U.S. patent application number 12/246325 was filed with the patent office on 2009-05-14 for anti-cd40 monoclonal antibody.
Invention is credited to Xingjie Chen, Walker R. Force, TOSHIFUMI MIKAYAMA, Nobuaki Takahashi, Hitoshi Yoshida.
Application Number | 20090123466 12/246325 |
Document ID | / |
Family ID | 32601004 |
Filed Date | 2009-05-14 |
United States Patent
Application |
20090123466 |
Kind Code |
A1 |
MIKAYAMA; TOSHIFUMI ; et
al. |
May 14, 2009 |
ANTI-CD40 MONOCLONAL ANTIBODY
Abstract
An antibody or a functional fragment thereof, acting
agonistically or antagonistically on CD40.
Inventors: |
MIKAYAMA; TOSHIFUMI; (Tokyo,
JP) ; Yoshida; Hitoshi; (La Jolla, CA) ;
Force; Walker R.; (La Jolla, CA) ; Chen; Xingjie;
(Milpitas, CA) ; Takahashi; Nobuaki; (Tokyo,
JP) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN LLP
ATTENTION: DOCKETING DEPARTMENT, P.O BOX 10500
McLean
VA
22102
US
|
Family ID: |
32601004 |
Appl. No.: |
12/246325 |
Filed: |
October 6, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11633716 |
Dec 5, 2006 |
|
|
|
12246325 |
|
|
|
|
10693629 |
Oct 23, 2003 |
7193064 |
|
|
11633716 |
|
|
|
|
10040244 |
Oct 26, 2001 |
|
|
|
10693629 |
|
|
|
|
09844684 |
Apr 27, 2001 |
7063845 |
|
|
10040244 |
|
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/130.1; 530/387.1; 530/387.3 |
Current CPC
Class: |
C07K 2319/30 20130101;
A61P 35/00 20180101; A61P 37/04 20180101; C07K 2317/21 20130101;
A61P 7/00 20180101; A61P 7/04 20180101; A61P 37/08 20180101; C07K
16/2878 20130101; A61P 37/06 20180101; A61P 37/02 20180101; A61K
2039/505 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
424/133.1 ;
530/387.1; 530/387.3; 424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2001 |
JP |
2001-142482 |
Oct 5, 2001 |
JP |
2001-310535 |
Apr 26, 2002 |
JP |
PCT/JP02/04292 |
Claims
1.-30. (canceled)
31. An antibody or a functional fragment thereof, having the amino
acid sequences of a heavy chain variable region and a light chain
variable region of an antibody that is produced by, 2105 (Accession
No: FERM BP-8024) or F1-102 (Accession No: ATCC PTA-3337).
32. An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma F2-103, which are respectively encoded by plasmid DNAs
with Accession Nos. ATCC PTA-3302 and ATCC PTA-3303; a heavy chain
variable region and a light chain variable region of the antibody
produced by a hybridoma F5-77, which are respectively encoded by
plasmid DNAs with Accession Nos. ATCC PTA-3304 and ATCC PTA-3305;
or a heavy chain variable region and a light chain variable region
of the antibody produced by a hybridoma F5-157, which are
respectively encoded by plasmid DNAs with Accession Nos. ATCC
PTA-3306 and ATCC PTA-3307.
33. An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma 2105, which are respectively represented by SEQ ID NOS:
32 and 34; a heavy chain variable region and a light chain variable
region of the antibody produced by a hybridoma 110, which are
respectively represented by SEQ ID NOS: 36 and 38; a heavy chain
variable region and a light chain variable region of the antibody
produced by a hybridoma 115, which are respectively represented by
SEQ ID NOS: 40 and 42; a heavy chain variable region and a light
chain variable region of the antibody produced by a hybridoma
KM643-4-11, which are respectively represented by SEQ ID NOS: 52
and 54; a heavy chain variable region and a light chain variable
region of the antibody produced by a hybridoma F2-103, which are
respectively represented by SEQ ID NOS: 60 and 62; or a heavy chain
variable region and a light chain variable region of the antibody
produced by a hybridoma F5-77, which are respectively represented
by SEQ ID NOS: 64 and 66.
34. An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma 2105, which are respectively
represented by SEQ ID NOS: 31 and 33; a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma 110, which are respectively
represented by SEQ ID NOS: 35 and 37; a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma 115, which are respectively
represented by SEQ ID NOS: 39 and 41; a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma KM643-4-11, which are
respectively represented by SEQ ID NOS: 51 and 53; a heavy chain
variable region and a light chain variable region that are encoded
by nucleic acid sequences isolated from a hybridoma F2-103, which
are respectively represented by SEQ ID NOS: 59 and 61; or a heavy
chain variable region and a light chain variable region that are
encoded by nucleic acid sequences isolated from a hybridoma F5-77,
which are respectively represented by SEQ ID NOS: 63 and 65.
35. An antibody or a functional fragment thereof, having amino acid
sequences of a heavy chain variable region and a light chain
variable region of the antibody produced by a hybridoma 2053 or
285.
36. An antibody or a functional fragment thereof, having amino acid
sequences of a heavy chain variable region and a light chain
variable region of the antibody produced by a hybridoma KM281-1-10
(Accession No: FERM BP-7579), 4D11 (Accession No: FERM
BP-7758).
37. An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma KM281-1-10, which are respectively represented by SEQ ID
NOS: 44 and 46; a heavy chain variable region and a light chain
variable region of the antibody produced by a hybridoma 4D11, which
are respectively represented by SEQ ID NOS: 48 and 50.
38. An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma KM281-1-10, which are
respectively represented by SEQ ID NOS: 43 and 45; a heavy chain
variable region and a light chain variable region of an antibody
produced by a hybridoma 4D11, which are respectively represented by
SEQ ID NOS: 47 and 49.
39. An antibody or a functional fragment thereof, having amino acid
sequences of a heavy chain variable region and a light chain
variable region of the antibody produced by a hybridoma
KM281-2-10.sup.-1-2, 1, KM283-5, KM292-1-24, <M225-2-56, 5H10,
11 E1 or 5G3.
40. The antibody or the functional fragment thereof of any one of
claims 1 to 9, which is a human antibody.
41. A pharmaceutical composition, containing as an active
ingredient the antibody or the functional fragment thereof of any
one of claims 1 to 9.
42. An immunopotentiating agent, anti-tumor agent or
anti-autoimmune disease agent, containing as an active ingredient
the antibody or the functional fragment thereof of any one of
claims 1 to 5.
43. An immunosuppressive agent, anti-autoimmune disease agent,
therapeutic agent against allergies or therapeutic agent against
blood coagulation factor VIII-inhibiting syndrome, containing as an
active ingredient the antibody or the functional fragment thereof
of any one of claims 6 to 9.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/844,684 filed on Apr. 27, 2001, application Ser. No.
10/040,244 filed Oct. 26, 2001 and PCT/JP02/04292 having an
international filing date of Apr. 26, 2002, which designated the
United States of America. This application and application Ser. No.
10/040,244 are also each a continuation-in-part of application Ser.
No. 09/844,684. This application also claims priority under 35
U.S.C. .sctn.119(a) on Japanese Patent Applications Nos.
2001-142482 filed May 11, 2001 and 2001-310535 filed Oct. 5, 2001.
The entire contents of all of the above-identified applications are
hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to an antibody or a functional
fragment thereof that recognizes a human CD40 antigen present on
the surface of human B cells, dendritic cells (DC) and the like.
Specifically, the present invention relates to an anti-human CD40
antibody or a functional fragment thereof that is substantially
antagonistic to a human CD40 antigen on the dendritic cell (DC)
surface, and an agonistic anti-human CD40 antibody or a functional
fragment thereof that is expected to have a therapeutic effect
higher than those of conventional anti-human CD40 antibodies.
BACKGROUND ART
1. CD40
[0003] CD40 is an antigen with a molecular weight of 50 kDa that is
present on the cell membrane surface. CD40 is expressed on B cells,
dendritic cells (DC), certain types of cancer cells, and thymic
epithelial cells. CD40 is known to play a key role in proliferation
and differentiation of B cells and DC. CD40 has been identified as
an antigen that is expressed on the human B cell surface (E. A.
Clark et. al., Proc. Natl. Acad. Sci. USA 83: 4494, 1986, I.
Stamenkovic et. al., EMBO J. 8:1403, 1989). Based on the amino acid
sequence homology, CD40 is thought to be a member of the TNF
receptor family, to which a low affinity NGF receptor, TNF
receptor, CD27, OX40, CD30 and the like belong. The gene of a
ligand (CD40L) for human and mouse CD40 has been cloned recently,
revealing that it is a type II membrane protein, and is expressed
on activated CD4+T cells. It has also been shown that CD40L
introduces strong activation signals into human and mouse B
cells.
[0004] The expression of CD40 has been confirmed more often on
dendritic cells than on B cells, so that it has become clear that
CD40 plays an important role. The binding of CD40 with CD40L causes
the activation of antigen-presenting cells (APC). Specifically, it
enhances the expression of co-stimulation molecules such as CD80
(B7-1) and CD86 (B7-2), or the production of IL-12 (Caux, C., et
al.: Activation of human dendritic cells through CD40
cross-linking. J. Exp. Med., 180:1263, 1994), (Shu, U., et al:
Activated T cells induce interleukin-12 production by monocyte via
CD40-CD40 ligand interaction. Eur. J. Immunol. 25: 1125, 1995).
Dendritic cells show strong antigen-presenting ability, and have
strong helper T (Th) cell-activating ability. Furthermore, it is
thought that dendritic cells control the differentiation of naive
Th cells into Th1 or Th2 cells. When dendritic cells (DC1) are made
to mature by culturing peripheral blood monocytes that are myeloid
dendritic cells in the presence of GM-CSF and IL-4 and using CD40L,
the DC1 in vitro are capable of producing IL-12, stimulate and
activate allo-naive Th cells, and thus induce IFN.gamma.-producing
T cells (specifically, promotes differentiation into Th1). Since
this action is inhibited by anti-IL-12 antibodies, the reaction may
be mediated by IL-12. On the other hand, when lymphocyte-dendritic
cells (DC2) are prepared by culturing lymphatic tissue T regions or
plasmacytoid T cells present in peripheral blood with IL-3 and CD40
ligands, DC2 are incapable of producing IL-12, stimulate and
activate allo-naive Th cells, induce IL-4-producing T cells, and
thus promote differentiation into Th2. It is thought that Th1 cells
are involved in the activation of cellular immunity, and Th2 cells
are involved in enhancement of the ability for humoral immunity as
well as the suppression of the ability for cellular immunity.
Cytotoxic T cells (CTL) activated with the help of Th1 cells can
remove causative factors (many viruses, Listeria monocytogenes,
tubercle bacillus, toxoplasma protozoa and the like) multiplying in
the cytoplasm and tumor cells.
[0005] It has been shown that anti-CD40 monoclonal antibodies that
recognize CD40 expressed on the membrane surfaces exert a variety
of biological activities on B cells. Anti-CD40 monoclonal
antibodies are largely classified into agonistic and antagonistic
antibodies impacting the interaction between CD40 and CD40L.
2. Agonistic Antibody
[0006] The activation of B cells is known as an action of agonistic
antibodies. For example, anti-CD40 antibodies have been reported to
induce cell adhesion (Barrett et al., J. Immunol. 146: 1722, 1991;
Gordon et al., J. Immunol. 140: 1425, 1988), enhance cell size
(Gordon et al., J. Immunol. 140: 1425, 1988; Valle et al., Eur. J.
Immunol. 19: 1463, 1989), induce the division of B cells that are
activated only with anti-IgM antibodies, anti-CD20 antibodies or
phorbol ester (Clark and Ledbetter, Proc. Natl. Acad. Sci. USA 83:
4494, 1986; Gordon et al., LEUCOCYTE TYPING III. A. J. McMicheal
ed. Oxford University Press. Oxford, p. 426; Paulie et al., J.
Immunol. 142: 590, 1989), induce the division of B cells in the
presence of IL4 (Valle et al., Eur. J. Immunol. 19: 1463, 1989;
Gordon et al., Eur. J. Immunol. 17: 1535, 1987), induce the
expression of IgE (Jabara et al., J. Exp. Med. 172: 1861, 1990;
Gascan et al., J. Immunol. 147: 8, 1991), IgG and IgM (Gascan et
al., J. Immunol. 147: 8, 1991) of cells stimulated with IL-4 and
cultured without T cells, enhance the secretion and the on-the-cell
expression (Challa A, Allergy, 54: 576, 1999) of soluble
CD23/Fc.epsilon. RII from B cells by IL-4 (Gordon and Guy, Immunol.
Today 8: 339, 1987; Cairns et al., Eur. J. Immunol. 18: 349, 1988),
and promote IL-6 production (Clark and Shu, J. Immunol. 145: 1400,
1990). Furthermore, it has been reported that B cell clones are
established from human primary culture B cells by adding IL-4 and
anti-CD40 antibodies in the presence of CDw32+ adhesion cells
(Bancherau et al., Science 241:70, 1991), and the inhibition of the
apoptosis of germinal center cells is mediated by CD40, regardless
of the function of antigen receptors (Liu et al., Nature 342: 929,
1989). As described above, CD40 has been identified as an antigen
expressed on the human B cell surface. Thus, most of the isolated
antibodies have been evaluated mainly using function to induce the
proliferation and differentiation of human B cells and activity to
induce cell death in cancer cells as indicators (Katira, A. et.
al., LEUKOCYTE TYPING V. S. F. Schlossossman, et al. eds. p. 547.
