U.S. patent application number 11/370898 was filed with the patent office on 2006-07-13 for treating autoimmune diseases with humanized anti-cd401 antibodies.
Invention is credited to Amelia Black, Nabil Hanna, Roland A. Newman, Eduardo A. Padlan.
Application Number | 20060153837 11/370898 |
Document ID | / |
Family ID | 25451580 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060153837 |
Kind Code |
A1 |
Black; Amelia ; et
al. |
July 13, 2006 |
Treating autoimmune diseases with humanized anti-CD401
antibodies
Abstract
The present invention is directed to humanized antibodies which
bind human gp39 and their use as therapeutic agents. These
humanized antibodies are especially useful for treatment of
autoimmune diseases; and an immunosuppressant during
transplantation of heterologous cells, tissues or organs, cell
therapy, and gene therapy.
Inventors: |
Black; Amelia; (Los Gatos,
CA) ; Hanna; Nabil; (Rancho Santa Fe, CA) ;
Padlan; Eduardo A.; (Kensington, MD) ; Newman; Roland
A.; (San Diego, CA) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Family ID: |
25451580 |
Appl. No.: |
11/370898 |
Filed: |
March 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10171681 |
Jun 17, 2002 |
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11370898 |
Mar 9, 2006 |
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08925339 |
Sep 8, 1997 |
6440418 |
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10171681 |
Jun 17, 2002 |
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08554840 |
Nov 7, 1995 |
6001358 |
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08925339 |
Sep 8, 1997 |
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Current U.S.
Class: |
424/133.1 ;
530/388.15 |
Current CPC
Class: |
A61K 9/008 20130101;
C07K 2319/00 20130101; C07K 16/467 20130101; A61P 27/00 20180101;
C07K 2317/73 20130101; G01N 33/564 20130101; C07K 2317/92 20130101;
A61P 37/08 20180101; A61P 11/06 20180101; A61P 35/02 20180101; A61P
35/00 20180101; A61K 9/0019 20130101; A61P 37/04 20180101; A61K
39/395 20130101; C07K 2317/76 20130101; G01N 2333/70578 20130101;
A61K 9/0048 20130101; G01N 2800/24 20130101; A61P 37/06 20180101;
A61P 29/00 20180101; A61P 3/10 20180101; C07K 16/2875 20130101;
A61P 37/02 20180101; A61P 1/04 20180101; A61K 9/0014 20130101; A61P
37/00 20180101; A61K 2039/505 20130101; C07K 2317/24 20130101; A61P
1/00 20180101; A61P 17/00 20180101; A61P 11/00 20180101; A61P 17/06
20180101; A61P 3/00 20180101; G01N 2800/245 20130101; A61K 38/00
20130101 |
Class at
Publication: |
424/133.1 ;
530/388.15 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/44 20060101 C07K016/44 |
Claims
1-24. (canceled)
25. A method of treatment of a disease treatable by modulating gp39
expression or inhibiting the gp39/CD40 interaction which comprises
administering a therapeutically effective amount of a humanized
antibody which is capable of competing with the murine 24-31
antibody for inhibiting CD40 binding to gp39.
26. The method of claim 25, wherein said disease is an autoimmune
disorder.
27. The method of claim 26, wherein said autoimmune disorder is
selected from the group consisting of rheumatoid arthritis,
psoriasis, multiple sclerosis, diabetes, systemic lupus
erythematosus and ITP.
28. The method of claim 25, wherein the disease is a non-autoimmune
disorder.
29. The method of claim 25, wherein the disease is
graft-versus-host disease or graft rejection.
30. A method of suppressing humoral and/or cellular immune
responses against cells or vectors administered during cell or gene
therapy comprising further administering prior, during, or after
gene therapy an amount of a humanized antibody derived from murine
antibody 24-31. sufficient to suppress humoral and/or cellular
immune responses against the cell or vector used during cell or
gene therapy.
31. The method of claim 30, wherein the vector is a viral vector, a
DNA or an antisense RNA.
32. The method of claim 30, wherein the viral vector is an
adenovirus or retrovirus.
33. The method of claim 30, wherein said humanized antibody
contains a sequence set forth in at least one of FIGS. 5-8.
34. An improved method of treatment which involves the
transplantation of cells, tissues or organs of the same or
different species into a subject in need of such treatment, wherein
the improvement comprises administering a humanized antibody
derived from murine antibody 24-31; prior, during, or after
transplantation; in an amount sufficient to suppress immune
responses against said transplanted cell, tissue or organ or to
suppress immune responses elicited by the transplanted cell, tissue
or organ against the host.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of copending
application Ser. No. 08/554,840, filed Nov. 7, 1995.
FIELD OF THE INVENTION
[0002] The present invention is directed to humanized antibodies
specific for human gp39, DNA encoding such antibodies, methods for
their production, pharmaceutical compositions containing, and the
use of such humanized antibodies as therapeutic agents. These
antibodies have particular application in the treatment of
autoimmune diseases including, e.g., rheumatoid arthritis, multiple
sclerosis, diabetes, and systemic lupus erythematosus as well as
non-autoimmune diseases including, e.g., graft-versus-host disease
and for preventing graft rejection.
BACKGROUND OF THE INVENTION
[0003] The immune system is capable of producing two types of
antigen-specific responses to foreign antigens. Cell-mediated
immunity is the term used to refer to effector functions of the
immune system mediated by T lymphocytes. Humoral immunity is the
term used to refer to production of antigen-specific antibodies by
B lymphocytes. It has long been appreciated that the development of
humoral immunity against most antigens requires not only
antibody-producing B lymphocytes but also the involvement of helper
T (hereinafter Th) lymphocytes. (Mitchison, Eur. J. Immunol.,
1:18-25 (1971); Claman and Chaperon, Transplant Rev., 1:92-119
(1969); Katz et al, Proc. Natl. Acad. Sci. USA, 70:2624-2629
(1973); Reff et al, Nature, 226:1257-1260 (1970)). Certain signals,
or "help", are provided by Th cells in response to stimulation by
Thymus-dependent (hereinafter TD) antigens. While some B lymphocyte
help is mediated by soluble molecules released by Th cells (for
instance lymphokines such as IL-4 and IL-5), activation of B cells
also requires a contact-dependent interaction between B cells and
Th cells. (Hirohata et al, J. Immunol., 140:3736-3744 (1988);
Bartlett et al, J. Immunol., 143:1745-1765 (1989)). This indicates
that B cell activation involves an obligatory interaction between
cell surface molecules on B cells and Th cells. Such an interaction
is further supported by the observation that isolated plasma
membranes of activated T cells can provide helper functions
necessary for B cell activation. (Brian, Proc. Natl. Acad. Sci.
USA, 85:564-568 (1988); Hodgkin et al, J. Immunol., 145:2025-2034
(1990); Noelle et al, J. Immunol., 146:1118-1124 (1991)).
[0004] It is further known that in a contact-dependent process
termed "T cell helper function", CD4.sup.+ T lymphocytes direct the
activation and differentiation of B lymphocytes and thereby
regulate the humoral immune response by modulating the specificity,
secretion and isotype-encoded functions of antibody molecules
(Mitchell et al, J. Exp. Med., 128:821 (1968); Mitchison, Eur. J.
Immunol., 1:68 (1971); White et al, J. Exp. Med., 14:664 (1978);
Reinherz et al, Proc. Natl. Acad. Sci. USA, 74:4061 (1979); Janeway
et al, Immunol. Rev., 101:39 (1988); O'Brien et al, J. Immunol.,
141:3335 (1988); Rahemtulla et al, Nature, 353:180 (1991); and
Grusby et al, Science, 253:1417 (1991)).
[0005] The process by which T cells help B cells to differentiate
has been divided into two distinct phases; the inductive and
effector phases (Vitetta et al, Adv. Immunol., 45:1 (1989); Noelle
et al, Immunol. Today, 11:361 (1990)). In the inductive phase,
resting T cells contact antigen-primed B cells and this association
allows clonotypic T cell receptor (TCR)-CD4 complexes to interact
with Ia/Ag complexes on B cells (Janeway et al, Immunol. Rev.,
101:39 (1988); Katz et al, Proc. Natl. Acad. Sci., 70:2624 (1973);
Zinkernagel, Adv. Exp. Med., 66:527 (1976); Sprent, J. Exp. Med.,
147:1159 (1978); Sprent, Immunol. Rev., 42:158 (1978); Jones et al,
Nature, 292:547 (1981); Julius et al, Eur. J. Immunol., 18:375
(1982); Chestnut et al, J. Immunol., 126:1575 (1981); and
Rogozinski et al, J. Immunol., 126:735 (1984)). TCR/CD4 recognition
of Ia/Ag results in the formation of stable T-B cognate pairs and
bi-directional T and B cell activation (Sanders et al, J. Immunol.,
137:2395 (1986); Snow et al, J. Immunol., 130:614 (1983);
Krusemeier et al, J. Immunol., 140:367 (1988); Noelle et al, J.
Immunol., 143:1807 (1989); Bartlett et al, J. Immunol., 143:1745
(1989); and Kupfer et al, Annu. Rev. Immunol., 7:309 (1987)). In
the effector phase, activated T cells drive B cell differentiation
by secreting lymphokines (Thompson et al, J. Immunol., 134:369
(1985)) and by contact-dependent stimuli (Noelle et al, J.
Immunol., 143:1807 (1989); Clement et al, J. Immunol., 140:3736
(1984); Crow et al, J. Exp. Med., 164:1760 (1986); Brian, Proc.
Natl. Acad. Sci., USA, 85:564 (1988); Hirohata et al, J. Immunol.
140:3736 (1988); Jover et al, Clin. Immunol. Immun., 53:90 (1989);
Whalen et al, J. Immunol., 141:2230 (1988); Pollok et al, J.
Immunol., 146:1633 (1991); and Bartlett et al, J. Immunol.,
143:1745 (1990)), both of which are required for T cells to drive
small resting B cells to terminally differentiate into Ig secreting
cells (Clement et al, J. Immunol., 132:740 (1984); Martinez et al,
Nature, 290:60 (1981); and Andersson et al, Proc. Natl. Acad. Sci.,
USA, 77:1612 (1980)).
[0006] Although the inductive phase of T cell help is Ag-dependent
and MHC-restricted (Janeway et al, Immun. Rev., 101:34 (1988); Katz
et al, Proc. Natl. Acad. Sci., USA, 10:2624 (1973); Zinkernagle,
Adv. Exp. Med. Biol., 66:527 (1976)); the effector phase of T cell
helper function can be Ag-independent and MHC-nonrestricted
(Clement et al, J. Immunol., 132:740 (1984); Hirohata et al, J.
Immunol., 140:3736 (1988); Whalen et al, J. Immunol., 143:1715
(1988)). An additional contrasting feature is that the inductive
phase of T cell help often requires CD4 molecules and is inhibited
by anti-CD4 mAb (Rogozinski et al, J. Immunol., 126:735 (1984)),
whereas helper effector function does not require CD4 molecules
(Friedman et al, Cell Immunol., 103:105 (1986)) and is not
inhibited by anti-CD4 mabs (Brian, Proc. Natl. Acad. Sci., USA,
85:564 (1988); Hirohata et al, J. Immunol., 140:3736 (1988); Whalen
et al, J. Immunol., 143:1745 (1988); and Tohma et al, J. Immunol.,
146:2547 (1991)). The non-specific helper effector function is
believed to be focused on specific B cell targets by the localized
nature of the T-B cell interactions with antigen specific, cognate
pairs (Bartlett et al, J. Immunol., 143:1745 (1989); Kupfer et al,
J. Exp. Med., 165:1565 (1987) and Poo et al, Nature, 332:378
(1988)).
[0007] Although terminal B cell differentiation requires both
contact- and lymphokine-mediated stimuli from T cells, intermediate
stages of B cell differentiation can be induced by activated T cell
surfaces in the absence of secreted factors (Crow et al, J. Exp.
Med., 164:1760 (1986); Brian, Proc. Natl. Acad. Sci., USA, 85:564
(1988); Sekita et al, Eur. J. Immunol., 18:1405 (1988); Hodgkin et
al, J. Immunol., 145:2025 (1990); Noelle et al, FASEB J, 5:2770
(1991)). These intermediate effects on B cells include induction of
surface CD23 expression (Crow et al, Cell Immunol., 121:94 (1989)),
enzymes associated with cell cycle progression (Pollok et al, J.
Immunol., 146:1633 (1991)) and responsiveness to lymphokines
(Noelle et al, FASEB J, 5:2770 (1989); Pollok et al, J. Immunol.,
146:1633 (1991)). Recently some of the activation-induced T cell
surface molecules that direct B cell activation have been
identified. Additionally, functional studies have characterized
some features of activation-induced T cell surface molecules that
direct B cell activation. First, T cells acquire the ability to
stimulate B cells 4-8 h following activation (Bartlett et al, J.
Immunol., 145:3956 (1990) and Tohma et al, J. Immunol., 146:2544
(1991)). Second, the B cell stimulatory activity associated with
the surfaces of activated T cells is preserved on paraformaldehyde
fixed cells (Noelle et al, J. Immunol., 143:1807 (1989); Cros et
al, J. Exp. Med., 164:1760 (1986); Pollok et al, J. Immunol.,
146:1633 (1991); Tohma et al, J. Immunol., 146:2544 (1991); and
Kubota et al, Immunol., 72:40 (1991)) and on purified membrane
fragments (Hodgkin et al, J. Immunol., 145:2025 (1990) and Martinez
et al, Nature, 290:60 (1981)). Third, the B cell stimulatory
activity is sensitive to protease treatment (Noelle et al, J.
Immunol., 143:1807 (1989); Sekita et al, Eur. J. Immunol., 18:1405
(1988); and Hodgkin et al, J. Immunol., 145:2025 (1990). Fourth,
the process of acquiring these surface active structures following
T cell activation is inhibited by cycloheximide (Tohma et al, J.
Immunol., 196:2349 (1991) and Hodgkin et al, J. Immunol., 195:2025
(1990)).
[0008] A cell surface molecule, CD40, has been identified on
immature and mature B lymphocytes which, when crosslinked by
antibodies, induces B cell proliferation. Valle et al, Eur. J.
Immunol., 19:1463-1467 (1989); Gordon et al, J. Immunol.,
140:1425-1430 (1988); Gruder et al, J. Immunol., 142:4144-4152
(1989).
[0009] CD40 has been molecularly cloned and characterized
(Stamenkovic et al, EMBO J., 8:1403-1410 (1989)).
[0010] CD40 is expressed on B cells, interdigitating dendritic
cells, macrophages, follicular dendritic cells, and thymic
epithelium (Clark, Tissue Antigens 36:33 (1990); Alderson et al, J.
Exp. Med., 178:669 (1993); Galy et al, J. Immunol. 142:772 (1992)).
Human CD40 is a type I membrane protein of 50 kDa and belongs to
the nerve growth factor receptor family (Hollenbaugh et al,
Immunol. Rev., 138:23 (1994)). Signaling through CD40 in the
presence of IL-10 induces IgA, IgM and IgG production, indicating
that isotype switching is regulated through these interactions. The
interaction between CD40 and its ligand results in a primed state
of the B cell, rendering it receptive to subsequent signals.
