides to its alpha chain is all that needs to be
assayed since the alpha chain is the portion of FCEH which binds
IgE.
[0035] Differential binding means that the polypeptide will bind to
one of FCEL or FCEH to the extent of at least about 75% of the
degree with which the homologous native IgE binds to that receptor,
but will not bind to the other receptor at more than about 20% of
the degree that the homologous IgE binds to the other receptor.
Binding is determined by the assays of Example 3. Included within
this invention are polypeptides that are capable of binding to one
of the two receptors to a greater degree than native IgE.
FCEL-Specific Polypeptides
[0036] These polypeptides preferentially bind to the low affinity
receptor. They typically contain Fc.epsilon.3 sequences in which
residues within the .beta.-strand D domain or loop EF have been
substituted or deleted, and/or an additional residue inserted
adjacent to one of such residues. For the purposes herein, the beta
strand D domain extends from N418-X431 (FIG. 1, wherein X indicates
a residue omitted from U266 IgE but found in the Kabat sequence)
and loop EF extends from G444 to T453. A preferred FCEL-specific
embodiment is mutant 6 (Table 6), in which the substitution of 4
residues within the human IgE heavy chain sequence K423-R428
substantially abolished FCEH binding. Other FCEL-specific
embodiments comprising EF loop variants are mutants 85, 89 and the
combination of 49, 51, 52, 83, 86 and 87. These sites (the D and EF
domains) are believed to be the principal sites involved in binding
IgE to FCEL. However, those skilled in the art will be able to
routinely screen for optimal FCEL-specific polypeptides using the
methods shown in the examples once it is understood that the
beta-strand D and loop EF domains are the prinicipal mutagenesis
targets.
[0037] The preferred FCEL-specific polypeptide is one in which a
residue has been substituted or deleted from within the
.beta.-strand D domain or loop EF, or both. For example, four
residues were substituted in generating mutation 6, and any one or
more of these substitutions may be responsible for the loss in FCEH
binding while retaining FCEL binding. As for loop EF, which is
involved in both FCEL and FCEH binding, it is desirable to screen
both activities in order to select the FCEL-specific IgE variants.
For example, mutant 85 (in which 9 IgE residues are substituted by
analogously positioned IgG residues) is not detectably capable of
binding to FCEH, but does bind to FCEL (see Table 11). On the other
hand, conversion of site 444 from Gly to Leu abolishes binding to
either receptor, while sites 447 and 452 are involved in binding to
both receptors since changes at these locations prevent binding to
FCEL but do not abolish FCEH binding.
Beta-Strand D Variants for FCEL Specificity
[0038] In general, D domain substitutions will be nonconservative,
i.e., substituted residues generally will differ substantially from
those found within the homologous native IgE in terms of charge,
hydrophobicity or bulk. Typically, a maximum of 4 of 14
.beta.-strand D domain residues are varied (and are usually
residues 423, 424, 426 and/or 428), although typically any 1 to 5
of these residues are suitable for variation. In general, no more
than 4 residues need to be varied and optimally only one will be
varied.
[0039] K423 and/or K426 are substituted with any of a residue
selected from the group of Arg, His, Cys, Met, Phe, Tyr, Trp, Pro,
Gly, Ala, Val, Ile, Leu, Ser, Thr, Asp, Glu, Gln and Asn,
preferably Gly, Pro, Glu, Gln and Asp and most preferably Pro or
Gln.
[0040] E424 and/or E425 are substituted with any of a residue
selected from Asp, Asn, Gln, His, Lys, Arg, Cys, Met, Phe, Tyr,
Trp, Pro, Gly, Ala, Val, Leu, Ile, Ser and Thr, preferably Arg,
Lys, Pro, Gly and His and most preferably Arg.
[0041] R428 and/or R422 are substituted with Cys, Met, Phe, Tyr,
Trp, Pro, Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Glu, Asn, Gln,
His, and Lys, preferably Cys, Met, Phe, Tyr, Trp, Pro, Gly, Ala,
Val, Leu, Ile, Ser, Thr, Asp, Glu, Asn and Gln, and most preferably
Tyr.
[0042] T421 is substituted with Cys, Met, Phe, Tyr, Trp, Pro, Gly,
Ala, Val, Len, Ile, Ser, Asp, Glu, Asn, Gln, His and Lys,
preferably Met, Phe, Tyr, Trp, Pro, Gly, Ala, Val, Leu, Ile, Asp,
Glu, Asn, Gln, His and Lys, and most preferably Phe, Trp, Pro, Gly,
Ala, Val, Len and Ile.
[0043] S420 is substituted with Met, Phe, Tyr, Trp, Pry, Gly, Ala,
Val, Leu and Ile, and preferably Pro or Gly.
[0044] X429 is substituted with any other naturally occurring amino
acid residue.
[0045] It is likely that optimal differential and FCEL binding
activity will be achieved by a combination of mutations.
Preferably, FCEH or FCEL binding, as the case may be, will be less
than 10% of native homologous IgE, and optionally will range from
undetectable to 3% of native homologous IgE, while binding to the
other receptor ranges from at least about 75% of native homologous
IgE to 90%, and preferably 95% to greater than 100%, e.g. 125%. The
mutations should be as conservative as possible, i.e., involve as
modest changes in hydrophobicity, charge or bulk as possible, yet
still result in a polypeptide exhibiting these differential binding
characteristics.
[0046] Any one or more of the .beta.-strand D domain residues also
may be deleted. Deletion of residues may possess the advantage of
not introducing potentially immunogenic sites into the IgE
analogue.
[0047] Examples of candidate .beta.-strand D domain substitutional
or deletional variants are set forth in the following Table 1. To
determine the sequence of each variant, identify the residue for
each variant number under each site. For example, the sequence of
compound 19 comprises C388 E389 E390, etc. TABLE-US-00001 TABLE 1
HuIgE Site AA.sup.1 423 K 424 E 425 E 426 K 427 Q 428 R C 19 20 37
55 M 18 21 38 56 F 8, 80 22 39 57, 88 Y 7 23 40 4, 75, 83-84, 89,
97 W 6 24 41 58, 85 P 1, 74, 78-79, 25, 97 42 59 89, 103 G 5, 76-77
26 43 60 A 12, 98-99 27, 98, 100 44, 98, 101 61, 98, 102 V 13, 97
28 45 62 L 14, 81 29 46 63 I 15, 82 30 47 64 S 16 31 48 65, 103 T
17 32 49 66, 104, 105 D 9 79 50 67, 86 E 9, 94 1, 3-19, 37-54,
1-72, 74, 76-78, 51 68, 87 55-72, 75, 88, 89, 80-88, 93-94, 99,
90-93, 99, 101, 100-105 102, 105 N 10 33 52, 79, 84 79 69 Q 11 34
3, 54, 75, 80, 1-72, 75, 77, 70 82-83, 85-89, 78, 80-95, 103-104
97-103, 105 H 83, 104 35, 78, 84 53 71 K 2-4, 20-72, 75, 36, 77,
79, 94 1-2, 5-36, 104 72, 79 85-88, 91-93, 55-72, 74, 76, 100-102,
105 77-90, 91, 93-95, 97, 99, 100, 102, 105 R 84 2, 74, 76, 80, 81
83, 89 1-3, 5-54, 85-87, 103-104 74, 76-78, 80-82, 90-92, 94, 99,
100-101 .DELTA..sup.2 90, 95, 96 91, 95, 96 91, 96 92, 96 96 93,
95, 96 .sup.1amino acid residues substituted into the variant
.sup.2signifies a deletion
[0048] Insertion of one or more extraneous residues adjacent to a
residue within the .beta.-strand D domain also falls within the
scope of this invention. Typically, only one residue will be
inserted, although from 2 to 4 or more residues can be inserted
adjacent to any one site within the domain. Smaller numbers of
inserted residues will be preferred in order to avoid the
introduction of immunogenic sites. This, however, is merely a
matter of choice. In general, insertions will be made at a single
site, although insertions can be made adjacent to any two or more
.beta.-strand D domain residues.
[0049] Insertions typically are made between the following
residues: 422 and 423, 423 and 424, 424 and 425, 425 and 426, 426
and 427, 427 and 428 and/or 428 and 429. The inserted residue or
residues generally will exhibit charge, bulk or hydrophobicity
character which is distinct from that of the flanking residues. For
example, candidate insertions can be selected from the following
Table 2. TABLE-US-00002 TABLE 2 Insertion .beta.-strand D domain
site.sup.1 Q 1, 2, 3, 4, 5, 7 or 8 D 1, 2, 3, 4, 5, 6 or 7 E 1, 2,
3, 4, 5, 6 or 7 F 1, 2, 3, 4, 5, 6 or 7 W 1, 2, 3, 4, 5, 6 or 7 P 1
or 2 K 2 or 3 R 2 or 3 EK 2 or 7 ER 2 or 7 DK 2 or 7 DR 2 or 7 G 1
or 2 A 8 Y 6 or 7 N 1, 2, 3, 4, 5, 7 or 8 H 1, 2, 3, 4, 5, 7 or 8 I
1, 2, 3, 4, 5, 7 or 8 .sup.1422R - site 1 - 423K - site 2 - 424E -
site 3 - 3425E - site 4 - 426K - site 5 - 427Q - site 6 - 428R -
site 7 - 429X y - site 8. Absence of a site indicates no insertion
at that site.
[0050] The FCEL-specific polypeptides need only contain so much of
the IgE Fc.epsilon. AB-B and loop EF domain sequences as are
required to substantially achieve FCEL binding. This is readily
determinable by preparing polypeptides comprising the AB-B and loop
EF domains and incrementally increasing numbers of flanking or
normally interposed residues, e.g., .beta.-strand A (N-terminal) or
loop BC, .beta.-strand C, loop CD, .beta.-strand D, loop DE,
.beta.-strand E, .beta.-strand F, loop EF, loop FG, .beta.-strand
G, and Fc.epsilon.4 (C-terminal). In general, the entire IgE
sequence from Fc.epsilon.3-Fc.epsilon.4 is used, although fragments
of Fc.epsilon.3 containing the AB-B domain may be satisfactory,
particularly if they contain the AB-B domain, loop EF and
intervening sequence, otherwise than as varied according to the
teachings herein to achieve specificity for FCEL.
[0051] The FCEL-specific polypeptides are provided as linear or
conformationally restrained polypeptides. Conformational restraint
is accomplished by cross-linking the polypeptide, preferably at the
N- and C-termini so as to produce a cyclic structure. In preferred
embodiments the cyclic forms have the following structure: ##STR1##
wherein (a3-a11) is a bond or the sequence--R373-F381; a12 and a18
are hydrophobic amino [0052] acid residues; a13 and a14 are basic
amino acid residues; and a15, a17 and a19 are hydrophilic amino
acid residues; R.sub.1 is selected from
[0053] (a) hydroxy,
[0054] (b) C.sub.1-C.sub.8 alkoxy,
[0055] (c) C.sub.3-C.sub.12 alkenoxy,
[0056] (d) C.sub.6-C.sub.12 arlyoxy,
[0057] (e) acylamino-C.sub.1-C.sub.8-alkoxy
[0058] (f) pivaloyloxyethoxy,
[0059] (g) C.sub.6-Cl.sub.12 aryl-C.sub.1-C.sub.8-alkoxy where the
aryl group is unsubstituted or substituted with one or more of the
groups nitro, halo, C.sub.1-C.sub.4-alkoxy, and amino;
[0060] (h) hydroxy substituted C.sub.2-C.sub.8 substituted alkoxy;
and
[0061] (i) dihydroxy substituted C.sub.3-C.sub.8 alkoxy;
R.sub.2,R.sub.3,R.sub.5,R.sub.7,R.sub.8 are the same or different
and are selected from
[0062] (a) hydrogen,
[0063] (b) C.sub.6-C.sub.12 aryl where the aryl group is
unsubstituted or substituted by one or more of the groups nitro,
hydroxy, halo, C.sub.1-C.sub.8 alkyl, halo-C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, amino, phenyl, acetamido, benzamido,
di-C.sub.1-C.sub.8 alkylamino, C.sub.6-C.sub.12 aroyl,
C.sub.1-C.sub.8 alkanoyl, and hydroxy substituted C.sub.1-C.sub.8
alkyl,
[0064] (c) C.sub.1-C.sub.12 alkyl or alkenyl; C.sub.3-C.sub.10
cycloalkyl or C.sub.3-C.sub.12 substituted with any of halo,
C.sub.1-C.sub.8 alkoxy, C.sub.6-C.sub.12 aryloxy, hydroxy, amino,
acetamido, C.sub.1-C.sub.8 alkylamino, carboxy or carboxamide;
[0065] R.sub.2 and R.sub.3, R.sub.5 and R.sub.6, or R.sub.7 and
R.sub.8 may optionally and independently be joined together to form
a carbocyclic or heterocyclic ring of from four to seven atoms
where the heteroatoms are selected from O, S, or NR.sub.10 where
R.sub.10 is selected from:
[0066] hydrogen, C.sub.1-C.sub.8-alkyl, C.sub.2-C.sub.8-alkenyl,
C.sub.6-C.sub.12-aryl, C.sub.3-C.sub.10 cycloalkyl,
C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkanoyl, and C.sub.6-C.sub.12 aroyl, [0067]
R.sub.4 is selected from:
[0068] hydrogen, C.sub.1-C.sub.8-alkyl, C.sub.2-C.sub.8-alkenyl,
C.sub.6-C.sub.12-aryl, C.sub.3-C.sub.10 cycloalkyl,
C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkanoyl, and C.sub.6-C.sub.12 aroyl; [0069]
R.sub.2 or R.sub.3 may be optionally joined with R.sub.4 to form a
piperidine, pyrrolidine or thiazolidine ring; [0070] X is selected
from:
[0071] an O or S atom,
[0072] NR.sub.9 wherein R.sub.9 is hydrogen, C.sub.1-C.sub.8-alkyl,
C.sub.3-C.sub.8-alkenyl, C.sub.3-C.sub.10 cycloalkyl,
C.sub.6-C.sub.12-aryl, C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkanoyl, or C.sub.6-C.sub.12 aroyl;
C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.8 alkanoyl, and
(CH.sub.2).sub.k where k is an integer from 0 to 5; and
pharmaceutically acceptable salts thereof.
[0073] As used herein and unless specified otherwise: alkyl and
alkenyl denote straight or branched, saturated or unsaturated
hydrocarbon chains, respectively; C.sub.6-C.sub.12 aryl groups
denote unsubstituted aromatic rings or fused aromatic rings such
as, for example, phenyl or naphthyl; halo denotes F, Cl, Br, or I
atoms; alkoxy denotes an alkyl group bonded through O to the
indicated site. Examples of C.sub.1-C.sub.8 alkyl or
C.sub.2-C.sub.8 alkenyl groups include methyl, ethyl, propyl,
isopropyl, butyl, t-butyl, pentyl, isopentyl, hexyl, vinyl, allyl,
butenyl and the like; examples of C.sub.3-C.sub.10-cycloalkyl
groups include cyclopropyl, cyclopentyl, cyclohexyl, and the like;
heterocyclic rings include but are not limited to pyridyl, thienyl,
furyl, indolyl, benzthienyl, imidazolyl, thiazolyl, quinolinyl and
isoquinolinyl. Hydrophobic amino acid residues include naturally
occurring or synthetic residues having hydrophobic side chains,
e.g. Phe, Leu, Ile, Val, Norleu, and the like. Hydrophilic amino
acid residues include naturally occurring or synthetic residues
having charged or uncharged hydrophilic side chains, e.g.
ornithine, Ser, Thr, Tyr, His, Asp, Glu, Lys and Arg. Preferably
a15, a17 and a19 are unchanged and bear normal, secondary or
tertiary mono or di-hydroxy substituted alkyl side chains. Basic
residues have guanidino or amino-substituted side chains for the
most part.