Oxford University Press. Oxford, W. C. Flansow et al., LEUKOCYTE
TYPING V. S. F. Schlossossman, et al. eds. p. 555. Oxford
University Press. Oxford, J. D. Pound et al., International
Immunology, 11: 11, 1999).
[0007] Anti-CD40 antibodies were shown to cause the maturation of
DC (Z. H. Zhou et. al., Hybridoma, 18: 471 1999). Moreover, the
role of CD4T cells in antigen-specific CD8T cell priming has been
reported to activate DC via CD40-CD40L signaling. It was shown that
the role of CD4 helper T cells in activation of dendritic cells
(DC) can be replaced by that of anti-CD40 monoclonal antibodies
(mAb) (Shoenberger, S. P., et al.: T-cell help for cytotoxic T
lymphocytes is mediated by CD40-CD40L interactions. Nature, 480,
1998). Furthermore, it was shown in mice that the organism can be
protected not only from tumor cells expressing CD40 but also from
tumor cells not expressing the same by the administration of
anti-CD40 antibodies (French, R. R., et. al.: CD40 antibody evokes
a cytotoxic T-cell response that eradicates lymphoma and bypasses
T-cell help. Nature Medicine, 5, 1999).
[0008] Most antibodies reported to date have not been isolated
using the effect on DC as an indicator. However, in terms of the
modification of DC functions, antibodies selected by their action
on B cells are likely to be insufficient as therapeutic agents. It
was reported that among monoclonal antibodies against mouse CD40,
there are clones that react to DC, but do not react to vascular
endothelial cells, and, conversely, clones that do not react to DC,
but react to vascular endothelial cells, depending on epitopes that
the antibodies recognize (Van Den Berg, T K, et. al., Immunology,
88: 294, 1996). It is also assumed that the binding and action of
human CD40 antibodies to DC differ depending on epitopes.
[0009] It is known that anti-CD40 antibodies or CD40 ligands can
suppress the proliferation of CD40-expressing lymphoma cell lines
and thus can induce the cell death (Funakoshi S et al., Blood, 83:
2782, 1994; Funakoshi S et al., Journal of Immunotherapy, 19, 93,
1996; Z. H. Zhou et. al., Hybridoma, 18: 471 1999; and Joseph A et
al., Cancer Research, 60: 3225, 2000). What is interesting about
agonistic antibodies is that the function of the antibody does not
always coincide always with that of CD40L. Action to activate B
cells does not also coincide with action to suppress B cell tumor
growth. It is desired to develop antibodies having both
DC-activating ability and tumor cell proliferation-suppressing
action. Moreover, among agonistic antibodies, both antibodies that
inhibit and those that do not inhibit the binding of CD40L to CD40
are present (Challa A et al., Allergy, 54: 576, 1999). For example,
antibodies produced by G28-5 (ATCC No. HB-9110) compete with CD40L,
so that there is no effect resulting from the combined use with
CD40L. The degree of activation of CD40-expressing cells differs
depending on antibodies. Even when antibodies exhibit independently
weak agonistic activity, the combined use of the antibodies with
CD40 ligands may more significantly promote the activity in the
presence of the antibodies, than the activity resulting from CD40
ligands alone. In contrast, even when antibodies exhibit
independently agonistic activity, inhibition of CD40 ligands may
lower the activity in the presence of the antibodies to a greater
extent than the activity resulting from CD40 ligands alone (Pound
et al., International Immunology, 11:11, 1999). It was shown that
with antibodies that do not compete with CD40 ligands, stronger
suppression of proliferation can be achieved in the presence of
CD40 ligands, although the tumor cell proliferation-suppressing
action of the antibody itself is weak (Joseph A et al., Cancer
Research, 60: 3225, 2000). Accordingly, it is desired to develop
antibodies that bind to CD40 to suppress independently cell
proliferation, but that do not inhibit the binding of CD40 ligands
to CD40. By taking full advantage of such characteristics, there is
a possibility of developing a therapeutic agent that is more
efficient than a soluble CD40L. For example, the soluble CD40L is
activated by binding with CD40, and at the same time, it suppresses
the function of CD40L present in vivo. An antibody that does not
compete with CD40L, does not cause such suppression, and has better
therapeutic effects can be expected by synergistic effect.
3. Antagonistic Antibody
[0010] In the meantime, as described above, it is expected that
because CD40 plays an important role in immune reaction,
therapeutic agents for immune suppression upon organ
transplantation and against autoimmune disease can be developed by
inhibiting the binding of CD40 with its ligand. Sawada-Hase et al.,
have reported that the proportion of cells strongly expressing CD40
was increased in the peripheral blood monocytes of Crohn's disease
patients. However, antibodies that inhibit the binding of CD40 with
its ligand have not been well understood For example, such
antibodies that inhibit the binding may be effective for the
functional analysis of CD40, and therapy against disease, for which
activation of CD40 is required. Moreover, antibodies that inhibit
CD40 ligands have been also shown to have the potential of being
effective as agents against diseases with which the binding of CD40
with CD40 ligands is involved. However, it has been reported that
CD40L is expressed in activated blood platelets (V. Henn et. al.,
Nature 391: 591, 1998). Thus, it has been reported that there is a
risk of causing thrombi, if anti-CD40L antibodies are used as a
therapeutic agent (T. Kawai et. al., Nat. Medi. 6: 114, 2000). From
such a point of view, antibodies against CD40 can be expected to be
safer than anti-CD40L antibodies, as an antibody therapeutic agent
that inhibits the binding of CD40 with its ligand. Anti-CD40
antibodies are required to suppress the binding of CD40L to CD40,
and not to activate CD40 by the antibody itself.
[0011] Although a huge number of studies have been conducted in the
past concerning antibodies that bind specifically to human CD40 and
suppress the binding of CD40L to CD40 without activating CD40, only
a single case, that is a mouse anti-human CD40 antibody, named
5D12, has been reported (J. Kwekkeboom et al., Immunology 79: 439,
1993). In addition, it has not been known whether or not antibodies
showing neutralization activity for B cells can also show the same
for DC that is, if the antibodies can neutralize the action of CD40
ligands. Furthermore, it has been reported that the action of
biotinylated anti-mouse CD40 antibodies is enhanced by
cross-linking with avidin (Johnson et al., Eur J Immunol, 24: 1835,
1994). We enhanced the action of soluble CD40 ligands against a B
cell line (Ramos cells) using antibodies (M2) against tags (FLAG),
which had been previously provided by genetic engineering
techniques to the soluble ligands, and measured the neutralization
activity. Thus, we confirmed that 5D12 (ATCC No. HB-11339) exhibits
only slight neutralization activity.
[0012] We have newly found that 5D12, an antagonistic antibody, has
agonistic activity on its own, as a result of cross-linking even in
the absence of CD40L. Conventionally, it has been reported that the
action of mouse CD40 antibodies is enhanced by cross-linking of
biotin with avidin (Johnson et al., Eur J Immunol, 24: 1835, 1994).
Furthermore, it has been known that solid-phasing of CD40
antibodies using anti-immunoglobulin antibodies solid-phased on a
plate leads to an increase in activity to suppress the
proliferation of tumor cells. This has been thought to be an effect
resulting from solid-phasing. However, it has not been known that
when anti-immunoglobulin antibodies are added to a culture solution
for cross-linking of anti-CD40 antibodies, it may become possible
even for antagonistic antibodies to show agonistic activity. If
antibodies to be used for therapy have antigenicity, a completely
opposite effect may occur, such that antibodies which bind to CD40
antibodies in a human body are produced, and with which CD40
antibodies are cross-linked, so that activity seemingly the same as
that of CD40 ligands is produced. Accordingly, in view of the
safety of a therapeutic agent, it is very important to keep the
antigenicity of antibodies at a low level. Consider a case wherein
a therapeutic agent is developed by humanization technology based
on the sequence of a variable region of a mouse antibody. Since
humanized antibodies are known to have immunogenicity,
anti-humanized anti-CD40 antibodies may be produced after
administration. Specifically, there may be a risk that the
antibodies would become agonistic antibodies. Even if the
antigenicity is low, anti-CD40 antibodies may be cross-linked with
antibody receptors (FcR). From these points, a preferred
antagonistic antibody is a human antibody, which binds specifically
to CD40, suppresses the binding of CD40L, and does not activate
CD40 even by cross-linking, and exhibits weak binding to FcR.
SUMMARY OF THE INVENTION
[0013] As described above, the functions of DC have been
increasingly analyzed recently, so that CD40 has begun to be
recognized as a gene important in controlling the functions of DC.
Starting from this background, the purpose of the present invention
is to provide by employing an evaluation system using DC, an
anti-human CD40 antibody or a functional fragment thereof, which is
substantially antagonistic also to a human CD40 antigen on the
dendritic cell (DC) surface, and an agonistic anti-human CD40
antibody or a functional fragment thereof that is expected to have
a therapeutic effect higher than that of the conventional
anti-human CD40 antibody.
[0014] As a result of intensive studies concerning the preparation
of antibodies against human CD40, we have completed the present
invention by succeeding in producing a novel agonistic antibody and
antagonistic antibody that are thought to have a therapeutic effect
against disease higher than that of the conventionally known
anti-CD40 antibody. That is, the present invention is as
follows.
(1) An antibody against a human CD40, or a functional fragment
thereof, having at least one property selected from the following
properties (a) to (f) of: (a) acting on dendritic cells to produce
IL-12 in the presence of LPS and IFN.gamma.; (b) having activity to
act on dendritic cells causing the cells to mature, which is higher
than that of a G28-5 antibody; (c) having activity to promote an
established B cell line to express CD95, which is higher than that
of the G28-5 antibody; (d) having activity to suppress the
proliferation of an established B cell line, which is higher than
that of the G28-5 antibody; (e) inducing cell death of an
established B cell line; and (f) not inhibiting the binding of CD40
ligands to CD40. (2) The above antibody or the functional fragment
thereof of the present invention, wherein the maturation of
dendritic cells is performed at a concentration of 20 .mu.g/ml or
less. In addition, the antibody or the functional fragment thereof
promote the established B cell line to express CD95 at the antibody
concentration of 20 .mu.g/ml or less. Examples of the established B
cell line include Ramos, HS-Sulton or the like. (3) Furthermore,
the above antibody or the functional fragment thereof of the
present invention leads to the production of 100 pg/ml or more
IL-12 when the antibodies with a concentration of 0.1 .mu.g/ml or
more are added to dendritic cells with a concentration of
1.times.10.sup.6 cells/ml, and the production of 1000 pg/ml or
more, preferably 10000 pg/ml or more IL-12 when the antibodies with
a concentration of 1 .mu.g/ml or more are added. (4) Furthermore,
within the antibody concentration range between 0.01 .mu.g/ml and
10 .mu.g/ml, the above antibody or the functional fragment thereof
of the present invention, promoting the established B cell line
(Ramos cell) to express CD95 with approximately 2 to 3 times or
more greater effectiveness than that expressed by a G28-5 antibody
as a control. For example, with an antibody concentration of 0.01
.mu.g/ml, the expression is promoted with approximately 2 to 6
times or more greater effectiveness than that expressed by the
G28-5 antibody as a control. With an antibody concentration of 0.1
.mu.g/ml, the expression is promoted with approximately 2 to 7
times or more greater effectiveness than that expressed by the
G28-5 antibody as a control. With an antibody concentration of 1
.mu.g/ml, the expression is promoted with approximately 2 to 7
times or more greater effectiveness than that expressed by the
G28-5 antibody as a control. With an antibody concentration of 10
.mu.g/ml, the expression is promoted with approximately 2 to 6
times or more greater effectiveness than that expressed by the
G28-5 antibody as a control. (5) An antibody or a functional
fragment thereof, having the amino acid sequences of a heavy chain
variable region and a light chain variable region of an antibody
that is produced by a hybridoma KM302-1 (Accession No: FERM
BP-7578), KM341-1-19 (Accession No: FERM BP-7759), 2105 (Accession
No: FERM BP-8024) or F1-102 (Accession No: ATCC PTA-3337).
TABLE-US-00001 Name Accession No. Deposition date Deposited with:
KM302-1 FERM BP-7578 May 9, 2001 International Patent KM341- FERM
BP-7759 Sep. 27, Organism Depositary, 1-19 2001 National Institute
of 2105 FERM BP-8024 Apr. 17, 2002 Advanced Industrial Science and
Technology (Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan)
F1-102 ATCC Apr. 24, 2001 American Type Culture PTA-3337 Collection
(10801 University Blvd. Manassas, Virginia, 20110-2209, U.S.A.)
(6) An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma F2-103, which are respectively encoded by plasmid DNAs
with Accession Nos. ATCC PTA-3302 and ATCC PTA-3303; a heavy chain
variable region and a light chain variable region of the antibody
produced by a hybridoma F5-77, which are respectively encoded by
plasmid DNAs with Accession Nos. ATCC PTA-3304 and ATCC PTA-3305;
or a heavy chain variable region and a light chain variable region
of the antibody produced by a hybridoma F5-157, which are
respectively encoded by plasmid DNAs with Accession Nos. ATCC
PTA-3306 and ATCC PTA-3307.