[0011] Also, a ligand for CD40, gp39 (also called CD40 ligand or
CD40L) has recently been molecularly cloned and characterized
(Armitage et al, Nature, 357:80-82 (1992); Lederman et al, J. Exp.
Med., 175:1091-1101 (1992); Hollenbaugh et al, EMBO J.,
11:4313-4319 (1992)). The gp39 protein is expressed on activated,
but not resting, CD4.sup.+ Th cells. Spriggs et al, J. Exp. Med.,
176:1543-1550 (1992); Lane et al, Eur. J. Immunol., 22:2573-2578
(1992); and Roy et al, J. Immunol., 151:1-14 (1993). Cells
transfected with gp39 gene and expressing the gp39 protein on their
surface can trigger B cell proliferation and, together with other
stimulatory signals, can induce antibody production. Armitage et
al, Nature, 357:80-82 (1992); and Hollenbaugh et al, EMBO J.,
11:4313-4319 (1992). In particular, the ligand for CD40, gp39, has
been identified for the mouse (Noelle et al, Proc. Natl. Acad. Sci.
USA, 89:6550 (1992); Armitage et al, Nature, 357:80 (1992)) and for
humans (Hollenbaugh et al, Embo. J. 11:4313 (1992); Spriggs et al,
J. Exp. Met., 176:1543 (1992)). gp39 is a type II membrane protein
and is part of a new gene super family which includes TNF-.alpha.,
TNF-.beta. and the ligands for FAS, CD27, CD30 and 4-1BB.
[0012] Expression of gp39 can be readily induced in vitro on
CD4.sup.+ T cells using either anti-CD3 antibody or phorbol
myristate acetate (PMA) plus ionomycin. Expression is rapid and
transient, peaking at 6-8 hours and returning to near resting
levels between 24 and 48 hours (Roy et al, J. Immunol., 151:2497
(1993)). In vivo, gp39 has been reported in humans to be present on
CD4.sup.+ T cells in the mantle and centrocytic zones of lymphoid
follicles and the periarteriolar lymphocyte sheath of the spleen,
in association with CD40.sup.+ B cells (Lederman et al, J.
Immunol., 149:3807 (1992)). gp39.sup.+ T cells produce IL-2, IL-4
and IFN-.gamma. (Van der Eetwegh et al, J. Exp. Med., 178:1555
(1993)).
[0013] Unique insights into the novel role of gp39 in the
regulation of humoral immunity have been provided by studies of a
human disease, X-linked hyper-IgM syndrome (HIM). HIM is a
profound, X-linked immunodeficiency typified by a loss in thymus
dependent humoral immunity, the inability to produce IgG, IgA and
IgE. Mutations in the gp39 gene were responsible for the expression
of a non-functional gp39 protein and the inability of the helper T
cells from HIM patients to activate B cells (Allen et al, Science,
259:990 (1993); Aruffo et al, Cell, 72:291 (1993); DiSanto et al,
Nature, 361:541 (1993); Korthauer et al, Nature, 361:539 (1993)).
These studies support the conclusion that early after T cell
receptor engagement of the peptide/MHC class II complex, gp39 is
induced on the cognate helper T cell, and the binding of gp39 to
CD40 on the B cell induces the B cell to move into the cell cycle
and differentiate to immunoglobulin (Ig) secretion and isotype
switching.
[0014] Functional studies have shown that treatment of mice with
anti-gp39 completely abolished the antibody response against thymus
dependent antigens (SRBC and TNP-KLH), but not thymus independent
antigens (TNP-Ficoll) (Foy et al, J. Exp. Med., 178:1567 (1993)).
In addition, treatment with anti-gp39 prevented the development of
collagen-induced arthritis (CIA) in mice injected with collagen
(Durie et al, Science, 261:1328 (1993)). Finally, anti-gp39
prevented formation of memory B cells and germinal centers in mouse
spleen (Foy et al, J. Exp. Med., 180:157 (1994)). Collectively,
these data provide extensive evidence that the interaction between
gp39 on T cells and CD40 on B cells is essential for antibody
responses against thymus dependent antigens.
[0015] Recently, a number of murine models of autoimmune disease
have been exploited to evaluate the potential therapeutic value of
anti-gp39 administration on the development of disease. A brief
discussion of the results of studies in these models are provided
below:
Collagen-Induced Arthritis:
[0016] CIA is an animal model for the human autoimmune disease
rheumatoid arthritis (RA) (Trenthorn et al, J. Exp. Med., 146:857
(1977)). This disease can be induced in many species by the
administration of heterologous type II collagen (Courtenay et al,
Nature, 283:665 (1980); Cathcart et al, Lab. Invest., 54:26
(1986)).
[0017] To study the effect anti-gp39 on the induction of CIA (Durie
et al, Science, 261:1328 (1993)) male DBA1/J mice were injected
intradermally with chick type II collagen emulsified in complete
Freund's adjuvant at the base of the tail. A subsequent challenge
was carried out 21 days later. Mice were then treated with the
relevant control antibody or anti-gp39. Groups of mice treated with
anti-gp39 showed no titers of anti-collagen antibodies compared to
immunized, untreated control mice. Histological analysis indicated
that mice treated with anti-gp39 antibody showed no signs of
inflammation or any of the typical pathohistological manifestations
of the disease observed in immunized animals. These results
indicated that gp39-CD40 interactions are absolutely essential in
the induction of CIA. If the initial cognate interaction between
the T cell and B cell is not obtained, then the downstream
processes, such as autoantibody formation and the resulting
inflammatory responses, do not occur.
[0018] Recently it has been shown that gp39 is important in
activating monocytes to produce TNF-.alpha. and IL-6 in the absence
of GM-CSF, IL-3 and IFN-.gamma. (Alderson et al, J. Exp. Med.,
178:669 (1993)). TNF-.alpha. has been implicated in the CIA disease
process (Thorbecke et al, Eur. J. Immunol., 89:7375 (1992) and in
RA (DiGiovane et al, Ann. Rheum. Dis., 47:68 (1988); Chu et al,
Arthrit. Rheum., 39:1125 (1991); Brennan et al, Eur. J. Immunol.,
22:1907 (1992). Thus, inhibition of TNF-.alpha. by anti-gp39 may
have profound anti-inflammatory effects in the joints of arthritic
mice. Both inhibition of TNF-.alpha. and of T cell-B cell
interactions by anti-gp39 may be contributory to manifestations of
CIA.
Experimental Allergic Encephalomyelitis (EAE):
[0019] EAE is an experimental autoimmune disease of the central
nervous system (CNS) (Zamvil et al, Ann. Rev. Immunol., 8:579
(1990) and is a disease model for the human autoimmune condition,
multiple sclerosis (MS) (Alvord et al, "Experimental Allergic Model
for Multiple Sclerosis," NY 511 (1984)). It is readily induced in
mammalian species by immunizations of myelin basic protein purified
from the CNS or an encephalitogenic proteolipid (PLP). SJL/J mice
are a susceptible strain of mice (H-2.sup.S) and, upon induction of
EAE, these mice develop an acute paralytic disease and an acute
cellular infiltrate is identifiable within the CNS.
[0020] Classen and co-workers (unpublished data) have studied the
effects of anti-gp39 on the induction of EAE in SJL/J mice. They
found that EAE development was completely suppressed in the
anti-gp39 treated animals. In addition, anti-PLP antibody responses
were delayed and reduced compared to those obtained for control
animals.
[0021] EAE is an example of a cell-mediated autoimmune disease
mediated via T cells, with no direct evidence for the requirement
for autoantibodies in disease progression. Interference with the
interaction between gp39 and CD40 prevents disease induction and
the adoptive transfer of disease.
Chronic (c) and Acute (a) Graft-Versus-Host-Disease (GVHD):
[0022] Chronic and acute GVHD result from donor cells responding to
host disparate MHC alleles. In cGVHD (H-2.sup.d->H-2.sup.bd),
heightened polyclonal immunoglobulin production is due to the
interaction of allospecific helper T cells and the host B cells. In
vivo administration of anti-gp39 antibody blocked cGVHD-induced
serum anti-DNA autoantibodies, IgE production, spontaneous
immunoglobulin production in vitro, associated splenomegaly and the
ability to transfer disease. Durie F. H. et al, J. Clin. Invest.,
94:133 (1994). Antibody production remained inhibited for extended
periods of time after termination of anti-gp39 administration.
Anti-allogeneic cytotoxic T lymphocyte (CTL) responses induced in
GVHD were also prevented by the in vivo administration of
anti-gp39. These data suggest that CD40-gp39 interactions are
critical in the generation of both forms of GVHD. The fact that CTL
responses were inhibited and a brief treatment with anti-gp39
resulted in long-term prevention of disease suggest permanent
alterations in the T cell compartment by the co-administration of
allogeneic cells and anti-gp39 antibody.
[0023] Various research groups have reported the production of
murine antibodies specific to gp39, which are disclosed to possess
therapeutic utility as immunosuppressants. For example, WO
93/09812, published May 27, 1993, and assigned to Columbia
University; EP 0,555,880, published Aug. 18, 1993, and PCT
US/94/09872, filed Sep. 2, 1994 by Noelle et al and assigned to
Dartmouth College, describe murine antibodies specific to gp39 and
their use as therapeutics and immunosuppressants.
[0024] Also, Scaria et al, Gene Therapy, 4:611-617 (1997) report
the use of an antibody to gp39 to inhibit humoral and cellular
immune responses to a DNA (adenoviral/vector) However, while murine
antibodies have applicability as therapeutic agents in humans, they
are disadvantageous in some respects. Specifically, murine
antibodies, because of the fact that they are of foreign species
origin, may be immunogenic in humans. This often results in a
neutralizing antibody response, which is particularly problematic
if the antibodies are desired to be administered repeatedly, e.g.,
in treatment of a chronic or recurrent disease condition. Also,
because they contain murine constant domains they may not exhibit
human effector functions.
[0025] In an effort to eliminate or reduce such problems, chimeric
antibodies have been disclosed. Chimeric antibodies contain
portions of two different antibodies, typically of two different
species. Generally, such antibodies contain human constant and
another species, typically murine variable regions. For example,
some mouse/human chimeric antibodies have been reported which
exhibit binding characteristics of the parental mouse antibody, and
effector functions associated with the human constant region. See,
e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Shoemaker et al.,
U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. No. 4,975,369;
and Boss et al., U.S. Pat. No. 4,816,397, all of which are
incorporated by reference herein. Generally, these chimeric
antibodies are constructed by preparing a genomic gene library from
DNA extracted from pre-existing murine hybridomas (Nishimura et al,
Cancer Research, 47:999 (1987)). The library is then screened for
variable region genes from both heavy and light chains exhibiting
the correct antibody fragment rearrangement patterns.
Alternatively, cDNA libraries are prepared from RNA extracted from
the hybridomas and screened, or the variable regions are obtained
by polymerase chain reaction. The cloned variable region genes are
then ligated into an expression vector containing cloned cassettes
of the appropriate heavy or light chain human constant region gene.
The chimeric genes are then expressed in a cell line of choice,
usually a murine myeloma line. Such chimeric antibodies have been
used in human therapy.
[0026] In a commonly assigned application, Ser. No. 07/912,292,
"Primatized,".TM. antibodies are disclosed which contain human
constant and Old World monkey variable regions. These
Primatized.TM. antibodies are well tolerated in humans given their
low or weak immunogenicity.
[0027] Also, humanized antibodies are known in the art. Ideally,
"humanization" results in an antibody that is less immunogenic,
with complete retention of the antigen-binding properties of the
original molecule. In order to retain all the antigen-binding
properties of the original antibody, the structure of its
combining-site has to be faithfully reproduced in the "humanized"
version. This can potentially be achieved by transplanting the
combining site of the nonhuman antibody onto a human framework,
either (a) by grafting the entire nonhuman variable domains onto
human constant regions to generate a chimeric antibody (Morrison et
al, Proc. Natl. Acad. Sci., USA, 81:6801 (1984); Morrison and Oi,
Adv. Immunol., 44:65 (1988) (which preserves the ligand-binding
properties, but which also retains the immunogenicity of the
nonhuman variable domains); (b) by grafting only the nonhuman CDRs
onto human framework and constant regions with or without retention
of critical framework residues (Jones et al, Nature, 321:522
(1986); Verhoeyen et al, Science, 239:1539 (1988)); or (c) by
transplanting the entire nonhuman variable domains (to preserve
ligand-binding properties) but also "cloaking" them with a
human-like surface through judicious replacement of exposed
residues (to reduce antigenicity) (Padlan, Molec. Immunol., 28:489
(1991)).
[0028] Essentially, humanization by CDR grafting involves
transplanting only the CDRs onto human fragment onto human
framework and constant regions. Theoretically, this should
substantially eliminate immunogenicity (except if allotypic or
idiotypic differences exist). However, it has been reported that
some framework residues of the original antibody also need to be
preserved (Riechmann et al, Nature, 332:323 (1988); Queen et al,
Proc. Natl. Acad. Sci. USA, 86:10,029 (1989)).
[0029] The framework residues which need to be preserved can be
identified by computer modeling. Alternatively, critical framework
residues may potentially be identified by comparing known antibody
combining site structures (Padlan, Molec. Immun., 31(3):169-217
(1994)).
[0030] The residues which potentially affect antigen binding fall
into several groups. The first group comprises residues that are
contiguous with the combining site surface which could therefore
make direct contact with antigens. They include the amino-terminal
residues and those adjacent to the CDRs. The second group includes
residues that could alter the structure or relative alignment of
the CDRs either by contacting the CDRs or the opposite chains. The
third group comprises amino acids with buried side chains that
could influence the structural integrity of the variable domains.
The residues in these groups are usually found in the same
positions (Padlan, 1994 (Id.) according to the adopted numbering
system (see Kabat et al, "Sequences of proteins of immunological
interest, 5th ed., Pub. No. 91-3242, U.S. Dept. Health & Human
Services, NIH, Bethesda, Md., 1991).
[0031] However, while humanized antibodies are desirable because of
their potential low immunogenicity in humans, their production is
unpredictable. For example, sequence modification of antibodies may
result in substantial or even total loss of antigen binding
function, or loss of binding specificity. Alternatively, "humanized
antibodies" may still exhibit immunogenicity in humans,
irrespective of sequence modification.
[0032] Thus, there still exists a significant need in the art for
novel humanized antibodies to desired antigens. More specifically,
there exists a need in the art for humanized antibodies specific to
gp39, because of their potential as immunotherapeutic agents.
OBJECTS OF THE INVENTION
[0033] Toward this end, it is an object of the invention to provide
humanized antibodies which are specific to human gp39.
[0034] More specifically, it is an object of the invention to
provide humanized antibodies derived from murine antibodies to gp39
and in particular 24-31, a specific murine antibody which binds to
human gp39.
[0035] It is also an object of the invention to provide
pharmaceutical compositions containing humanized antibodies which
are specific to human gp39.
[0036] It is a more specific object of the invention to provide
pharmaceutical compositions containing humanized antibodies derived
from 24-31, a murine antibody which specifically binds to human
gp39.