[0074] The AB-B domain and/or loop EF--containing, FCEL-specific
polypeptides of this invention optionally are associated with other
substances or are fused to additional polypeptide sequences. The
polypeptides generally contain only IgE-homologous sequences,
although they also or alternatively are labelled for diagnostic use
(employing enzymes, radioisotopes, biotin or avidin, stable free
radicals, and chemiluminescent or fluorescent moeities in
conventional fashion). Also the polypeptides are fused to non-IgE
polypeptides such as cytotoxic or immunosuppressive polypeptides,
to other IgE polypeptides (e.g. Fv regions), or to polypeptides
capable of binding to a predetermined ligand or antigen.
[0075] Cytotoxic polypeptides include IgG Fc effector sequences and
polypeptide toxins such as diphtheria toxin or ricin A chain (U.S.
Pat. Nos. 4,714,749 and 4,861,579). A preferred fusion is one in
which the FCEL-specific sequence (such as that of the
Fc.epsilon.3-Fc.epsilon.4 sequence of mutant 6) is fused at its
N-terminus (i.e., at approximately D360) to the C-terminus of an
immunoglobulin, or an immunoglobulin fragment terminating at the
C-terminus of IgG Fc.gamma.2 or IgG Fc.gamma.3. Alternatively the
FCEL specific polypeptide is fused to an effector IgG sequence in
place of one or both of the IgG Fv domains in analogous fashion to
known immunoadhesins.
[0076] The polypeptides herein optionally are fused to polypeptides
which are capable of binding a predetermined antigen or ligand.
Generally, these additional polypeptides will be IgE or other
immunoglobulin Fv domains, although they optionally are
heterologous polypeptides such as receptor extracellular domains
(produced in the known fashion of immunoadhesions, e.g. as has been
accomplished with CD4). Immunoglobulin sequences fused to the
FCEL-specific polypeptides herein include Fc or variable sequences
of the heavy chains of IgG1, IgG2, IgG3, IgG4, IgE, IgM, IgD or
IgA. Any FCEL-specific heavy chain fusion optionally is disulfide
bonded in the ordinary fashion to heavy chains having the same
specificity (thereby forming homopolymers) or to different heavy
chains (thereby forming heteropolymers), including different heavy
chains having specificity for a different antigen. Such
heteropolymeric heavy chains include heavy chains which are not
FCEL-specific, e.g., these comprise native IgE sequences which bind
to FCEL and FCEH in the ordinary fashion, or the heavy chains
optionally include at least one heavy chain that is FCEL specific
and at least one that is FCEH specific. Heteropolymeric heavy
chains also may include the heavy chains of non-IgE
immunoglobulins, e.g., IgG, IgD, IgM and the like. In addition, the
heavy chain hetero- or homopolymers optionally are disulfide bonded
to light chains in the fashion of native immunoglobulins so as to
cooperatively bind to predetermined antigen in the usual way.
Unless the heteropolymeric heavy chains comprise IgM heavy chains
they generally will be heterodimeric.
[0077] In some embodiments, immunoglobulins comprising an
FCEL-specific polypeptide will also comprise an immunoglobulin
variable region, preferably (if at all) an IgE Fv domain. The
antigenic specificity of the variable region may vary widely,
including those which bind haptens, or which bind polypeptides or
proteins from human, animal, plant, fungal, bacterial or insect
sources. The specificity may be unknown or the variable region may
have the ability to bind to a predetermined antigen. If the
immunoglobulin is to have a functional variable domain (as opposed
to a deleted Fv in the case of Fce3 or Fce4 fragments) it is
preferred that it have a known antigenic specificity. Antigenic
specificity may include the ability to bind antigens associated
with a cytotoxic or immune response particularly lymphoid cell
antigens such as CD3 or CD8, integrins, B-cell surface antigens,
helper or suppressor cell surface antigens, or epitopes located in
the variable region of effector subtypes of IgG. FCEL-specific Fc
domains also are usefully employed in combination with F.sub.v
domains capable of binding a particular allergen to which a patient
is allergic. These generally are human IgEs directed against
allergens and which contain an FCEL-specific Fc domain.
Alternatively, the immunoglobulin specificity is directed against
the Fc region of effector subtypes of IgG, in this case however it
being preferable that the FCEL-specific polypeptide not suppress
complement binding or ADCC functions of the IgG.
[0078] The polypeptides of this invention that contain antigen or
ligand binding capability contain one or more sites capable of
binding to the antigen or ligand. For example, the polypeptides
herein comprise one or more IgE or other immunoglobulin Fv domain
to produce monovalent or polyvalent immunoglobulins. For the most
part such polypeptides will be monovalent for antigen or ligand, as
in the case when the immunoglobulin comprises a heavy-light chain
pair that has a deleted or inactivated Fv or CDR so as to not be
able to bind to antigen. Alternatively, they will be bivalent in
the predominant instance, and will be monospecific or
bispecific.
[0079] In another embodiment, FCEL-specific polypeptides are
covalently bound to a cytotoxic agent. For example, the polypeptide
ricin D toxin isolated from the Ricinus communis plant is bound to
the carboxy terminus of the Fc domain, either by chemical means or,
most preferably, by production of a fusion protein using standard
recombinant DNA methods. This provides a means to selectively
deliver the toxin only to cells expressing FCEL on their
surfaces.
[0080] The FCEL-specific polypeptides need only contain so much of
the IgE Fc.epsilon. sequence as is required to substantially
maintain FCEL binding. This is readily determinable by synthesizing
or expressing the product and determining its activity. In general,
the entire IgE sequence extending from Fc.epsilon.2-Fc.epsilon.4
can be used, although fragments containing only FcE3 and FcE4 are
generally satisfactory.
[0081] In general the immunoglobulin sequences and the
FCEL-specific sequence will be derived from the same species which
is to be treated with the IgE analogue. Preferably, the
immunoglobulin sequences are human.
[0082] The FCEL-specific polypeptides of this invention (when
employed as such without fusion to non-IgE sequences) exclude the
linear polypeptide sequences disclosed by Nio et al. (supra), as
well as other prior art polypeptides which include the native IgE
AB-B domain or loop EF (Burt et al., supra).
FCEH-Specific Polypeptides
[0083] These polypeptides are amino acid sequence variants of IgE
or its fragments in which a residue within the AB-B or loop EF
domains have been deleted, substituted or another residue inserted
so that the AB-B or loop EF domains are no longer capable of
binding to FCEL, and which contain sufficient beta strand D
sequence and (optionally) loop EF sequence to bind to the high
affinity receptor. As disclosed above, the AB-B and loop EF domains
have been implicated in binding to FCEL since mutations in these
domains have a serious impact on the binding of the IgE variants to
the low affinity receptor. In particular, mutations in loop EF or
the C-terminal half of the AB loop and in the N-terminal half of
beta strand B produce a divergence in IgE FCEL/FCEH specificity
wherein the variant continues to bind to the high affinity receptor
but largely fails to bind to the low affinity receptor. In
addition, we have found that the IgE loop EF and the heavy chain
beta strand D domains participate in binding to the high affinity
receptor. Therefore, FCEH-specific differential binding
polypeptides will comprise at least the FCEH-binding sequence of
beta strand D and preferably also will contain a variant AB-B or
loop EF domain sequence that binds substantially only to FCEH.
[0084] In preferred embodiments amino acid sequence variation is
introduced into the low affinity receptor binding functionality of
the AB-B or loop EF domains. Preferably, one or more of residues
I382, R383, K384, S385, T387, 1388, T389, C390, R446, D447, W448,
I449, E150, G151, E152 or T153 are varied, although modifications
optionally are introduced into loop AB N-terminal to the designated
loop AB residues. Only one of R383, K384, S385, T387, T-389, or
R446-T453 need be mutated, although it is preferable to vary 1, 2
or 3 residues from each domain.
[0085] When substituted at all, I382 and/or I388 generally are
independently substituted with Asn, Gln, Leu, Val, His, Lys, Arg,
Met, Phe, Tyr, Trp, Pro, Gly, Ala, Ser, Thr, Asp or Glu, preferably
Trp, Pro, Gly, Ser, Thr, Asp or Glu. Ordinarily these two residues
are not modified.
[0086] R383 typically is substituted with Cys, Met, Phe, Tyr, Trp,
Pro, Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Glu, Asn, Gln, His, or
Lys, preferably Met, Phe, Tyr, Trp, Pro, Gly, Ala, Val, Leu, Ile,
Ser, Thr, Asp, Glu, Asn or Gln and most preferably Ala, Glu, Asp or
Ser.
[0087] K384 typically is substituted with Arg, His, Cys, Met, Phe,
Tyr, Trp, Pro, Gly, Ala, Val, Ile, Leu, Ser, Thr, Asp, Glu, Gln and
Asn, preferably Ala, Gly, Pro, Glu, Gln or Asp and most preferably
Ala, Glu or Asp.
[0088] S385 is substituted with Asp, Asn, Gln, His, Lys, Arg, Cys,
Met, Phe, Tyr, Trp, Pro, Gly, Ala, Val, Leu, Ile, Glu and Thr,
preferably Ala, Tyr, Val, Ile, Leu, Phe, Arg, Lys and His and most
preferably Ala, Val, Ile, Leu, Phe and Tyr. When substituted, P386
usually is substituted by Gly, Ala, Cys, Val, Leu, Ile, Ser, Thr,
Asp, Glu, Asn, Gln, His, Lys, Arg, Phe, Tyr, or Trp, and preferably
Gly, Ala, Ser, Thr, Asp, Glu, Asn, Gln, His, Lys, Arg or Trp.
Ordinarily, P386 is not modified.
[0089] T387 and/or T389 generally are independently substituted by
Gly, Ala, Val, Leu, Ile, Ser, Asp, Pro, Glu, Asn, Gln, His, Lys,
Arg, Cys, Phe, Tyr and Trp, preferably Gly, Ala, Val, Leu, Ile,
Asp, Glu, Asn, Gln, His, Lys, Arg, Phe, Tyr and Trp, and most
preferably Ala.
[0090] C390 ordinarily is not substituted except when employed as a
component of a cyclizing group as shown in Formula I.
[0091] The differential FCEH-binding polypeptides of this invention
will comprise the sequence of functional FCEH-binding beta strand D
and loop EF domains, as defined above. In general, it is expected
that the functional domains need not contain all of the beta strand
D or loop EF domain residues. However, any modifications of the
beta strand D domain residues will need to be conservative, if made
at all, in order to preserve FCEH binding. Since loop EF is
involved in both FCEL and FCEH binding, it likely will be necessary
to screen these variants in order to determine their activity as
shown in Example 5. However, a number of loop EF mutants already
have been identified that substantially abolish FCEL binding
without apparently interfering with FCEH binding, e.g. mutants 50
and 52. Thus, loop EF variants may belong in either the FCEL or
FCEH specific category, or may equally affect binding to each
receptor.
[0092] A particularly preferred embodiment of a FCEH-specific
polypeptide is one which contains a beta strand D domain together
with additional C-terminal sequence. The sequence of this
embodiment extends from about T421 to about T440. Generally, the
N-terminus of this embodiment is S420 or T421, while the C-terminus
is T440, L441 or P442. In addition, one or more residues extraneous
to this sequence are fused to its N- or C-termini. These extraneous
residues are particularly useful in forming covalent or noncovalent
bonds between the N- and C-termini of this polypeptide. The N-
and/or C-termini preferably are covalently bonded through a side
chain of a residue or through the polypeptide backbone. For
example, cysteine residues are fused to the N- and C-termini and,
upon oxidation, a polypeptide having a terminal disulfide bond is
formed which joins the terminal ends of the polypeptide, thereby
conformationally restraining the polypeptide. Alternatively, the
alpha amino group of the polypeptide (or that of an extraneous
N-terminally located residue) is covalently bonded to the sulfur
atom of an extraneous C-terminally located cysteine residue to form
thioether cyclic compounds analogous to those depicted in Formula
I. Other cyclic compounds are prepared in the same fashion as
described elsewhere herein. Also within the scope of this
embodiment are amino acid sequence variants of native IgE sequences
corresponding to the sequence of this embodiment. Beta strand D
variants are selected to enhance binding to FCEH, while the
sequence outside of the beta strand D domain need only retain
sufficient conformational structure to properly juxtapose the N-
and C-termini in substantially the same position as is the case
with the native IgE sequence.
[0093] The FCEH-specific polypeptides herein optionally comprise
non-IgE polypeptides exactly as described above for the
FCEL-specific polypeptides, except that it is not prefered that the
FCEH-specific polypeptides comprise cytotoxic functionalities. In
addition, conformationally restrained (typically cyclic)
polypeptides comprising the FCEH-binding sequence of the beta
strand D domain are included within the scope hereof. Such
polypeptides are identical to those shown in Formula I above except
that the FCEH-binding beta strand D domain replaces the (a3)-(a19)
moiety. Exemplary replacement moieties include S420-R428,
T421-N430, S420-G433 and R422-R428 (note that sequences such as
T421-N430 from U266 that omit a residue from the Kabat sequence can
contain a residue at that site or may have a deletion at the same
location, in the latter case here the Asn residue would occupy site
429).
[0094] Any one or more of the AB-B domain residues also may be
deleted in order to substantially reduce or eliminate FCEL binding.
Residue deletion may be preferred for the same reason noted above
with respect to the beta strand D domain.
[0095] Examples of candidate AB-B domain substitutional or
deletional variants are set forth in the following Table 3. To
determine the sequence of each variant, identify the residue for
each variant number under each site. For example, the sequence of
compound 98 comprises A383 A384 A385, and represents the class of
mutations to which mutant 7 belongs. TABLE-US-00003 TABLE 3 HuIgE
Site AA.sup.1 350 I 351 R 352 K 353 S C 55 19 37 M 56 18 38 F 57,
88 8, 80 39 Y 4, 75, 83-84, 7, 73 40 89, 97 W 58, 85 6 41 P 59 1,
74, 78-79 42 G 60, 73 5, 76-77 43 A 61, 98, 102 12, 98-99 44, 98,
101 V 72 62 13, 97 45 L 73 63 14, 81 46 I 75 64 15, 82 47 S 65, 103
16 1-2, 5-36, 55-72, 74, 76-91, 93-95, 97, 99-100, 102, 105 T 66,
104, 105 17 49 D 67, 86 9 50 E 68, 87 89, 94 51 N 79 69 10 52, 79,
84 Q 1-71, 77, 78, 70 11, 103 3, 54, 75, 80, 80-95, 82-83, 85-89,
97-103, 105 103-104 H 71 83, 104 4, 53 K 104 72, 79 2-4, 20-72, 48
75, 85-88, 91-93, 100-102, 105 R 1-3, 5-54, 74, 84 73 76-78, 80-82,
90-92, 94, 99-101 .DELTA..sup.2 96 93, 95, 96 90, 95, 96 92, 96
.sup.1Amino acid residue substituted into the analogue
.sup.2Signifies a deletion.
[0096] Insertion of one or more extraneous residues adjacent to a
residue within the AB-B domain also falls within the scope of this
invention, although substitutions or deletions are preferred.
Typically, only one residue will be inserted, although from 2 to 4
or more residues can be inserted adjacent to any one site within
the AB-B domain. Smaller numbers of inserted residues will be
preferred in order to avoid the introduction of immunogenic sites.
This, however, is merely a matter of choice. In general, insertions
will be made at a single site, although insertions can be made
adjacent to any two or more AB-B domain residues.
[0097] Insertions typically are made between the following
residues: S385 and P386, P386 and T387, T387 and 1388, and 1388 and
T389. The inserted residue or residues generally will exhibit
charge, bulk or hydrophobicity character which is distinct from
that of the flanking residues. For example, candidate insertions
can be selected from the following Table 4. TABLE-US-00004 TABLE 4
Insertion AB-B domain site.sup.1 Q 1, 2, 3, 4 or 5 D 1, 2, 3, 4 or
5 E 1, 2, 3, 4 or 5 F 1, 2, 3, 4 or 5 W 1, 2, 3, 4 or 5 P 1 or 2 K
2 or 3 R 2 or 3 T 3 or 4 EK 2 or 4 ER 2 or 4 DK 2 or 4 DR 2 or 4 G
1 or 2 A 5 Y 3 or 4 N 1, 2, 3, 4 or 5 H 1, 2, 3, 4 or 5 I 1, 2, 3,
4 or 5 .sup.11382 site 1 - R383 - site 2 - K384 - site 3 - S385 -
site 4 - P386 - site 5 - T387. Absence of a site indicates no
insertion site.