TABLE-US-00002 Name Accession No. Deposition date Deposited with:
F2-103 ATCC Apr. 19, 2001 American Type Culture heavy chain
PTA-3302 Collection (10801 (F2-103-H) University Blvd. F2-103 light
ATCC Apr. 19, 2001 Manassas, Virginia, chain PTA-3303 20110-2209,
U.S.A.) (F2-103-L) F5-77 heavy ATCC Apr. 19, 2001 chain PTA-3304
(F5-77-H) F5-77 light ATCC Apr. 19, 2001 chain PTA-3305 (F5-77-L)
F5-157 ATCC Apr. 19, 2001 heavy chain PTA-3306 (F5-157-H) F5-157
light ATCC Apr. 19, 2001 chain PTA-3307 (F5-157-L)
(7) An antibody or a functional fragment thereof, laving amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma KM34'-1-19, which are respectively represented by SEQ ID
NOS: 28 and 30; a heavy chain variable region and a light chain
variable region of the antibody produced by a hybridoma 2105, which
are respectively represented by SEQ ID NOS: 32 and 34; a heavy
chain variable region and a light chain variable region of the
antibody produced by a hybridoma 110, which are respectively
represented by SEQ ID NOS: 36 and 38; a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma 115, which are respectively represented by SEQ ID NOS: 40
and 42; a heavy chain variable region and a light chain variable
region of the antibody produced by a hybridoma KM643-4-11, which
are respectively represented by SEQ ID NOS: 52 and 54; a heavy
chain variable region and a light chain variable region of the
antibody produced by a hybridoma F2-103, which are respectively
represented by SEQ ID NOS: 60 and 62; or a heavy chain variable
region and a light chain variable region of the antibody produced
by a hybridoma F5-77, which are respectively represented by SEQ ID
NOS: 64 and 66. (8) An antibody or a functional fragment thereof,
having amino acid sequences of the mature portions of a heavy chain
variable region and a light chain variable region that are encoded
by nucleic acid sequences isolated from a hybridoma KM341-1-19,
which are respectively represented by SEQ ID NOS: 27 and 29; a
heavy chain variable region and a light chain variable region that
are encoded by nucleic acid sequences isolated from a hybridoma
2105, which are respectively represented by SEQ ID NOS: 31 and 33;
a heavy chain variable region and a light chain variable region
that are encoded by nucleic acid sequences isolated from a
hybridoma 110, which are respectively represented by SEQ ID NOS: 35
and 37; a heavy chain variable region and a light chain variable
region that are encoded by nucleic acid sequences isolated from a
hybridoma 115, which are respectively represented by SEQ ID NOS: 39
and 41; a heavy chain variable region and a light chain variable
region that are encoded by nucleic acid sequences isolated from a
hybridoma KM643-4-11, which are respectively represented by SEQ ID
NOS: 51 and 53; a heavy chain variable region and a light chain
variable region that are encoded by nucleic acid sequences isolated
from a hybridoma F2-10).sub.3, which are respectively represented
by SEQ ID NOS: 59 and 61; or a heavy chain variable region and a
light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma F5-77, which are respectively
represented by SEQ ID NOS: 63 and 65. (9) An antibody against a
human CD40, or a functional fragment thereof, having at least one
property selected from the following properties (g) to (j) of: (g)
neutralizing the action of ligands on CD40; (h) neutralizing or
alleviating one or more effects that ligands, which are for CD40 on
an established B cell line, have on CD40-expressing cells, and
having agonistic action on CD40 on the above established B cell
line weaker than that of 5D12 due to cross-linking by
anti-immunoglobulin antibodies; (i) alleviating or neutralizing the
action of CD40 ligands on the established B cell line to increase
CD95 expression; and (j) having antagonistic action on CD40
expressed on dendritic cells. (10) The antibody or the functional
fragment of (9) above can suppress the expression of CD95 in Ramos
cells to a level approximately 10% or less than that of a control,
when antibodies with a concentration of 0.1 .mu.g/ml are added to
the Ramos cells with a concentration of 1.times.10.sup.6 cells/ml
supplemented with a saturated amount of CD40L-expressing cells; can
suppress the expression of CD95 in Ramos cells to the same level as
that of a negative control, when the antibodies with a
concentration of 1 .mu.g/ml are added; and can suppress the
expression of CD95 in the Ramos cells to the same level as that of
the negative control, when the antibodies with a concentration of
10 .mu.g/ml are added. (11) The antibody or the functional fragment
thereof of (9) above, wherein the proliferation of tonsillar B
cells is suppressed in vitro by approximately 80 to 95% or more,
when the antibodies with a concentration between 0.001 .mu.g/ml and
10 .mu.g/ml are added to 1.times.10.sup.5 tonsillar B cells
supplemented with soluble CD40L (1 .mu.g/ml). For example, when the
antibodies with a concentration between 0.01 .mu.g/ml and 10
.mu.g/ml are added, the proliferation of tonsillar B cells is
suppressed by approximately 95% or more. In particular, when the
antibodies with a concentration of 0.001 .mu.g/ml are added, the
proliferation of tonsillar B cells is suppressed by approximately
80% or more. (12) An antibody or a functional fragment thereof,
having amino acid sequences of a heavy chain variable region and a
light chain variable region of the antibody produced by a hybridoma
KM281-1-10 (Accession No: FERM BP-7579), 4D11 (Accession No: FERM
BP-7758) or F4-465 (Accession No: ATCC PTA-3338).
TABLE-US-00003 Name Accession No. Deposition date Deposited with:
KM281-1-10 FERM BP-7579 May 9, 2001 International Patent 4D11 FERM
BP-7758 Sep. 27, 2001 Organism Depositary, National Institute of
Advanced Industrial Science and Technology (Central 6, 1-1-1,
Higashi, Tsukuba, Ibaraki, Japan) F4-465 ATCC Apr. 24, 2001
American Type PTA-3338 Culture Collection (10801 University Blvd.
Manassas, VA 20110-2209, U.S.A.)
(13) An antibody or a functional fragment thereof, having amino
acid sequences of the mature portions of a heavy chain variable
region and a light chain variable region of the antibody produced
by a hybridoma KM281-1-10, which are respectively represented by
SEQ ID NOS: 44 and 46; a heavy chain variable region and a light
chain variable region of the antibody produced by a hybridoma 4D11,
which are respectively represented by SEQ ID NOS: 48 and 50; or a
heavy chain variable region and a light chain variable region of
the antibody produced by a hybridoma F4-465, which are respectively
represented by SEQ ID NOS: 56 and 58. (14) An antibody or a
functional fragment thereof, having amino acid sequences of the
mature portions of a heavy chain variable region and a light chain
variable region that are encoded by nucleic acid sequences isolated
from a hybridoma KM281-1-10, which are respectively represented by
SEQ ID NOS: 43 and 45; a heavy chain variable region and a light
chain variable region of an antibody produced by a hybridoma 4D11,
which are respectively represented by SEQ ID NOS: 47 and 49; or a
heavy chain variable region and a light chain variable region that
are encoded by nucleic acid sequences isolated from a hybridoma
F4-465, which are respectively represented by SEQ ID NOS: 55 and
57. (15) Examples of the antibody or the functional fragment
thereof of (1) to (14) above include human antibodies. (16) A
pharmaceutical composition, containing as an active ingredient the
antibody or the functional fragment thereof of any one of (1) to
(15) above. (17) An immunopotentiating agent, anti-tumor agent or
anti-autoimmune disease agent, containing as an active ingredient
the antibody or the functional fragment thereof of any one of (1)
to (8) above. (18) An immunosuppressive agent, anti-autoimmune
disease agent, therapeutic agent against allergies or therapeutic
agent against blood coagulation factor VIII-inhibiting syndrome,
containing as an active ingredient the antibody or the functional
fragment thereof of any one of (9) to (14) above. (19) Here, an
epitope of a human CD40 that the monoclonal antibody of the present
invention recognizes can be determined by a known method, such as
by examining the binding to overlapping synthetic oligopeptides
obtained from the primary amino acid sequence of human CD40 (e.g.,
Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual,
1988 Cold Spring Harbor Laboratory Press; U.S. Pat. No. 4,708,871).
A peptide library kit with the phage display process (New England
BioLabs) can also be used for epitope mapping. The present
invention also encompasses an antibody or a functional fragment
thereof that recognizes a novel epitope of human CD40 that the
antibody or the functional fragment thereof produced by each of the
above hybridomas recognizes. (20) The present invention further
provides a nucleic acid (RNA or cDNA) encoding at least the
variable region of a heavy chain and/or light chain of an antibody
isolated from each of the above hybridomas, a vector containing the
nucleic acid, and a host cell carrying the nucleic acid.
[0015] The present invention will be described in detail. This
specification includes part or all of the contents as disclosed in
the specification and/or drawings of PCT Application PCT/US01/13672
(filed on Apr. 27, 2001), Japanese Patent Application No.
2001-142482 (filed on May 11, 2001), Japanese Patent Application
No. 2001-310535 (filed on Oct. 5, 2001), and U.S. patent
application U.S. Ser. No. 10/040,244 (filed on Oct. 26, 2001) which
are priority documents of the present application.
[0016] As described later, we have found that a known monoclonal
antibody 5D12 (ATCC No. HB-11339) that is antagonistic to CD40 on B
cells is not antagonistic to CD40 on DC. We have further found that
many monoclonal antibodies show agonistic activity on their own as
a result of cross-linking by anti-immunoglobulin antibodies, even
if they are antagonistic antibodies that block the action of
CD40L.
1. DEFINITION
[0017] The terms used in this specification are defined as
follows.
[0018] The term "human CD40" in the present invention means a
polypeptide having an amino acid sequence shown by Clark et al. (E.
A. Clark et al., Proc. Natl. Acad. Sci. USA 83: 4494, 1986) or
Stamenkovic et al. (1. Stamenkovic et al., EMBO J. 8: 1403, 1989).
Specifically, the human CD40 is an antigen polypeptide that is
expressed on the surface of a B cell, DC, macrophage, endothelial
cell, epithelial cell or tumor cells of these cells.
[0019] The term "anti-CD40 monoclonal antibody" means any
monoclonal antibody against CD40 expressed by a cell, full-length
CD40 or partial length CD40. A more preferred anti-CD40 monoclonal
antibody binds to the extracellular portion of CD40 and provides
agonistic or antagonistic action on the cells expressing CD40.
[0020] Furthermore, the term "antibody" in the present invention is
derived from a gene (generically called an "antibody gene")
encoding a heavy chain variable region, a heavy chain constant
region, a light chain variable region and a light chain constant
region composing an immunoglobulin. The antibody of the present
invention encompasses an antibody that is of any immunoglobulin
class and has any isotype. The term "functional fragment" of the
antibody in the present invention is a part (a partial fragment) of
an antibody as defined above, and means a fragment retaining one or
more actions of the antibody on an antigen. Specific examples of
such functional fragment include F(ab').sub.2, Fab', Fab, Fv, FVs
with disulfide bond, single-stranded FV(scFV), and polymers thereof
(D. J. King., Applications and Engineering of Monoclonal
Antibodies., 1998 T. J. International Ltd).
[0021] The term "human antibody" in the present invention means an
antibody which is the expression product of a human-derived
antibody gene.
[0022] The term "agonistic" means an action to promote the binding
of CD40 ligands to CD40 expressed on the surfaces of cells such as
B cells, tumor cells or dendritic cells, or an action to provide
CD40-expressing cells with one or more effects that are provided by
CD40 ligands to CD40-expressing cells. The term "agonistic
antibody" means an antibody having such an agonistic action.
[0023] The term "antagonistic" means an action to inhibit the
binding of CD40 ligands to CD40 expressed on the surfaces of cells
such as B cells, tumor cells or dendritic cells, or an action to
neutralize one or more effects that are provided by CD40 ligands to
CD40-expressing cells. The term "antagonistic antibody" means an
antibody having such an action.
[0024] The term "dendritic cells (DC)" in the present invention
indicates a group of cells which are also referred to as dendritic
leukocytes having a strong antigen-presenting function. Dendritic
cells used herein are induced by culturing CD34 positive precursor
cells contained in, for example, bone marrow, umbilical cord blood
or peripheral blood. Alternatively, the dendritic cells can be
obtained by culturing CD14 positive monocytes in peripheral blood
in the presence of GM-CSF and IL-4.
[0025] The term "immature DC" means DC that are CD14 negative, CD1a
strongly positive, CD83, CD86 positive, and MHC class II
positive.
[0026] The term "mature DC" means DC that are CD14 negative, CD1a
positive, and have become CD83, CD86 and MHC class II strongly
positive.
[0027] The term "activate DC" in the present invention means a
change that DC induce by responding to the stimulation by CD40. For
example, it also means to cause the maturation of immature DC, the
high expression of CD80, CD86 and HLA-Class II, and the enhancement
of IL-12 production. Alternatively, when T cells co-exist, it also
means to stimulate T cells to promote their proliferation.
[0028] The term "activate B cells and a B cell line" in the present
invention means a change that cells induce by responding to the
stimulation by CD40. For example, it means to cause DNA synthesis,
promote the incorporation of thymidine, and thus to increase the
expression amount of CD95.
2. OBTAINMENT OF ANTIBODY
[0029] To obtain the antibody of the present invention, it is
preferred to immunize mice using as an antigen a gene recombinant
mouse cell line expressing a human CD40 or a soluble human CD40
that has been produced and purified with recombinants. Mice to be
used for immunization are preferred to produce human antibodies
(Tomizuka. et al., Proc Natl Acad Sci USA., 2000 Vol 97: 722). By
selecting monoclonal antibodies that bind to soluble human CD40
that has been produced and purified with recombinants, antibodies
that react also to CD40 expressed on cells other than B cells may
be more easily obtained than by a case wherein clones reacting
specifically to B cells are selected. Hybridomas can be produced by
the method of Kohler and Milstein et al. (Nature, 1975 Vol. 256:
495) generally used in monoclonal antibody production using the
lymphnode cells or splenocytes of immunized mice.