[0037] It is another specific object of the invention to provide
methods of using humanized antibodies to human gp39 for treatment
of human disease conditions, which are treatable by modulation of
gp39 expression and/or inhibition of the gp39/CD40 binding
interaction including, e.g., autoimmune diseases such as systemic
lupus erythematosus, rheumatoid arthritis, multiple sclerosis,
idiopathic thrombocytopenic purpura (ITP), diabetes and
non-autoimmune conditions such as graft-versus-host disease and
transplantation.
[0038] It is still another object of the invention to provide
nucleic acid sequences which encode for humanized antibodies to
human gp39.
[0039] It is a more specific object of the invention to provide
nucleic acid sequences which encode humanized antibodies derived
from 24-31, a murine antibody which specifically binds to human
gp39 antigen.
[0040] It is another object of the invention to provide vectors
which provide for the expression of humanized antibodies to human
gp39, in particular humanized antibodies derived from 24-31, a
murine antibody which specifically binds to human gp39 antigen.
SUMMARY OF THE INVENTION
[0041] In its broadest embodiment, the present invention is
directed to humanized antibodies which retain not less than about
one-tenth and more preferably not lower than one-third the gp39
antigen binding affinity of the murine 24-31 antibody and/or which
retain not less than about one-tenth and more preferably not less
than about one-third the in vitro functional activity of the murine
antibody 24-31, e.g., in B-cell assays which measure T-cell
dependent antibody production. More particularly, the present
humanized antibodies retain at least one-tenth and more preferably
at least about one-third the half-maximal potency in in vitro
functional activity in a B cell assay at a concentration of not
more than three times the concentration of the 24-31 antibody.
[0042] The present invention is further directed to humanized
antibodies which bind to the same epitope as the murine 24-31
antibody and/or which are capable of competing with the murine
24-31 antibody for inhibiting the binding of CD40 to gp39 and/or
which contain the CDR's of the 24-31 antibody.
[0043] The present invention is more preferably directed to
humanized antibodies derived from murine 24-31 which possess the
humanized variable light sequences and/or humanized variable heavy
sequences set forth below: TABLE-US-00001 (1)
DIVMTQSPSFLSASVGDRVTITC KASQNVITAVA WYQQKPGKSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSLQPEDFADYFC QQYNSYPYT FGGGTKLEIK; (2)
DIVMTQSPDSLAVSLGERATINC KASQNVITAVA WYQQKPGQSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSLQAEDVADYFC QQYNSYPYT FGGGTKLEIK; (3)
DIVMTQSPSFMSTSVGDRVTITC KASQNVITAVA WYQQKPGKSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSMQPEDFADYFC QQYNSYPYT FGGGTKLEIK; (4)
DIVMTQSPDSMATSLGERVTINC KASQNVITAVA WYQQKPGQSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSMQAEDVADYFC QQYNSYPYT FGGGTKLEIK
[0044] and a humanized variable heavy sequence selected from the
following group: TABLE-US-00002 (1) EVQLQESGPGLVKPSETLSLTCTVSGDSIT
NGFWI WIRKPPGNK LEYMG YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAA
DTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS; (2)
EVQLQESGPGLVKPSQTLSLTCTVSGDSIT NGFWI WIRKHPGNK LEYMG
YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAA DTGVYYCAC RSYGRTPYYFDF
WGQGTTLTVSS; (3) EVQLQESGPGLVKPSQTLSLTCAVSGDSIT NGFWI WIRKHPGNK
LEYMG YISYSGSTYYNPSLKS RISISRDTSNNQFSLNLNSVTRA DTGVYYCAC
RSYGRTPYYFDF WGQGTTLTVSS; (4) EVQLQESGPGLVKPSETLSLTCAVYGDSIT NGFWI
WIRKPPGNK LEYMG YISYSGSTYYNPSLKS RISISRDTSKNQFYLKLSSVTAA DTGVYYCAC
RSYGRTPYYFDF WGQGTTLTVSS
as well as variants and equivalents thereof. Variants and
equivalents thereof in the present invention are intended to
embrace humanized immunoglobulin sequences wherein one or several
of the amino acid residues in the above identified humanized
variable heavy and/or variable light sequences are modified by
substitution, addition and/or deletion in such manner that does not
substantially effect gp39 antigen binding affinity. In particular,
the present invention embraces variants and equivalents which
contain conservative substitution mutations, i.e., the substitution
of one or more amino acids by similar amino acids. For example,
conservative substitution refers to the substitution of an amino
acid within the same general class, e.g., an acidic amino acid, or
a basic amino acid, a neutral amino acid by another amino acid
within the same class. What is intended by a conservative amino
acid substitution is well known in the art. Preferably, such
variants and equivalents will retain not less than about one-tenth
and more preferably not less than about one-third the gp39 antigen
binding affinity as the parent murine 24-31 antibody and more
preferably not less than about one-third the gp39 antigen binding
affinity as the murine 24-31 antibody. Additionally, such variants
and equivalents will preferably retain not lower than one-tenth and
more preferably retain at least about one-third the in vitro
functional activity of murine antibody 24-31, e.g., in B-cell
assays which measure T-cell dependent antibody production. More
preferably, these variants and equivalents will retain at least
about one-third the in vitro functional activity of murine antibody
24-31, for example, in B-cell assays which measure T-cell dependent
antibody production. More specifically, these antibodies will
retain the half-maximal potency in in vitro functional activity in
a B cell assay at a concentration of not more than about three
times the concentration of the parent 24-31 antibody.
[0045] The present invention is further directed to nucleic acid
sequences which encode for the expression of such humanized
antibodies, as well as expression vectors which provide for the
production of humanized antibodies in recombinant host cells. In
the most preferred embodiments these DNA sequences will encode for
the humanized variable heavy and/or humanized variable light
sequences set forth below: TABLE-US-00003 (1)
DIVMTQSPSFLSASVGDRVTITC KASQNVITAVA WYQQKPGKSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSLQPEDFADYFC QQYNSYPYT FGGGTKLEIK; (2)
DIVMTQSPDSLAVSLGERATINC KASQNVITAVA WYQQKPGQSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSLQAEDVADYFC QQYNSYPYT FGGGTKLEIK; (3)
DIVMTQSPSFMSTSVGDRVTITC KASQNVITAVA WYQQKPGKSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSMQPEDFADYFC QQYNSYPYT FGGGTKLEIK; (4)
DIVMTQSPDSMATSLGERVTINC KASQNVITAVA WYQQKPGQSP KLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSMQASDVADYFC QQYNSYPYT FGGGTKLEIK
[0046] and a humanized variable heavy sequence selected from the
following group: TABLE-US-00004 (1) EVQLQESGPGLVKPSETLSLTCTVSGDSIT
NGFWI WIRKPPGNK LEYMG YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAA
DTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS; (2)
EVQLQESGPGLVKPSQTLSLTCTVSGDSIT NGFWI WIRKHPGNK LEYMG
YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAA DTGVYYCAC RSYGRTPYYFDF
WGQGTTLTVSS; (3) EVQLQESGPGLVKPSQTLSLTCAVSGDSIT NGFWI WIRKHPGNK
LEYMG YISYSGSTYYNPSLKS RISISRDTSNNQFSLNLNSVTRA DTGVYYCAC
RSYGRTPYYFDF WGQGTTLTVSS; (4) EVQLQESGPGLVKPSETLSLTCAVYGDSIT NGFWI
WIRKPPGNK LEYMG YISYSGSTYYNPSLKS RISISRDTSKNQFYLKLSSVTAA DTGVYYCAC
RSYGRTPYYFDF WGQGTTLTVSS.
[0047] Moreover, the present invention also embraces equivalent and
variants thereof as defined supra.
[0048] The present invention is further directed to the use of the
above-identified humanized antibodies specific to gp39 as
pharmaceuticals. The present invention is also directed to the use
of the subject humanized anti-gp39 antibodies for treating diseases
treatable by modulation of gp39 expression or by inhibition of the
gp39/CD40 interaction. The present invention is more particularly
directed to the use of humanized antibodies of the above-identified
humanized antibodies specific to gp39 for the treatment of
autoimmune disorders, for example, rheumatoid arthritis, multiple
sclerosis, diabetes, systemic lupus erythematosus and ITP. The
present invention is further directed to the use of the subject
humanized antibodies to gp39 for the treatment of non-autoimmune
disorders including graft-versus-host disease and for inhibiting
graft rejection.
[0049] Also, the subject invention is further directed to usage of
the subject humanized antibodies as immunosuppressants, in
particular during gene or cellular therapy. The subject humanized
antibodies should enhance the efficacy of gene therapy or cellular
therapy by inhibiting adverse immunogenic reaction to vectors and
cells used therein. For example, they may be used to inhibit
humoral and cellular immune responses against viral vectors, e.g.,
retro-viral vectors, adenoviral vectors. Also, the use of such
antibodies should enable such cells or vectors to be administered
repeatedly, which will facilitate treatment of chronic diseases
such as cancers and autoimmune diseases.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIG. 1 depicts the IDEC expression vector N5KG1 used to
express humanized and chimeric antibodies derived from 24-31.
[0051] FIG. 2a contains results of a B cell proliferation assay
which contacts human PBLs with soluble gp39-CD8, recombinant human
IL-4 and the murine 24-31 antibody or control murine IgG1
monoclonal antibody which demonstrate that 24-31 antibody inhibits
B cell proliferation induced by gp39.
[0052] FIG. 2B contains results of B cell differentiation assay
using mitomycin treated T cells activated with immobilized anti-CD3
cultured in the present of IGD.sup.+ B cells and different
concentrations of the 24-31 antibody which demonstrate that 24-31
antibody inhibits T-cell dependent polyclonal antibody production
by human B cells.
[0053] FIG. 3 contains FACS of non-transfected CHO cells and a gp39
transfectant.
[0054] FIG. 4 contains the amino acid sequence and DNA sequence
corresponding to a preferred humanized variable light sequence
(including the complementarity determining regions) referred to as
VL#1 or preferred humanized variable light sequence (1).
[0055] FIG. 5 contains the amino acid and DNA sequence
corresponding to a preferred humanized variable ligand sequence
(including the complementarity determining regions) referred to as
VL#2 or preferred humanized variable light sequence (2).
[0056] FIG. 6 contains the amino acid and DNA sequence
corresponding to a preferred humanized variable heavy sequence
(including the complementarity determining regions) referred to as
VH#1 of preferred humanized variable heavy sequence (1).
[0057] FIG. 7 contains the amino acid and DNA sequence of the
variable light sequence of 24-31 (non-humanized) FIG. 8 contains
the amino acid and DNA sequence of the variable heavy sequence of
24-31 (non-humanized) FIG. 9 compares binding of murine 24-31,
chimeric 24-31 and a humanized 24-31 antibody to gp39 expressing
CHO cells.
[0058] FIG. 10 contains results of a competition assay comparing
the binding of 24-31 (biotin) and humanized, chimeric and 24-31 to
gp39 expressing CHO cells.
[0059] FIG. 11 contains results of an assay which measures effects
of murine 24-31 and a humanized 24-31 antibody of the invention on
human IgM production by B cells cultured in the presence of
mitomycin C treated T cells.
[0060] FIG. 12 contains results of an assay comparing binding of
two humanized antibodies of the present invention to gp39
expressing CHO cells.
[0061] FIG. 13 contains the Scatchard plot for murine 24-31.
[0062] FIG. 14 contains the Scatchard plot for humanized Version
1.
[0063] FIG. 15 contains the Scatchard plot for humanized Version
2.
DETAILED DESCRIPTION OF THE INVENTION
[0064] Prior to setting forth the invention, definitions of certain
terms which are used in this disclosure are set forth below:
[0065] Humanized antibody--This will refer to an antibody derived
from a non-human antibody, typically murine, that retains or
substantially retains the antigen-binding properties of the parent
antibody but which is less immunogenic in humans. This may be
achieved by various methods including (a) grafting the entire
non-human variable domains onto human constant regions to generate
chimeric antibodies, (b) grafting only the non-human CDRs onto
human framework and constant regions with or without retention of
critical framework residues, or (c) transplanting the entire
non-human variable domains, but "cloaking" them with a human-like
section by replacement of surface residues. Such methods are
disclosed in Jones et al, Morrison et al, Proc. Natl. Acad. Sci.,
81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92
(1988); Verhoeyen et al, Science, 239:1534-1536 (1988); Padlan,
Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun.,
31(3):169-217 (1994), all of which are incorporated by
reference.
[0066] Complementarity Determining Region or CDR--The term CDR, as
used herein, refers to amino acid sequences which together define
the binding affinity and specificity of the natural Fv region of a
native immunoglobulin binding site as delineated by Kabat et al
(1991).
[0067] Framework Region--The term FR, as used herein, refers to
amino acid sequences interposed between CDRs. These portions of the
antibody serve to hold the CDRs in appropriate orientation (allows
for CDRs to bind antigen).
[0068] Constant Region--The portion of the antibody molecule which
confers effector functions. In the present invention, murine
constant regions are substituted by human constant regions. The
constant regions of the subject chimeric or humanized antibodies
are derived from human immunoglobulins. The heavy chain constant
region can be selected from any of the five isotypes: alpha, delta,
epsilon, gamma or mu. Further, heavy chains of various subclasses
(such as the IgG subclasses of heavy chains) are responsible for
different effector functions and thus, by choosing the desired
heavy chain constant region, chimeric antibodies with desired
effector function can be produced. Preferred constant regions are
gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4). More preferred
is an Fc region of the gamma 1 (IgG1) isotype. The light chain
constant region can be of the kappa or lambda type, preferably of
the kappa type.
[0069] Chimeric antibody--This is an antibody containing sequences
derived from two different antibodies, which typically are of
different species. Most typically chimeric antibodies comprise
human and murine antibody fragments, generally human constant and
murine variable regions.
[0070] Immunogenicity--A measure of the ability of a targeting
protein or therapeutic moiety to elicit an immune response (humoral
or cellular) when administered to a recipient. The present
invention is concerned with the immunogenicity of the subject
humanized antibodies or fragments thereof.
[0071] Humanized or chimeric antibody of reduced
immunogenicity--This refers to an antibody or humanized antibody
exhibiting reduced immunogenicity relative to the parent antibody,
e.g., the 24-31 antibody.
[0072] Humanized antibody substantially retaining the binding
properties of the parent antibody--This refers to a humanized or
chimeric antibody which retains the ability to specifically bind
the antigen recognized by the parent antibody used to produce such
humanized or chimeric antibody. Humanized or chimeric antibodies
which substantially retain the binding properties of 24-31 will
bind to human gp39. Preferably the humanized or chimeric antibody
will exhibit the same or substantially the same antigen-binding
affinity and avidity as the parent antibody. Ideally, the affinity
of the antibody will not be less than 10% of the parent antibody
affinity, more preferably not less than about 30%, and most
preferably the affinity will not be less than 50% of the parent
antibody. Methods for assaying antigen-binding affinity are well
known in the art and include half-maximal binding assays,
competition assays, and Scatchard analysis. Suitable antigen
binding assays are described in this application.
[0073] The present invention is directed to novel humanized
monoclonal antibodies which bind human gp39 and their use as
therapeutic agents. The present invention is further directed
toward nucleic acid sequences which encode said humanized
antibodies, and their expression in recombinant host cells.
[0074] More specifically, the present invention is directed toward
humanized antibodies derived from murine antibody 24-31 which
specifically binds to human gp39.