One or more of the AB-B domain residues are substituted or deleted,
or additional residues inserted adjacent to such residues. In
general, no more than 4 residues or sites are varied and optimally
only one will be varied. Variations herein include combinations of
insertions, deletions or substitutions. Excluded from the scope of
FCEH specific polypeptides are the linear IgE polypeptide fragments
disclosed by Nio et al. (or the naturally occurring sequence
variants of such fragments, e.g. alleles and the like), together
with any other such fragments disclosed by the prior art. Loop EF
Variants
[0098] Loop EF is defined above. Loop EF variants not described in
the examples may require screening against both FCEH and FCEL
assays since loop EF is involved in both FCEL and FCEH binding.
However, this screening will be routine and well within the
ordinary skill when following the directions and principles herein.
In general, FCEH or FCEL-binding differential polypeptides will
comprise substitutions or deletions of (or insertions adjacent to)
one or more of residues 446, 447, 448, 449, 450, 452 and 453. It
should be noted that sites such as 446 and 447, while shown in the
case of Ala substitution to lead to loss of FCEL binding (Example
5), also serve as sites for selecting variants which bind FCEL to a
greater degree than native IgE. For the most part, however, sites
446 and 447 are not prefered for introducing variants in which the
objective is FCEL binding. For this, one should focus on the region
extending from residue 448 to 453, and preferably residues 450, 452
and 453. In general, loop EF variants are employed with variants
introduced into loop AB--beta strand B or beta strand D or
both.
[0099] R446 typically is substituted by Gly, Ala, Val, Leu, Ile,
Ser, His, Lys, Met, Thr, Asp, Pro, Glu, Asn, Gln, Cys, Phe, Tyr or
Trp, preferably Ala for FCEH specificity.
[0100] D447 generally is substituted by Gly, Ala, Val, Leu, Ile,
Met, Cys, Ser, Thr, Pro, Glu, Asn, Gln, His, Lys, Arg, Phe, Tyr or
Trp, preferably Ala for FCEH specificity.
[0101] W448 also generally is not substituted, but if so then Gly,
Ala, Val, Leu, Ile, Met, Cys, Ser, Thr, Pro, Glu, Asn, Asp, Gln,
His, Lys, Arg, Phe or Tyr are employed.
[0102] I449 likewise generally is not substituted, but if so then
Gly, Ala, Val, Leu, Met, Cys, Ser, Thr, Pro, Glu, Asn, Asp, Gln,
His, Lys, Arg, Phe, Tyr or Trp are employed.
[0103] E450 typically is substituted with Gly, Ala, Val, Ile, Leu,
Met, Cys, Ser, Thr, Pro, Gln, Asn, Asp, His, Lys, Arg, Phe, Tyr or
Trp, preferably Ala for FCEH specificity.
[0104] G151 generally is not substituted, but if so then Ala, Val,
Leu, Met, Cys, Ser, Thr, Pro, Glu, Asn, Ile, Asp, Gln, His, Lys,
Arg, Phe, Tyr or Trp are employed.
[0105] E452 also generally is substituted with Ala, Val, Leu, Met,
Cys, Ser, Thr, Pro, Gly, Asn, Ile, Asp, Gln, His, Lys, Arg, Phe,
Tyr or Trp.
[0106] T453 typically is substituted with Ala, Val, Leu, Met, Cys,
Ser, Pro, Gly, Asn, Glu, Ile, Asp, Gln, His, Lys, Arg, Phe, Tyr, or
Trp.
[0107] Exemplary IgE variants are set forth in Table 5. It will be
understood that this table may contain variants that bind to both
receptors, differentially to one or the other, or to neither
receptor. TABLE-US-00005 TABLE 5 HuIgE Site AA.sup.1 446 R 447 D
450 E 452 E 453 T C 47 46 45 44 43 M 34 F 33 25 Y 41 30 W 26 36, 38
36, 38 P 49 G A 13, 17 16 12, 15 12, 14 12 V 31 L 40 I 48 S 29 T 43
1-3, 5-7, 9, 10, 13-17, 24-26, 28, 33, 34, 37, 39, 44-48, 50, 51 D
39 1, 2, 4-15, 5, 8, 11, 18, 1, 29, 30, 42 17-23, 23, 27, 32, 34,
50 31-45, 47, 33, 35, 40, 49-52 42, 52 E 9, 20 24, 29, 30 1-5, 7,
9, 3, 4, 6, 7, 8, 11, 10, 13, 14, 9, 10, 13, 18-23, 16, 17, 15-17,
27, 35 24-28, 24-26, 30, 31, 34, 28, 31-33, 37, 39, 43, 37, 39, 43,
44, 46, 47, 45-49, 52 48, 51 N 19, 22, 40 3 50 51 Q 10, 11, 23, 2
52 35, 36, 42 H 21, 30 27 36 K 18, 28, 29, 28 8, 11, 18-23, 32 52
27, 35, 40, 42 R 1-8, 12, 7 6 5 4 14-16, 24-27, 31, 32, 38, 44-46,
48-51 .DELTA..sup.2 37 38 .sup.1amino acid residue substituted into
the variant .sup.2signifies a deletion
Variant Anti-huIgE Antibodies
[0108] Variant anti-huIgE antibodies were produced by first
obtaining a group of murine monoclonal antibodies which were
capable of binding to FCEL but not to FCEH. 8 such murine
monoclonal antibodies, designated MAE10, MAE11, MAE12, MAE13,
MAE14, MAE15, MAE16 and MAE17, were obtained by conventional
methods involving immunizing mice with human IgE or a polypeptide
consisting of residues 315-547 of huIgE and screening for anti-IgE
activity.
[0109] MAE11/15 and MAE13 recognize different epitopes. It appears
that the MAE13 epitope is located three-dimensionally adjacent to a
key component of the FCEH binding site of IgE (but does not
directly occupy that site) since a slight amount of histamine
release will occur at high concentrations of MAE 13 suggesting that
some limited antibody mediated crosslinking of FCEH occurs with MAE
13. MAE17 was most effective in suppressing B-cell IgE synthesis
despite the fact that MAE11 and MAE13 exhibited greater IgE
affinity. This may be attributed to its ability to mediate
complement fixation (it possessed an IgG2a isotope, thus containing
an Fc capable of eliciting effector function).
[0110] MAE11 and MAE15 are believed to recognize the same IgE
epitope. Each antibody shared certain unusual features in its amino
acid sequence. For example, CDR1 of the light chain of each
contained 3 aspartic acid residues. CDR3 of the heavy chains of
MAE11 and MAE15 contained 3 histidine residues (and contained two
arginine residues, respectively).
[0111] Antibodies such as the foregoing having desired IgE binding
characteristics may be further modified. Such modifications fall
into two general classes. In the first class the antibodies are
modified so that they are monovalent for IgE. This means that only
one "arm" of the antibody, i.e., one light-heavy chain fork of the
antibody, shall be capable of binding IgE. The remaining Fv "arm"
of the antibody (or arms in the case of IgM) is specific for a
second (non-IgE) antigen, is not capable of binding any antigen, or
is deleted entirely. Thus, the term IgE monovalent covers
polyvalent antibodies that are monovalent for IgE. The best results
may be obtained with the second alternative, since this would
preserve the structure of the antibody most faithfully and would
likely confer the longest circulating half-life on the antibody.
IgE-monovalent antibodies specific for FCEL bound IgE optimally
will comprise sufficient Fc domains of the heavy chains to be
capable of complement binding and Ig effector functions.
[0112] The second antigen recognized by one embodiment of IgE
monovalent antibody is one which, when indirectly cross-linked to
FCEL by the antibody herein, will not produce any toxic or
deleterious response, i.e. the second antigen is not FCEH, and
generally is one which is not found in the animal to be treated (in
order to avoid undesired absorption of the antibody onto tissues or
proteins within the body). Thus, the second antigen ordinarily will
not (but may be) FCEL. However, in some circumstances the second
antigen will be a protein present in the patient to be treated,
e.g. where such proteins are to serve as carriers or depot releases
for the therapeutic antibodies herein.
[0113] Such IgE-monovalent antibodies are made by methods known per
se. For example, DNA encoding the anti-IgE Fv heavy and light
chains is ligated to DNA encoding the Fc of a human recipient
antibody. In addition, DNA is provided that encodes heavy and light
chains for an antibody capable of binding second antigen or an
unidentified antigen, or that encodes heavy and light chain having
sufficient residues deleted from the CDRs that non-IgE antigen
binding no longer can occur. A conventional recombinant host is
transformed with all four DNAs and the products recovered. Assuming
random chain assortment, a subpopulation of antibody products will
contain one arm with anti-IgE heavy and light chain and at least
another arm having specificity for second antigen or no antigen.
The desired subpopulation then is purified by conventional methods,
e.g., immunoaffinity absorption or by molecular sieving. These
antibodies also can be made by reduction of the starting antibodies
followed by oxidative chain recombination, as has heretofore been
employed in the preparation of monovalent antibodies (see for
example Glennie et al., Nature 295:712 [1982]).
[0114] In addition to IgE-monovalency, in other embodiments the
antibodies are modified so that they contain a maximum proportion
of human sequence (commensurate with retention of required or
desired activity), i.e., they are converted to chimeras or are
humanized. In both instances the functional effect is to place the
anti-IgE binding capability of the murine or other donor antibody
into a human background to make it as non-immunogenic as possible.
General methods are known for making chimeras and for humanizing
antibodies (as noted above). A minimal amount of non-human antibody
sequence is substituted into the recipient human antibody.
Typically, the non-human residues are substituted into the V.sub.H,
V.sub.L, V.sub.H-V.sub.L interface or framework of the recipient
human antibody. Generally, the Kabat CDR's of the humanized
antibodies are about 80% and more typically about 90% homologous
with the non-human donor CDR's. The V.sub.H-V.sub.L interface and
framework residues of the humanized antibody, on the other hand,
are about 80%, ordinarily 90% and preferably about 95% homologous
with the recipient human antibody. Homology is determined by
maximal alignment of identical residues. The resulting antibody is
(a) less immunogenic in humans than a murine antibody and (b)
capable of binding to FCEL-bound huIgE but substantially incapable
of binding to FCEH-bound huIgE. Such antibodies typically comprise
a human antibody which is substituted by an amino acid residue from
a complementarity determining region (CDR), VL-VH interface or a
framework region of a non-human anti-IgE antibody which is capable
of binding. One or more, and preferably all, of the nonhuman CDR's
L1, L2, L3, H1, H2 or H3 are substituted into the human antibody
recipient.
[0115] The characteristics possessed by the MAE11 antibody were
preferred for therapeutic use. Since MAE11 bound to soluble IgE,
bound to MIge bearing B cells, blocked IgE binding to the low and
high affinity IgE receptor, inhibited in vitro IgE production and
failed to bond to IgE coated basophils, it was chosen as the donor
antibody for humanization. The recipient antibody was Kabat human
kappa (light) subgroup I and human subgroup III heavy chain,
although it will be understood that any other human antibody can be
suitably employed. Surprisingly, optimal results were not obtained
by simply substituting the murine CDRs in place of the CDRs in a
recipient human antibody (FIG. 3; Table 8 infra). Instead, it was
necessary to restore donor framework hydrophobic residues such as
V.sub.H 78, 48, 49, 63, 67, 69; 82 or 82c, or V.sub.L 13, 19, 58,
78 or 104, in order to achieve a degree of inhibition of IgE
binding similar to that of the donor antibody. While these residues
function to establish the conformation of CDRs, they generally are
not exposed to the exterior of the antibody so use of the murine
residues should not exert a significant impact on immunogenicity.
Other non-CDR residues exerting an effect on binding included
V.sub.H60, 61, 37, 24, and V.sub.H50, 52, 58 and 95 (non-CDR by
Chothia), and V.sub.L4, V.sub.L33 (non-CDR by Chothia) and
V.sub.L53 (non-CDR by Chothia). The human framework hydrophobic
residues generally are substituted with other hydrophobic residues
(especially those from the donor antibody) such as valine,
isoleucine, leucine, phenylalanine or methionine. The remaining
non-CDR residues are substituted with any other amino acid residue,
but again preferably the murine residue found at the analogous
site.
[0116] In general, the character of the anti-IgE antibody is
improved by substituting, deleting or inserting a residue at or
adjacent to V.sub.L sites 30, 30b, 30d, 33, 55, 57, 58, 78, 93, 94,
or 104 and/or V.sub.H residues 24, 37, 48, 49, 54, 57, 60, 61, 63,
65, 67, 69, 78, 82, 82c, 97, 100a or 100c.
[0117] Position V.sub.H-78 is most preferably substituted with
phenylalanine. However, it also is substituted with leucine,
valine, isoleucine, methionine, alanine or any other residue which
results in an improvement in the characteristics of the antibody
(see infra).
[0118] Position V.sub.H-60 is most preferably substituted with
asparagine, although substitution with glutamine, histidine,
lysine, arginine or any other residue which improves the
characteristics of the antibody shall fall within the scope of this
invention.
[0119] Position V.sub.H-61 is most preferably substituted with
proline, although glycine, alanine, valine, leucine, isoleucine or
any other residue which results in an improvement in the
characteristics of the antibody also is suitable.
[0120] CDR residues were imported from the donor MaE11. These
included four inserts in V.sub.L1, 30a-30d, as well as 91-94
(V.sub.L3), V.sub.H1 27-29, 29a, 31, 33 and 34, V.sub.H253-55, and
V.sub.H397-101. V.sub.L 30, 30b or 30d, as well as V.sub.H97, 100a
or 100c, are important in conferring on the CDR ability to bind
IgE.
[0121] V.sub.H positions 97, 100a and 100c in humae11 (humanized
Mae11) are all histidine, and 2 are arginine in MaE15. These
residues are important in IgE binding. One, two or three of these
are modified by substitution with basic residues, particularly
lysine or arginine, but also with alanine, glycine, valine,
isoleucine, serine, threonine, aspartic acid, glutamic acid,
asparagine, glutamine, methionine, phenylalanine, tyrosine,
tryptophan or proline.
[0122] V.sub.L positions 30, 30b and 30d of humae11 also are
important for IgE binding. In humae11 each of these positions are
occupied by the acidic residue, aspartic acid. They are substituted
in other embodiments by glutamic acid, but also may be substituted
with alanine, glycine, valine, isoleucine, serine, threonine,
asparagine, glutamine, methionine, phenylalanine, tyrosine,
tryptophan or proline. It is within the scope of this invention to
reverse the charges on positions V.sub.L 30, 30b and 30d with those
on V.sub.H 97, 100a and 100c, e.g. by employing aspartic acid
residues in the three V.sub.H sites (2 in the case of humanized
MaE15) and histidine in the three V.sub.L sites.
[0123] Residues also may be inserted adjacent to V.sub.H positions
97, 100a, 100c, 61 or 61, or V.sub.L residues at positions 30, 30b,
30d or 78. Inserted residues generally will be of like kind, e.g.
an acid residue would be inserted adjacent to V.sub.L-30, 30b or
30d, while a basic residue is inserted adjacent to V.sub.H-97, 100
or 100c. The residues at these sites also may be deleted.
[0124] Humanized IgE-monovalent antibodies also are included within
the scope of this invention. In this instance humanization extends
to the anti-IgE arm as well, if necessary, to the remaining arm(s).
Non-IgE binding arms of course can originate from human antibodies
and in such case will not require humanization.
[0125] The foregoing variations are made by introducing mutations
into the DNA encoding the precursor form of the antibody and
expressing the DNA in recombinant cell culture or the like. This is
accomplished by conventional methods of site directed mutagenesis.