[0030] Furthermore, the binding of soluble CD40L to CD40 is
analyzed using a surface plasmon resonance system such as BIAcore
2000 (Biacore), and then antibodies that do not compete with CD40L
are selected. In addition, antibodies that suppress independently
the suppression of the cell growth of B lymphoma are selected.
Furthermore, antibody selection is performed using the condition of
whether or not they act on DC as an indicator. This enables the
production and selection of antibodies with advantages of acting on
dendritic cells or B cells without competing with CD40L, and
suppressing the proliferation of CD40-expressing cancer cells.
[0031] The antibody of the present invention is obtained by
culturing the thus obtained hybridoma. Further, a gene encoding a
human monoclonal antibody or a variable region thereof is cloned
from an antibody-producing cell such as a B cell or a hybridoma,
the cloned gene is incorporated into an appropriate vector, and
then the vector is introduced into a host (e.g., a mammalian cell
line, Escherichia coli, yeast cell, insect cell or plant cell), so
that a recombinant antibody produced by gene recombination
technology can be prepared (P. J. Delves., ANTIBODY PRODUCTION
ESSENTIAL TECHNIQUES., 1997 WILEY, P. Shepherd and C. Dean.,
Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS; J. W.
Goding., Monoclonal Antibodies: principles and practice., 1993
ACADEMIC PRESS). Moreover, transgenic cattle, goat, sheep or pigs,
wherein a target antibody gene is incorporated into the endogenous
gene by transgenic animal generation techniques are generated. From
the milk of these transgenic animals, monoclonal antibodies derived
from the antibody gene can be obtained in large quantities. When
hybridomas are cultured in vitro, they are grown, maintained and
stored in a way suitable for various conditions such as the
properties of cell species to be cultured, purposes of experiments
and studies, and culturing methods. Then, hybridomas can be
cultured using any nutrient medium that is induced and prepared
from a known nutrient medium or known basic medium that is used for
the production of monoclonal antibodies in the culture
supernatant.
3. SCREENING
[0032] Screening for agonistic antibodies is performed by analysis
using human B lymphoma, so that antibodies that promote CD95
expression can be selected. Antibodies are further added to a
purified DC culture solution, and then antibodies causing
maturation are selected. Alternatively, antibodies showing activity
to proliferate T cells in a mixed-lymphocyte reaction using
immature DC are selected. Furthermore, antibodies are added to
mature DC, and then antibodies having action to promote IL-12
production are selected. Furthermore, antibodies having activity to
suppress the growth of tumor cells expressing CD40 or activity to
induce cell death of the tumor cells are selected. Competition with
CD40L can be distinguished from other cases based on whether or not
the antibody inhibits the binding of soluble CD40 with soluble CD40
ligands using, for example, a surface plasmon resonance system
(BIOCore). Alternatively, it can also be distinguished from other
cases based on whether or not the antibody enhances the action of
CD40 ligands on a B cell line.
[0033] Screening for antagonistic antibodies is performed by
analysis using human B lymphoma. Further addition of soluble CD40L
having FLAG as a tag in the presence of anti-FLAG antibody enables
screening for antibodies that inhibit more strongly the binding of
soluble CD40L to CD40 on the human B lymphoma cell. By the
introduction of a gene encoding CD40L instead of soluble CD40L; it
is also possible to use recombinant cells expressing many CD40
ligands on the cell surface. Subsequently, human antibodies are
cross-linked with anti-human IgG antibodies, so that clones that
activate B lymphoma by cross-linking are removed. Furthermore,
antibodies showing activity to suppress the T cell proliferation in
a mixed-lymphocyte reaction using purified and matured DC, or
antibodies having action to suppress IL-12 production when CD40
ligands are added to mature DC are selected.
[0034] Antibodies that are obtained as described above have at
least any of the following properties that are thought to be
therapeutically effective.
(1) In the Case of Agonistic Antibody
[0035] (a) The antibody acts on dendritic cells to cause IL-12
production in the presence of LPS (lipopolysaccharide) and
IFN.gamma.. The LPS concentration in this case ranges from 10 pg/ml
to 10 .mu.g/ml and the IFN.gamma. concentration ranges from
10.sup.-4 M to 10.sup.-2 M. With an antibody concentration of 1
.mu.g/ml or more, or preferably 0.1 .mu.g/ml or more, the
production amount of IL-12 is greater than that in a test using a
G28-5 antibody as a control, the known agonistic anti-CD40
antibody. When the antibodies with a concentration of 0.1 .mu.g/ml
or more are added to dendritic cells with a concentration of
1.times.10.sup.6 cells/ml, 100 pg/ml or more IL-12 is produced, or
when the same with a concentration of 1 .mu.g/ml or more are added,
1,000 pg/ml or more, or preferably, 10,000 pg/ml or more IL-12 is
produced (see Examples 9 and 13).
[0036] (b) The antibody has action of binding to dendritic cells
and thus to cause the maturation of the dendritic cells. Moreover,
when the antibodies with a concentration of 20 .mu.g/ml or less,
preferably 0.1 to 15 .mu.g/ml; further preferably 5 to 15 .mu.g/ml
were cultured with dendritic cells, the activity to cause
maturation is higher than that of the G28-5 antibody (see Example
9).
[0037] (c) The antibody has activity to promote CD95 expression of
an established B cell line, which is greater than that of the G28-5
antibody. In this case, with an antibody concentration of 10
.mu.g/ml or more, preferably 1 .mu.g/ml or more, further preferably
0.1 .mu.g/ml or more, still further preferably 0.01 .mu.g/ml or
more, and most preferably 0.001 .mu.g/ml or more, the activity to
promote CD95 expression is higher than that of the G28-5 antibody
that is used as a control in a test. The ratios of the activity of
the G28-5 antibody, which was used in a test as a control, to
promote CD95 expression to the same of the antibody with
concentrations of 10 .mu.g/ml, 1 .mu.g/ml, 0.1 .mu.g/ml and 0.01
.mu.g/ml are as shown below (Table 1).
TABLE-US-00004 TABLE 1 Antibody concentration Ratio 10 .mu.g/ml
Approximately 2-fold, preferably approximately 3-fold, more
preferably 4.5-fold, and further preferably 6-fold 1 .mu.g/ml
Approximately 2-fold, preferably approximately 5-fold, more
preferably approximately 6-fold, and further more preferably 7-fold
0.1 .mu.g/ml 2-fold, preferably 6-fold, more preferably
approximately 7-fold 0.01 .mu.g/ml 2-fold, preferably 4-fold, more
preferably 5-fold, further preferably approximately 6-fold
[0038] The promoted expression of CD95 means that the antibody
activates the established B cell line. Here, examples of the
established B cell line include Ramos cells and HS-Sulton cells. In
addition, Ramos cells are of Burkitt's lymphoma, which are model
cells of human centroblastic B cells. HS-Sulton cells are of
Burkitt's lymphoma (see Examples 6 and 12).
[0039] (d) The antibody has activity to suppress the DNA synthesis,
thymidine incorporation, and proliferation of the established B
cell line (Ramos cells or HS-Sulton cells), which is higher than
that of G28-5 antibody. The antibody concentration in this case is
at least 0.05 .mu.g/ml, or preferably 0.1 to 15 .mu.g/ml (see
Example 8).
[0040] (e) The antibody induces cell death of the established B
cell line (see Example 16).
[0041] (f) The antibody does not inhibit the binding of CD40
ligands to CD40. The term "does not inhibit" means that CD40L can
bind to CD40 to the same degree as that when the antibody is
absent, even when the antibody previously binds to CD40 (that is,
in the presence of the antibody). Either one of or both CD40
ligands and CD40 may be a type of a protein that is expressed on
the membrane or a soluble protein (see Example 11).
[0042] Antibodies having the above properties are produced, for
example, by a hybridoma KM302-1 (FERM BP-7578) and a hybridoma
KM341-1-19 (FERM BP-7759).
[0043] The nucleotide sequences and amino acid sequences of the
heavy chain (H chain) and light chain (L chain) variable regions of
a monoclonal antibody produced by the hybridoma KM341-1-19 were
determined (Example 17). The present invention provides DNA
encoding at least the heavy chain variable region, or the
full-length heavy chain, and DNA encoding the light chain variable
region of the monoclonal antibody produced by the hybridoma
KM341-1-19. The DNAs also include other DNAs encoding the same
amino acid sequences due to codon degeneration in addition to those
described in Example 17. Moreover, the present invention provides
monoclonal antibodies or functional fragments thereof as specified
by the amino acid sequences of at least the heavy chain variable
regions or the amino acid sequences of the full-length heavy
chains, and the amino acid sequences of the light chain variable
regions, as disclosed in Example 17.
(2) In the Case of Antagonistic Antibody
[0044] (g) The antibody neutralizes the action of ligands for CD40.
Here, the term "the action of ligands" means both the action of
ligands expressed on T cells or other cells, and the action of free
ligands for CD40 (see Examples 7 and 14).
[0045] (h) The antibody neutralizes one or more effects that
ligands for CD40 on the established B cell line have on
CD40-expressing cells, and do not show agonistic action to CD40 on
the above established B cell line by cross-linking by
anti-immunoglobulin antibodies. This action is weaker than that of
5D12. The "effects that ligands have on CD40-expressing cells" mean
the activation of the CD40-expressing cells. Specifically in B
cells, the effect means the activation of thymidine incorporation
and B cell proliferation, and the activation of the enhanced
expression of CD95 in the established B cell line. Furthermore, in
DC, the effect means the activation of DC maturation, the
activation of the enhanced expression of CD86 and HLA-DR, the
activation of thymidine incorporation by the co-existing T cells,
the promotion of the proliferation, the activation of IL-12 and
IL-10 production, and the like. Cross-linking by
anti-immunoglobulin antibodies is performed by causing the presence
of 0.1 .mu.g/ml or more of anti-immunoglobulin antibodies in a
culture solution (see Example 7).
[0046] (i) The antibody alleviates or neutralizes the activity of
cross-linked CD40L or CD40L expressed by cells to enhance the
expression of CD95 in the established B cell line. The antibody
also alleviates or neutralizes the activity of CD40L, the action of
which is enhanced by cross-linking by antibodies and the like
against tags. The binding of ligands (including both free ligands
and ligands expressed by specific cells) for CD40 to
CD40-expressing cells causes intracellular signal transduction, and
finally causes the cells to express CD95 (Fas) on the cell
surfaces. Accordingly, the antagonistic antibody of the present
invention inhibits the above signal transduction by binding to
CD40, thereby neutralizing the expression of CD95. The antibody
concentration in this case is 1 .mu.g/ml or more, or preferably 0.1
.mu.g/ml or more (see Examples 7 and 14).
[0047] (j) The antibody is antagonistic to CD40 on DC.
Specifically, the antibody alleviates or neutralizes the activity
of CD40L to activate DC. When DC are stimulated by ligands on T
cells co-existing with the DC, T cells are activated, so that
thymidine incorporation and the like are promoted. In the
mixed-lymphocyte reaction, wherein DC and T cells that are both
derived from different individuals are allowed to co-exist, DC
interact with T cells, thereby causing T-cell activation. The
antagonistic antibody of the present invention inhibits the above
interaction by binding to CD40, resulting in suppressed
incorporation of thymidine. The antibody concentration in this case
is at least 0.001 .mu.g/ml, or preferably 0.1 to 10 .mu.g/ml (see
Example 10).
[0048] The above antagonistic antibody is produced by, for example,
hybridomas KM281-1-10 (FERM BP-7579) and KM281-2-10.sup.-1-2 (FERM
BP-7580) (May 9, 2001, the International Patent Organism Depositary
(IPOD) at the National Institute of Advanced Industrial Science and
Technology (Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki) and 4D11
(FERM BP-7758).
(3) The antibody of the present invention can be altered to an
antibody of a different subclass (for example, see EP314161
publication), by modification by genetic engineering techniques
known by a person skilled in the art, specifically by substituting
a region that defines the subclass of an antibody heavy chain with
a region that defines another subclass. A heavy chain variable
region and the constant region of another subclass can be directly
linked. For example, an alteration of the subclass of the antibody
of the present invention to IgG2 or IgG4 makes it possible to lower
the binding degree of the antibody to a Fc receptor. Specifically,
Nhe I site (GCTAGC) is introduced into a human antibody heavy
chain, EU index 118 (Ala), 119 (Ser) site according to Kabat et al
(Sequence of Proteins of Immunological Interest, 5.sup.th Ed.
Public Health Service, National Institute of Health, Bethesda, Md.
(1991)). By digestion using the restriction enzyme, switching to
another subclass, IgG, can be performed without altering the amino
acid. Moreover, artificial alteration of the amino acid sequence of
a constant region, or the binding of a constant region sequence
having such an altered sequence with the variable region of the
antibody of the present invention can lower the binding degree to a
Fc receptor (Lund J., et al., J. Immunol. 1991 vol 147: 2657-2662),
or can also increase or decrease CDC activity (Tao M., et al., J.
Exp. Med. 1991 vol 1025-1028, Idusogie E E., et al., J. Immunol.