[0075] Murine antibody 24-31 is a murine antibody raised against
human gp39 which functionally inactivates gp39 both in vitro and in
vivo. Therefore, it possesses properties which render it
potentially useful for treatment of diseases wherein gp39
inactivation and/or modulation or inhibition of the gp39/CD40
interaction is desirable. In particular, such diseases include
autoimmune diseases such as, e.g., rheumatoid arthritis, multiple
sclerosis, ITP, diabetes, and systemic lupus erythematosus as well
as non-auto-immune diseases such as graft-versus-host disease and
graft rejection.
[0076] However, while murine antibody 24-31 possesses functional
properties which render it potentially suitable as a therapeutic
agent, it possesses several potential disadvantages. Namely,
because it is of murine origin it potentially will be immunogenic
in humans. Also, because it contains murine constant sequences, it
will likely not exhibit the full range of human effector functions
and will probably be more rapidly cleared if administered to
humans. While such disadvantages should not be problematic in the
treatment of some disease conditions or persons, they pose
substantial concern if the disease treated is of a chronic or
recurrent nature. Examples of recurrent or chronic diseases
include, e.g., autoimmune diseases, wherein the host continually or
chronically exhibits an autoimmune reaction against
self-antigens.
[0077] Therefore, in order to alleviate the disadvantages
associated with murine antibody 24-31, namely potential
immunogenicity in humans and decrease of human effector functions,
the present inventors desired to produce improved, humanized
derivatives of the murine 24-31 antibody. While this was the goal
of the present invention, the desired result was not of a routine
or predictable nature. Humanization of antibodies requires the
careful selection of amino acid residues which are to be modified,
and the judicious selection of residues which are to be substituted
therefor. This is because modification of antibody variable
regions, even those involving a few amino acid residues, may cause
substantial deleterious effects on antigen binding. For example,
humanized antibodies may exhibit substantially reduced antigen
affinity and/or antigen-specificity in relation to the parent
antibody.
[0078] As noted supra, different methods of humanization of
antibodies, including murine antibodies have been reported in the
literature. See, e.g., Padlan, Molec. Immunol., 31(3):169-217
(1994); Padlan, Molec. Immunol., 28:484-498 (1991); Morrison and
Oi, Adv. Immunol., 44:65-92 (1988), all of which references are
incorporated by reference in their entirety herein. These methods
include in particular humanization by CDR grafting (Jones et al,
Nature, 321:522-525 (1986); Verhoeyen et al, Science, 239:1534-1539
(1988); and the more tailored approach of Padlan, Molec. Immunol.,
28:489 (1991) and Padlan, Molec. Immunol., 31:169 (1994) which
involves the selection of non-essential framework amino acid
residues and their modification by appropriate substitution
mutation. These references are incorporated by reference in their
entirety herein.
[0079] As noted, CDR grafting techniques, while successful in some
instances, may substantially adversely affect the affinity of the
resultant humanized antibodies. This is believed to occur because
some framework residues affect or are essential for and at least
affect antigen binding. Our technique; Padlan (1994) (Id.)) is more
refined because we retain only those murine framework residues
which we deem critical to the preservation of the antibody
combining site while keeping the surface properties of the molecule
as human as possible. Accordingly, this technique has the potential
of producing humanized antibodies which retain the antigen-binding
characteristics of the parent antibody. Because of this, this
technique was selected by the present inventors as the means by
which humanized antibodies derived from murine antibody 24-31
specific to human gp39 would potentially be obtained.
[0080] The cloning of the variable regions of 24-31 (described in
detail in the examples infra) resulted in the identification of the
V.sub.L and V.sub.H sequences utilized by the 24-31 antibody
respectively shown in FIG. 7 and FIG. 8. After sequencing, the
variable regions were then humanized. As noted, this was effected
substantially according to the method of Padlan (1994) (Id.),
incorporated by reference supra.
[0081] This method generally comprises replacement of the non-human
framework by human framework residues, while retaining only those
framework residues that we deem critical to the preservation of
antigen binding properties. Ideally, this methodology will confer a
human-like character on the surface of the xenogeneic antibody thus
rendering it less immunogenic while retaining the interior and
contacting residues which affect its antigen-binding
properties.
[0082] More specifically, the 24-31 V.sub.K and V.sub.H sequences
set forth in FIGS. 7 and 8 were humanized by comparison to human
antibodies of reported sequence, which are referred to as
"templates."
[0083] Specifically, the 24-31 V.sub.K was humanized using as
templates:
[0084] (a) For VL#1, the human V-Kappa subgroup I sequences, e.g.,
DEN and the like, as well as the germline 012 (see Cox et al, Eur.
J. Immunol. 24:827-836 (1994)), and for VL#2, the human V-Kappa
subgroup IV sequences, e.g., LEN. Such template sequences are known
and are reported in Kabat et al (1991) (Id.) or GenBank.
[0085] The 24-31 V.sub.H #1 was humanized using as templates
[0086] (a) the human V.sub.H subgroup IV sequence, 58p2 and
[0087] (b) (GenBank Accession No.) Z18320 and the germline 3d75d
(S. van der Maarel et al, J. Immunol., 150:2858-2868 (1993).
[0088] Such template variable heavy antibody sequences are also
known and are reported in Kabat et al, "Sequences of Proteins of
Immunological Interest," 5th Ed., NIH (1991) and in GenBank.
[0089] The template human variable heavy and light sequences were
selected based on a number of different criteria, including, in
particular, high degree of sequence similarity with 24-31 overall,
as well as similarity in the "important" residues, i.e., those
which are believed to be comprised in the V.sub.L:V.sub.H
interface; those which are in contact with the complementarity
determining regions, or which are inwardly pointing. Also, the
templates were selected so as to potentially preserve the
electrostatic charge of the 24-31 F.sub.v as much as possible, and
also so as to preserve glycines, prolines and other specific amino
acid residues which are believed to affect antigen binding.
[0090] This methodology resulted in the following preferred
humanized V.sub.L and V.sub.H heavy sequences derived from the
24-31 antibody which are set forth below in Table 1 and Table 2. As
discussed above, the invention further embraces equivalents and
variants of these preferred humanized sequences, e.g., those
containing one or more conservative amino acid substitutions which
do not substantially affect gp39 binding. The complementarity
determining regions are identified in FIGS. 7 and 8 which contain
the entire variable heavy and light chain CDR sequences of the
parent (non-humanized) 24-31 antibody. TABLE-US-00005 TABLE 1
HUMANIZED 24-31 VL SEQUENCES 10 20 40 60 70 80 24-31
DIVMTQSQKFMSTSVGDRVSITC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISNMQSEDLADYFC 100 QQYNSYPYT FGGGTKLEIK (1)
DIVMTQSPSFLSASVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSLQPEDFADYFC QQYNSYPYT FGGGTKLEIK (2)
DIVMTQSPDSLAVSLGERATINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSLQAEDVADYFC QQYNSYPYT FGGGTKLEIK (3)
DIVMTQSPSFMSTSVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRT
GVPDRFSGSGSGTDFTLTISSMQPEDFADYFC QQYNSYPYT FGGGTKLEIK (4)
DIVMTQSPDSMATSLGERVTINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT
GVPDRFSGSGSGTDFTLTISSMQAEDVADYFC QQYNSYPYT FGGGTKLEIK
[0091] TABLE-US-00006 TABLE 2 HUMANIZED 24-31 VH SEQUENCES 10 20 30
40 70 82abc 24-31 EVQLQESGPSLVKPSQTLSLTCSVTGDSIT NGFWI
WIRKFPGNKLEYMG YISYSGSTYYNPSLKS RISITRDTSQNQFYLQLNSVTTE 90 110
DTGTYYCAC RSYGRTPYYFDF WGQGTTLTVSS (1)
EVQLQESGPGLVKPSETLSLTCTVSGDSIT NGFWI WIRKPPGNKLEYMG
YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAA DTGVYYCAC RSYGRTPYYFDF
WGQGTTLTVSS (2) EVQLQESGPGLVKPSQTLSLTCTVSGDSIT NGFWI WIRKHPGNKLEYMG
YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAA DTGVYYCAC RSYGRTPYYFDF
WGQGTTLTVSS (3) EVQLQESGPGLVKPSQTLSLTCAVSGDSIT NGFWI WIRKHPGNKLEYMG
YISYSGSTYYNPSLKS RISISRDTSNNQFSLNLNSVTRA DTGVYYCAC RSYGRTPYYFDF
WGQGTTLTVSS (4) EVQLQESGPGLVKPSETLSLTCAVYGDSIT NGFWI WIRKPPGNKLEYMG
YISYSGSTYYNPSLKS RISISRDTSKNQFYLKLSSVTAA DTGVYYCAC RSYGRTPYYFDF
WGQGTTLTVSS
[0092] As can be seen therefrom, four preferred humanized framework
sequences were designed for both the V.sub.H and V.sub.L chains.
Therefore, there are 16 different possible humanized 24-31
antibodies which may be synthesized using the above-identified
humanized V.sub.H and V.sub.L 24-31 sequences, excluding variants
and equivalents containing conservative modifications.
[0093] Humanized 24-31 antibodies containing these humanized
variable heavy and light sequences may be obtained by recombinant
methods. It is expected that humanized sequences which contain any
combination of the above preferred humanized variable sequences
will result in humanized antibodies which bind human gp39.
Moreover, based on these sequences, the order of preference using
the numbering set forth in Table 1 and Table 2 is expected to be as
follows:
[0094] (1) #1 V.sub.L with #1 V.sub.H (Version 1)
[0095] (2) #2 V.sub.L with #1 V.sub.H (Version 2)
[0096] (3) #1 V.sub.L with #2 V.sub.H (Version 3)
[0097] (4) #2 V.sub.L with #2 V.sub.H (Version 4)
[0098] The above-identified humanized V.sub.H and V.sub.L sequences
may be further modified, e.g., by the introduction of one or more
additional substitution modifications and also by the addition of
other amino acids. Additional modifications will be selected which
do not adversely affect antigen (gp39) binding. For example, the
inventors contemplate further modification of the V.sub.H chain by
substitution of one or more of residues 34, 43, 44 and 68
(according to Kabat numbering scheme) Kabat et al (1991) (Id.).
Also, the inventors contemplate modification of residue 85 of the
V.sub.L chain. Based on the structural features of the antibody
combining site, it is believed that modification of such residues
should also not adversely impact antigen binding. Moreover, it is
expected that the introduction of one or more conservative amino
acid substitutions should not adversely affect gp39 binding.
[0099] So as to better describe the subject humanized 24-31,
V.sub.H and V.sub.L sequences, the preferred humanized framework
sequences are also set forth in Table 3 below, which compares these
sequences to the template human variable heavy and light framework
sequences, i.e., human DEN VK1, Human o12/V36 germline, human LEN
VKIV, human 58p2, human Z18320, and human 3d75d as well as to the
unhumanized murine 24-31 V.sub.H and V.sub.L framework sequences.
TABLE-US-00007 TABLE 3 VK Framework Region Comparisons - Humanized
Anti-gp39 FR1 FR2 Human 012/V3b DIQMTQSPSFLSASVGDRVTITC
WYQQKPGKAPKLLIY germline Human DEN VKI ---------T-------------
-------E---V--- Murine 24-31 --V----QK-M-T------S---
-------QS------ Padlan VL#1 --V-------------------- --------S------
humanized FR3 FR4 Human 012/V3b GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
Human DEN VKI -------------E---------SD------- FGQGTKLEIK Murine
24-31 ---D----------------NM-SE-L-D-F- --G------- Padlan VL#1
---D------------------------D-F- --G------- FR1 FR2 Human LEN VKIV
DIVMTQSPDSLAVSLGERATINC WYQQKPGQPPKLLIY Murine 24-31
-------QKFMST-V-D-VS-T- --------S------ Padlan VL#2
----------------------- --------S------ humanized FR3 FR4 Human LEN
VKIV GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC FGQGTKLEIK Murine 24-31
--------------------NM-S--L-D-F- --G------- Padlan VL#2
----------------------------D-F- --G------- FR1 Human 58p2
QVQLQESGPGLVKPSETLSLTCTVSGGSIS Murine 24-31
E---------S----Q------S-T-D--T Padlan VH#1
E-------------------------D--T humanized Human Z18320
---------------Q-------------- Genflank Human 3d75d
---------------Q-------------- germline Padlan VH#2
E--------------Q----------D--T humanized FR2 FR3 Human 58p2
WIRQPPGKGLEWIG RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR Murine 24-31
---KF--NK--YM- -IS-TR---Q---Y-Q-N---TE--GT----C Padlan VH#1
---K---NK--YM- -IS--R-------------------G-----C Human Z18320
-----A-------- -------------------------------- Human 3d75d
----H--------- -------------------------------- Padlan VH#2
---KH--NK--YM- -IS--R-------------------G-----C Human 58p2
WGQGTMVTVSS Murine 24-31 -----TL---- Padlan VH#1 -----TL---- Human
Z18320 ----------- Padlan VH#2 -----TL----
[0100] In order to produce humanized antibodies, DNA sequences are
synthesized which encode for the afore-identified humanized V.sub.L
and V.sub.H sequences. As noted, taking into account these four
humanized V.sub.L sequences, and four humanized V.sub.H sequences,
there are 16 potential humanized antigen combining sites which may
be synthesized. Also, there are even more potential humanized
antigen combining sites taking into account the potential
substitution of residues 34, 43, 44 and 68 of the humanized V.sub.H
and residue 85 of the humanized V.sub.L by other amino acid
residues and/or the potential incorporation of conservative
substitution mutations. Two of the preferred humanized variable
light sequences (1) and (2) and a preferred humanized variable
heavy sequence (1) including the complementarity determining
regions and corresponding DNA sequences are set forth in FIGS. 4, 5
and 6, respectively.
[0101] Methods for synthesizing DNA encoding for a protein of known
sequence are well known in the art. Using such methods, DNA
sequences which encode the subject humanized V.sub.L and V.sub.H
sequences are synthesized, and then expressed in vector systems
suitable for expression of recombinant antibodies. This may be
effected in any vector system which provides for the subject
humanized V.sub.L and V.sub.H sequences to be expressed as a fusion
protein with human constant domain sequences and associate to
produce functional (antigen binding) antibodies.
[0102] Expression vectors and host cells suitable for expression of
recombinant antibodies and humanized antibodies in particular, are
well known in the art.
[0103] The following references are representative of methods and
vectors suitable for expression of recombinant immunoglobulins
which are incorporated by reference herein: Weidle et al, Gene,
51:21-29 (1987); Dorai et al, J. Immunol., 13(12):4232-4241 (1987);
De Waele et al, Eur. J. Biochem., 176:287-295 (1988); Colcher et
al, Cancer Res., 49:1738-1745 (1989); Wood et al, J. Immunol.,
145(a):3011-3016 (1990); Bulens et al, Eur. J. Biochem.,
195:235-242 (1991); Beggington et al, Biol. Technology, 10:169
(1992); King et al, Biochem. J., 281:317-323 (1992); Page et al,
Biol. Technology, 9:64 (1991); King et al, Biochem. J., 290:723-729
(1993); Chaudary et al, Nature, 339:394-397 (1989); Jones et al,
Nature, 321:522-525 (1986); Morrison and Oi, Adv. Immunol.,
44:65-92 (1988); Benhar et al, Proc. Natl. Acad. Sci. USA,
91:12051-12055 (1994); Singer et al, J. Immunol., 150:2844-2857
(1993); Cooto et al, Hybridoma, 13(3):215-219 (1994); Queen et al,
Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989); Caron et al,
Cancer Res., 32:6761-6767 (1992); Cotoma et al, J. Immunol. Meth.,
152:89-109 (1992). Moreover, vectors suitable for expression of
recombinant antibodies are commercially available.