The variants then are screened for the desired character in assays
conventional per se. In the case of anti-huIgE, desired character
includes increasing the antibody affinity for huIgE, increasing its
capacity and specificity for FCEL bound IgE, increasing the
concentration of antibody required to stimulate histamine release
from mast cells or basophils, reducing immunogenicity in humans,
and other improvements apparent to the ordinary artisan. Optimizing
these characteristics frequently will require balancing one
improvement against another and therefore is a matter of judgment
and is dependent upon the performance parameters dictated by the
use intended for the antibody.
[0126] It is preferable to use a human IgG1 (or other complement
fixing antibody) as the recipient immunoglobulin for humanization,
although hu IgG2, IgG3, IgG4, IgE, IgM, IgD or IgA also can be used
as recipient. Preferably the recipient is a complement fixing IgG
antibody or an IgG antibody capable of participating in ADCC.
Therapeutic, Diagnostic and Preparatory Uses
[0127] The anti-IgE antibodies herein are useful in identifying IgE
amino acid sequence variants in which the FCEL or FCEH-binding
domains have been modified. Candidate FCEL or FCEH-specific
polypeptides are incubated with these antibodies, and analogues to
which these antibodies fail to bind are selected for further
evaluation, e.g., determination, respectively of their FCEH and
FCEL receptor binding characteristics. Any antibody, whether of
murine, human, or another animal species in origin, or a variant
thereof such as the humanized immunoglobulins described above,
which has the epitopic specificity of any of antibodies MAE10-MAE17
(especially MAE11/15, MAE13 or MAE17) will be equally acceptable.
Such antibodies are easily identified by immunizing a suitable
animal or using an in vitro Fv selection system, e.g. phagemid,
with IgE of the appropriate animal origin and screening the animals
or products for antibodies having the ability to compete for IgE
with MAE11/15, 13, 17 or other antibodies which map to
substantially the same epitopic site(s) as those described herein.
As noted, the antibodies desirably are monovalent for FCEL-bound
IgE when employed therapeutically. They may be bivalent and/or
bispecific when used to purify IgE from plasma, serum or
recombinant cell culture.
[0128] The FCEH and FCEL-specific, differential binding
polypeptides are useful for diagnostics and therapeutics. In in
vitro diagnostic assays they are employed as specific binding
reagents in assays for FC.epsilon.RI or FC.epsilon.RII,
respectively. The polypeptides of this invention are labelled with
a detectable substance such as an enzyme, fluorescent or
chemiluminescent group, radioisotope or a specific binding moiety
that binds to a detectable substance (such as an enzyme). A typical
specific binding moiety is an immunoglobulin variable domain which
is capable of binding to the detectable substance. FCEL and FCEH
specific polypeptides comprising immunoglobulin variable domains
are described in more detail above.
[0129] Assay systems that employ the FCEL or FCEH specific
polypeptides of this invention are analogous to the sandwich-type
systems heretofore generally used in the immunoassay field. Here,
the specific polypeptide is employed in the same fashion as
labelled antibodies directed against antigen (the FCEL or FCEH
receptor) or as an absorption agent insolubilized on a matrix for
the isolation of receptor from test sample. Redox, proteolytic,
esterolytic or other conventional enzyme labels are conjugated to
the polypeptides of this invention for use in conventional assay
systems.
[0130] The differential binding polypeptides of this invention also
are useful for the isolation of FCEL or FCEH from cell culture in
preparing FCEL or FCEH for therapeutic or research purposes. The
polypeptide is covalently bonded or noncovalently adsorbed to a
matrix such as an ion exchange resin, an immunoaffinity column
(containing an antibody capable of binding a polypeptide fused to
the FCEH or FCEL-specific polypeptide), an immobilized antigen
(where the FCEH or FCEL-specific polypeptide comprises an
immunoglobulin variable region capable of binding to the antigen)
or a cyanogen bromide activated polysaccharide. The immobilized
FCEH or FCEL-specific polypeptide then is contacted with the
receptor preparation under conditions such that the receptor is
bound to the FCEH or FCEL-specific polypeptide. The receptor then
is eluted by changing the pH or ionic conditions and separating the
polypeptide preparation from the receptor.
[0131] The differential binding polypeptides herein are useful in
preparing antibodies specific to the FCEH or FCEL-binding domain of
IgE. For example, antibodies capable of binding specifically to the
FCEH or FCEL-binding domains of IgE are selected by first
immunizing a subject with IgE. Monoclonal antibodies then are
selected in the ordinary way for native IgE binding, and the
monoclonal antibodies then screened to identify those that bind to
a FCEH or FCEL-specific polypeptide of this invention. Preferably
the FCEH or FCEL-specific polypeptide will be identical in sequence
to the corresponding sequence of the IgE used as immunogen except,
of course, for the minimal mutations need to confer FCEH or FCEL
differential binding specificity. For example, the IgE monoclonal
antibodies can be selected for their inability to bind to mutation
6. If they are unable to bind to mutation 6 one can conclude that
they bind to the FCEH-binding site and are therefore promising for
use in diagnostic or therapeutic procedures that depend upon an
antibody that fails to bind to FCEH-bound IgE but which binds to
FCEL-bound IgE. Confirmation is obtained by determining that the
antibody selected in fact binds to IgE bound to FCEL. Since the
selected antibody is highly specific for the key site(s) involved
in receptor binding it is then possible to reduce the size of the
antibody; the bulk of the antibody is not needed for steric
hinderance of the IgE-receptor interaction. Thus, it becomes
feasible in allergy therapy to use anti-IgE monovalent antibodies
or other anti-IgE fragments such as Fab, Fab' and the like.
[0132] Similarly, the FCEL or FCEH-specific polypeptides are useful
as immunogens for raising antibodies capable of cross-reacting with
native IgE only at epitopic sites outside of the domains varied in
creating the FCEH or FCEL-specific polypeptides. For example,
mutations 6 and 7 are useful for raising antibodies specific for
IgE epitopes except for the mutated AB-B or beta strand B domains
as the case may be.
[0133] The FCEH and FCEL-specific polypeptides and anti-IgE
antibodies (especially those with reduced immunogenicity) are
useful in therapies for the treatment or prophylaxis of allergies,
although the FCEH specific polypeptide subgroup which bears
cytotoxic functionalities is not considered suitable for therapy
since it could lead to degranulation of mast cells and basophils.
Otherwise, the polypeptides typically are administered to a patient
who is known to be sensitized to an allergen, preferably prior to
an acute allergic response. The dosages and administration route
will depend upon the accessory functionalities accompanying the
polypeptides (e.g. cytotoxic agents, immunoglobulin effector
functions, etc.), the condition of the patient (including the
population of B cells or mast cells and basophils), the half-life
of the polypeptide, the affinity of the polypeptide for its
receptor and other parameters known to the clinician. As a general
guide in the case of FCEH-specific polypeptide, one will determine
from blood tests the amount of target cells circulating in the
patient and determine the amount of polypeptide to displace or
effectively compete with endogenous IgE taking into account the
population of FCEH receptors as well as the half life and affinity
of the polypeptide for FCEH. An excess of polypeptide calculated to
be necessary to substantially displace native FCEH-bound IgE over a
reasonable therapeutic interval will then be administered. Similar
analysis used to determine the dosage of anti-IgE antibody or FCEL
polypeptide.
[0134] Therapeutic polypeptides are administered by intravenous
intrapulmonary, intraperitoneal subcutaneous or other suitable
routes. Preferably the polypeptides are administered s.c. or i.v.
over a period of about from 1 to 14 days as required. In the case
of FCEL-specific polypeptide or anti-FCEL-bound IgE one would
determine the amount needed to inhibit, suppress or kill a
substantial portion of the IgE-secreting B cell population.
Inhibition or suppression of the B cell population includes either
or both of reductions in IgE secretion and attenuation of the total
number of IgE secreting B cells. Candidate doses are readily
determined by the use of in vitro cell cultures or animal
models.
[0135] Therapy of allergic disorders with anti-FCEL bound IgE and
FCEL or FCEH polypeptides optionally is accomplished with other
known therapies for allergies. These include administration of
gamma interferon, allergen desensitization, reduction in exposure
to allergen, treatment with anti-histamines and the like.
Preparation of FCEH- and FCEL-Specific Polypeptides
[0136] The FCEH- or FCEL-specific polypeptides of this invention
are made in conventional fashion, i.e., modifications of amino acid
sequence are accomplished by commonly available DNA mutagenesis
methods such as PCR amplification using primers bearing the
mutants, or by M13 mutagenesis, followed by expression of the
mutated DNA in recombinant host cells. The polypeptides also can be
made by Merrifield or other in vitro methods of synthesis if they
are sufficiently small (generally, under about 100 residues).
However, the polypeptides preferably are made by recombinant
methods. Selection of recombinant host cells, vectors, culture
conditions and other parameters are not believed to be critical. In
general, hosts, vectors and methods heretofore used in the
recombinant expression of immunoglobulins (generally, IgGs) are
also useful for the preparation of the polypeptide sequences of
this invention. Preferably, mammalian cells such as myelomas, CHO,
Cos, 293s and the like are employed as hosts, and the vectors are
constructed for secretory expression of the polypeptide.
Recombinant expression systems facilitate the preparation of
functional immunoglobulin variants containing FCEL- or
FCEH-specific sequences since the host cells can be transformed
with DNA encoding one heavy chain containing the FCEL- or
FCEH-specific sequences and one light chain, each of which contains
a variable domain for binding a first antigen, and an
immunoglobulin that binds antigen and FCEL or FCEH recovered.
Similarly, the same process is used with DNA encoding in addition
another heavy chain containing the FCEL- or FCEH-specific domain
and another light chain, each of which contain a variable domain
for binding a second antigen, and a bivalent immunoglobulin
recovered. Properly assembled immunoglobulin analogues are
recovered by affinity chromatography on a matrix containing the two
antigen(s).
[0137] The polypeptides of this invention are recovered from lysed
recombinant cell culture or (when secreted) the culture
supernatant. Substantial purification is achieved by passing cell
free extracts which contain the polypeptides over an immobilized
FCEL or FCEH receptor affinity matrix. Other methods heretofore
used to purify IgE or other appropriate immunoglobulins are equally
acceptable here, including immunoaffinity and (when appropriate)
absorption on immobilized antigen.
[0138] Polypeptides of this invention which contain short sequences
preferably are prepared using solid-phase synthesis, e.g. the
method of Merrifield, J. Am. Chem. Soc., 85:2149 (1963). However,
other equivalent chemical syntheses known in the art are
acceptable. The recombinant or in vitro synthesized polypeptides
then are cross-linked to matrices (for use in diagnostic or
preparatory procedures) or are placed into conformationally
restrained structures. Known cyclizing procedures such as those
described in PCT 90/01331 or Lys/Asp cyclization using
N.alpha.-Boc-amino acids on solid-phase support with
Fmoc/9-fluorenylmethyl (Ofm) side-chain protection for Lys/Asp,
followed by piperidine treatment and cyclization, are useful.
Methods which depend upon cross-linking or cyclization through
residue side chains may require that an extraneous residue be
inserted at the C and/or N terminus of the AB-B or beta stand D
domains, as the case may be, to provide a suitable cyclizing or
cross-linking site.
[0139] Glu and Lys side chains also have been crosslinked in
preparing cyclic or bicyclic peptides: the peptide is synthesized
by solid phase chemistry on a p-methylbenzhydrylamine resin, the
peptide is cleaved from the resin and deprotected. The cyclic
peptide is formed using diphenylyphosphorylazide in diluted
methylformamide. For an alternative procedure, see Schiller et al.,
Peptide Protein Res. 25:171-77 (1985). See also U.S. Pat. No.
4,547,489.
[0140] Disulfide crosslinked or cyclized peptides are generated by
conventional methods. The method of Pelton et al., J. Med. Chem.,
29:2370-2375 (1986) is suitable. Also useful are thiomethylene
bridges (Tetrahedron Letters 25:2067-2068 (1984). See also Cody et
al., J. Med. Chem. 28:583 (1985). The C390 residue found in the
C-terminal sequence of the AB-B domain is useful in cross-linking
or cyclizing this domain.
[0141] Typically, extraneous residues which are to participate in
cyclization or cross-linking are inserted at the N- and C-termini
of the chosen AB-B or beta strand D sequence as part of the
synthesis of the polypeptide precursor to be employed in the
procedure. The desired cyclic or cross-linked peptides are purified
by gel filtration followed by reversed-phase high pressure liquid
chromatography or other conventional procedures. The peptides are
sterilized by 0.2 .mu.m filtration and formulated into conventional
pharmacologically acceptable vehicles.
[0142] The compounds described in this invention may be the free
acid or base or converted to salts of various inorganic and organic
acids and bases. Such salts are within the scope of this invention.
Examples of such salts include ammonium, metal salts like sodium,
potassium, calcium and magnesium; salts with organic bases like
dicyclohexylamine-N-methyl-D-glucamine and the like; and salts with
amino acids such as arginine or lysine. Salts with inorganic and
organic acids may be like prepared, for example, using
hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoroacetic,
methanesulfonic, maleic, fumaric and the like. Non-toxic and
physiologically compatible salts are particularly useful although
other less desirable salts may have use in the processes of
isolation and purification.
[0143] A number of methods are useful for the preparation of the
salts described above and are known to those skilled in the art.
For example, reaction of the free acid or free base form of a
compound of Formula I with one or more molar equivalents of the
desired acid or base in a solvent or solvent mixture in which the
salt is insoluble; or in a solvent like water after which the
solvent is removed by evaporation, distillation or freeze drying.
Alternatively, the free acid or base form of the product may be
passed over an ion exchange resin to form the desired salt, or one
salt form of the product may be converted to another using the same
general process.
[0144] Additional pharmaceutical methods may be employed to control
the duration of action of the polypeptides of this invention.
Controlled release preparations are achieved through the use of
polymers which complex with or absorb the subject polypeptides.
Controlled delivery is achieved by formulating the polypeptides
into appropriate macromolecular articles (for example, those
prepared from polyesters, polyamino acids, polyvinyl,
polypyrrolidone, ethylenevinylacetate, methlycellulose,
carboxymethylcellulose, or polyamine sulfate).
[0145] Alternatively, instead of entrapping the polypeptides in
polymeric matrices, it is possible to entrap these materials in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization. Hydroxymethylcellulose or gelatin
microcapsules and poly-(methylmethacrylate) microcapsules,
respectively, are useful, as are in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules). See Remington's Pharmaceutical
Sciences (1980).
EXAMPLE 1
Preparation of Monoclonal Antibodies to IgE
[0146] Eight monoclonal antibodies with the ability to block the
binding of IgE to the FCEH were used. These monoclonal antibodies,
referred to as MAE10-MAE17, were made in the following manner.
Purified human IgE was prepared from supernatants of U266B1 cells
(ATCC TIB 196) using affinity chromatography on a previously
isolated anti-IgE antibody (Genentech MAE1, although other
anti-huIgE antibodies are equally useful). For MAE12, five BALB/c
female mice, age six weeks, were immunized in their foot pads with
10 .mu.g of the purified IgE in Ribi's adjuvant. Subsequent
injections were done in the same manner one and three weeks after
the initial immunizations. Three days after the final injection,
the inguinal and popliteal lymph nodes were removed and pooled, and
a single cell suspension was made by passing the tissue through
steel gauze. For MAE14, MAE15, and MAE13 the immunizations were
done in a similar manner except that for MAE13 30 .mu.g of IgE per
injection were used and IgE 315-547 was used as a prefusion boost;
for MAE14 and MAE15 five injections of 50 .mu.g each were used; and
the IgE immunogen for MAE17 was IgE 315-547. For MAE10 and MAE11,
injections were given subcutaneously in two doses of 100 .mu.g and
a final booster of 50 .mu.g, and spleen cells were used for the
fusions. The cells were fused at a 4:1 ratio with mouse myeloma
P3X63-Ag8.653 (ATCC CRL 1580) in high glucose (DMEM) containing 50%
w/v polyethylene glycol 4000.