2001 vol 166: 2571-5). Furthermore, to avoid the action of ADCC,
CDC or the like, only IgG2 or IgG4 subclass antibodies can be
previously selected. In addition, the binding of a radionuclide,
bacterial toxin, chemotherapeutant, prodrug or the like with the
antibody of the present invention can further enhance the
therapeutic effect against disease such as cancer.
4. Pharmaceutical Composition
[0049] A pharmaceutical composition containing a pharmaceutical
preparation that is the purified antibody of the present invention
is also encompassed by the scope of the present invention. Such a
pharmaceutical composition preferably contains a physiologically
acceptable diluent or carrier in addition to the antibody, or may
be a mixture with other antibodies or other drugs, such as
antibiotics. Examples of the appropriate carrier include, but are
not limited to, a physiological saline solution, a phosphate
buffered saline solution, a phosphate buffered saline glucose
solution and a buffered physiological saline. Alternatively, the
antibody is freeze-dried, and then used when necessary by adding
the above buffered aqueous solution for reconstruction. Examples of
the route of administration include an oral route and a parenteral
route including intravenous, intramuscular, hypodermic and
intraperitoneal injections or drug delivery.
[0050] In this case, the effective dose to be administered as a
combination of the effective dose of the antibody of the present
invention, an appropriate diluent, and a pharmacologically
acceptable carrier ranges from 0.1 mg to 100 mg per kg of body
weight per administration. Administration is performed at intervals
of 2 days to 8 weeks.
[0051] When a pharmaceutical composition containing the antibody of
the present invention is used, and particularly, when the agonistic
antibody is used, the composition is used as an immunopotentiating
drug (anti-viral agent and anti-infective drug), anti-tumor agent
or anti-autoimmune disease agent. Multiple examples of these
diseases may occur together. Alternatively, the antibody can also
be used as an adjuvant in combination with a vaccine such as a
cancer-specific peptide. When the composition contains the
antagonistic antibody, it is useful as an immunosuppressive agent
(prophylactic or therapeutic agent against immunological rejection
or GVHD upon transplantation of islets of Langerhans, kidneys or
the like) upon organ transplantation, or an anti-autoimmune disease
agent (e.g., against rheumatism, or as a therapeutic agent against
arterial sclerosis, disseminated sclerosis, systemic erythematodes,
idiopathic thrombocythemia or Crohn's disease), therapeutic agent
against allergies such as asthma, or therapeutic agent against
blood coagulation factor VIII-inhibiting syndrome. Multiple
examples of these diseases may occur together.
[0052] When the anti-CD40 antibody is used as a therapeutic means
against a disease in which CD40 is involved, it can be expected
that antibodies providing a better therapeutic effect can be
obtained by selecting the antibodies using the function of DC as an
indicator.
[0053] In the case of the agonistic antibody, it can be expected
that antibodies having strong immunopotentiation action can be
obtained by selecting antibodies that can activate DC more
effectively. Furthermore, by using the promotion of IL-12
production by mature DC as an indicator, antibodies having strong
CTL-inducing action can be obtained. By the CTL induction,
antibodies that are highly effective for removing cells infected
with viruses or tumor cells can be obtained. Moreover, since
synergistic effects can be expected, preferred antibodies bind to
CD40 without inhibiting the binding of CD40 ligands to CD40. When
cancer treatment is considered, if antibodies that directly induce
cell death of CD40-expressing cancer cells or suppress their
proliferation, and effectively activate DC are present, synergistic
effects are expected therefrom, and such antibodies can be a
therapeutic agent that can be used against tumors that do not
express CD40. These antibodies are considered to be useful as a
therapeutic agents against viral diseases or anti-tumor agents.
[0054] In the meantime, antibodies that specifically bind to CD40
and suppress the binding of CD40L without activating CD40 are also
expected to be able to suppress not only the action of ligands for
B cells, but also the action on DC. However, antibodies have been
so far obtained using as an indicator their effect on B cells.
Thus, it is highly significant to obtain antibodies that have
strong suppressive action also on dendritic cells and to develop
them as a pharmaceutical product. Further, it is a concern that the
anti-CD40 antibody can have a totally opposite action by
cross-linking as described above. Thus, antibodies that do not
activate CD40 even by cross-linking are required. It is also a
concern that monoclonal antibodies derived from a non-human mammal
such as a mouse, chimeric antibodies consisting of the variable
region of a mouse monoclonal antibody and a constant region of a
human immunoglobulin and humanized antibodies resulting from CDR
grafting, which have been so far reported as antibodies against
human CD40, have antigenicity. Therefore, a human antibody is
desirable as an antibody to inhibit the binding with CD40
ligands.
BRIEF DESCRIPTION OF DRAWINGS
[0055] FIG. 1 shows that KM302-1 antibodies promoted CD95
expression.
[0056] FIG. 2A shows that the antagonistic antibodies neutralized
the action of CD40 ligands on Ramos cells.
[0057] FIG. 2B shows that the antagonistic antibodies neutralized
the action of CD40 ligands on Ramos cells.
[0058] FIG. 3 shows that KM281-1-10 antibodies neutralized the
action of CD40 ligands on Ramos cells.
[0059] FIG. 4 shows that cross-linked KM281-1-10 antibodies did not
promote CD95 expression.
[0060] FIG. 5 shows that cross-linked 5D12 antibodies promoted CD95
expression.
[0061] FIG. 6 shows the proliferation suppressive effect of KM302-1
antibodies on tumor cells.
[0062] FIG. 7 shows that KM302-1 antibodies promoted the maturation
of DC.
[0063] FIG. 8 shows that KM302-1 antibodies promoted the IL-12
production of DC.
[0064] FIG. 9 shows that KM281-1-10 antibodies neutralized the
action of CD40 ligands on DC.
[0065] FIG. 10 shows that KM281-1-10 antibodies neutralized the
action of CD40 ligands on DC.
[0066] FIG. 11 shows that KM302-1 antibodies activated immature
DC-MLR.
[0067] FIG. 12 shows that KM341-1-19 antibodies and the like
promoted CD95 expression of Ramos cells.
[0068] FIG. 13 shows that KM341-1-19 antibodies promoted IL-12
production of mature DC.
[0069] FIG. 14 shows that KM341-1-19 antibodies promoted IL-10
production of mature DC.
[0070] FIG. 15 shows that 4D11 antibodies and the like neutralized
the action of CD 40 ligands on Ramos cells.
[0071] FIG. 16 shows that KM302-1 antibodies showed anti-tumor
effect on the human tumor cell-transplanted mouse model.
[0072] FIG. 17 shows that KM341-1-19 antibodies showed a
proliferation suppressive effect against tumor cells.
[0073] FIG. 18 shows that F4-465, 4D11 and KM28'-1-10 suppressed
antigen-specific IgG production.
[0074] FIG. 19 shows that F4-465, 4D11 and KM281-1-10 suppressed
antigen-specific IgM production.
[0075] FIG. 20 shows that F4-465 suppressed the proliferation of
tonsillar B cells.
BEST MODE FOR CARRYING OUT THE INVENTION
[0076] The present invention will be further described in detail by
referring to the examples. However, the technical scope of the
invention is not limited by these examples.
Example 1
Preparation of Antigen
(1) Cell
[0077] EL-4 cells are of a mouse-derived established T cell line,
and can be easily obtained (ATCC No.: TIB-39). Ramos B cells (ATCC
No.: CRL-1596) and mouse anti-CD40 antibody-producing hybridoma
G28-5 (HB-9110) and 5D12 (HB-11339) were purchased from ATCC.
(2) Expression and Purification of Antigen
[0078] Extracellular regions were amplified by PCR using human CD40
cDNA (Genbank Accession Number: NM.sub.--001250) as a template and
the following primers under conditions of 20 cycles of 95.degree.
C. for 5 seconds, 55.degree. C. for 30 seconds and 72.degree. C.
for 30 seconds.
TABLE-US-00005 Primer 1: (SEQ ID NO: 1)
5'-CCCAGATCTGTCCATCCAGAACCACCCACTGCATGCAGAG-3' Primer 2: (SEQ ID
NO: 2) 5'-ACAAGATCTGGGCTCTACGTATCTCAGCCGATCCTGGGGAC-3'
[0079] The amplified cDNA was inserted following the melittin
signal sequence and before the human IgG1-derived FC or mouse
IgG2a-derived FC region of a pFastBac vector (Gibco BRL). To
produce CD40, recombinant baculoviruses were prepared according to
the instruction. Th5 cells were infected with the recombinant
viruses, and then cultured for 4 days. The supernatant was treated
with a 0.22 nm filter, Protein G sepharose (Amersham Pharmacia) was
added thereto, and then the mixture was gently shaken at 4.degree.
C. After one night, sepharose was transferred to a column and then
washed with a 20.times. volume of PBS. A human CD40 FC protein was
eluted with a 20 mM glycine buffer (pH 3.0). The vector to express
CD40 on cell surfaces was obtained from Randolph J. Noelle (Inui, S
et al., EJI, 20, 1747-1753, 1990). The full-length cDNA was cleaved
with the Xba I enzyme, and then inserted into pCDNA3 (INVITROGEN).
The vector was introduced into EL-4 cells, and then the cells were
cultured in the presence of 0.5 mg/ml G418 (Gibco BRL), thereby
obtaining a stable expression strain. The expression of CD40 was
confirmed by FACS analysis using FITC-conjugated anti-human CD40
antibodies (Pharmingen).
Example 2
Generation of Mice for Immunization
[0080] The mice used for immunization had a genetic background
whereby they were homozygotes for both disrupted endogenous Ig
heavy chain and K light chain, and the mice harbored at the same
time chromosome 14 fragment (SC20) containing human Ig heavy chain
gene locus, and human Ig.kappa. chain transgene (KCo5). These mice
were generated by crossing mice of a line A having a human Ig heavy
chain gene locus with mice of a line B having a human Ig.kappa.
chain transgene. The mice of line A are homozygous for both
disrupted endogenous Ig heavy chain and K light chain, and harbor
chromosome 14 fragment (SC20), which is transmittable to progeny,
as is described, for example, in the report of Tomizuka et al.
(Tomizuka. et al., Proc Natl Acad Sci USA., 2000 Vol 97: 722). The
mice of the line A were immunized, so that the following hybridomas
F2-103 and F5-77 were obtained. Furthermore, the mice of line B
(transgenic mice) are homozygotes for both disrupted endogenous Ig
heavy chain and .kappa. light chain, and harbor a human Ig.kappa.
chain transgene (KCo5), as described, for example, in the report of
Fishwild et al. (Nat. Biotechnol., 1996 Vol 14:845).
[0081] Individuals obtained by crossing male mice of the line A
with female mice of the line B, or female mice of the line A with
male mice of the line B, and having human Ig heavy chain and
.kappa. light chain detected simultaneously in the sera (Ishida
& Lonberg, IBC's 11th Antibody Engineering, Abstract 2000) were
used for the following immunization experiment. In addition, the
above human antibody-producing mice are available from Kirin
Brewery Co., Ltd via contract. By immunizing the above mice, the
following hybridomas KM302-1, KM341-1-19, KM643-4-11, 2053, 2105,
3821, 3822, 285, 110, 115, KM281-1-10, KM281-2-10-1-2, KM283-5,
KM292-1-24, KM225-2-56, KM341-6-9, 4D11, 5H10, 11E1, 5G3, 3811,
3411 and 3417 were obtained. Moreover, chimeric mice (Kuroiwa et
al., Nat. Biotechnol., 2000 vol 18:1086) harboring human antibody
Lambda chain reported by Kuroiwa et al. were also used for the
following immunization experiment. A hybridoma F4-465 was obtained
from the mouse.
Example 3
Preparation of Human Monoclonal Antibody Against Human CD40
[0082] Monoclonal antibodies in this example were prepared
according to a general method described in the Introduction of
Experimental Procedures for Monoclonal Antibodies (written by Tamie
ANDO et al., KODANSHA, 1991). The human CD40 used as an immunogen
herein were the human CD40 human FC and CD40-expressing EL-4 cells
prepared in Example 1. Animals used herein for immunization were
human antibody-producing mice that produce the human immunoglobulin
prepared in Example 2.
[0083] The human antibody-producing mice were immunized with 2 to
100 .mu.g/immunization of CD40: hFc per mouse. Excluding the first
immunization, an antigen solution was mixed with an equivalent
volume of Freund's incomplete adjuvant (Sigma), and then injected
subcutaneously into several separate positions. Immunization was
performed 3 to 4 times approximately every 10 days to 3 weeks. For
the first immunization, Freund's incomplete adjuvant (Sigma) was
used. Blood was collected from the mouse tail, and then human
antibody .gamma. and .kappa. against CD40 in the serum were
measured using ELISA. 3 to 4 days before excision of the spleen,
final immunization was performed by injecting 20 .mu.g of CD40: Fc
dissolved in PBS via the caudal vein.
[0084] The human antibody-producing mice were immunized with human
CD40-expressing mouse EL-4 cells. EL-4 cells (10.sup.8 cells/ml)
were suspended in PBS, and then gently mixed with an equivalent
volume of RIBI adjuvant previously emulsified with PBS.
Immunization was performed with the cells 3 to 5 times
approximately every 10 days to 3 weeks. When the adjuvant was not
used, the cells were irradiated with X-rays with 8000 rad for
use.
[0085] The spleen was surgically obtained from the immunized mice.