[0104] Host cells known to be capable of expressing functional
immunoglobulins include by way of example mammalian cells such as
Chinese Hamster Ovary (CHO) cells, COS cells, myeloma cells,
bacteria such as Escherichia coli, yeast cells such as
Saccharomyces cerevisiae, among other host cells. Of these, CHO
cells are used by many researchers given their ability to
effectively express and secrete immunoglobulins.
[0105] Essentially, recombinant expression of humanized antibodies
are effected by one of two general methods. In the first method,
the host cells are transfected with a single vector which provides
for the expression of both heavy and light variable sequences fused
to selected constant regions. In the second method, host cells are
transfected with two vectors, which respectively provide for
expression of either the variable heavy or light sequence fused to
selected constant regions.
[0106] Human constant domain sequences are well known in the art,
and have been reported in the literature. Preferred human V.sub.L
sequences includes the Kappa and lambda constant light sequences.
Preferred human heavy constant sequences include human gamma 1,
human gamma 2, human gamma 3, human gamma 4 and mutated versions
thereof which provide for altered effect or function, e.g. enhanced
in vivo half-life and reduced Fc receptor binding.
[0107] Preferred modifications of the human gamma 4 constant domain
include P and/or E modifications, which respectively refer to the
change of a leucine to a glutamic acid at position 236 and/or the
change of a serine to a proline (Kabat numbering) at position 229
such as described in commonly assigned Attorney Docket No.
012712-165 filed on Sep. 6, 1995 and incorporated by reference in
its entirety herein.
[0108] A particularly preferred vector system comprises the
expression vectors described in commonly assigned U.S. Ser. No.
08/476,237 filed Jun. 7, 1995, Ser. No. 08/397,072, filed Jan. 25,
1995 and 07/912,122 filed Jul. 10, 1992, Ser. No. 07/886,281 filed
Mar. 23, 1992, and Ser. No. 07/735,064 filed Jul. 25, 1991, all
incorporated by reference in their entirety.
[0109] In particular, these applications describe vector systems
for the production of recombinant antibodies, referred to as TCAE
5.2 and TCAE 6 which comprise the following:
[0110] 1) Four transcriptional cassettes in tandem order: [0111]
(a) a human immunoglobulin light chain constant region. In TCAE 5.2
this is the human immunoglobulin Kappa light chain constant region
(Kabat numbering amino acids 108-214, allotype Km 3) and in TCAE 6
the human immunoglobulin light chain lambda constant region (Kabat
numbering amino acids 108-215, genotype Oz minus, Mcg minus, Ke
minus allotype). [0112] (b) a human immunoglobulin heavy chain
constant region; in both constructs the human immunoglobulin heavy
chain is a gamma/constant region (Kabat numbering amino acids
114-478 allotype Gm1a, Gm12). [0113] (c) DHFR; containing its own
eukaryotic promoter and polyadenylation region; and [0114] (d) NEO;
also containing its own eukaryotic promoter and polyadenylation
region.
[0115] 2) The human immunoglobulin light and heavy chain cassettes
contain synthetic signal sequences for secretion of the
immunoglobulin chains; and
[0116] 3) The human immunoglobulin light and heavy chain cassettes
contain specific DNA links which allow for the insertion of light
and heavy immunoglobulin variable regions which maintain the
translational reading frame and do not alter the amino acids
normally found in immunoglobulin chains.
[0117] These vectors are preferably utilized in CHO cells. The
subject antibodies are preferably expressed in the above-described
vector systems.
[0118] However, the subject humanized antibody sequences derived
from the number 24-31 antibody may be expressed in any vector
system which provides for the expression of functional antibodies,
i.e., those which bind gp39 antigen.
[0119] In particular, the inventors elected to express the subject
humanized V.sub.L and V.sub.H sequences, as well as the native
(unmodified) V.sub.L and V.sub.H sequences derived from 24-31 in
CHO cells using the N5KG1 expression vector which contains human
Kappa and human gamma 1 constant regions. The N5KG1 expression
vector is depicted schematically in FIG. 1. As hoped, the chimeric
antibody derived from 24-31, when expressed in CHO cells binds gp39
(by demonstrated binding to CHO-gp39 transfectant). Also, several
humanized anti-bodies of the invention derived from 24-31 when
expressed using this vector system resulted in functional (gp39
binding) antibodies.
[0120] The present invention is further described through
presentation of the following examples. These examples are offered
by way of illustration and not by way of limitation.
EXAMPLE 1
Selection of 24-31 Antibody for Humanization.
[0121] Accumulating evidence in animal models indicates that
anti-gp39 administration prevents a variety of autoimmune processes
and interferes with allograft rejection. These results provide
compelling evidence that antibodies to human gp39 may have
significant therapeutic value in the management of autoimmune
disease and the transplantation of allogeneic tissue and organs in
humans. A monoclonal antibody (mAb) specific for human gp39 has
been reported (Lederman et al, J. Immunol., 199:3817 (1992)), and
its functional activity in blocking gp39-CD40 interactions in vitro
has been evaluated. To gain greater insights into the functional
impact of anti-gp39 antibodies on the human immune system, a panel
of anti-human gp39 mAbs was generated. From this panel, one mAb
appeared superior and was extensively tested for functional
inactivation of gp39 in vitro and in vivo.
[0122] More specifically, a panel of 6 murine (all IgG1) anti-gp39
antibodies was generated by immunization with a soluble fusion
protein of human gp39 (gp39-CD8), followed by challenge with
activated human peripheral blood T cells. Flow cytometric analysis
of human peripheral blood T cells demonstrated that the mAbs
recognized a cell surface molecule expressed on activated
(PMA/ionomycin), but not resting, CD3.sup.+ T cells, and that the
pattern of reactivity was similar to that seen with a recombinant
CD40 fusion protein (CD40-Ig) (data not shown). Immunoprecipitation
of [.sup.35S] metabolically labeled activated human peripheral
blood T cells revealed that each of the 6 mAbs precipitated a
molecule of similar size (33 kDa) to that precipitated by CD40-Ig.
Finally, binding of CD40-Ig to gp39 was blocked in the presence of
the antibodies indicating recognition of the same molecule, further
confirming their specificity. Although all 6 mAbs were capable of
blocking gp39 function, one mAb, 24-31, was selected for extensive
analysis.
EXAMPLE 2
T Cell-Dependent B Cell Proliferation and Differentiation (Ig
Production) is Blocked by Anti-gp39.
[0123] A number of studies have provided evidence that signals
delivered through CD40 by its ligand, gp39, induce B cell
activation, proliferation, differentiation, and iso-type switching.
To determine if the anti-gp39 24-31 mAb blocked gp39 function, B
cells were cultured with a soluble fusion protein of gp39
(gp39-CD8) in the presence or absence of 24-31, and the B cell
proliferative response was assessed by .sup.3H-thymidine
incorporation. The results, shown in FIG. 2A, demonstrate that
gp39-CD8 induced vigorous proliferation of B cells. The presence of
anti-gp39 24-31 mAb completely ablated B cell proliferation induced
by gp39-CD8 at concentrations as low as 2.5 .mu.g/ml. To determine
whether 24-31 interfered with T cell-induced B cell
differentiation, B cells were co-cultured with anti-CD3 activated T
cells in the presence or absence of 24-31. Polyclonal IgM, IgG, and
IgA production was assessed after 12 days. As shown in FIG. 2B, the
addition of 24-31 inhibited polyclonal IgM, IgG, and IgA antibody
production (90-99%). These results confirm previous reports
establishing the requirement for gp39-CD40 interactions in T
cell-dependent B cell differentiation (Nishioka et al, J. Immunol.,
153:1027 (1994), and further demonstrate the use of newly
characterized anti-human gp39 24-31 mAb in blocking gp39
function.
EXAMPLE 3
Anti-gp39 Blocks In Vivo Tetanus Toxoid Specific Antibody
Production in SCID Mice Reconstituted with Human PBL.
[0124] Numerous studies have established that the human immune
system can be studied in vivo under experimental conditions through
the use of severe combined immunodeficiency (scid) mice engrafted
with human peripheral blood lymphocytes (hu-PBL-scid mice) (Mosler
et al, Nature, 335:256 (1988); McCune et al, Science, 241:1632
(1988). Long-term chimerism is achieved in scid mice by injection
with human PBL, and antigen-specific secondary antibody responses
are detected in hu-PBL-scid mice challenged in vivo with antigen
(Carlsson et al, J. Immunol., 148:1065 (1992); Duchosal et al, Cell
Immunol., 139:468 (1992)). This system was exploited to evaluate
the immunosuppressive effects of in vivo anti-gp39 administration
on the immune responses elicited by human T and B-cells.
[0125] Experiments, the results of which are contained in FIG. 2B,
demonstrated that blockade of gp39 function by 24-31 inhibited T
cell-dependent polyclonal Ig production by human B cells in vitro.
To determine whether 24-31 could also inhibit antigen specific B
cell antibody production in vivo, C.B-17 scid/scid mice injected
i.p. with human PBL (hu-PBL-scid) and immunized with tetanus toxoid
(TT) were treated with 24-31 or PBS, and the secondary (IgG)
anti-TT antibody response was assessed. Immunization of hu-PBL-scid
with TT resulted in detectable levels of IgG anti-TT antibody
within 14 days post immunization in most animals (Table 4).
However, treatment with anti-gp39 (24-31; 250 .mu.g/day, twice
weekly) completely ablated the secondary anti-TT antibody response
in 9/10 mice examined, demonstrating that in vivo blockade of gp39
function also resulted in inhibition of antigen specific humoral
responses. TABLE-US-00008 TABLE 4 Ablation of the secondary
anti-tetanus antibody response following engraftment of human PBL
in C.B-17 scid/scid mice immunized with tetanus toxoid. *Four-six
week old C.B-17-scid/scid mice were injected i.p. with 20 .times.
10.sup.6 human PBL and 0.25 ml tetanus toxoid. Anti-Tetanus
Antibody (O.D. .+-. SE) .sctn. (Frequency of Mice Containing Recip-
Anti-Tetanus Antibody) ient Treat- days post immunization Strain*
ment 7 d 14 d 21 d 28 d C.B-17 PBS <0.02 2.30 .+-. .042 .224
.+-. .040 .137 + .007 scid/ (0/10) (7/10)* (8/10)** (4/10) scid
anti- .162 <0.02 (0/10) <0.02 (0/10) <0.02 (0/10) gp39
(1/10) Anti-gp39 24-31 or PBS (250 .mu.g/injection) was
administered i.p. twice weekly throughout the entire experiment.
.sctn.The level of human anti-tetanus toxoid antibody in the serum
was determined weekly by ELISA. All mice with serum levels of human
anti-tetanus toxoid antibody > 0.100 O.D. at a 1:10 dilution
were considered positive. Only positive mice were used in the
calculation of the mean .+-. SE values included in the table. The
level of human anti-tetanus toxoid in sera from pre-immune mice not
immunized with tetanus toxoid was < 0.02 O.D. Data are presented
as mean .+-. SE. *Significantly different (p = 0.222) than the
anti-gp39 treated group. **Significantly different (p < 0.001)
than the anti-gp39 treated group.
EXAMPLE 4
Anti-gp39 Treatment does not Inhibit the Antigen-Specific T Cell
Proliferative Response of hu-PBL-scid Spleen Cells.
[0126] To determine whether treatment of hu-PBL-scid mice with
anti-gp39 altered the responsiveness of antigen-specific T cells in
vivo, the proliferative response of spleen cells from hu-PBL-scid
mice immunized with TT and treated with 24-31 was assessed in
vitro. Spleen cells from control or anti-gp39 treated hu-PBL-scid
mice were cultured with TT or medium alone, and the proliferative
response was assessed by .sup.3H-thymidine incorporation after 6
days. Table 5 summarizes the results of one such experiment.
Hu-PBL-scid mice treated with anti-gp39 responded similarly to in
vitro stimulation with TT as did hu-PBL-scid mice which were
untreated (5/10 vs. 3/10 responding mice). Experiments using
NOD/LtSz-scid/scid mice as recipients yielded similar results,
although anti-TT antibodies were undetectable in these mice (data
not shown). These data demonstrate that treatment with anti-gp39
does not result in deletion or functional inactivation of
antigen-specific T cells in hu-PBL-scid mice and support the
contention that inhibition of TT specific antibody responses by
anti-gp39 is due to blockade of gp39-CD40 interactions and
subsequent B cell responses rather than T cell inactivation.
TABLE-US-00009 TABLE 5 Anti-gp39 treatment does not alter the
anti-tetanus T cell proliferative response following engraftment of
human PBL in C.B-17-scid/scid or NOD/LtSz-scid/scid mice immunized
with tetanus toxoid. *Four-six week old C.B-17-scid/scid or
NOD/LtSz-scid/scid mice were injected i.p. with 20 .times. 10.sup.6
human PBL and 0.25 ml tetanus toxoid. Frequency of Responding
Recipient Strain* Treatment Mice.sctn. C.B-17 scid/scid PBS 3/10
anti-gp39 5/10 NOD/LtSz-scid/scid PBS 5/10 anti-gp39 6/10 Anti-gp39
24-31 or PBS (250 .mu.g/injection) was administered i.p. twice
weekly throughout the entire experiment. .sctn.Spleen cells from
mice injected with human PBL and immunized with tetanus toxoid were
cultured at a concentration of 1 .times. 10.sup.5 cells/ml in the
presence of media alone or tetanus toxoid (2.5 or 5.0 .mu.g/ml).
Proliferation was assessed by .sup.3H-thymidine incorporation after
6 d. Stimulation indices were calculated by the following formula''
S.I. = cpm tetanus - cpm medium/cpm medium. S.I. of > than 2.0
was considered positive.
EXAMPLE 5
Generation of a gp39 CHO Transfectant Cell Line.
[0127] Recently, a CHO transfectant that constitutively expresses
cell-surface gp39 was generated to use as a reagent for the
humanized anti-gp39 24-31 binding studies proposed in this
application. The full-length gp39 gene (Hollenbaugh et al, Immunol.
Rev., 138:23 (1994)) was amplified by polymerase chain reaction
(PCR) of phytohemag-glutinin-activated human PBL and cloned into
IDEC's INPEP4 vector under the transcriptional control of the
cytomegalo-virus (CMV) promoter and enhancer elements. A CHO
transfectant was established and amplified in 50 nM methotrexate.
The transfectant, 50D4, was shown to express cell-surface gp39 by
ELISA (data not shown) and FACS analysis (FIG. 3).
EXAMPLE 6
High-Level Expression of Antibodies Using a CHO Expression
System.
[0128] IDEC's proprietary NSKG1 expression vector is used in CHO
cells for expression of the humanized anti-gp39 24-31 antibody.