[0147] Fused cells were plated at a density of 2.times.10.sup.5 per
well in 96 well tissue culture plates. After 24 hours HAT selective
medium (hypoxanthine/aminopterin/thymidine, Sigma Chemical Company,
# H0262) was added. Of 1440 wells plated, 365 contained growing
cells after HAT selection.
[0148] Fifteen days after the fusion, supernatants were tested for
the presence of antibodies specific for human IgE using an
enzyme-linked immunosorbent assay (ELISA). The ELISA was performed
as follows, with all incubations done at room temperature. Test
plates (Nunc Immunoplate) were coated for 2 hours with rat
anti-mouse IgG (Boehringer Mannheim, # 605-500) at 1 .mu.g/ml in 50
Mm sodium carbonate buffer, Ph 9.6, then blocked with 0.5% bovine
serum albumin in phosphate buffered saline (PBS) for 30 minutes,
then washed four times with PBS containing 0.05% Tween 20 (PBST).
Test supernatants were added and incubated two hours with shaking,
then washed four times with PBST. Human IgE (purified from U266
cells as described above) was added at 0.5 .mu.g/ml and incubated
for one hour with shaking, then washed four times in PBST.
Horseradish peroxidase conjugated goat anti-human IgE (Kirkegaard
& Perry Labs, # 14-10-04, 0.5 mg/ml) was added at a 1:2500
dilution and incubated for one hour, then washed four times with
PBST. The plates were developed by adding 100 .mu.l/well of a
solution containing 10 mg. of o-phenylenediamine dihydrochloride
(Sigma Chemical Company # P8287) and 10 .mu.l of a 30% hydrogen
peroxide solution in 25 ml of phosphate citrate buffer Ph 5.0, and
incubating for 15 minutes. The reaction was stopped by adding 100
.mu.l/well of 2.5 M sulfuric acid. Data was obtained by reading the
plates in an automated ELISA plate reader at an absorbance of 490
nm. For MAE12, 365 supernatants were tested and 100 were specific
for human IgE. Similar frequencies of IgE specificity were obtained
when screening for the other antibodies. All of the monoclonal
antibodies described herein were of the IgG1 isotype except for
MAE17, which was IgG2b, and MAE14, which was IgG2a.
[0149] Each of the IgE specific antibodies was further tested in
cell-based and plate assays to select for antibodies which bound to
IgE in such a way as to inhibit IgE binding to FCEH and which are
not capable of binding to FCEH-bound IgE. The results of these
assays are set forth in Table 6 and Table 6a below. TABLE-US-00006
TABLE 6 SUMMARY OF MURINE Anti-Hu IgE mAb CHARACTERISTICS Kabat
Residue No. Human IgeE Human IgG1 Mutant (Structure).sup.1
Fc.epsilon.3 Seq. Fc.gamma.2 Seq. 1 377-385 (1AB) FDLFIRKS
KDTLMISRT (SEQ ID NO: 10) (SEQ ID NO: 11) 2 396-401 (1BC) APSKGT
SHEDPQ (SEQ ID NO: 12) (SEQ ID NO: 13) 3 407-420 (1CD) SRASGKPVNHS
YVDGVQVHNAK (SEQ ID NO: 14) (SEQ ID NO: 15) 4 444-453 (1EF)
GTRDWIEGET LHQDWLDGKE (SEQ ID NO: 16) (SEQ ID NO: 17) 5 465-469
(1FG) RALM APIE (SEQ ID NO: 18) (SEQ ID NO: 19) 6 423-428 (.beta.D)
KEEKQR PREQQY (SEQ ID NO: 20) (SEQ ID NO: 21) 7 383-385 (1AB) RKS
[AAA].sup.2 8 387, 389 (.beta.B) T(I)T [A(I)A].sup.2 9 403, 405
(.beta.C) N(L)T [A(L)A].sup.2 10 438-440 (.beta.E) T(S)T
[A(S)A].sup.2 11 455, 457, 459 (.beta.F) Q(C)R(V)T
[A(C)A(V)A].sup.2 (SEQ ID NO: 22) (SEQ ID NO: 23) 12 471, 473
(.beta.G) S(T)T [A(T)A].sup.2 13 329-331, 334-336 QKH(WL)SDR
[AAA(WL)AAA].sup.2 (SEQ ID NO: 24) (SEQ ID NO: 25)
[0150] TABLE-US-00007 TABLE 6a Summary of murine Anti-Hu IgE mAb
(continued) % Binding to % Binding of IgE Blocks 1 .mu.g Inhibition
of Membrane IgE on FcErII(CD23) IgE binding to in-vitro IgE
Affinity constant mAb on U266BL (EC50).sup.4 IM9 (EC50).sup.5 FcER
II (EC 50).sup.6 synthesis.sup.7 for IgE.sup.8 (Kd) MaE 1 0.4
.mu.g/ml .05 .mu.g/ml >100 .mu.g (-) 5.4 .times. 10.sup.-8 MaE
10 0.5 .mu.g/ml No binding at 10 .mu.g/ml 2.5 .mu.g (-) 7 .times.
10.sup.-9 MaE 11 0.15 .mu.g/ml No binding at 10 .mu.g/ml 0.6 .mu.g
(+) 3 .times. 10.sup.-8 MaE 12 >10 .mu.g/ml 1 .mu.g/ml 5.0 .mu.g
(-) 4 .times. 10.sup.-7 MaE 13 1 .mu.g/ml No binding at 10 .mu.g/ml
0.7 .mu.g (++) 5 .times. 10.sup.-8 MaE 14 6 .mu.g/ml No binding at
10 .mu.g/ml 2.5 .mu.g (.+-.) 1.4 .times. 10.sup.-8 MaE 15 6
.mu.g/ml No binding at 10 .mu.g/ml 0.6 .mu.g (.+-.) 7 .times.
10.sup.-8 MaE 16 10 .mu.g/ml <.05 .mu.g/ml 5 .mu.g (+) ND MaE 17
10 .mu.g/ml No binding at 10 .mu.g/ml 5 .mu.g (++) ND
1. FACS based assays for analysis of murine anti-human IgE
monoclonals. Screen of murine anti-human IgE monoclonal binding to
IgE on CHO 3D10 (FcERI alpha +)
[0151] a. CHO 3D10 cells (FcERI alpha chain stable transfectant;
Hakimi et al., J. Biol. Chem. 265:22079) at 5.times.10.sup.5 cells
per sample are incubated with U266 IgE standard (lot no. 13068-46)
at 10 .mu.g/ml in 100 .mu.l FACS buffer (0.1% BSA 10 mN sodium
azide in PBS pH 7.4) for 30 minutes at 4.degree. C. followed by one
wash with FACS buffer. The amount of IgE binding is determined by
incubating an aliquot of IgE loaded cells with a polyclonal FITC
conjugated rabbit anti-human IgG (Accurate Chem. Co. AXL-475F, lot
no 16) at 50 .mu.g/ml for 30 minutes at 4.degree. C. followed by
three washes with FACS buffer.
[0152] b. IgE loaded cells are incubated with 100 .mu.l of murine
anti-human IgE hybridoma supernatant (murine IgG concentration
ranging from 1 to 20 .mu.g/ml) for 30 min. at 4.degree. C. followed
by one wash with FACS buffer. A Genentech monoclonal anti-human IgE
(MAE1) at 10 .mu.g/ml is used as a positive control for binding.
Genentech monoclonal (MAD 6P) which does not recognize IgE is used
at 10 .mu.g/ml as a negative control.
[0153] c. Monoclonal binding to human IgE on CHO cells is detected
by incubating cells with 20 .mu.g/ml FITC-conjugated, affinity
purified F(ab) 2 Goat anti-mouse IgG (Organon Teknica cat. no.
10711-0081) for 30 minutes at 4.degree. C. followed by three washes
with FACS buffer. Cells are added to 400 .mu.l buffer contain 2
.mu.g/ml propidium iodide (Sigma cat no. P4170) to stain dead
cells.
[0154] d. Cells are analyzed on a Becton Dickinson FACSCAN flow
cytometer. Forward light scatter and 90 degree side scatter gates
are set to analyze a homogeneous population of cells. Dead cells
which stain with propidium iodide are excluded from analysis.
Hybridoma supernatants which do not bind IgE on CHO 3D10 cells were
considered candidates for further screening.
[0155] 2. Histamine release from peripheral blood basophils:
Heparinized blood was obtained from normal donors and diluted 1:4
in a modified Tyrodes buffer (25 mM tris, 150 mM NaCl, 10 mM
CaCl.sub.2, MgCl.sub.2, 0.3 mg/ml HSA, pH 7.35) then incubated with
1 nM human IgE (ND) at 4.degree. C. for 60 minutes. Cells were then
added to Tyrodes buffer containing either the murine monoclonal
anti-IgE Abs (10 mg/ml) or a polyclonal anti-human antiserum as the
positive control, and incubated at 37C for 30 minutes. Cells were
pelleted, histamine in supernatants was acetylated and histamine
content was determined using an RIA kit (AMAC, Inc. Wesbrook,
Main). Total histamine was determined from cells subjected to
several rounds of freezed thawing. Percent histamine release was
calculated as nM histamine content in supernatant--nM histamine
spontaneously released divided by nM total histamine in the
sample.
3. Blocking of Fitc conjugated IgE binding to FcERI alpha
chain.
[0156] The effect of the antibodies on IgE binding was studied by
preincubating Fitc labelled IgE with the various Mae antibodies at
37.degree. C. for 30 minutes in PBS containing 0.1% BSA and 10 mM
Sodium Azide pH 7.4, then incubating the complex with
5.times.10.sup.5 3D10 cells at 4.degree. C. for 30 minutes. The
cells were then washed three times and mean channel fluorescence at
475 nM was measured. A murine anti-human IgE mAb (Mae1) which does
not block IgE binding to the FcERI alpha chain was used as a
control.
4. Analysis of Murine Anti-Human IgE Binding to Membrane IgE
Positive B Cell U266
[0157] a. U266 B1 cells (membrane IgE +) are cultured in base
medium supplemented with 15% head inactivated fetal calf serum
(Hyclone cat No. A-1111-L), penicillin, streptomycin (100 units/ml)
and L-glutamine (2 mM).
[0158] b. Cells (5.times.10.sup.5/aliquot) are incubated in 100
.mu.l FACS buffer containing murine anti-Human IgE monoclonals at
10, 5, 1, 0.5, and 0.1 .mu.g/ml for 30 minutes on ice in 96 well
round bottom microtiter plates followed by two washes with FACS
buffer. The Genentech monoclonal MAE1 is used as a positive
control.
[0159] c. Cells are incubated in 100 .mu.l FACS buffer containing
50 .mu.g/ml (1:20 stock) FITC conjugated F(ab') 2 affinity purified
goat anti-mouse IgG (Organon Teknika Cat. No. 1711-0084) for 30
minutes on ice followed by three washes with FACS buffer. Cells are
added to 400 .mu.l FACS buffer containing propidium iodide at 2
.mu.g/ml to stain dead cells.
5. FACS based binding assays to FcERII(CD23+) B cell IM9
[0160] a. FACS analysis of IgE binding to FcERII(CD23) (+) B cell
line IM9. The IM9 human B cell myeloma ATCC CCL 159. (Ann. N.Y.
Acad. Sci., 190:221-234 [1972]) was maintained in GIF base medium
with 10% heat inactivated fetal bovine serum, penicillin,
streptomycin (100 units/ml) and L-glutamine (2 mM).
[0161] b. Cells (5.times.10.sup.5 aliquot) were incubated in 100
.mu.l of FACS buffer containing U266 IgE standard at 2 .mu.g/ml for
30 minutes at 4.degree. C. in 96 well microtiter plates followed by
2 washes with FACS buffer. As a control, cells were incubated in
buffer alone or buffer containing 2 .mu.g/ml human IgG1 (Behring
Diagnostics Cat. no. 400112, lot no. 801024).
[0162] c. Cells were then incubated with murine anti-human IgE
monoclonals at 0.1 to 10 .mu.g/ml for 30 minutes on ice. Genentech
monoclonal MAE1 was used as a positive control.
[0163] d. Cells were incubated in 100 .mu.l FACS buffer containing
FITC conjugated F(ab').sub.2 goat anti-mouse IgG at 50 .mu.g/ml
(Organon Teknika Ca #1711-0084) for 30 minutes at 4.degree. C.
followed by 3 washes with FACS buffer.
[0164] e. Cells were added to 400 .mu.l buffer containing propidium
iodide at 2 .mu.g/ml to stain dead cells.
[0165] f. Cells were analyzed on a Becton Dickinson FACSCAN flow
cytometer. Forward light scatter and 90 degree side scatter gates
were set to analyze a homogeneous population of cells and dead
cells which stained with propidium iodide were excluded from
analysis. FITC positive cells (IgE binding) were analyzed relative
to cells stained with FITC rabbit anti-Human IgE alone.
[0166] g. As a positive control to determine the level of CD 23 on
the surface of IM9 cells in each experiment, an aliquot of cells
was stained with Becton Dickinson murine monoclonal Leu 20
(anti-CD23) at 10 .mu.g/ml for 30 minutes at 4.degree. C. followed
by 2 washes. The cells were then incubated with FITC conjugated
F(ab).sub.2 affinity purified goat anti-murine IgG at 50
.mu.g/ml.
6. Antibody blocking of Fitc conjugated IgE binding to the low
affinity IgE receptor.
[0167] The binding of 40 nM FITC labelled IgE to the low affinity
IgE receptor (CD23) expressed on the B lymphoblast cell IM-9 was
analyzed by flow cytometry on a FACSCAN flow cytometer. The effect
of the antibodies on Fitc IgE binding was studied by preincubating
Fitc IgE with the murine anti-human antibodies at 0.1 to 10
.mu.g/ml. chimera at 37.degree. C. for 30 minutes in PBS containing
0.1% BSA and 10 mM Sodium Azide pH 7.4, then incubating the complex
with 5.times.10.sup.5 cells at 4.degree. C. for 30 minutes. The
cells were then washed three times and mean channel fluorescence at
475 nM was measured.
7. IgE In Vitro Assay Protocol
[0168] a. Peripheral blood mononuclear cells were separated from
normal donors.
[0169] b. Cells were washed extensively with phosphate buffered
saline to remove as many platelets as possible.
[0170] c. Mononuclear cells were counted and resuspend in media at
1.times.10.sup.6 cells/ml. (Media=DMEM+pen/strep+15% horse
serum+IL-2 (25U/ml)+IL-4 (20 ng/ml)).
[0171] d. Antibodies were added at appropriate concentrations on
day 0, 5, and 8.
[0172] e. Cultures were incubated in 24 well Falcon tissue culture
plates for 14 days.
[0173] f. On day 14 supernatants were removed and assayed for IgE
concentrations by an IgE specific ELISA protocol.
8. Affinity constant (kd) of murine mAb for human IgE was
determined by equilibrium binding (Scatchard analysis as
follows:
[0174] a. IgE (ND and PS allotypes were iodinated by the chloramine
T method and separated from free .sup.125I Na with a PD10 sephadex
G25 column (Pharmacia cat. no. 17-0851-01) in RIA buffer:PBS, 0.5%
bovine serum albumin (Sigma cat. no. A-7888), 0.05% Tween 20 (Sigma
cat. no. P-1379), 0.01% thimerosal (Sigma cat. no. T-5125), pH 7.4.
Approximately 78-95% of the post column counts were precipitated
with 50% trichloroacetic acid and specific activity of iodinated
IgE preparations ranged from 1.6 to 13 .mu.Ci/.mu.g assuming 70%
counting efficiency.
[0175] b. A fixed concentration of .sup.125I, IgE (approximately
5.times.10.sup.4 cpm) was added to varying concentrations of
unlabelled IgE (1 to 200 nM) in a final volume of 0.1 ml RIA buffer
in 12.times.75 mm polypropylene test tubes. Murine anti-human IgE
mABs (20 nM final concentration) in 0.1 ml RIA buffer were then
added for a final assay volume of 0.2 ml.