The collected splenocytes were mixed with mouse myeloma SP2/0 (ATCC
No.: CRL1581) at a ratio of 5 to 1. The cells were fused using
polyethylene glycol 1500 (Boehringer Mannheim) as an agent for cell
fusion, thereby preparing a large number of hybridomas. The
selection of hybridomas was performed by culturing in
HAT-containing DMEM media (Gibco BRL) supplemented with 10% fetal
calf serum (FCS), hypoxanthine (H), aminopterin (A) and thymidine
(T). Furthermore, single clones were obtained by the limiting
dilution method using HT-containing DMEM media. Culturing was
performed in a 96-well microtiter plate (Beckton Dickinson).
Screening for hybridma clones producing anti-human CD40 human
monoclonal antibodies was performed by measurement using
enzyme-linked immuno adsorbent assay (ELISA) and fluorescence
activated cell sorter (FACS), as described later in Example 4.
[0086] Screening for the human monoclonal antibody-producing
hybridoma by ELISA was performed by 3 types of ELISA and FACS
analyses as described below. Thus, a large number of hybridomas
producing human monoclonal antibodies that had human immunoglobulin
.gamma. chain (hIg.gamma.) and human immunoglobulin light chain K,
and had reactivity specific to human CD40 were obtained. In any of
the following examples including this example, and tables and
figures showing the test results of the examples, each hybridoma
clone producing the human anti-human CD40 monoclonal antibody of
the present invention was denoted using symbols. A clone
represented by the symbols followed by "antibody" means an antibody
that is produced by each of the hybridomas, or a recombinant
antibody that is produced by a host cell carrying an antibody gene
(full-length or a variable region) isolated from the hybridoma. In
addition, within a contextually clear range, the name of a
hybridoma clone may express the name of an antibody.
[0087] The following hybridoma clones represent single clones.
Agonistic Antibody:
KM302-1, KM341-1-19, KM643-4-11, 2053, 2105, 3821, 3822, 285, 110,
115, F1-102, F2-103, F5-77 and F5-157
Antagonistic Antibody:
KM281-1-10, KM281-2-10.sup.-1-2, KM283-5, KM292-1-24, KM225-2-56,
KM341-6-9, 4D11, 5H10, 11E1, 5G3, 3811, 3411, 3417 and F4-465
[0088] 3 hybridoma clones KM 302-1, KM 281-1-10 and KM
281-2-10.sup.-1-2 among them were deposited on May 9, 2001, clones
KM341-1-19 and 4D11 were deposited on Sep. 27, 2001, and clone 2105
was deposited on Apr. 17, 2002, with the International Patent
Organism Depositary at the National Institute of Advanced
Industrial Science and Technology (Central 6, 1-1-1, Higashi,
Tsukuba, Ibaraki, Japan) under the Budapest Treaty. Plasmids having
the heavy chain and light chain variable regions of F2-103, F5-77
and F5-157, were deposited on Apr. 19, 2001, and hybridoma clones
F1-102 and F4-465 were deposited on Apr. 24, 2001, with ATCC
(American Type Culture Collection, University Blvd, Manassas, Va.,
U.S.A.) under the Budapest Treaty (Table 2).
TABLE-US-00006 TABLE 2 Name Accession No. KM302-1 FERM BP-7578
KM281-1-10 FERM BP-7579 KM281-2-10-1-2 FERM BP-7580 KM341-1-19 FERM
BP-7759 4D11 FERM BP-7758 2105 FERM BP-8024 F1-102 ATCC PTA-3337
F4-465 ATCC PTA-3338 F2-103 heavy chain ATCC PTA-3302 (F2-103-H)
F2-103 light chain ATCC PTA-3303 (F2-103-L) F5-77 heavy chain
(F5-77-H) ATCC PTA-3304 F5-77 light chain (F5-77-L) ATCC PTA-3305
F5-157 heavy chain ATCC PTA-3306 (F5-157-H) F5-157 light chain ATCC
PTA-3307 (F5-157-L)
Example 4
Screening for Hybridoma
[0089] Detection of monoclonal antibody having human immunoglobulin
.gamma. chain
[0090] The human CD40 mouse FC (1 .mu.g/ml) prepared in Example 1
was added at 50 .mu.l/well to each well of a 96-well microplate for
ELISA (Maxisorp, Nunc) and incubated at 4.degree. C. for the human
CD40 mouse FC to be adsorbed to the microplate. Next, the
supernatant was discarded, and then a blocking reagent (Block Ace,
DAINIPPON PHARMACEUTICAL) was added to each well, followed by
incubation at room temperature for blocking. The culture
supernatant (50 .mu.l) of each hybridoma was added to each well for
reaction, and then each well was washed with a 0.1%
Tween20-containing phosphate buffer (PBS-T). Goat anti-human IgG
(.gamma.) antibody (Sigma, A0170) labeled with peroxydase was then
diluted 5,000-fold with 1% FBS-containing PBS-T. The solution was
added (50 .mu.l/well) to each well, and then incubation was
performed. The microplate was washed 3 times with PBS-T, and then a
chromogenic substrate solution (TMB, 50 .mu.l/well, SUMITOMO
BAKELITE) was added to each well, followed by incubation at room
temperature for 30 minutes. A stop solution was added (50
.mu.l/well) to each well to stop reaction. Absorbance at a
wavelength of 450 nm was measured with a microplate reader. The
culture supernatant of positive wells was analyzed by FACS, an then
the wells wherein Ramos cells were stained were selected. The cells
in the wells were cloned by the limiting dilution method, and then
the cells of 1 clone were obtained per well. h.kappa.-positive
status was confirmed by ELISA using the human CD40 mouse FC. As a
result, anti-human CD40 antibodies of 173 clones were obtained from
20 mice. Some of these antibodies were shown in Table 3 (agonistic
antibodies) and Table 4 (antagonistic antibodies). Among the
agonistic antibodies, at least KM341-1-19 and 2105 did not
significantly compete with ligands in a competitive test using
CD40L-expressing cells, CD40-expressing cells and the
antibodies.
TABLE-US-00007 TABLE 3 Agonistic antibody Hybridoma Antigen
Subclass DC Tumor cell KM302-1 CD40 mouse FC IgG4 activated
suppressed proliferation KM341-1-19 human IgG2 activated suppressed
CD40-expressing proliferation EL-4 KM643-4-11 CD40 mouse FC IgG1
not not implemented implemented 2053 CD40 mouse FC IgG2 not not
implemented implemented 2105 CD40 mouse FC IgG2 not not implemented
implemented 3821 human IgG3 not not CD40-expressing implemented
implemented EL-4 3822 human IgG3 not not CD40-expressing
implemented implemented EL-4 285 CD40 mouse FC IgG1 not not
implemented implemented 110 CD40 mouse FC IgG4 not not implemented
implemented 115 CD40 mouse FC IgG4 not not implemented implemented
F2-103 CD40 mouse FC IgG1 not not implemented implemented F5-77
CD40 mouse FC IgG1 not not implemented implemented
TABLE-US-00008 TABLE 4 Antagonistic antibody Effect of cross-
Hybridoma Antigen Subclass linking DC-MLR KM281-1-10 CD40 mouse FC
IgG1 low suppressed KM281-2- CD40 mouse FC IgG1 low not 10-1-2
implemented KM283-5 CD40 mouse FC IgG4 significant not suppressed
KM225-2-56 CD40 mouse FC IgG4 significant not implemented
KM292-1-24 CD40 mouse FC IgG2 significant not implemented KM341-6-9
human IgG1 significant not CD4-expressing implemented EL-4 4D11
CD40 mouse FC IgG1 low not implemented 5H10 CD40 mouse FC IgG1 low
not implemented 11E1 CD40 mouse FC IgG1 low not implemented 5G3
CD40 mouse FC IgG2 significant not implemented 3811 human IgG1
significant not CD40-expressing implemented EL-4 3411 human IgG2
significant not CD40-expressing implemented EL-4 3417 human IgG2
significant not CD40-expressing implemented EL-4 F4-465 human IgG1
not not CD40-expressing implemented implemented EL-4
[0091] Monoclonal antibodies having human immunoglobulin light
chain .kappa. (Ig.kappa.) were detected in a manner similar to the
above described ELISA method for human immunoglobulin .gamma. chain
except that goat anti-human Ig.kappa. antibodies (diluted
1,000-fold, 50 .mu.l/well, Southern Biotechnology) labeled with
peroxydase were used.
[0092] The subclass of each monoclonal antibody was identified in a
manner similar to the above ELISA method for human immunoglobulin
.gamma. chain, except that a sheep anti-human IgG1 antibody, sheep
anti-human IgG2 antibody, sheep anti-human IgG3 antibody or sheep
anti-human IgG4 antibody (each diluted 2,000-fold, 50 .mu.l/well,
The Binding Site) labeled with peroxydase, was used.
Reaction Test of Each Monoclonal Antibody Against Human
CD40-Expressing Cells
[0093] The reactivity of each monoclonal antibody against a Ramos
cell line reported to express CD40 was studied by FACS
analysis.
[0094] The Ramos cell line was suspended at a concentration of
2.times.10.sup.6/ml in a staining buffer (SB) of 0.1% NaN.sub.3 and
2% FCS-containing PBS. The cell suspension (100 .mu.l/well) was
apportioned to a 96-well round bottom plate (Beckton Dickinson).
The culture supernatant (50 .mu.l) of each hybridoma was added, and
then incubation was performed at ice temperature for 30 minutes.
Human IgG1 antibodies against human serum albumin were used as a
negative control, and prepared at a concentration of 2 .mu.g/ml
with a hybridoma culture medium. 50 .mu.l of the solution was
added, and then incubation was performed at ice temperature for 15
minutes. After washing with SB, 50 .mu.l of R-PE
fluorescence-labeled anti-human antibody (Southern Biotechnology)
diluted 250-fold was added, and then incubation was performed at
ice temperature for 15 minutes. After washing twice with SB, the
product was suspended in 300 to 500 .mu.l of a FACS buffer, and
then the fluorescence intensity of each cell was measured by FACS
(FACSort and FACScan, Beckton Dickinson). As a result, antibodies
having binding activity for the Ramos cell line were selected.
Example 5
Preparation of Each Antibody
[0095] The culture supernatant containing monoclonal antibodies was
prepared by the following method.
[0096] A G28-5 antibody-producing hybridoma was obtained from ATCC
(ATCC No. HB-9110). Anti-CD40 antibody-producing hybridomas were
acclimatized in eRDF media (Kyokutoseiyaku) containing bovine
insulin (5 .mu.g/ml, Gibco BRL), human transferrin (5 .mu.g/ml,
Gibco BRL), ethanolamine (0.01 mM, Sigma) and sodium selenite
(2.5.times.10.sup.-5 nM, Sigma). The hybridomas were cultured in a
spinner flask. When the viable cell rate of the hybridomas reached
90%, the culture supernatant was collected. The collected
supernatant was applied to a 10 .mu.m and 0.2 .mu.m filters (German
Science) so as to eliminate miscellaneous debris such as
hybridomas.
[0097] Anti-CD40 antibodies were purified from the above culture
supernatant by the following method. The culture supernatant
containing the anti-CD40 antibodies was subjected to affinity
purification using a Hyper D Protein A column (NGK INSULATORS, LTD)
or a Protein G column (for purifying mouse IgG1, Amersham Pharmacia
Biotech) according to the attached instruction using PBS (-) as an
adsorption buffer and 0.1 M sodium citrate buffer (pH 3) as an
elution buffer. 1 M Tris-HCl (pH 8.0) or Na.sub.2HPO.sub.4 solution
was added to adjust the elution fraction to have a pH of around
7.2. The prepared antibody solution was substituted with PBS (-)
using a dialysis membrane (10000 cut, Spectrum Laboratories) or SP
column (Amersham Pharmacia Biotech), and then sterilization by
filtration was performed using a membrane filter MILLEX-GV
(MILLIPORE) with a pore size of 0.22 .mu.m. The concentration of
the purified antibody was found by measuring absorbance at 280 nm
and then calculating with 1 mg/ml at 1.45 OD.
Example 6
Promotion of CD95 Expression in Ramos Cells by Anti-CD40 Agonistic
Antibody
[0098] A 5.0.times.10.sup.5 cells/ml Ramos cell suspension was
inoculated at 100 .mu.l/well (5.times.10.sup.4 cells per well) to a
96-well plate. The hybridoma culture supernatant or the purified
antibody was diluted to 20 .mu.g/ml with a medium, and then the
solution was added at a concentration of 100 .mu.l/well to a
96-well plate. After overnight culture, the cells were collected
and then analyzed by FACSCan or FACSsort (Beckton Dickinson) using
R-PE-labeled anti-CD95 antibodies (Pharmingen N.J.). FIG. 1 shows
the result. The horizontal axes in FIG. 1 indicate the expression
intensity of CD95. Addition of antibodies is indicated with a thick
line, and non addition of antibodies is indicated with a thin line.
It was shown that the KM302-1 antibodies promoted CD95 expression
better than G28-5 antibodies, which were the known antibodies. That
is, the KM302-1 antibody was shown to be more effectively
agonistic.