This vector is depicted schematically in FIG. 1. High-level
expression of recombinant antibodies is consistently obtained in
CHO cells using this vector and similar vectors. Using these
vectors, a high percentage of G418 resistant clones, 5-10%, are
found to express significant amounts of recombinant proteins (1-10
mg/l of antibody). These are usually single plasmid copy
integrants, and can easily be amplified using methotrexate to
obtain 30-100 pg/cell/day of secreted immunoglobulin. Table 6 lists
the antibody levels obtained before and after gene amplification of
3 antibodies expressed in CHO cells utilizing this system.
TABLE-US-00010 TABLE 6 Antibody production levels using IDEC's CHO
expression technology. after after before amplification
amplification amplification in spinner in fermentor Antibody mg/l
flask mg/l mg/l Anti-CD4 .gamma.1 1-2 100-110 950 Anti-CD4 .gamma.4
3-4 125-150 N.D. Anti-CD20 5-10 200-300 650
EXAMPLE 7
Cloning of 24-31 V.sub.k and V.sub.H DNA Sequences
[0129] The anti-gp39 24-31 V.sub.k and V.sub.H gene segments were
cloned and sequenced. Following analyses of their sequences,
humanized versions of the V region gene segments were designed. The
corresponding DNA sequences were synthesized and cloned into a
high-level expression vector containing human constant region
genes. A CHO transfectant producing the humanized 24-31 antibody is
then established. To confirm that the humanized version of the
anti-gp39 antibody retains its gp39 binding affinity, the relative
affinities of the murine and humanized antibodies were compared in
direct binding and competition assays. In addition, the ability of
the humanized 24-31 to block CD40 binding to gp39 and to inhibit T
cell-dependent antibody production is evaluated.
[0130] 1. Cloning of the 24-31 V.sub.k and V.sub.H Gene
Segments
[0131] a. Preparation of cDNA. PolyA.sup.+ mRNA was prepared from
2.times.10.sup.6 cells each of the 24-31 hybridoma and the NS1 cell
line, (Carroll et al, Mol. Immunol., 10:991 (1988)), the fusion
partner used in the generation of the 24-31 hybridoma, utilizing an
Invitrogen Corporation Micro-Fast Track.TM. mRNA isolation kit,
according to the manufacturer's protocol. First strand cDNA was
synthesized utilizing 50 pmoles oligo-dT and 5 units M-MLV reverse
transcriptase (Promega) (Sambrook et al, Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press
(1989)) followed by Sephadex G-25 chromatography.
[0132] b. PCR amplification Of V.sub.k and V.sub.H cDNA. 24-31 and
NS1 cDNA were amplified by PCR using a panel of 5' primers specific
for V.sub.k or V.sub.H leader sequences in combination with 3'
constant region primers. The panel of 5'V.sub.H primers are
identical to those described by Jones and Bendig (Bio/Technol.,
9:88 (1991); Errata, Bio/Technol., 9:579 (1991)). The panel of
5'V.sub.k primers (Jones et al, (Id.)) were modified to convert the
Sal I cloning site recognition sequences (GTCGAC) into Bgl II
recognition sequences (AGATCT) to facilitate the cloning of the
amplified gene segments into IDEC's N5KG1 expression vector (See
FIG. 1). The 3' V.sub.k and V.sub.H primers contain a Bsi WI
cloning site sequence at amino acid positions 108-109 (numbering
according to Kabat et al, "Sequences of Proteins of Immunological
Interest," 5th Ed., NIH (1991)) and a Nhe I cloning site sequence
at positions 114-115, respectively, and have the following
sequences: TGCAGCATCCGTACGTTTGATTCCAGCTT (Ck) and
GGGGGTGTCGTGCTAGCTG (A/C)(G/A) GAGAC(G/A)GTGA (C.gamma.1). This
primer panel has been previously used by the Assignee to amplify
and clone the C2B8 anti-CD20 antibody (Nishioka et al, J. Immunol.,
153:1027 (1994)) and numerous other mouse V.sub.k and V.sub.H gene
segments (data not shown).
[0133] In order to determine the correct primer pair for the
amplification of the 24-31 V.sub.k and V.sub.H gene segments, the
24-31 cDNA were amplified in 23 individual reactions containing one
of the 11 5'V.sub.k primers in combination with the C.sub.K primer
or one of the 12 5'V.sub.H primers in combination with the
C.gamma.1 primer. For comparison, NS1 cDNA was amplified using the
same panel of primers. 1 .mu.l cDNA (1/50 of the cDNA sample) was
amplified in a 100 .mu.l final volume containing 5 units Tag DNA
polymerase (Perkin Elmer), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5
mM MgCl.sub.2, 0.25 mM each of dCTP, dGTP, DATP, and TTP, 50 pmoles
3' constant region primer, and 50 pmoles 5' primer.
The-amplification cycle consisted of denaturation for 1 minute at
95.degree. C., annealing for 2 minutes at 50.degree. C., and
extension for 2 minutes at 72.degree. C., repeated 34 times. The
amplified products were analyzed by agarose gel electrophoresis.
The 24-31 PCR reactions yielding a unique amplified product for
V.sub.k and for V.sub.H were repeated and the products from
duplicate PCR reactions cloned. PCR amplified products are agarose
gel-purified (Sambrook et al, Molecular Cloning: A Laboratory
Manual, 2nd Ed. (1989)) and digested with Bgl II and Bsi WI (for
Vk) or Sal I and Nhe I (for V.sub.H). The products are ligated
(Ausabel et al, Current Protocols in Molecular Biology, Vol. 2,
Greene Publ. Assoc. (1992)) sequentially into IDEC's vector,
N5KG1.
[0134] Following transformation of E. coli XL1-blue cells
(Stratagene), plasmid DNA was prepared, and the V.sub.k and V.sub.H
sequences obtained from the duplicate constructs (sequencing
performed by Scripps Research Institute Core Facility, La Jolla,
Calif.). The sequences of the endogenous light and heavy chains of
the NS1 fusion partner are known (Carroll et al, Mol. Immunol.,
10:991 (1988); Kabat et al, (1991) (Id.)) and were used to
distinguish PCR products resulting from the amplification of the
24-31 versus the NS1 fusion partner V regions.
EXAMPLE 8
Synthesis of Gene Segments Encoding Humanized 24-31 V Regions.
[0135] Humanized versions containing the most preferred humanized
24-31 V.sub.k and V.sub.H sequences identified in Tables 1 and 2 as
humanized V.sub.L and V.sub.H (1) were synthesized. Specifically,
four pairs of overlapping, complementary olionucleotides (oligos)
encoding the above-identified humanized V.sub.k or V.sub.H regions
were synthesized (Midland Chemicals) and purified by denaturing
polyacrylamide gel electrophoresis (Ausubel et al, Current
Protocols in Molecular Biology, Vol. 2, Greene Publ. Assoc.
(1992)). Each oligo is approximately 100 bases in length and
overlap by 20 bases the adjacent complementary oligonucleotide. The
V.sub.k and V.sub.H 5' oligos contain Bgl II and Sal I cloning
sites and the 3' oligos possess Bsi WI and Nhe I cloning sites,
respectively. Each variable region gene segment was assembled from
the synthetic oligos, diagrammed below, using the following
procedure (summarized in Watson et al, Recombinant DNA, 2nd Ed.,
Scientif. Amer. Books, NY, N.Y. (1992)). Complementary oligo pairs
(A+E, B+F, C+G, D+F) were kinased using 300 pmoles of each primer
and T4 polynucleotide kinase (Promega) according to the
manufacturer's protocol. The oligos were annealed by heating to
95.degree. C. and slow cooling to room temperature. The annealed
oligo pairs were ligated (A/E with B/F and C/G with D/H) utilizing
6 units T4 DNA ligase (New England Biolabs). After digestion with
the appropriate 5' or 3' cloning site restriction endonuclease, the
approximately 200 base pair DNA fragments were purified by
electroelution following polyacrylamide gel electrophoresis
(Sambrook et al, (Id.)). The synthetic gene fragments were then
inserted into IDEC's proprietary high-level expression vector,
N5KG1, under the transcriptional control of the CMV promoter and
enhancer elements. The ligation reaction contains the 2
gel-purified fragments (A/E/B/F and C/G/D/H) and N5KG1 at a molar
ratio of 100:100:1, respectively. After transformation of XL1-blue
cells, plasmid DNA was prepared and the sequences of the synthetic
gene segments confirmed. The resulting construct, h24-31, encodes
the humanized 24-31 V region segments and human kappa and gamma 1
constant regions. As indicated, this antibody contains the
humanized variable heavy and humanized-variable light sequences
identified in Table 1 and Table 2 as the "(1)" sequences, which are
predicted to provide for humanized antibody having optimal gp39
properties. In addition, a construct was generated which contains
V.sub.L#2 in combination with V.sub.H#1 (version 2 of humanized
24-31). Similar constructs utilizing IDEC's proprietary vectors
have been used for. high-level expression of IDEC's anti-CD20 (Reff
et al, Blood, 83:425 (1994)) and anti-CD4 (Newman et al, Biol.
Technology, 10:1455 (1992)) antibodies. TABLE-US-00011 A B C D 5'
.sub.---------- .sub.---------- .sub.---------- .sub.---------- 3'
.sub.---------- .sub.---------- .sub.---------- .sub.---------- E F
G H
EXAMPLE 9
[0136] 2. Production and Characterization of Humanized 24-31
[0137] a. Generation of CHO Transfectants Producing Humanized 24-31
(Version 1 and Version 2).
[0138] CHO transfectants expressing humanized 24-31 (version 1 or
version 2) were generated by electroporation of 4.times.10.sup.6
CHO cells with linearized h24-31 DNA (version 1 or version 2)
followed by selection in G418. The cell culture supernatants from
G418 resistant clones were assayed for immunoglobulin production by
sandwich ELISA employing a goat anti-human kappa to capture the
immunoglobulin. Immunoglobulin binding was measured by incubating
with a horse radish peroxidase (HRP)-conjugated goat antibody
specific for human IgG, followed by HRP substrate, 0.4 mg/ml
O-Phenylene-diamine (OPD) in a citrate buffer (9-34 g/l
C.sub.6H.sub.8O.sub.7, and 14.2 g/l Na.sub.2HPO.sub.4), pH 5.0,
including 0.0175% H.sub.2O.sub.2. The plate was read in a Molecular
DeviCes "Vmax, kinetic microplate reader" spectrophotometer at 490
nm.
EXAMPLE 10
[0139] b. Characterization of Humanized 24-31 (Version 1).
[0140] The humanized anti-gp39 24-31 antibody is evaluated
initially for direct binding to cell surface gp39 expressed on
50D4, the gp39 CHO transfectant described in Example 5.
Supernatants from the G418-resistant h24-31 CHO transfectants that
produce immunoglobulin are tested for binding to 50D4 cells and, as
negative control, to CHO cells. In this assay 50D4,
1.times.10.sup.5 /well, are bound to the bottom of 96 well,
poly-L-lysine coated polystyrene plates. The cells are fixed in
0.5% glutaraldehyde in phosphate buffered saline (PBS) for 15
minutes. Plates coated with CHO cells are generated similarly. The
cell culture supernatants are added and antibody binding measured
using a HRP-conjugated goat anti-human IgG, as described above.
[0141] Two assays are used to determine if the humanized 24-31
antibody retains its affinity to gp39 relative to the original
murine 24-31 antibody, (i) half-maximal binding concentration and
(ii) a competition assay using 50D4 cells. For this purpose the
antibodies will be purified on protein A and the concentration of
each antibody determined by ELISA by a comparison to isotype
matched controls. Half-maximal binding (i) are determined by
incubating humanized 24-31 with 50D4 cells at various
concentrations from 2 .mu.g/ml to 0.1 ng/ml. The concentration
resulting in a half-maximal OD 490 reading, as described above, is
compared with the half-maximal binding of murine 24-31. In the
competition assay (ii) the humanized 24-31 antibody and the murine
24-31 antibody are mixed in various molar ratios ranging from 100:1
to 1:100, and their ability to compete for binding to 50D4 cells
measured. Two sets are run, one where the binding of the humanized
antibody will be measured using goat-anti-human IgG (anti-mouse IgG
depleted)-HRP and one where the binding of murine antibody is
measured using goat-anti-mouse IgG (anti-human IgG depleted)-HRP.
Binding curves, one for the murine and one for the humanized
antibody, based on molar ratios, are generated and their relative
affinities calculated. These assays will confirm the anti-gp39
binding properties of the subject humanized antibodies derived from
24-31.
EXAMPLE 11
Blocking of CD40-Ig Binding to gp39 by Humanized 24-31.
[0142] After establishing that humanized anti-gp39 binds to gp39,
an assay is effected to confirm that the humanized anti-gp39
retains its ability to block the binding of the ligand to its
receptor. For this purpose, activated human peripheral blood T
cells, or the gp39-transfected CHO cells, 50D4, are pretreated with
graded concentrations of murine 24-31 or with humanized 24-31 for
15 minutes at 4.degree. C. Following this preincubation,
CD40-Ig-biotin is added and the binding determined by flow
cytometry using PE-avidin. Concentrations of mAbs to achieve a 50%
reduction in CD40-Ig binding are determined.
EXAMPLE 12
Blocking of B Cell Proliferation and Differentiation by Humanized
24-31.
[0143] To confirm that humanized 24-31 blocks gp39 function, B
cells are cultured with a soluble fusion protein of gp39 (gp39-CD8)
in the presence or absence of a range of doses of murine 24-31 or
humanized 24-31. B cell proliferative response is assessed by
.sup.3H-thymidine incorporation as shown in FIG. 2A.
[0144] T cell dependent B cell differentiation (Ig production) is
blocked by mAbs to gp39. To confirm that the subject humanized
24-31 antibodies are effective in blocking the function of native
gp39 expressed on the surface of activated human T cells, the
ability of the subject humanized 24-31 antibodies inhibit T
cell-induced B cell differentiation is assessed. B cells are
co-cultured with anti-CD3 activated T cells in the presence or
absence of humanized 24-31 and murine 24-31. Polyclonal IgM, IgG,
and IgA production is assessed after 12 days (see FIG. 2B). These
results will confirm that humanized anti-gp39 can block CD40
binding and interfere with T-cell-dependent B cell activation via
CD40.
EXAMPLE 13
Binding Capacity
[0145] This experiment was effected to determine the reactivity of
the murine, chimeric, and humanized (version 1) 24-31 antibodies to
the gp39 antigen relative to the concentration of antibody.
Protocol:
Plate Preparation
[0146] 1. Add 50 of poly-1-lysine to each well on the 96 well
plate. Incubate for 30 minutes at room temperature. Flick plates to
remove poly-1-lysine. [0147] 2. Wash mgp39-CHO cells (Chinese
hamster ovary cells expressing cell surface, membrane gp39) 3 times
with HBSS by centrifuging at 1500 rpm for 5 minutes. Resuspend
cells in HBSS to 2.times.10.sup.6 cells ml. [0148] 3. Add 50 .mu.l
of cell suspension to each well and centrifuge plates at 2000 rpm
for 5 minutes. [0149] 4. Add 50 .mu.l/well of ice cold 0.5%
glutaraldehyde and incubate for 15 minutes at room temperature.