[0176] c. Samples were incubated 16-18 hours at 25.degree. C. with
continuous agitation.
[0177] d. Bound and free .sup.125I IgE was separated by the
addition of a 0.3 ml mixture of affinity purified goat anti-mouse
IgG (Boehringer Mannheim cat. no. 605 208) coupled to CN Br
activated Sepharose 4B (cat No. 17-0430-01) and carrier protein A
sepharose (Repligen cat. No. IPA 300) in RIA buffer and incubated 1
to 2 hours at 25.degree. C. with continuous agitation. RIA buffer
(1 ml) was then added, and tubes were centrifuged 5 min.
400.times.g. Samples were counted to determine total counts.
Supernatants were aspirated with a finely drawn pasteur pipet,
samples were recounted and bound versus free counts were
calculated.
[0178] e. Scatchard analysis was performed utilizing a Fortran
program (scanplot) based on the Ligand program written by P. Munson
at NIH. Scatplot uses a mass action equation fitting bound as a
function of total using the Rodbard type regression analysis.
EXAMPLE 2
Preparation of Variant IgE
[0179] Based on the model of IgE Fc by Padlan & Davies (Mol.
Immunol. 23:1063 (1986), which is based on the crystal structure of
human IgG1 Fc (Deisenhofer, Biochem. 20:2361-2370 [1981]), a series
of mutants were designed which could be used to test the binding of
human IgE to its receptors. These mutants are designated Emut 1-13,
and are listed in Table 7 below. The Fc.epsilon.3 domain is
comprised of seven .beta.-strands which form a .beta.-sheet
structure representative of all immunoglobulin domains; there are
six loops which connect these seven .beta.-strands. We refer to
these loops by the 2 .beta.-strands they connect, e.g. loop AB
connects .beta.-stands A and B. We have constructed mutants of
human IgE in which we have substituted five of the Fc.epsilon.3
domain loops with their counterparts from human IgG1 (Table 7,
1-5). The sixth loop contains the glycosylation site in both IgE
and IgG and hence was not altered. One mutant, (Table 7, 6), was
made by exchanging human Fc.epsilon.3 .beta.-strand D with its
human IgG1 Fcgamma2 counterpart. Seven additional mutants, (Table
7, 7-13), consisted of the substitution of Ala residues into
Fc.epsilon.3 .beta.-strands and a loop in Fc.epsilon.2.
[0180] A human IgE gene was cloned from U266, a publicly available
cell line. The gene was cloned into a previously described phagemid
vector containing the human cytomegalovirus enhancer and promoter,
a 5' intron and sv40 polyadenylation signal (Gorman et al., DNA and
Prot. Eng. Techn., 2:3-10 [1990]). Mutagenesis was performed by the
Kunkel method (T. A. Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492
(1985) using buffers and enzymes supplied with the BioRad Muta-gene
phagemid in vitro mutagenesis kit, together with oligonucleotides
encoding the human IgG1 sequences shown in Table 7 below. Sequences
of the mutant IgE DNAs were checked only at the site of mutation
using .sup.35S dideoxy sequencing. TABLE-US-00008 TABLE 7 Kabat
Residue No. Human IgeE Human IgG1 Mutant (Structure).sup.1
Fc.epsilon.3 Seq. Fc.gamma.2 Seq. 1 377-385 (1AB) FDLFIRKS
KDTLMISRT (SEQ ID NO: 10) (SEQ ID NO: 11) 2 396-401 (1BC) APSKGT
SHEDPQ (SEQ ID NO: 12) (SEQ ID NO: 13) 3 407-420 (1CD) SRASGKPVNHS
YVDGVQVHNAK (SEQ ID NO: 14) (SEQ ID NO: 15) 4 444-453 (1EF)
GTRDWIEGET LHQDWLDGKE (SEQ ID NO: 16) (SEQ ID NO: 17) 5 465-469
(1FG) RALM APIE (SEQ ID NO: 18) (SEQ ID NO: 19) 6 423-428 (.beta.D)
KEEKQR PREQQY (SEQ ID NO: 20) (SEQ ID NO: 21) 7 383-385 (1AB) RKS
[AAA].sup.2 8 387, 389 (.beta.B) T(I)T [A(I)A].sup.2 9 403, 405
(.beta.C) N(L)T [A(L)A].sup.2 10 438-440 (.beta.E) T(S)T
[A(S)A].sup.2 11 455, 457, 459 (.beta.F) Q(C)R(V)T
[A(C)A(V)A].sup.2 (SEQ ID NO: 22) (SEQ ID NO: 23) 12 471, 473
(.beta.G) S(T)T [A(T)A].sup.2 13 329-331, 334-336 QKH(WL)SDR
[AAA(WL)AAA].sup.2 (SEQ ID NO: 24) (SEQ ID NO: 25) .sup.1loop = 1
B-strand = .beta. .sup.2Sequences in brackets are from mutants in
which alanine residues rather than IgG sequences were used to
replace the IgE target sequence. Residues in parentheses were not
altered in these mutants.
[0181] The mutant IgEs were transiently expressed in human
embryonic kidney 293 cells (Gorman et al., supra), purified on a
mouse anti-human IgE antibody affinity column and samples run using
SDS-PAGE to ascertain that the mutant proteins were of the proper
molecular weight.
EXAMPLE 3
Soluble FCEH Binding Assay
[0182] This assay is a sequential inhibition ELISA which measures
binding to the FCEH only. In this assay, a monoclonal antibody
against the FCEH is coated onto ELISA plates at a concentration of
1 .mu.g/ml in 50 mM sodium carbonate pH 9.6 for two hours at room
temperature, and blocked for two hours with PBS containing 0.5%
bovine serum albumin (PBSA), then washed three times with ELISA
wash buffer (0.05% Tween 20 in PBS). Recombinantly produced soluble
FCEH is added at a concentration of 50 units/ml and incubated for
one hour, then washed five times in ELISA wash buffer. Mutant IgE
samples are then added to the wells and incubated for one to two
hours. The excess mutant IgE is removed by aspiration, and
biotinylated IgE is then added at 50 ng/ml for 15 minutes followed
by five washes with ELISA wash buffer. Streptavidin conjugated to
horseradish peroxidase (Sigma Chemical Company #S5512) was added at
a 1:5000 dilution for 15 minutes, then washed three times with
ELISA wash buffer. Color was developed with a tetramethyl benzidine
peroxidase substrate system (Kirkegaard &. Perry Labs #
50-76-00, Lot. no. NA 18) for seven minutes at 25.degree. C. The
reaction was stopped by the addition of 1 M HCl. The ability of the
mutant IgE to bind the FCEH is assessed by the degree to which the
biotinylated IgE is prevented from binding. This assay is designed
to test for any FCEH binding by the mutant IgE and is not meant to
determine the affinity of the mutant for the FCEH relative to
native IgE.
FACS Based Binding Assays for U266 IgE Mutants
[0183] Tissue culture supernatants from 293s cells transfected with
U266 IgE cDNA were harvested at either 48 or 96 hours post
transfection. Tissue culture supernatants were concentrated 5-X
with Amicon Centriprep 30.RTM. centrifugal concentrators (30,000 MW
cutoff). Concentrated supernatants were passed through a mouse
monoclonal anti-U266 IgE affinity column (Genentech MAE1 coupled to
CnBr-Sepharose). U266 IgE was eluted from the column with 3.O M
potassium cyanate in 50 mM tris buffer Ph 7.8. Eluate fractions
containing protein as determined by 0.D.280 nm were pooled and
placed in Amicon Centricon 30.RTM. concentrators. Eluate buffer was
exchanged for PBS by passing multiple volumes of PBS through the
concentrator. The final volume of affinity purified supernatant
ranged from 0.5-1 ml. Structural integrity of recombinant IgE
mutants was analyzed on 1-12% SDS PAGE gels and compared with U266
IgE standard obtained from the U266 cell line. Mutants were also
analyzed for the ability to bind to a series of monoclonal and IgE
antibodies to further ascertain proper folding and structural
identity with native IgE. The concentration of immunoreactive IgE
for each IgE mutant was determined by a human IgE capture ELISA as
follows. Nunc Immunoplate Maxisorp.RTM. plates (Nunc # 4-39451)
were coated overnight at 4.degree. C. with a Genentech murine IgG1
anti-U266 IgE (MAE1) at 1 .mu.g/ml in coat buffer (50 mM sodium
carbonate buffer pH 9.6). Coat antibody was removed by three washes
with ELISA wash buffer (0.05% Tween 20 (US Biochemical Corporation
# 20605) in PBS). Non-specific sites were blocked with ELISA
diluent buffer (50 mM tris buffered saline containing 0.5% BSA
(Sigma Chemical Company # A-7888), 6.05% Tween 20 and 2 mM EDTA)
for two hours at 25.degree. C. on an orbital shaker. Diluent buffer
was removed with 3 washes of ELISA wash buffer. Serial two-fold
dilutions of IgE mutants in ELISA diluent buffer were added to the
plate. U266 IgE standard (lot 13068-46) was added at 1000, 500,
250, 125, 62.5, 31.3, and 15.6 ng/ml in duplicate as standards.
Samples and standard were incubated two hours at 25.degree. C.
followed by three washes with ELISA wash buffer. IgE was detected
with HRP conjugated Sheep anti-human IgE (ICN # N060-050-1) at
1:8000 in ELISA diluent buffer for 90 min. at 25C followed by 3
washes with ELISA wash buffer. HRP conjugate was developed with a
tetramethyl benzidine peroxidase substrate system (Kirkegaard &
Perry Labs. # 50-76-00, Lot. no. NA 18) for 7 minutes at 25.degree.
C. The reaction was stopped by the addition of 1 M HCl. The
reaction product was analyzed with a dual wavelength
spectrophotometer at 450 nm minus absorption at 570 nm. The U266
IgE standards were used to generate a standard curve and IgE
concentrations of the sample were extrapolated by non-parametric
linear regression analysis.
[0184] FcERI alpha (+) CHO 3D10 (FCEH expressing) and FcERII(CD23)
(+) IM9 (FCEL expressing) B cell lines were used for the binding
assays. The stably transfected CHO (duk -) cell clone 3D10 (JBC
265, 22079-22081, 1990) was maintained in Iscove's modified
Dulbecco's media supplemented with 10% heat inactivated fetal calf
serum, 80 .mu.g/ml gentamicin sulfate and 5.times.10.sup.-7 M
methotrexate. The IM9 human B cell myeloma ATCC CCL 159. (Ann. N.Y.
Acad. Sci. 190:221-234, 1972) was maintained in GIF base medium
with 10% heat inactivated fetal bovine serum, penicillin,
streptomycin (100 units/ml) and L-glutamine (2 mM). As a positive
control to determine the level of CD23 on the surface of IM9 cells
in each experiment, an aliquot of cells was stained with Becton
Dickinson murine monoclonal Leu 20 (anti-CD23) at 10 .mu.g/ml for
30 minutes at 4.degree. C. followed by two washes in FACS buffer.
The cells were then incubated with FITC conjugated F(ab').sub.2
affinity purified goat anti-murine IgG at 5 .mu.g/ml. Adherent
CHO3D10 cells were removed from tissue culture dishes by incubation
with 10 mM EDTA in PBS for 2 minutes at 37.degree. C. Cells were
counted, then resuspended in FACS buffer (0.1% BSA, 10 mM Na azide
in PBS pH 7.4) at a concentration of 5.times.10.sup.6/m]. CHO3D10
and Im9 cells (5.times.10.sup.5/aliquot) were incubated in 100
.mu.l of FACS buffer containing U266 IgE standard or IgE mutants at
2 .mu.g/ml for 30 minutes at 4.degree. C. in 96 well microtiter
plates followed by two washes with FACS buffer. As a control, cells
were incubated in buffer alone or buffer containing 2 .mu.g/ml
human IgG1 (Behring Diagnostics # 400112, lot no. 801024). Cells
were then incubated in 100 .mu.l FACS buffer containing FITC
conjugated rabbit anti-human IgE at 20 .mu.g/ml (Accurate Chem. Co.
# AXL 475F, lot. no. 040A) for 30 minutes at 4.degree. C. followed
by 3 washes with FACS buffer. 400 .mu.l of buffer containing
propidium iodide at 2 .mu.g/ml was added to the cell suspension to
stain dead cells. Cells were analyzed on a Becton Dickinson FACSCAN
flow cytometer. Forward light scatter and 90 degree side scatter
gates were set to analyze a homogeneous population of cells and
dead cells which stained with propidium iodide were excluded from
analysis. FITC positive cells (IgE binding) were analyzed relative
to cells stained with FITC rabbit anti-H IgE alone.
[0185] The foregoing assays were used to determine the ability of
the example 2 IgE analogues to bind to FCEH and FCEL. The results
are set forth in Table 8. TABLE-US-00009 TABLE 8 BINDING OF IGE AND
IGE ANALOGUES TO FCEH AND FCEL Conc. FCEH alpha FCEL (CD23)
Sample/Mutant (ug/ml) % CHO 3D10(+) % IM9 (+) U266 IgE 10 90.3 92.5
U266 IgE 5 89.9 82.6 U266 IgE 0.5 59.6 4.6 U266 IgE 0.1 15.8 1.7 1
1.65.sup.1 1.7 4.3 2 1.65 34.3 48.9 3 1.65 32.3 1.2 4 1.65 4.9 9.2
5 1.65 60.5 73.9 6 1.65 1.4 71.6 7 1.65 76.4 4.6 8 1.65 70.3 16.3 9
1.65 84.2 94.3 10 1.65 67.5 84.8 11 1.65 70.8 61.5 12 1.65 84.7
90.3 13 1.65 85.7 96.1 dh 184 (+) 1.65 83.8 21.1 PA13.sup.2
(control) 10 1.3 .sup.1Values based on quantitative Elisa. U266 was
used as the standard and murine anti-F.sub.C.epsilon. monoclonal
antibody to capture. .sup.2A CDR grafted human IgG.
[0186] Three mutant IgEs exhibited complete loss of binding to the
FCEH receptor: mutants 1, 4 and 6. Mutant 6 altered .beta.-strand D
at the end of Fc.epsilon.3 close to the Fc.epsilon.2 domain.
Mutants 1 and 4 involved alteration of two Fc.epsilon.3 loops which
are adjacent and near the Fc.epsilon.4 domain. Note that mutant 7
is subset of mutant 1 in which the three C-terminal residues of
loop AB have been changed to alanines (Table 7, 1 vs. 7). However,
mutant 7 does not affect binding to FCEH. We interpret this to mean
that either 1) Fc.epsilon.RI binds at least one of IgE residues
377-381 or 2) the extra residue in IgG1 loop AB (9 residues)
substituted for IgE loop AB (8 residues) effected deformation of
some adjacent binding determinant, possibly loop EF. That mutants 8
and 10 had no affect on Fc.epsilon.RI binding most likely means
that the FCEH receptor does not protrude into the cavity bounded by
loop AB and .beta.-strand D.
[0187] Although mutant 4 had a Leu replacing Gly444 (Table 7), this
should not affect the conformation of loop EF. Residue 444 is prior
to the N-terminus of this .alpha.-helix. In addition, murine IgE
has a Val at position 444 and rat IgE has an Asp. The two buried
hydrophobic residues in the middle of the .alpha.-helix, W448 and
1449, are retained in the substituted IgG1 loop (W448, L449) as is
G451 which terminated the .alpha.-helix. Hence the conformation of
loop EF should be similar in IgE and IgG1.
[0188] Mutants 2 and 3 exhibited decreased binding to FCEH. Since
loop BC lies near .beta.-strand D and loop CD is in the vicinity of
loop EF, it is conceivable that one or two residues in loops BC and
CD contact FCEH.
[0189] Five mutant IgEs exhibited loss of binding to the FCEL
receptor: mutants 1, 3 4, 7 and 8. Mutants 1 and 4 were discussed
above. Mutant 3 involved alteration of loop CD; in contrast to
FCEH, loop CD evidently plays a major role in FCEL binding. Mutant
7, a subset of mutant 1 as discussed above, comprises the
C-terminal portion of loop AB and is proximal to loop EF.