Example 7
Suppression of CD95 Expression in Ramos Cell by Anti-CD40
Antagonistic Antibody
[0099] A 1.0.times.10.sup.6 cells/ml Ramos cell suspension was
inoculated at 50 .mu.l/well to a 96-well plate. The hybridoma
culture supernatant or the purified antibody was adjusted at 2
.mu.g/ml with a medium, and then added at 100 .mu.l/well to a
96-well plate. Soluble CD40 ligands (4 .mu.g/ml, ALEXIS
CORPORATION) and anti-FLAG antibodies (4 .mu.g/ml, M2, Sigma) were
added to media, and then the media were added at 50 .mu.l/well to
the 96-well plate. After overnight culture, the cells were
collected and then analyzed by FACS using R-PE-labeled anti-CD95
antibodies (Pharmingen N.J.). FIGS. 2A and 2B, and 3 show the
results. The horizontal axes in the figures indicate the expression
intensity of CD95. CD95 expression was suppressed to the same
degree as that of a negative control by the antibodies produced by
each of the following hybridomas: KM281-1-10, KM281-2-10.sup.-1-2,
KM283-5, KM292-1-24 and KM225-2-56.
[0100] In FIG. 3, KM281-1-10 antibodies (lower panel) suppressed
CD95 expression more effectively than that the 5D12 antibodies
(central panel), the known antibody only slightly suppressed CD95
expression. Specifically, the KM281-1-10 antibody was shown to be
more effectively antagonistic. Thus, the human monoclonal antibody
was shown to be an antagonistic antibody.
[0101] Effect of Cross-Linking by Anti-Immunoglobulin Antibody
[0102] A 1.0.times.10.sup.6 cells/ml Ramos cell suspension was
inoculated at 50 .mu.l/well to a 96-well plate. The hybridoma
culture supernatant or the purified antibody was adjusted to 2
.mu.g/ml with a medium, and then added at 100 .mu.l/well to a
96-well plate. Anti-human IgG antibodies (Sigma, 13382) or
anti-mouse IgG antibodies (Biosource, AMI3401) were added at 4
.mu.g/ml to media, and then the media were added at 50 .mu.l/well
to a 96-well plate. After overnight culture, the cells were
collected, and then analyzed by FACS using R-PE-labeled anti-CD95
antibodies (Pharmingen N.J.). FIGS. 4 and 5 show the results. The
horizontal axes in the figures indicate the expression intensity of
CD95. CD95 expression was suppressed by the antibodies produced by
each of the hybridomas KM281-1-10 and KM281-2-10.sup.-1-2.
Conversely, CD95 expression was enhanced by the antibodies produced
by each of the following hybridomas, 5D12, KM283-5, KM292-1-24 and
KM225-2-56.
Example 8
Proliferation Suppression in Ramos Cells by Anti-CD40 Agonistic
Antibody
[0103] A 1.0.times.10.sup.5 cells/ml Ramos and HS-Sulton cell
suspension was inoculated at 100 .mu.l/well to a 96-well plate. A
mixture of equivalent amount of the purified antibodies or soluble
CD40 ligands, and anti-FLAG antibodies (M2) was added to media.
After 2 days of culturing, 10 .mu.l of 100 .mu.Ci/ml
.sup.3H-Thymidine (Amersham Pharmacia) was added. After 18 hours,
the culture product was harvested in a Printed Filtermat A (Wallac)
using a Macro 96 Harvester (SKATRON), dried, and then immersed well
in Betap; Scint (Wallac). After packaging, activity was measured
using a 1205 BETAPLATE liquid scintillation counter. FIG. 6 shows
the results. In the figure, the longitudinal axes indicate the
amount of .sup.3H thymidine incorporated by cells, and the
horizontal axes indicate the concentration of the antibody or CD40L
in the culture solution. When the KM302-1 antibodies were added to
Ramos cells and HS-Sulton cells, the amount of thymidine
incorporated was lower than the conventional G28-5 antibodies and
CD40L. Thus, it was shown that the KM302-1 antibody is an agonistic
antibody that can effectively suppress the proliferation of tumor
cells.
Example 9
Activation Of Dendritic Cell by CD40 Agonistic Antibody
[0104] (1) Materials and methods
[0105] Recombinant human IL-4 was purchased from Genzyme techne.
Anti-human CD14 MACS beads were purchased from Miltenyi Biotech
GmbH. Lymphoprep was purchased from Nycomed Pharma AS. The medium
used for culturing was RPMI1640 (Gibco BRL) supplemented with 10%
heat inactivated FCS (Cell Culture Technologies), 10 mM HEPES
(Sigma), 55 .mu.M 2-mercaptoethanol (Gibco BRL) and streptomycin
sulfate (MEIJI SEIKA KAISHA, LTD.), when DC were induced. The cells
in a staining process were washed with PBS (Sigma) supplemented
with 2% FCS (Cell Culture Technologies) and 0.02% Azaid. When the
cells were frozen, Cell banker (Nippon Zenyaku Kogyo) was used.
(2) Induction of Monocyte-Derived DC
[0106] Mononuclear cells were prepared (PBMC) from peripheral blood
by density gradient centrifugation using Lymphoprep. The cells were
subjected to positive selection using anti-human CD14 MACS beads,
so as to separate the cells into a CD14 positive fraction and
negative fraction. Recombinant human GM-CSF (50 ng/ml) and
recombinant human IL-4 (100 ng/ml) were added to the positive
fraction, followed by culturing in RPMI1640 media supplemented with
10% FCS in a 6-well plate. At the start of culturing, the cells
were cultured at a concentration of 1.times.10.sup.6/ml (3 ml per
well). During culturing, the media were exchanged once every 2
days. Medium exchange was performed by sampling 10% of the culture
solution in a centrifugation tube, centrifuging the solution,
removing the supernatant, suspending with a new culture solution
(containing cytokine and the like at the above concentration) in a
volume 2-fold greater than the sampled culture solution, and then
returning the suspension to each well. On day 6 of culturing, the
cells were collected, the cell number was calculated, and then the
cells were suspended at a concentration of 1.times.10.sup.6/ml in
the above media. Anti-CD40 antibodies or the isotype controls
thereof were added to the media, and then cultured for further 4
days in a 24-well plate. During this period, no culture exchange
was performed (cell number per well of 1.times.10.sup.6 cells, and
cell concentration of 1.times.10.sup.6/ml).
(3) Cell Staining and Analysis by Flow Cytometer
[0107] For staining, anti-HLA-DR antibodies (isotype control: rat
IgG2a), anti-CD86 antibodies (isotype control: rat IgG1) and
anti-CD83 antibodies (isotype control: rat IgG2b) were used. First,
the antibodies were added, and then incubation was performed at
4.degree. C. for 30 minutes. After 3 washings, analysis was
performed using the FACS Calibur (Beckton Dickinson).
(4) Increase in IL-12 Secretion Ability of Mature DC
[0108] After immature DC were obtained as described above, LPS (400
pg/ml) and IFN.gamma. (10.sup.-3M) were added, and then culturing
was performed for 2 days, thereby obtaining mature DC. To the
mature DC, 10 .mu.g/ml anti-CD40 antibodies or the isotype control
was added. For the supernatant after 24 hours, IL-12 production was
measured using ELISA (Pharmingen).
(5) Results and Discussion
[0109] FIG. 7 shows the effect of KM302-1 antibodies, the agonistic
antibodies, on DC maturation, and FIG. 8 shows the effect of
KM302-1 antibodies on IL-12 production of mature DC. The degree of
maturation was compared with G28-5 antibodies as a control. When
the expression of CD86 and that of HLA-DR were examined, the
expression was further elevated, that is, the degree of maturation
was increased in the case of KM302-1 antibodies compared with the
case of G28-5 antibodies. It was also shown that IL-12 secretion
was increased by the treatment of mature DC with KM302-1
antibodies. Accordingly, it was shown that the KM302-1 antibodies
acted as agonistic antibodies on DC.
Example 10
DC-MLR
[0110] Blood (peripheral blood) collected from a normal human was
centrifuged at 2000 rpm for 10 minutes, and then the serum was
absorbed. The blood cell fraction was re-suspended with PBS, and
then gently placed on Ficoll (Amersham Pharmacia). Centrifugation
was performed at 2000 rpm for 30 minutes, so that a PBMC portion in
the intermediate layer was collected, washed twice with PBS, and
then used for a certain cell separation process using MACS.
[0111] Monocyte separation for culturing DC was performed according
to the attached instruction using MACS (Miltenyi Biotec GmbH). This
is briefly explained as follows. 800 .mu.l of MACS Buffer and 200
.mu.l of MACS CD14 (Miltenyi Biotec GmbH, 502-01) were added to
PBMC (1.times.10.sup.8), and then treated at 4.degree. C. for 15
minutes. The cells were adsorbed to a MACS LS column, and then
washed. The cells adsorbed to the column were collected as
monocytes. MACS HLA-DR (Miltenyi Biotec GmbH, 461-01) was added to
the cells that were not adsorbed to the column. HLR-DR positive
cells were removed with a BS column, thereby preparing a T cell
fraction. The proportion of CD3 positive cells was measured by
FACS, and then substantial number of T cells was calculated from
the total cell number in the T cell fraction. The obtained
monocytes were cultured in R0 media (PPMI medium supplemented with
.beta.-mercapto ethanol (Gibco) and HEPES (SIGMA)) containing 100
ng/ml IL-4 (R&D system), 50 ng/ml G-CSF (KIRIN) and 10% FCS
(SIGMA) at a concentration of 1.times.10.sup.6 cells/ml in a 6-well
culture plate. On day 5 after culturing, 10 ng/ml LPS (DIFCO) was
added for the cells to differentiate into mature DC.
[0112] MLR was performed by mixing T cells and mature DC, which had
been isolated from different humans. The cell ratio of T cells to
DC was determined as 1:80, and number of T cells was determined as
2.times.10.sup.5 cells/well. First, antibodies were added to DC for
reaction to proceed for 30 minutes. Subsequently, T cells were
added, culturing was performed for 4 days, and then 10 .mu.l of 100
.mu.Ci/ml .sup.3H-Thymidine (Amersham Pharmacia) was added. 14
hours later, the cells were harvested in Printed Filtermat A
(Wallac) using a Macro 96 Harvester (SKATRON), dried, and then
immersed well in Betap; Scint (Wallac). After packaging, activity
was measured using a 1205 BETAPLATE liquid scintillation counter.
MLR using immature DC was performed by mixing T cells with mature
DC, which had been isolated from different humans. With a cell
ratio of T cells to DC of 1:40, MLR was performed similarly. FIGS.
9 and 10 show the results. It was shown that the addition of
KM281-1-10 antibodies lowered thymidine incorporation, and thus MLR
could be suppressed. Furthermore, it was shown in FIG. 10 that
KM283-5 and 5D12 antibodies could not suppress DC-MLR. That is,
only the KM281-1-10 antibody is an antagonistic antibody that
neutralizes the action of CD40 ligands on DC. Moreover, FIG. 11
shows the results of examining the effect of KM302-1 antibodies,
which are agonistic antibodies, on MLR using immature DC.
Activation of DC promoted interaction with T cells, and caused an
increase in thymidine incorporation. These results indicated that
KM302-1 is an agonistic antibody that acts on immature DC.
Example 11
Effect of CD40 Antibody on the Binding of CD40L to CD40
[0113] Anti-CD40 antibodies were caused to bind to immobilized CD40
human FC using BIAcore 2000 (Biacore), and then changes in the
binding amount of soluble CD40L to CD40 were measured. According to
the instruction attached to the system, soluble CD40 human FC was
immobilized on a CM chip (CM5, Biacore). Next, 25 .mu.g/ml
anti-CD40 antibodies were added to bind to CD40. Further, 10
.mu.g/ml soluble CD40L was added for binding. A difference between
the binding amounts before and after addition of CD40L was
measured. When control IgG was added, the binding amount of CD40L
was 100 RU. After addition of KM302-1 antibodies, the binding
amount of CD40L was 110 RU, and after addition of KM283-5
antibodies the binding amount of CD40L was 18RU. Thus, it was shown
that the KM302-1 antibody does not inhibit the binding of CD40L to
CD40.
Example 12
Promotion of CD95 Expression in Ramos Cells by Anti-CD40 Agonistic
Antibody
[0114] Purified antibodies of the hybridomas obtained in Example 4
were analyzed according to the method of Example 6, and then clones
producing agonistic antibodies were selected (number of cells per
well: 5.times.10.sup.4; cell concentration: 2.5.times.10.sup.5/ml).
FIG. 12 shows the results. In the figure, the horizontal axis
indicates the antibody concentration in culture solutions, and the
longitudinal axis indicates average fluorescence intensities, that
is, CD95 expression intensities. At a concentration of 0.01
.mu.g/ml or more, KM341-1-19 and 2105 antibodies were shown to
promote CD95 expression of Ramos cells more effectively than G28-5
antibodies, which are known mouse antibodies. Specifically,
KM341-1-19 and 2105 antibodies were shown to be more effective
agonistic antibodies. Further, the agonistic activity (to increase
CD95 expression of Ramos cells) of KM341-1-19 and 2105 antibodies
(0.01 .mu.g/ml) was higher than that of G28-5 antibodies (10
.mu.g/ml) (FIG. 12). Table 5 summarizes that at each antibody
concentration, CD95 expression level is how many times greater or
less than that expressed by the addition of G28-5 antibodies.
TABLE-US-00009 TABLE 5 Antibody concentration (.mu.g/ml) KM341-1-19
2105 F5-77 F2-103 0.01 5.7 3.9 0.1 7.0 7.1 1.2 1.2 1 5.7 5.1 1.7
1.8 10 4.5 3.3 2.0 1.7
Example 13
Activation of Dendritic Cell by CD40 Agonistic Antibody
[0115] According to the method of Example 9, the effect of CD40
agonistic antibodies on IL-12 production and IL-10 production by
mature DC was examined. IL-10 was measured by the ELISA
(Pharmingen) method. FIGS. 13 and 14 show the results. It was shown
that IL-12 secretion was increased by treatment with KM341-1-19
antibodies. In contrast, even when CD40 ligand-expressing
recombinant L cells (2.times.10.sup.5 cells/ml) that had been
irradiated with X-rays (5000 rad) were allowed to co-exist, the
concentrations of IL-12 and IL-10 in culture solutions were 254 and
51 pg/ml, respectively. They were lower than that when 1 .mu.g/ml
KM341-1-19 antibodies were added.