[0150] 5. Flick plate and blot to remove excess glutaraldehyde. Add
150 .mu.l/well of 100 mM glycine with 0.1% BSA and incubate for 30
minutes at room temperature. Plates can be used immediately or
frozen at -20.degree. C. for future use. Binding Assay [0151] 1.
Thaw plate and remove glycine buffer. [0152] 2. Serially dilute,
1:2, the test antibodies in dilution buffer starting at 1 .mu.g/ml.
Transfer 50 .mu.g/well of each dilution in duplicate. Incubate 2
hours at room temperature. [0153] 3. Wash plate 10 times in flowing
tap water. [0154] 4. Add 50 .mu.l/well of 1:2000 dilution of goat
anti-human IgG HRP or goat anti-mouse IgG HRP. Incubate 1 hour at
room temperature. [0155] 5. Wash plate 10 times in flowing tap
water. [0156] 6. Add 50 .mu.l/well of ABTS substrate and develop
plate for 20-30 minutes. Read the plate at wavelength 405 mn with a
background wavelength of 490 run. [0157] 7. Plot graph of
absorbance vs antibody concentration. Results and Conclusions:
[0158] The binding capacities for the three anti-gp39 antibodies
(murine, chimeric and humanized version 1 of 24-31) relative to the
concentration of the antibodies, were essentially superimposable
(see FIG. 9). This is a good indication that these antibodies have
similar binding capacities for human gp39, indicating that the
humanized antibody has retained the gp39 binding affinity of murine
24-31.
EXAMPLE 14
Competition Between Biotin Labeled Murine 24-31 and Chimeric and
Humanized Version 1 24-31
[0159] The ability of the chimeric and humanized (version 1) 24-31
antibodies to compete with the murine 24-31 for binding to
mgp39-CHO cells basis was evaluated. The ability of the humanized
24-31 to compete with the murine 24-31 for binding to mgp39-CHO was
used to evaluate whether in the humanized antibody the exchanges of
the murine framework residues with their human counterparts
resulted in a significant loss (.gtoreq.3.times. decrease) of
affinity.
Protocol:
Plate Preparation
[0160] 1. Add 50 of poly-1-lysine to each well on the 96 well
plate. Incubate for 30 minutes at room temperature. Flick plates to
remove poly-1-lysine. [0161] 2. Wash mgp39-CHO cells 3 times with
HBSS by centrifuging at 1500 rpm for 5 minutes. Resuspend cells in
HBSS to 2.times.10.sup.6 cells/ml. [0162] 3. Add 50 .mu.l of cell
suspension to each well and centrifuge plates at 2000 rpm for 5
minutes. [0163] 4. Add 50 .mu.l/well of ice cold 0.5%
glutaraldehyde and incubate for 15 minutes at room temperature.
[0164] 5. Flick plate and blot to remove excess glutaraldehyde. Add
150 .mu.l/well of 100 mM glycine with 0.1% BSA and incubate for 30
minutes at room temperature. Plates can be used immediately or
frozen at -20.degree. C. for future use. Competition Assay [0165]
1. Thaw plate and remove glycine buffer. [0166] 2. Dilute mouse
anti-gp39 biotin to 200 ng/ml in PBS with 1% BSA. [0167] 3.
Serially dilute test antibodies (mouse, chimeric, and humanized
24-31) 1:2 starting at 10 .mu.g/ml in dilution buffer. [0168] 4.
Transfer 50 .mu.l of diluted test antibodies and mouse anti-gp39
biotin into each well in duplicate. Several wells should contain 50
.mu.l dilution buffer with the mouse anti-gp39 biotin as a maximal
control group. Incubate 2 hours at room temperature. [0169] 5. Wash
plates 10 times in flowing tap water. [0170] 6. Add 50 .mu.l/well
of 1:2000 dilution of streptavidin HRP and incubate 1 hour at room
temperature. [0171] 7. Wash plates 10 times in flowing tap water.
[0172] 8. Add 50 .mu.l/well of ABTS substrate and develop plate for
20-30 minutes. Read the plate at wavelength 405 nm with a
background wavelength of 490 nm. [0173] 9. Percent inhibition is
calculated using the average of the control wells. Results and
Conclusions:
[0174] All three antibodies competed equally well with the biotin
labeled 24-31 (see FIG. 10). The competition profiles are
essentially superimposable at all concentrations, within the
limitations of the assay. This demonstrates that the tested
humanized antibody (version 1) retains its gp39 binding
affinity.
EXAMPLE 15
Modulation of T Cell Dependent B Cell Differentiation
[0175] To confirm that the humanized 24-31 retains the in vitro
functional activity of murine 24-31, the humanized 24-31 was
compared to the murine 24-31 in a "Lipsky" assay. Donor peripheral
blood mononuclear cells were separated into two fractions, a T and
a B cell fraction. The T cells were first treated with mitomycin C,
to prevent mitosis, and then activated with an anti-CD3 antibody.
The B cells were added, together with either the murine or
humanized (version 1) 24-31 antibodies. A positive control without
antibody, and a negative control without B cells were included in
the experiment. After a 10 day incubation, the supernatants were
tested for the presence of human IgM.
Protocol:
[0176] 1. Coat a 96 well plate with 50 .mu.l/well of sterile 4
.mu.g/ml anti-CD3 antibody (diluted in 50 mM Tris, pH 9) for 2
hours at 37.degree. C. [0177] 2. Selectively purify T and B cells
from a buffy coat using Lympho-Kwik reagents. Activate the T cells
with 50 .mu.g/ml mitomycin C per 5.times.10.sup.6 cells for 30
minutes at 37.degree. C. [0178] 3. Wash plate wells several times
with sterile HBSS or media to remove non-adherent antibody. [0179]
4. Add 1.times.10.sup.5 purified T cells (2.times.10.sup.6/ml) to
each well. [0180] 5. Add 5.times.10.sup.5 purified B cells
(5.times.10.sup.6/ml) to each well. Add 50 .mu.l anti-gp39 antibody
(10-0.1 .mu.g/ml) to each well in quadruplicate. Control wells
should include:
[0181] a) 0 antibody, b) 0 antibody, no T cells, and c) 0 antibody,
no B cells. [0182] 6. Incubate plate at 37.degree. C./5% CO.sub.2
for 12 days. [0183] 7. Access cell growth after 7 days using 3H
thymidine or any other acceptable method on duplicate wells. [0184]
8. After 12 days, collect supernatants from duplicate wells and
perform ELISA assays to determine Ig production (IgM). Results and
Conclusions:
[0185] The results show that the production of human IgM is
inhibited 50% by the humanized 24-31 at a concentration below 0.01
.mu.g/ml, similar to the inhibition level obtained with the murine
24-31 (see FIG. 11). The humanized antibody retained its ability to
inhibit T cell dependent B cell differentiation (IgM production) in
this experiment.
EXAMPLE 16
Evaluation of Humanized 24-31, Version 2
[0186] This experiment was conducted to determine whether humanized
24-31 version 2, as compared to version 1, has a similar gp39
binding capacity in a direct binding assay.
Protocol:
[0187] Same as in Example 13 above.
Results and Conclusions.
[0188] The results show that the binding capacity of the two 24-31
versions are essentially superimposable (see FIG. 12). This
indicates that the two versions have comparable binding activity to
gp39.
EXAMPLE 17
[0189] This experiment was conducted to measure the Kd of 24-31,
and two humanized versions, 1 and 2.
Protocol:
[0190] A predetermined amount of each of the three antibodies
(murine, version 1 or version 2 24-31) was labeled with .sup.125I
using IODO-BEADS.RTM. (Pierce). Antibody bound-.sup.125I was
separated from free .sup.125I by size separation on a
Sephadex-G25/DEAE/Amberlite column.
[0191] Direct binding of the .sup.125I-labeled antibody to murine
gp39-CHO cells was tested in a dilution series, in order to
determine both counts/pg and the appropriate working concentration
(.apprxeq.half-maximal binding concentration).
[0192] .sup.125I-labeled antibody was mixed and incubated with
non-labeled antibody in a dilution series. Based on the total
amount of bound antibody and the amount of free antibody, a
Scatchard plot was generated from a bound vs. bound-free graph. The
total antibody concentration was based on a standard size of 75 kD
for one active site.
[0193] The Kd was calculated by generating a "best fit" line. The
inverse of the slope of the curve is the Kd. The correlation
coefficient, r.sup.2, was also computed.
Results:
[0194] The Scatchard plots were analyzed. The Kd's from this
analysis are: Version 2, Kd=14 nM; murine 24-31, Kd=8.51 nM;
version 1, Kd=5.6. The results are depicted in FIGS. 13, 14 and 15,
respectively. These results provide further evidence that the
subject humanized antibodies bind the gp39 antigen similarly to
24-31.
Use
[0195] The humanized anti-gp39 antibodies of the present invention
have potential in treating any disease condition wherein gp39
modulation and/or inhibition of the gp39-CD40 interaction is
therapeutically beneficial. Moreover, the subject humanized
anti-gp39 antibodies may be used in treatment of diseases wherein
suppression of antibody responses to antigens are desirable. Such
conditions include both autoimmune and non-autoimmune
disorders.
[0196] The ability of anti-gp39 antibodies to prevent CD40
signaling in B cells is functionally translated into marked
inhibition of T cell-dependent antibody responses in vivo.
Therefore, autoimmune diseases which are mediated by auto-antibody
production would be expected to benefit from anti-gp39 antibody
therapy. Such diseases include systemic lupus erythematosus,
idiopathic thrombocytopenic purpura, myasthenia gravis and a
subpopulation of diabetic patients with anti-insulin and
anti-insulin receptor antibodies. In addition, CD40 signaling in B
cells and dendritic cells is essential for upregulation of
co-signaling receptors such as B7.1 and B7.2 molecules. Blocking of
this CD40 signaling by anti-gp39 antibodies interferes with antigen
presentation to T cells, resulting in inhibition of T cell
activation and T cell-mediated responses. The therapeutic efficacy
of anti-gp39 antibodies in disease models such as CIA, EAE, NOD
mice, GVHD and graft rejection further confirms the antibody's
inhibitory effect on T cell-mediated responses. Based on this
mechanism of action supported by the efficacy in animal models, the
therapeutic potential of the subject humanized anti-gp39 antibodies
extend to such diseases as RA, MS, diabetes, psoriasis, GVHD and
graft rejection.
[0197] Specific conditions which are potentially treatable by
administration of the subject humanized antibodies include the
following:
[0198] Allergic bronchopulmonary aspergillosis; Autoimmune
hemolytic anemia; Acanthosis nigricans; Allergic contact
dermatitis; Addison's disease; Atopic dermatitis; Alopecia areata;
Alopecia universalis; Amyloidosis; Anaphylactoid purpura;
Anaphylactoid reaction; Aplastic anemia; Angioedema, hereditary;
Angioedema, idiopathic; Ankylosing spondylitis; Arteritis, cranial;
Arteritis, giant cell; Arteritis, Takayasu's; Arteritis, temporal;
Asthma; Ataxia-telangiectasia; Autoimmune oophoritis; Autoimmune
orchitis; Autoimmune polyendocrine failure; Behcet's disease;
Berger's disease; Buerger's disease; Bullous pemphigus;
Candidiasis, chronic mucocutaneous; Caplan's syndrome;
Post-myocardial infarction syndrome; Post-pericardiotomy syndrome;
Carditis; Celiac sprue; Chagas's disease; Chediak-Higashi syndrome;
Churg-Strauss disease; Cogan's syndrome; Cold agglutinin disease;
CREST syndrome; Crohn's disease; Cryoglobulinemia; Cryptogenic
fibrosing alveolitis; Dermatitis herpetifomis; Dermatomyositis;
Diabetes mellitus; Diamond-Blackfan syndrome; DiGeorge syndrome;
Discoid lupus erythematosus; Eosinophilic fascitis; Episcleritis;
Drythema elevatum diutinum; Erythema marginatum; Erythema
multiforme; Erythema nodosum; Familial Mediterranean fever; Felty's
syndrome; Fibrosis pulmonary; Glomerulonephritis, anaphylactoid;
Glomerulonephritis, autoimmune; Glomerulonephritis,
post-streptococcal; Glomerulonephritis, post-transplantation;
Glomerulopathy, membranous; Goodpasture's syndrome; Graft-vs.-host
disease; Granulocytopenia, immune-mediated; Granuloma annulare;
Granulomatosis, allergic; Granulomatous myositis; Grave's disease;
Hashimoto's thyroiditis; Hemolytic disease of the newborn;
Hemochromatosis, idiopathic; Henoch-Schoenlein purpura; Hepatitis,
chronic active and chronic progressive; Histiocytosis X;
Hypereosinophilic syndrome; Idiopathic thrombocytopenic purpura;
Job's syndrome; Juvenile dermatomyositis; Juvenile rheumatoid
arthritis (Juvenile chronic arthritis); Kawasaki's disease;
Keratitis; Keratoconjunctivitis sicca; Landry-Guillain-Barre-Strohl
syndrome; Leprosy, lepromatous; Loeffler's syndrome; Lyell's
syndrome; Lyme disease; Lymphomatoid granulomatosis; Mastocytosis,
systemic; Mixed connective tissue disease; Mononeuritis multiplex;
Muckle-Wells syndrome; Mucocutaneous lymph node syndrome;
Mucocutaneous lymph node syndrome; Multicentric
reticulohistiocytosis; Multiple sclerosis; Myasthenia gravis;
Mycosis fungoides; Necrotizing vasculitis, systemic; Nephrotic
syndrome; Overlap syndrome; Panniculitis; Paroxysmal cold
hemoglobinuria; Paroxysmal nocturnal hemoglobinuria; Pemphigoid;
Pemphigus; Pemphigus erythematosus; Pemphigus foliaceus; Pemphigus
vulgaris; Pigeon breeder's disease; Pneumonitis, hypersensitivity;
Polyarteritis nodosa; Polymyalgia rheumatica; Polymyositis;
Polyneuritis, idiopathic; Portuguese familial polyneuropathies;
Preeclampsia/eclampsia; Primary biliary cirrhosis; Progressive
systemic sclerosis (Scleroderma); Psoriasis; Psoriatic arthritis;
Pulmonary alveolar proteinosis; Pulmonary fibrosis, Raynaud's
phenomenon/syndrome; Reidel's thyroiditis; Reiter's syndrome,
Relapsing polychrondritis; Rheumatic fever; Rheumatoid arthritis;
Sarcoidosis; Scleritis; Sclerosing cholangitis; Serum sickness;
Sezary syndrome; Sjogren's syndrome; Stevens-Johnson syndrome;
Still's disease; Subacute sclerosing panencephalitis; Sympathetic
ophthalmia; Systemic lupus erythematosus; Transplant rejection;
Ulcerative colitis; Undifferentiated connective tissue disease;
Urticaria, chronic; Urticaria, cold; Uveitis; Vitiligo;
Weber-Christian disease; Wegener's granulomatosis; Wiskott-Aldrich
syndrome.
[0199] Of these, the preferred indications treatable or presentable
by administration of anti-gp39 antibodies include autoimmune
hemolytic anemia; aplastic anemia; arteritis, temporal; diabetes
mellitus; Felty's syndrome; Goodpasture's syndrome; graft-vs-host
disease; idiopathic thrombocytopenia pupura; myasthenia gravis;
multiple sclerosis; polyarteritis nodosa; psoriasis; psoriatic
arthritis; rheumatoid arthritis; systemic lupus erythematosus;
asthma; allergic conditions; and transplant rejection.