Additionally, mutant 8 consists of replacement of two Thr residues
(387,389) with Ala; these two residues are part of .beta.-strand B
which is at the bottom of the aforementioned cavity bounded by loop
AB and .beta.-strand D. Mutant 10 comprised two different residues
in this cavity (438,440) on .beta.-strand E, which is adjacent to
.beta.-strand B. Since mutant 10 did not affect FCEL binding, we
conclude that the FCEL receptor should have only a minimal
incursion into cavity while the high affinity receptor does not
intrude into the cavity.
[0190] In addition to a glycosylation site at Asn430 which
corresponds to the glycosylation site in IgG Fc, human IgE contains
another glycosylation site at Asn403. Mutant 9 converted Asn403 and
Thr405 to alanines (Table 6). Loss of carbohydrate did not affect
binding to either receptor.
[0191] Based on the information from mutants 1-13, we propose that
FCEH and FCEL have binding sites on IgE Fc which are distinct but
overlap. The low affinity receptor seems to interact with a
relatively smaller portion of the IgE Fc.epsilon.3 domain involving
three adjacent loops: AB, CD and EF. In contrast, the high affinity
receptor interacts with a larger portion of IgE Fc.epsilon.3, which
spans loop EF, .beta.-strand D and, possibly, the N-terminal
portion of loop AB. Portions of loops BC and CD in the vicinity of
loop EF and .beta.-strand D may also interact with FCEH. In
addition, FCEL may protrude into the cavity bounded by loop AB and
.alpha.-strand D whereas FCEH does not do so. Since we have not
evaluated any mutants in FC.epsilon.4 and only one in Fc.epsilon.2
(mutant 13), it is possible that portions of these two domains play
a role in IgE-receptor binding.
EXAMPLE 4
Preparation of Humanized MaE11
[0192] Residues were selected from MaE11 and inserted or
substituted into a human Fab antibody background (V.sub.H region
Kabat subgroup III and V.sub.L region kappa subgroup I). A first
version, humae11v1 or version 1, is described in Table 9.
TABLE-US-00010 TABLE 9 Changes in V.sub.H human subgroup III and
V.sub.L .kappa. subgroup I (Kabat) consensus sequences for
humanized MaE11 Version 1 Hu Residue CDR CDR Domain Residue No. V.
1 by Kabat by Chothia V.sub.L M 4 L Insert 30abcd YDGD L1 L1 (SEQ
ID NO: 26) L* 33 M L1 S 53 Y L2 Y 91 S L3 L3 N 92 H L3 L3 S 93 E L3
L3 L 94 D L3 L3 V.sub.H A 24 V F* 27 Y H1 H1 T 28 S H1 H1 F* 29 I
H1 H1 Insert 29a T H1 H1 D 31 G H1 H1 A 33 S H1 H1 M* 34 W H1 H1 V
37 I V 50 S H2 S 52 T H2 N 53 Y H2 H2 G 54 D H2 H2 S 55 G H2 H2 Y
58 N H2 L 78 F D 95 G H3 97-101 All Changed H3 H3 to MaE11 Sequence
*These residues typically do not vary despite their position within
CDRs. The remaining residues found in the KI and III CDR sequences
(particularly the CDRs by Chothia structural analysis), will vary
widely among recipient human antibodies.
[0193] The affinity of version 1 was assayed and found to be about
100 times lower than that of the donor antibody Mae11 (see FIGS. 4a
and 4b). Therefore, further modifications in the sequence of
version 1 were made as shown in Table 10. Determination was made of
the ability of these further modifications to inhibit the binding
of labelled huIgE to FCEH.
[0194] The 50% inhibition assays whose results are shown in Table
10 were conducted as follows:
[0195] A 96-well assay plate (Manufn Nunc.) was coated with 0.05 ml
of the Fc.epsilon.RI alpha chain IgG1 chimeric receptor in 1
.mu.g/ml coating buffer (50 nmol carbonate/bicarbonate, pH 9.6).
Assay was done for 12 hours at 4-8.degree. C. The wells were
aspirated and 250 .mu.l blocking buffer (PBS-1% BSA pH 7.2) was
added and incubated for one hour at 4.degree. C. In a separate
assay plate the samples and reference murine MaE11 antibody were
titered from 200 .mu.g/ml by 1 to 10-fold dilution with assay
buffer (0.5% BSA, 0.05% Tween 20, PBS, pH 7.2) and an equal volume
of 10 ng/ml biotinylated IgE at 10 ng/ml was added and the plate
incubated for 2-3 hours at 25.degree. C. The Fc.epsilon.RI-coated
wells were washed three times with PBS-0.05% Tween20, and then 50
.mu.l from the sample wells were transferred and incubated with
agitation for 30 minutes at 25.degree. C. 50 .mu.l/well of
streptavidin-HRP diluted 1:5000 in assay buffer was incubated for
15 minutes with agitation and then the plate was washed as before.
50 .mu.l/well of Microwell peroxidase substrate (Kirkgaard &
Parry Laboratories) was added and color was developed for 30
minutes. The reaction was stopped by adding an equal volume of 1
normal HCl and the adsorbance measured at 450 nm. The concentration
for 50% inhibition was calculated by plotting percent inhibition
versus concentration of blocking antibody with a nonlinear
4-parameter curve-fit for each antibody using INPLOT.
TABLE-US-00011 TABLE 10 Humanized MaE11 Variants Version Changes
from Conc. At 50% inh. S.D. for F(ab)-X [F(ab)-X] Domain
F(ab)-Version 1 Purpose (ng/ml)* Mean prev. col. F(ab)-1 1 -- -- --
6083 1279 1.0 2 V.sub.L L4M Packing; CDR-L1 9439 508 1.6 M33L 3
V.sub.L E55G Sequence usually 5799 523 1.0 G57E E55-X-G57 4 V.sub.H
I37V VL-VH interface 8622 107 1.4 5 V.sub.H V24A Packing; CDR-H1
9387 733 1.6 6 V.sub.H F78L Packing; CDR- 17537 4372 2.9 H1, H2 7
V.sub.L L4M remake version 1 >100000 >16.0# R24K to
accomplish a E55G direct exchange G57E of CDR residues V.sub.H V24A
I37V T57S A60N D61P V63L G65N F78L 7a V.sub.H As V. 7 except
V.sub.H 98000 16.0 L78 is F 8 V.sub.H A60N Extended Kabat 1224 102
0.20 D61P CDR-H2 & A60N is at V.sub.L-V.sub.H interface 8a
V.sub.H As V. 8 except V.sub.H CDR-H2; packing 416 66 0.07 V63 is L
and of L63 and I67 F67 is I 8b V.sub.H As V. 8 except CDR-H2;
packing 501 84 0.08 F67 is I of V63 and I67 9 V.sub.L A13V Repack
Version 1 842 130 0.14 V19A interior as in V58I murine MaE11 L78V
V104L V.sub.H V48M A49G A60N V63L F67I I69V M82L L82cA 23 V.sub.L
L4M Packing; CDR-L1 6770 349 1.1 1 -- -- -- 6083 1279 1.0 10
V.sub.L D30A CDR-L1 >100000 >16.0 D30bA modification D30dA 11
V.sub.L E93A CDR-L3 17456 7115 2.9 D94A modification 12 V.sub.H
D54A CDR-H2 2066 174 0.34 modification 13 V.sub.H H97A CDR-H3
>100000 >16.0 H100aA modification H100cA 14 V.sub.L D30A
CDR-L1 3452 183 0.57 modification 15 V.sub.L D30bA CDR-L1 6384 367
1.0 modification 16 V.sub.L D30dA CDR-L1 >100000 >16.0
modification 17 V.sub.H H97A CDR-H3 19427 8360 3.2 modification 18
V.sub.H H100aA CDR-H3 2713 174 0.45 modification 19 V.sub.H H100cA
CDR-H3 15846 8128 2.6 modification *Inhibition of fitc-IgE binding
to FCEH (FcERI). Full-length antibody and humanized fragment
versions: mean and standard deviation of three assays. #A
F(ab)-X/F(ab)-1 ratio of >16 means that this variant exhibited
no binding even at the highest F(ab) concentrations used.
[0196] As can be seen from Table 10 and FIGS. 4a and 4b, version 8
(in which human residues of version 1 at sites 60 and 61 in the
light chain were replaced by their Mae11 counterparts) demonstrated
substantially increased affinity. Further increases in affinity are
seen in versions 8a and 8b, where one or two murine residues
replaced human residues. Other increases, at least virtually to the
level of Mae 11, were accomplished by replacing hydrophobic human
residues found in the interior of V.sub.H and V.sub.H1 with their
MaE11 counterparts, resulting in the variant designated version 9
(see Table 10 and FIGS. 4a and 4b). Accordingly, the humanized
antibodies of this invention will possess affinities ranging about
from 0.1 to 100 times that of MAE11.
[0197] Table 11 explores the effects on FCEH affinity of various
combinations of humanized maE11 IgG1 variants. TABLE-US-00012 TABLE
11 Humanized MaE11 IgG1 Variants Conc. at 50% S.D. from inh.
(ng/ml) previous Var. X Var. X Variant Mean* column* {overscore
(IgL1H1)} {overscore (MaE11)} IgL1H1 7569 1042 1.0 16.9 IgL1H8 3493
1264 0.46 7.8 IgL9H9 1118 172 0.15 2.5 IgL1H9 608 364 0.08 1.4
IgL9H1 5273 2326 0.70 11.7 IgL1H8b 1449 226 0.19 3.2 MaE11 449 53
0.06 1.0 *L1 = V.sub.L as in F(ab)-1 (human buried residues--not
exposed to solvent); L9 = V.sub.L as in F(ab)-9 (murine buried
residues); H1 = V.sub.H as in F(ab)-1 (human buried residues); H8 =
V.sub.H as in F(ab)-8 (F(ab)-1 with AlaH60Asn, AspH61Pro); H9 =
V.sub.H as in F(ab)-9 (murine buried residues); H8b = V.sub.H as in
F(ab)-8b (F(ab)-8 with PheH67Ile).
EXAMPLE 5
Creation of IgE Mutants
[0198] IgE mutants (Table 12) were prepared to evalute their effect
on binding to anti-IgE, especially MaE11, and to Fc.epsilon.RI and
Fc.epsilon.RII. Some of the mutants were designed to substitute for
a specific amino acid residue another residue with either similar
or very different charge or size. The impact of these changes on
receptor binding is reflected in the table below.
[0199] The receptor assays are performed substantially as
follows:
[0200] A 96-well assay plate (Manufn Nunc.) was coated with 0.05 ml
of Fc.epsilon.RI or RII IgG1 chimeric receptor in 1 .mu.g/ml
coating buffer (50 nmol carbonate/bicarbonate, pH 9.6). Assay was
done for 12 hours at 4-8.degree. C. The wells were aspirated and
250 .mu.l blocking buffer (PBS--1% BSA pH 7.2) was added and
incubated for one hour at 4.degree. C. In a separate assay plate
the samples and reference murine MaE11 antibody were titered from
200 .mu.g/ml by 1 to 10-fold dilution with assay buffer (0.5% BSA,
0.05% Tween 20, PBS, pH 7.2) and an equal volume of 10 ng/ml
biotinylated IgE at 10 ng/ml was added and the plate incubated for
2-3 hours at 25.degree. C. The Fc.epsilon.RI-coated wells were
washed three times with PBS-0.05% Tween 20, and then 50 .mu.l from
the sample wells were transferred and incubated with agitation for
30 minutes at 25.degree. C. 50 .mu.l/well of streptavidin-HRP
diluted 1:5000 in assay buffer was incubated for 15 minutes with
agitation and then the plate was washed as before. 50 .mu.l/well of
Microwell peroxidase substrate (Kirkgaard & Parry Laboratories)
was added and color was developed for 30 minutes. The reaction was
stopped by adding an equal volume of 1 normal HCl and the
adsorbance measured at 450 nm. The absorbance was plotted versus
concentration of blocking antibody MaE11 and an inhibition standard
curve was generated using INPLOT. TABLE-US-00013 TABLE 12 Amino
acid sequences of IgE mutants Mutant Kabat Residue # Human IgE Fce3
seq. Mutant seq. Fc.epsilon.-RI* Fc.epsilon.RII* Loop AB 1 377-385
FDLFIRKS KDTLMISRT - - (SEQ ID NO: 27) (SEQ ID NO: 28) 7 383-385
RKS AAA +/-, - +, - 21 377, 381 F(DL)F Q(DL)H + + (SEQ ID NO: 29)
(SEQ ID NO: 30) 66 382 I A + + 67 383 R A + +/- 68 384 K A + + 69
385 S A 102 383, 384 RK DD .beta.-strand B 8 387, 389 T(I)T A(I)A
+/-, + - 70 387 T A + +/-, + 71 389 T A + + Loop BC 2 396-401
APSKGT SHEDPQ (SEQ ID NO: 31) (SEQ ID NO: 32) .beta.-strand C 9
403, 405 N(L)T A(L)A + + Loop CD 3 407-420 SRASGKPVNHS YVDGVQVHNAK
+/- - (SEQ ID NO: 33) (SEQ ID NO: 34) 55 407-415 SR(A)S(G)K
AA(A)A(G)A +/- + (SEQ ID NO: 35) (SEQ ID NO: 36) 59 407 S A + + 60
408 R A + - 61 411 S A + + 62 415 K A + - 63 418 N A +/- + 64 419 H
A + + 65 420 S A +/- + 100 408 R E 101 415 K D .beta.-strand D 6
423--428 KEEKQR PREQQY + + (SEQ ID NO: 37) (SEQ ID NO: 38) 35 422 R
A + + 36 4423 K A + + 37 424 E A + + 38 425 E A + + 39 426 K A + 40
427 Q A -, +/- + 41 428 R A + + 75 423-425 KEE AAA -, +/-, + + 76
426-428 KQR AAA 79 423, 425, 427 KEEKQR AEAKAR (SEQ ID NO: 39) (SEQ
ID NO: 40) 80 424, 426, 428 KEEKQR KAEAQA (SEQ ID NO: 41) (SEQ ID
NO: 42) 81 423, K P 82 423-427 KEEKQR AAEAQA (SEQ ID NO: 43) (SEQ
ID NO: 44) .beta.-strand E 10 438, 440 T(S)T A(S)A + + Loop EF 4
444-453 GTRDWIEGET LHQDWLDGKE - - (SEQ ID NO: 45) (SEQ ID NO: 46)
49 445 T A + + 50 336 R A + - 51 337 D A + +, - +/- 52 450 E A + -
53 452 E A + + + +/- 77 445, 446 TR AA - - 78 450, 452, 453 E(G)ET
A(G)AA + + (SEQ ID NO: 47) (SEQ ID NO: 48) + 83 444 G L + + 84 444
G A 85 445-453 TRDWIEGET HQDWLDGKE - + (SEQ ID NO: 49) (SEQ ID NO:
50) + 86 445 T H + 87 445, 446 TR HQ +/-, + 88 446 R E - 89 450,
452, 453 E(G)ET D(G)KE +/-, - +/- (SEQ ID NO: 51) (SEQ ID NO: 52) +
93 447 D R +/-, - 94 450 E R 95 452 E R 96 453 T R 97 447 D N 98
452 E Q 99 452 E D .beta.-strand F 11 445, 457, 459 Q(C)R(V)T
A(C)A(V)A (SEQ ID NO: 53) (SEQ ID NO: 54) Loop FG 5 465-469 RALM
APIE (SEQ ID NO: 55) (SEQ ID NO: 56) .beta.-strand G 12 471, 473
S(T)T A(T)A +, + Fc.epsilon.2 13 329-331, 334-336 QKH(WL)SDR
AAA(WL)AAA +, + (SEQ ID NO: 57) (SEQ ID NO: 58) Fc.epsilon.4 72
498-501 PRAA QPRE (SEQ ID NO: 59) (SEQ ID NO: 60) 73 594-599 ASPSQT
LHNHY (SEQ ID NO: 61) (SEQ ID NO: 62) 74 595-599 S(P)SQT A(P)AAA
(SEQ ID NO: 63) (SEQ ID NO: 64) *Positive receptor binding
indicated by "+", no binding by "-", and positive binding but less
than unaltered is shown by "+/-". Where more than one assay was
performed, results are separated by commas.