[0200] The amount of antibody useful to produce a therapeutic
effect can be determined by standard techniques well known to those
of ordinary skill in the art. The antibodies will generally be
provided by standard technique within a pharmaceutically acceptable
buffer, and may be administered by any desired route. Because of
the efficacy of the presently claimed antibodies and their
tolerance by humans it is possible to administer these antibodies
repetitively in order to combat various diseases or disease states
within a human.
[0201] The subject anti-gp39 humanized antibodies (or fragments
thereof) of this invention are also useful for inducing
immunomodulation, e.g., inducing suppression of a human's or
animal's immune system. This invention therefore relates to a
method of prophylactically or therapeutically inducing
immunomodulation in a human or other animal in need thereof by
administering an effective, non-toxic amount of such an antibody of
this invention to such human or other animal.
[0202] The fact that the antibodies of this invention have utility
in inducing immunosuppression means that they are useful in the
treatment or prevention of resistance to or rejection of
transplanted organs or tissues (e.g., kidney, heart, lung, bone
marrow, skin, cornea, etc.); the treatment or prevention of
autoimmune, inflammatory, proliferative and hyperproliferative
diseases, and of cutaneous manifestations of immunologically
mediated diseases (e.g., rheumatoid arthritis, lupus erythematosus,
systemic lupus erythematosus, Hashimoto's thyroiditis, multiple
sclerosis, myasthenia gravis, type 1 diabetes, uveitis, nephrotic
syndrome, psoriasis, atopical dermatitis, contact dermatitis and
further eczematous dermatitides, seborrheic dermatitis, Lichen
planus, Pemplugus, bullous pemphicjus, Epidermolysis bullosa,
urticaria, angioedemas, vasculitides, erythema, cutaneous
eosinophilias, Alopecia areata, etc.); the treatment of reversible
obstructive airways disease, intestinal inflammations and allergies
(e.g., Coeliac disease, proctitis, eosinophilia gastroenteritis,
mastocytosis, Crohn's disease and ulcerative colitis) and
food-related allergies (e.g., migraine, rhinitis and eczema). Also,
the subject antibodies have potential utility for treatment of
non-autoimmune conditions wherein immunomodulation is desirable,
e.g., graft-versus-host disease (GVHD), transplant rejection,
asthma, leukemia, lymphoma, among others.
[0203] Also, the subject antibodies can be used as
immunosuppressants during cellular or gene therapy. This
potentially will enable such cells or gene therapy constructs to be
administered repeatedly, or at higher dosages without an adverse
immunogenic response.
[0204] One skilled in the art would be able, by routine
experimentation, to determine what an effective, non-toxic amount
of antibody would be for the purpose of inducing immunosuppression.
Generally, however, an effective dosage will be in the range of
about 0.05 to 100 milligrams per kilogram body weight per day.
[0205] The antibodies of the invention may be administered to a
human or other animal in accordance with the aforementioned methods
of treatment in an amount sufficient to produce such effect to a
therapeutic or prophylactic degree. Such antibodies of the
invention can be administered to such human or other animal in a
conventional dosage form prepared by combining the antibody of the
invention with a conventional pharmaceutically acceptable carrier
or diluent according to known techniques. It will be recognized by
one of skill in the art that the form and character of the
pharmaceutically acceptable carrier or diluent is dictated by the
amount of active ingredient with which it is to be combined, the
route of administration and other well-known variables.
[0206] The route of administration of the antibody (or fragment
thereof) of the invention may be oral, parenteral, by inhalation or
topical. The term parenteral as used herein includes intravenous,
intramuscular, subcutaneous, rectal, vaginal or intraperitoneal
administration. The subcutaneous and intramuscular forms of
parenteral administration are generally preferred.
[0207] The daily parenteral and oral dosage regimens for employing
compounds of the invention to prophylactically or therapeutically
induce immunosuppression will generally be in the range of about
0.05 to 100, but preferably about 0.5 to 10, milligrams per
kilogram body weight per day.
[0208] The antibody of the invention may also be administered by
inhalation. By "inhalation" is meant intranasal and oral inhalation
administration. Appropriate dosage forms for such administration,
such as an aerosol formulation or a metered dose inhaler, may be
prepared by conventional techniques. The preferred dosage amount of
a compound of the invention to be employed is generally within the
range of about 10 to 100 milligrams.
[0209] The antibody of the invention may also be administered
topically. By topical administration is meant non-systemic
administration and includes the application of an antibody (or
fragment thereof) compound of the invention externally to the
epidermis, to the buccal cavity and instillation of such an
antibody into the ear, eye and nose, and where it does not
significantly enter the blood stream. By systemic administration is
meant oral, intravenous, intraperitoneal and intramuscular
administration. The amount of an antibody required for therapeutic
or prophylactic effect will, of course, vary with the antibody
chosen, the nature and severity of the condition being treated and
the animal undergoing treatment, and is ultimately at the
discretion of the physician. A suitable topical dose of an antibody
of the invention will generally be within the range of about 1 to
100 milligrams per kilogram body weight daily.
Formulations
[0210] While it is possible for an antibody or fragment thereof to
be administered alone, it is preferable to present it as a
pharmaceutical formulation. The active ingredient may comprise, for
topical administration, from 0.001% to 10% w/w, e.g., from 1% to 2%
by weight of the formulation, although it may comprise as much as
10% w/w but preferably not in excess of 5% w/w and more preferably
from 0.1% to 1% w/w of the formulation.
[0211] The topical formulations of the present invention, comprise
an active ingredient together with one or more acceptable
carrier(s) therefor and optionally any other therapeutic
ingredients(s) . The carrier(s) must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation
and not deleterious to the recipient thereof.
[0212] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of where treatment is required, such as
liniments, lotions, creams, ointments or pastes, and drops suitable
for administration to the eye, ear or nose.
[0213] Drops according to the present invention may comprise
sterile aqueous or oily solutions or suspensions and may be
prepared by dissolving the active ingredient in a suitable aqueous
solution of a bactericidal and/or fungicidal agent and/or any other
suitable preservative, and preferably including a surface active
agent. The resulting solution may then be clarified by filtration,
transferred to a suitable container which is then sealed and
sterilized by autoclaving or maintaining at 90.degree.-100.degree.
C. for half an hour. Alternatively, the solution may be sterilized
by filtration and transferred to the container by an aseptic
technique. Examples of bactericidal and fungicidal agents suitable
for inclusion in the drops are phenylmercuric nitrate or acetate
(0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate
(0.01%). Suitable solvents for the preparation of an oily solution
include glycerol, diluted alcohol and propylene glycol.
[0214] Lotions according to the present invention include those
suitable for application to the skin or eye. An eye lotion may
comprise a sterile aqueous solution optionally containing a
bactericide and may be prepared by methods similar to those for the
preparation of drops. Lotions or liniments for application to the
skin may also include an agent to hasten drying and to cool the
skin, such as an alcohol or acetone, and/or a moisturizer such as
glycerol or an oil such as castor oil or arachis oil.
[0215] Creams, ointments or pastes according to the present
invention are semi-solid formulations of the active ingredient for
external application. They may be made by mixing the active
ingredient in finely-divided or powdered form, alone or in solution
or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable machinery, with a greasy or non-greasy basis. The basis
may comprise hydrocarbons such as hard, soft or liquid paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural
origin such as almond, corn, arachis, castor or olive oil; wool fat
or its derivatives, or a fatty acid such as stearic or oleic acid
together with an alcohol such as propylene glycol or macrogels. The
formulation may incorporate any suitable surface active agent such
as an anionic, cationic or non-ionic surface active such as
sorbitan esters or polyoxyethylene derivatives thereof. Suspending
agents such as natural gums, cellulose derivatives or inorganic
materials such as silicaceous silicas, and other ingredients such
as lanolin, may also be included.
[0216] It will be recognized by one of skill in the art that the
optimal quantity and spacing of individual dosages of an antibody
or fragment thereof of the invention will be determined by the
nature and extent of the condition being treated, the form, route
and site of administration, and the particular animal being
treated, and that such optimums can be determined by conventional
techniques. It will also be appreciated by one of skill in the art
that the optimal course of treatment, i.e., the number of doses of
an antibody or fragment thereof of the invention given per day for
a defined number of days, can be ascertained by those skilled in
the art using conventional course of treatment determination
tests.
[0217] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following are,
therefore, to be construed as merely illustrative examples and not
a limitation of the scope of the present invention in any way.
Capsule Composition
[0218] A pharmaceutical composition of this invention in the form
of a capsule is prepared by filling a standard two-piece hard
gelatin capsule with 50 mg. of an antibody or fragment thereof of
the invention, in powdered form, 100 mg. of lactose, 32 mg. of talc
and 8 mg. of magnesium stearate.
Injectable Parenteral Composition
[0219] A pharmaceutical composition of this invention in a form
suitable for administration by injection is prepared by stirring
1.5 k by weight of an antibody or fragment thereof of the invention
in 10 k by volume propylene glycol and water. The solution is
sterilized by filtration.
Ointment Composition
[0220] Antibody or fragment thereof of the invention 1.0 g.
[0221] White soft paraffin to 100.0 g.
[0222] The antibody or fragment thereof of the invention is
dispersed in a small volume of the vehicle to produce a smooth,
homogeneous product. Collapsible metal tubes are then filled with
the dispersion.
Topical Cream Composition
[0223] Antibody or fragment thereof of the invention 1.0 g.
[0224] Polawax GP 200 20.0 g.
[0225] Lanolin Anhydrous 2.0 g.
[0226] White Beeswax 2.5 g.
[0227] Methyl hydroxybenzoate 0.1 g.
[0228] Distilled Water to 100.0 g.
[0229] The polawax, beeswax and lanolin are heated together at
60.degree. C. A solution of methyl hydroxybenzoate is added and
homogenization is achieved using high speed stirring. The
temperature is then allowed to fall to SOOC. The antibody or
fragment thereof of the invention is then added and dispersed
throughout, and the composition is allowed to cool with slow speed
stirring.
Topical Lotion Composition
[0230] Antibody or fragment thereof of the invention 1.0 g.
[0231] Sorbitan Monolaurate 0.6 g. Polysorbate 20 0.6 g.
[0232] Cetostearyl Alcohol 1.2 9. Glycerin 6.0 g.
[0233] Methyl Hydroxybenzoate 0.2 g.
[0234] Purified Water B.P. to 100.00 ml. (B.P.=British
Pharmacopeia)
[0235] The methyl hydroxybenzoate and glycerin are dissolved in 70
ml. of the water at 75.degree. C. The sorbitan monolaurate,
polysorbate 20 and cetostearyl alcohol are melted together at
75.degree. C. and added to the aqueous solution. The resulting
emulsion is homogenized, allowed to cool with continuous stirring
and the antibody or fragment thereof of the invention is added as a
suspension in the remaining water. The whole suspension is stirred
until homogenized.
Eye Drop Composition
[0236] Antibody or fragment thereof of the invention 0.5 g.
[0237] Methyl Hydroxybenzoate 0.01 g.
[0238] Propyl Hydroxybenzoate 0.04 g.
[0239] Purified Water B.P. to 100.00 ml.
[0240] The methyl and propyl hydroxybenzoates are dissolved in 70
ml. purified water at 75.degree. C. and the resulting solution is
allowed to cool. The antibody or fragment thereof of the invention
is then added, and the solution is sterilized by filtration through
a membrane filter (0.022 Am pore size), and packed aseptically into
suitable sterile containers.
Composition for Administration by Inhalation
[0241] For an aerosol container with a capacity of 15-20 ml: mix 10
mg. of an antibody or fragment thereof of the invention with
0.2-0.5 k of a lubricating agent, such as polysorbate 85 or oleic
acid, and disperse such mixture in a propellant, such as freon,
preferably in a combination of (1,2 dichlorotetrafluoroethane) and
difluorochloromethane and put into an appropriate aerosol container
adapted for either intranasal or oral inhalation administration.
Composition for Administration by Inhalation For an aerosol
container with a capacity of 15-20 ml: dissolve 10 mg. of an
antibody or fragment thereof of the invention in ethanol (6-8 ml.),
add 0.1-0.2 k of a lubricating agent, such as polysorbate 85 or
oleic acid; and disperse such in a propellant, such as freon,
preferably in combination of (1-2 dichlorotetrafluoroethane) and
difluorochloromethane, and put into an appropriate aerosol
container adapted for either intranasal or oral inhalation
administration.
[0242] The antibodies and pharmaceutical compositions of the
invention are particularly useful for parenteral administration,
i.e., subcutaneously, intramuscularly or intravenously. The
compositions for parenteral administration will commonly comprise a
solution of an antibody or fragment thereof of the invention or a
cocktail thereof dissolved in an acceptable carrier, preferably an
aqueous carrier. A variety of aqueous carriers may be employed,
e.g., water, buffered water, 0.4 k saline, 0.3% glycine, and the
like. These solutions are sterile and generally free of particulate
matter. These solutions may be sterilized by conventional,
well-known sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, etc. The concentration of the antibody or
fragment thereof of the invention in such pharmaceutical
formulation can vary widely, i.e., from less than about 0.5 k,
usually at or at least about 1% to as much as 15 or 20% by weight,
and will be selected primarily based on fluid volumes, viscosities,
etc., according to the particular mode of administration
selected.
[0243] Thus, a pharmaceutical composition of the invention for
intramuscular injection could be prepared to contain 1 mL sterile
buffered water, and 50 mg. of an antibody or fragment thereof of
the invention. Similarly, a pharmaceutical composition of the
invention for intravenous infusion could be made up to contain 250
ml. of sterile Ringer's solution, and 150 mg. of an antibody or
fragment thereof of the invention. Actual methods for preparing
parenterally administrable compositions are well-known or will be
apparent to those skilled in the art, and are described in more
detail in, e.g., Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa., hereby incorporated by reference
herein.
[0244] The antibodies (or fragments thereof) of the invention can
be lyophilized for storage and reconstituted in a suitable carrier
prior to use. This technique has been shown to be effective with
conventional immune globulins and art-known lyophilization and
reconstitution techniques can be employed.
[0245] Depending on the intended result, the pharmaceutical
composition of the invention can be administered for prophylactic
and/or therapeutic treatments. In therapeutic application,
compositions are administered to a patient already suffering from a
disease, in an amount sufficient to cure or at least partially
arrest the disease and its complications. In prophylactic
applications, compositions containing the present antibodies or a
cocktail thereof are administered to a patient not already in a
disease state to enhance the patient's resistance.
[0246] Single or multiple administrations of the pharmaceutical
compositions can be carried out with dose levels and pattern being
selected by the treating physician. In any event, the
pharmaceutical composition of the invention should provide a
quantity of the altered antibodies (or fragments thereof) of the
invention sufficient to effectively treat the patient.
[0247] It should also be noted that the antibodies of this
invention may be used for the design and synthesis of either
peptide or non-peptide compounds (mimetics) which would be useful
in the same therapy as the antibody. See, e.g., Saragovi et al,
Science, 253:792-795 (1991).
[0248] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without diverting from the scope of the invention. Accordingly, the
invention is not limited by the appended claims.
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