[0201]
Sequence CWU 1
1
68 1 109 PRT Homo sapiens 1 Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr
Leu Ser Arg Pro Ser 1 5 10 15 Pro Phe Asp Leu Phe Ile Arg Lys Ser
Pro Thr Ile Thr Cys Leu 20 25 30 Val Val Asp Leu Ala Pro Ser Lys
Gly Thr Val Asn Leu Thr Trp 35 40 45 Ser Arg Ala Ser Gly Lys Pro
Val Asn His Ser Thr Arg Lys Glu 50 55 60 Glu Lys Gln Arg Asn Gly
Thr Leu Thr Val Thr Ser Thr Leu Pro 65 70 75 Val Gly Thr Arg Asp
Trp Ile Glu Gly Glu Thr Gln Cys Arg Val 80 85 90 Thr His Pro His
Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys 95 100 105 Thr Ser Gly
Pro 2 111 PRT Mus musculus 2 Asp Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu 1 5 10 15 Gly Gln Arg Ala Thr Ile Ser Cys
Lys Ala Ser Gln Ser Val Asp 20 25 30 Tyr Asp Gly Asp Ser Tyr Met
Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Pro Pro Ile Leu Leu
Ile Tyr Ala Ala Ser Tyr Leu Gly Ser 50 55 60 Glu Ile Pro Ala Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Asn Ile
His Pro Val Glu Glu Glu Asp Ala Ala Thr Phe 80 85 90 Tyr Cys Gln
Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Ala Gly 95 100 105 Thr Lys
Leu Glu Ile Lys 110 3 134 PRT Mus musculus 3 Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15 Gln Ser Leu Ser
Leu Ala Cys Ser Val Thr Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr
Ser Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys 35 40 45 Leu Glu
Trp Met Gly Ser Ile Thr Tyr Asp Gly Ser Ser Asn Tyr 50 55 60 Asn
Pro Ser Leu Lys Asn Arg Ile Ser Val Thr Arg Asp Thr Ser 65 70 75
Gln Asn Gln Phe Phe Leu Lys Leu Asn Ser Ala Thr Ala Glu Asp 80 85
90 Thr Ala Thr Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His 95
100 105 Trp His Phe Ala Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser
110 115 120 Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Arg
125 130 4 124 PRT Mus musculus 4 Asp Ile Val Met Thr Gln Ser Gln
Lys Phe Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Val Thr
Cys Lys Ala Ser Gln Asn Val Ser 20 25 30 Ser Asn Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys 35 40 45 Ala Leu Ile Tyr Ser
Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp 50 55 60 Arg Phe Thr Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 Ser Asn Val
Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln 80 85 90 Tyr Tyr
Thr Tyr Pro Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu 95 100 105 Glu
Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro 110 115 120
Pro Ser Thr Arg 5 130 PRT Mus musculus 5 Asp Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15 Gln Ser Leu Ser Leu
Thr Cys Thr Val Thr Gly Tyr Thr Ile Thr 20 25 30 Ser Asp Asn Ala
Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys 35 40 45 Leu Glu Trp
Met Gly Tyr Ile Asn His Ser Gly Thr Thr Ser Tyr 50 55 60 Asn Pro
Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser 65 70 75 Lys
Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp 80 85 90
Thr Ala Thr Tyr Tyr Cys Ala Trp Val Val Ala Tyr Ala Met Asp 95 100
105 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 110
115 120 Thr Pro Pro Ser Val Tyr Pro Leu Ala Arg 125 130 6 106 PRT
Mus musculus 6 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val
Ser Leu 1 5 10 15 Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln
Ser Val Asp 20 25 30 Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Pro Gly 35 40 45 Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala
Ser Asn Leu Glu Ser 50 55 60 Gly Ile Pro Ala Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Asn Ile His Pro Val Glu
Glu Glu Asp Ala Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser Asn Glu
Asp Pro Phe Thr Phe Gly Ala Gly 95 100 105 Thr 7 137 PRT Mus
musculus 7 Asp Val Gln His Gln Glu Ser Glu Pro Asp Leu Val Lys Pro
Ser 1 5 10 15 Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser
Ile Thr 20 25 30 Ser Gly Tyr Asn Arg His Trp Ile Arg Gln Phe Pro
Gly Asn Lys 35 40 45 Leu Glu Trp Met Gly Tyr Ile His Tyr Ser Gly
Ser Thr Asn Tyr 50 55 60 Asn Pro Ser Leu Lys Arg Arg Ile Ser Ile
Thr Arg Asp Thr Ser 65 70 75 Lys Asn Gln Phe Phe Leu Gln Leu Asn
Ser Val Thr Thr Glu Asp 80 85 90 Thr Ala Thr Tyr Tyr Cys Ala Arg
Gly Ser Ile Tyr Tyr Tyr Gly 95 100 105 Ser Arg Tyr Arg Tyr Phe Asp
Val Trp Gly Ala Gly Thr Thr Val 110 115 120 Thr Val Ser Ser Ala Lys
Arg His Pro His Leu Ser Ile His Trp 125 130 135 Pro Gly 8 453 PRT
Artificial Sequence humanized maell, version 1 heavy chain 8 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 25
30 Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35
40 45 Leu Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr
50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser 65 70 75 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr
Phe Gly His 95 100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 110 115 120 Ser Ala Ser Thr Lys Gly Lys Gly Pro Ser
Val Phe Pro Leu Ala 125 130 135 Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 140 145 150 Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 155 160 165 Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 170 175 180 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 185 190 195 Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 200 205 210 Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 215 220 225 Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 230 235 240 Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280
285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290
295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
305 310 315 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser 320 325 330 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala 335 340 345 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 350 355 360 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 365 370 375 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn 380 385 390 Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 395 400 405 Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 410 415 420 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 425 430 435 Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 440 445 450 Pro Gly Lys 9 218
PRT Artificial Sequence humanized maell, version 1, light chain 9
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10
15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp 20
25 30 Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly
35 40 45 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu
Ser 50 55 60 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe 65 70 75 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr
Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala Pro Ser Val Phe 110 115 120 Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser 125 130 135 Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val 140 145 150 Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu 155 160 165 Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 170 175 180 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val 185 190 195 Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 200 205 210 Lys Ser Phe
Asn Arg Gly Glu Cys 215 10 8 PRT Homo sapiens 10 Phe Asp Leu Phe
Ile Arg Lys Ser 1 5 11 9 PRT Homo sapiens 11 Lys Asp Thr Leu Met
Ile Ser Arg Thr 1 5 12 6 PRT Homo sapiens 12 Ala Pro Ser Lys Gly
Thr 1 5 13 6 PRT Homo sapiens 13 Ser His Glu Asp Pro Gln 1 5 14 11
PRT Homo sapiens 14 Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser 1 5
10 15 11 PRT Homo sapiens 15 Tyr Val Asp Gly Val Gln Val His Asn
Ala Lys 1 5 10 16 10 PRT Homo sapiens 16 Gly Thr Arg Asp Trp Ile
Glu Gly Glu Thr 1 5 10 17 10 PRT Homo sapiens 17 Leu His Gln Asp
Trp Leu Asp Gly Lys Glu 1 5 10 18 4 PRT Homo sapiens 18 Arg Ala Leu
Met 1 19 4 PRT Homo sapiens 19 Ala Pro Ile Glu 1 20 6 PRT Homo
sapiens 20 Lys Glu Glu Lys Gln Arg 1 5 21 6 PRT Homo sapiens 21 Pro
Arg Glu Gln Gln Tyr 1 5 22 5 PRT Homo sapiens 22 Gln Cys Arg Val
Thr 1 5 23 5 PRT Artificial Sequence Modified IgG1-derived sequence
23 Ala Cys Ala Val Ala 1 5 24 8 PRT Homo sapiens 24 Gln Lys His Trp
Leu Ser Asp Arg 1 5 25 8 PRT Artificial Sequence Modified
IgG1-derived sequence 25 Ala Ala Ala Trp Leu Ala Ala Ala 1 5 26 4
PRT Mus musculus 26 Tyr Asp Gly Asp 1 27 8 PRT Homo sapiens 27 Phe
Asp Leu Phe Ile Arg Lys Ser 1 5 28 9 PRT Homo sapiens 28 Lys Asp
Thr Leu Met Ile Ser Arg Thr 1 5 29 4 PRT Homo sapiens 29 Phe Asp
Leu Phe 1 30 4 PRT Artificial Sequence Mutant IgE Fc(epsilon)3
sequence fragment 30 Gln Asp Leu His 1 31 6 PRT Homo sapiens 31 Ala
Pro Ser Lys Gly Thr 1 5 32 6 PRT Homo sapiens 32 Ser His Glu Asp
Pro Gln 1 5 33 11 PRT Homo sapiens 33 Ser Arg Ala Ser Gly Lys Pro
Val Asn His Ser 1 5 10 34 11 PRT Homo sapiens 34 Tyr Val Asp Gly
Val Gln Val His Asn Ala Lys 1 5 10 35 6 PRT Homo sapiens 35 Ser Arg
Ala Ser Gly Lys 1 5 36 6 PRT Artificial Sequence Mutant IgE
Fc(epsilon)3 sequence fragment 36 Ala Ala Ala Ala Gly Ala 1 5 37 6
PRT Homo sapiens 37 Lys Glu Glu Lys Gln Arg 1 5 38 6 PRT Homo
sapiens 38 Pro Arg Glu Gln Gln Tyr 1 5 39 6 PRT Homo sapeins 39 Lys
Glu Glu Lys Gln Arg 1 5 40 6 PRT Artificial Sequence Mutant
sequence substituted in place of IgE Fc(epsilon)3 fragment 40 Ala
Glu Ala Lys Ala Arg 1 5 41 6 PRT Homo sapiens 41 Lys Glu Glu Lys
Gln Arg 1 5 42 6 PRT Artificial Sequence Mutant sequence
substituted in place of IgE Fc(epsilon)3 fragment 42 Lys Ala Glu
Ala Gln Ala 1 5 43 6 PRT Homo sapiens 43 Lys Glu Glu Lys Gln Arg 1
5 44 6 PRT Artificial Sequence Mutant sequence substituted in place
of IgE Fc(epsilon)3 fragment 44 Ala Ala Glu Ala Gln Ala 1 5 45 10
PRT Homo sapiens 45 Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr 1 5 10
46 10 PRT Artificial Sequence Mutant sequence substituted in place
of IgE Fc(epsilon)3 fragment 46 Leu His Gln Asp Trp Leu Asp Gly Lys
Glu 1 5 10 47 4 PRT Homo sapiens 47 Glu Gly Glu Thr 1 48 4 PRT
Artificial Sequence Mutant sequence substituted in place of IgE
Fc(epsilon)3 fragment 48 Ala Gly Ala Ala 1 49 9 PRT Homo sapiens 49
Thr Arg Asp Trp Ile Glu Gly Glu Thr 1 5 50 9 PRT Artificial
Sequence Mutant sequence substituted in place of IgE Fc(epsilon)3
fragment 50 His Gln Asp Trp Leu Asp Gly Lys Glu 1 5 51 4 PRT Homo
sapiens 51 Glu Gly Glu Thr 1 52 4 PRT Artificial Sequence Mutant
sequence substituted in place of IgE Fc(epsilon)3 fragment 52 Asp
Gly Lys Glu 1 53 5 PRT Homo sapiens 53 Gln Cys Arg Val Thr 1 5 54 5
PRT Artificial Sequence Mutant sequence substituted in place of IgE
Fc(epsilon)3 fragment 54 Ala Cys Ala Val Ala 1 5 55 4 PRT Homo
sapiens 55 Arg Ala Leu Met 1 56 4 PRT Artificial Sequence Mutant
sequence substituted in place of IgE Fc(epsilon)3 fragment 56 Ala
Pro Ile Glu 1 57 8 PRT Homo sapiens 57 Gln Lys His Trp Leu Ser Asp
Arg 1 5 58 8 PRT Artificial Sequence Mutant sequence substituted in
place of IgE Fc(epsilon)e fragment 58 Ala Ala Ala Trp Leu Ala Ala
Ala 1 5 59 4 PRT Homo sapiens 59 Pro Arg Ala Ala 1 60 4 PRT
Artificial Sequence Mutant sequence substituted in place of IgE
Fc(epsilon)3 fragment 60 Gln Pro Arg Glu 1 61 6 PRT Homo sapiens 61
Ala Ser Pro Ser Gln Thr 1 5 62 5 PRT Artificial Sequence Mutant
sequence substituted in place of IgE Fc(epsilon)3 fragment 62 Leu
His Asn His Tyr 1 5 63 5 PRT Homo sapiens 63 Ser Pro Ser Gln Thr 1
5 64 5 PRT Homo sapiens 64 Ala Pro Ala Ala Ala 1 5 65 451 PRT
Artificial Sequence Full-length heavy chain sequence corresponding
to F(ab)8b of Table 9 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val
Ser Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile
Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile
Thr Tyr Asp Gly Ser Thr Asn Tyr 50 55 60 Asn Pro Ser Val Lys Gly
Arg Ile Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr Phe Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr
Tyr Cys Ala Arg Gly Ser His
Tyr Phe Gly His 95 100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr
Leu Val Thr Val Ser 110 115 120 Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser 125 130 135 Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val 140 145 150 Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly 155 160 165 Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 170 175 180 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser 185 190 195 Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 200 205 210 Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 215 220 225 Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 230 235 240 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265
270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275
280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln 305 310 315 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys 320 325 330 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 335 340 345 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu 350 355 360 Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 365 370 375 Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 380 385 390 Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp 395 400 405 Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg 410 415 420 Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 425 430 435 Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 440 445 450 Lys 66 451 PRT
Artificial Sequence Full-length heavy chain sequence corresponding
to F(ab)8a of Table 9 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val
Ser Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile
Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile
Thr Tyr Asp Gly Ser Thr Asn Tyr 50 55 60 Asn Pro Ser Leu Lys Gly
Arg Ile Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr Phe Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr
Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105 Trp His Phe
Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser 110 115 120 Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 125 130 135 Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 140 145 150
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 155 160
165 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 170
175 180 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
185 190 195 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro 200 205 210 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 215 220 225 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln 305 310 315 Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 320 325 330 Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 335 340 345 Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 350 355 360 Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 365 370 375 Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 380 385 390 Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 395 400 405
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 410 415
420 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 425
430 435 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
440 445 450 Lys 67 218 PRT Artificial Sequence Full-length light
chain sequence corresponding to F(ab)9 of Table 9 67 Asp Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val 1 5 10 15 Gly Asp
Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp 20 25 30 Tyr
Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45
Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser 50 55
60 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65
70 75 Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr
80 85 90 Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln
Gly 95 100 105 Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
Val Phe 110 115 120 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser 125 130 135 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val 140 145 150 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu 155 160 165 Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 170 175 180 Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val 185 190 195 Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr 200 205 210 Lys Ser Phe Asn Arg Gly
Glu Cys 215 68 451 PRT Artificial Sequence Full-length heavy chain
sequence corresponding to F(ab)9 of Table 9 68 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr
Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu
Trp Met Gly Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr 50 55 60 Asn
Asp Ser Leu Lys Gly Arg Ile Thr Val Ser Arg Asp Asp Ser 65 70 75
Lys Asn Thr Phe Tyr Leu Gln Leu Asn Ser Ala Arg Ala Glu Asp 80 85
90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His 95
100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser
110 115 120 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser 125 130 135 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val 140 145 150 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly 155 160 165 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 170 175 180 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser 185 190 195 Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro 200 205 210 Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp 215 220 225 Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 230 235 240 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 320 325 330
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 335 340
345 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 350
355 360 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
365 370 375 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln 380 385 390 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp 395 400 405 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg 410 415 420 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala 425 430 435 Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 440 445 450 Lys
* * * * *