[0116] As described above, it was shown that KM341-1-19 antibodies
act on DC as effective agonistic antibodies. The agonistic activity
of KM341-1-19 antibodies (0.1 .mu.g/ml) to cause mature DC to
secrete IL-12 was higher than that of G28-5 antibodies (100
.mu.g/ml). The agonistic activity of KM341-1-19 antibodies (1
.mu.g/ml) to cause mature DC to secrete IL-12 was 100 times or more
greater than that by G28-5 antibodies (100 .mu.g/ml) (FIG. 13).
Furthermore, the agonistic activity of KM341-1-19 antibodies (1
.mu.g/ml) to cause mature DC to secrete IL-10 was 10 times or more
greater than that by G28-5 antibodies (100 .mu.l/ml) (FIG. 14).
Moreover, since the subclass of KM341-1-19 antibody was IgG2, the
antibody has lower binding ability to the Fc receptor than that of
IgG1 or IgG3. Its ability to sensitize the killer activity of NK
cells and ability to activate the complement system are also weak.
Accordingly, there may be a low risk that the function of
CD40-expressing cells or the number of the cells themselves
decreases due to the antibody. Furthermore, the antibody is not
easily cross-linked by an Fc receptor, so that it can be expected
that the drug effect is easily controlled without any large
fluctuation in in vivo agonistic activity due to cross-linking.
Example 14
Suppression of CD95 Expression in Ramos Cells by Anti-CD40
Antagonistic Antibody
[0117] 1.0.times.10.sup.6 cells/ml Ramos cell suspension was
inoculated at 50 .mu.l/well to a flat bottom 96-well plate (number
of cells per well: 5.times.10.sup.4). Purified antibodies diluted
with media were added at 100 .mu.l/well to a 96-well plate. Human
CD40 ligand-expressing recombinant mouse L cells (see Spriggs, M.
K. et. al., J. Exp. Med., 176: 1543, 1992; Garrone, P. et. al., J.
Exp. Med., 182: 1265, 1995 and the like) were prepared at
1.0.times.10.sup.5 cells/ml. The prepared cells were added at 50
.mu.l/well (the number of Ramos cells per well: 5.times.10.sup.4;
Ramos cell concentration: 2.5.times.10.sup.4 cells/ml; the number
of mouse L cells per well: 5.times.10.sup.3; mouse cell
concentration: 2.5.times.10.sup.4 cells/ml). After overnight
culture, the cells were collected, and then analyzed by FACS using
R-PE-labeled anti-CD95 antibodies. FIG. 15 shows the results. In
the figure, the longitudinal axis indicates the average
fluorescence intensity, that is, CD95 expression intensity. Whereas
the known 5D12 antibodies suppressed the expression slightly, 4D11
antibodies suppressed, even at a concentration of 0.1 .mu.g/ml,
CD95 expression to the same degree as that of a case of a negative
control wherein no CD40L-expressing cells had been added. Moreover,
at a concentration of 1 .mu.g/ml, 4D11, F4-465 and KM281-1-10
suppressed CD95 expression to the same degree as that of the case
of the negative control wherein no CD40L-expressing cells had been
added. These results showed that 4D11, F4-465 and KM281-1-10
antibodies are more effective antagonistic antibodies. Table 6
shows relative values of the average fluorescence intensity
corresponding to each antibody concentration, when the value of the
control case wherein no antagonistic antibodies were added is
determined as 100.
TABLE-US-00010 TABLE 6 Antibody concentration (.mu.g/ml) 5D12 4D11
F4-465 KM281-1-10 0.1 77.6 11.8 49.6 60.1 1 72.3 0.01 2.5 7.3 10
69.5 0 1.1 2.6
Example 15
Anti-Tumor Effect in Ramos Cell Transplantation Model by Anti-CD40
Agonistic Antibody
[0118] Anti-asialo GM1 antibodies were intravenously injected to
5-week-old C.B.17/Icr-scidJc1 mice (CLEA JAPAN). 1 day later,
5.times.10.sup.6 Ramos cells per mouse were intravenously injected
as tumor cells. 1 day later, KM302-1 antibodies or anti-human
albumin human IgG antibodies as a negative control were
intravenously injected. The doses per mouse of KM302-1 antibodies
were 1, 10 and 100 .mu.g, and the same of the negative control
antibodies was 100 .mu.g. Each of these antibodies was administered
once to 5 mice. FIG. 16 shows the results. By day 34 after
transplantation, all the mice of the negative control-administered
group had died, whereas all the 5 mice each of the groups
administered with 10 .mu.g and 100 .mu.g of KM302-1 antibodies had
been administered to which survived. Thus, the anti-tumor effect of
KM302-1 antibodies was confirmed. The KM302-1 antibody is of the
IgG4 subclass, so that the Fc receptor-mediated antibody dependent
cellular cytotoxicity (ADCC) and activation of the complement
system are weak. Despite these characteristics, it was observed
that single administration of 10 .mu.g of KM302-1 antibodies
prolonged the survival time of tumor-bearing mice.
Example 16
Ramos Cell Proliferation Suppression by Anti-CD40 Agonistic
Antibody
[0119] A Ramos cell suspension was prepared at 1.times.10.sup.4
cells/ml in an RPMI1640 medium supplemented with 10% FBS, and then
100 .mu.l of the suspension was apportioned to a 96-well plate. A
KM341-1-19 antibody or soluble ligand solution prepared at 20
.mu.g/ml using media was added. Anti-FLAG antibodies (M2) with the
same concentration as that of the ligands were allowed to co-exist
with the soluble ligands (the concentration in the reaction
solution was 10 .mu.g/ml), thereby enhancing the activity. After 5
days of culturing, 20 .mu.l of MTS reagent (Promega) was added to
each well, and then allowed to react for 2 to 3 hours. Differences
in absorbance between the cell-free and antibody-free wells and the
cell- and antibody-containing wells were measured at a wavelength
of 490 nm, thereby measuring viable cell count. Furthermore, the
proliferation-suppressing action was compared with that of G28-5
antibodies using a 96-well U-bottomed plate similarly. KM341-1-19
antibodies or G28-5 antibodies prepared at 2 .mu.g/ml using media
were added. FIG. 17 shows the results. In wells to which KM341-1-19
antibodies had been added, dead cells were observed, the cell
number was significantly lower than those of wells to which G28-5
antibodies or the ligands had been added, and the absorbance was
also low. These results indicate that the proliferation of tumor
cells was suppressed, and cell death was induced.
Example 17
cDNA Cloning of Antibody Gene
[0120] Hybridomas producing KM341-1-19, 2105, 110, 115, KM281-1-10,
4D11, KM643-4-11, F4-465, F2-103 and F5-77 antibodies were
cultured, and then the cells were collected by centrifugation.
TRIZOL (Gibco BRL) was added to the cells, and then Total RNA was
extracted according to the attached instructions. Cloning of the
variable regions of the antibody cDNA was performed according to
the attached instructions using a SMART RACE cDNA amplification Kit
(CLONTECH). Using 5 .mu.g of total RNA as a template, 1st Strand
cDNA was constructed. To amplify the heavy chains (H chain) of
KM341-1-19, 2105, 110, 115, KM281-1-10, 4D11, KM643-4-11, F2-103
and F5-77, Z-Taq (Takara) and UMP and hh6 primers were used, and a
cycle of 98.degree. C. for 1 second and 68.degree. C. for 30
seconds was repeated 30 times. Furthermore, using 1 .mu.l of the
reaction solution as a template and NUMP and hh3 primers, a cycle
of 98.degree. C. for 1 second and 68.degree. C. for 30 seconds was
repeated 20 times. To amplify a F4-465 heavy chain, UMP and hh2
primers and an Advantage 2 PCR kit (Clonthech, cat#1910) were used,
and 5 cycles of 94.degree. C. for 5 seconds and 72.degree. C. for 3
minutes, 5 cycles of 94.degree. C. for 5 seconds, 70.degree. C. for
0 seconds and 72.degree. C. for 3 minutes, and 25 cycles of
94.degree. C. for 5 seconds, 68.degree. C. for 10 seconds and
72.degree. C. for 3 minutes were performed.
TABLE-US-00011 hh6 primer: (SEQ ID NO: 3) 5'-GGT CCG GGA GAT CAT
GAG GGT GTC CTT-3' hh3 primer: (SEQ ID NO: 4) 5'-GTG CAC GCC GCT
GGT CAG GGC GCC TG-3' hh2 primer: (SEQ ID NO: 5) 5'-GCT GGA GGG CAC
GGT CAC CAC GCT G-3'
[0121] Subsequently, the amplified PCR product was purified using a
PCR purification kit (QIAGEN), and then the nucleotide sequence was
determined using hh4 as a primer. Alternatively, the product was
subcloned to PCR-Script (Stratagene, Lajolla, Calif.) or PCR-Blunt
(Invitrogene, Carlsbad, Calif.), and then sequencing was performed.
Based on the sequence information, antibody heavy-chain-specific
primers were synthesized. A 341H primer was synthesized in the case
of KM341-1-19, a 2105Hsa1 primer in the case of 2105, a 110Hsa1
primer in the case of 110 and 115, a 2811Hsa1 primer in the case of
KM281-1-10, a 4D11Sa1 primer in the case of 4D11, a 643Hsa1 primer
in the case of KM643-4-11, H11-95' primer in the case of F4-465, a
F2-103H primer in the case of F2-103 and F5-77H primer in the case
of F5-77. Using the antibody heavy chain specific primers and hh4,
cDNA was amplified from the 1st Strand cDNA, and then the sequence
from the opposite direction was determined using the amplified
product as a template and the antibody-specific primers.
TABLE-US-00012 hh4 primer: (SEQ ID NO: 6)
5'-GGTGCCAGGGGGAAGACCGATGG-3' 341 H primer: (SEQ ID NO: 7)
5'-atatgtcgacGCTGAATTCTGGCTGACCAGGGCAG-3' 2105Hsal: (SEQ ID NO: 8)
atatgtcgacTCCCAGGTGTTTCCATTCAGTGATCAG 110Hsal: (SEQ ID NO: 9)
atatgtcgacTTCCATTCGGTGATCAGCACTGAACAC 281Hsal: (SEQ ID NO: 10)
atatgtcgacTTTGAGAGTCCTGGACCTCCTGTG 4D11Sal: (SEQ ID NO: 11)
atatgtcgacGAGTCATGGATCTCATGTGCAAG 643Hsal: (SEQ ID NO: 12)
atatgtcgacCCAGGGCAGTCACCAGAGCTCCAGAC H11-9 5': (SEQ ID NO: 13) ACC
GTG TCG ACT ACG CGG GAG TGA CT F2-103 H: (SEQ ID NO: 14)
accgtgtcgacgctgatcaggactgcaca F5-77 H: (SEQ ID NO: 15)
accgtgtcgacggtgatcaggactgaacag
[0122] The light chains (L chains) of KM341-1-19, 2105, 110, 115,
KM281-1-10, 4D11, KM643-4-11, F2-103 and F5-77 were amplified using
UMP and hk2 primers and by repeating 30 times a cycle of 98.degree.
C. for 1 second and 68.degree. C. for 30 seconds. The light chain
of F4-465 was amplified using UMP and hL2 primers and by repeating
30 times a cycle of 98.degree. C. for 1 second and 68.degree. C.
for 30 seconds. The amplified PCR product was purified using a PCR
purification kit, and then the nucleotide sequence was determined
using hk6 or hL2 primers. Based on the sequences, light chain
specific primers were synthesized. A 341K primer was synthesized in
the case of KM341-1-19, 2053 KBg1 primer in the case of 2105, 110
KBg1 primer in the case of 110 and 115, 281 KBg1 primer in the case
of KM281-1-10, 4D11KBg1 in the case of 4D11, 643 KBg1 primer in the
case of KM643-4-11, Lamda 5' primer in the case of F4-465, and
F2-103K primer in the case of F-103 and F5-77.
[0123] In the case of 341-1-19, 110, 115, KM643-4-11, KM281-1-10,
4D11 and 2105, cDNA was amplified from the 1st Strand cDNA using
the light chain specific primer and hk6 primer. The sequence was
then determined from both directions using the amplified product as
a template. For F4-465, F2-103 and F5-77, subcloning to PCR-Script
(Stratagene, Lajolla, Calif.) or PCR-Blunt (Invitrogene, Carlsbad,
Calif.) was performed to determine the sequence.
TABLE-US-00013 hk2 primer: (SEQ ID NO: 16) 5'-GTT GAA GCT CTT TGT
GAC GGG CGA GC-3' hL2 primer: (SEQ ID NO: 17) 5'-TCT TCT CCA CGG
TGC TCC CTT CAT-3' 341K primer: (SEQ ID NO: 18)
5'-atatagatctGAACTGCTCAGTTAGGACCCAGAGG-3' 2053KBgl: (SEQ ID NO: 19)
atatagatctCGCGGGGAAGGAGACTGCTCAGTT 110KBgl: (SEQ ID NO: 20)
atatagatctAGTCAGACCCAGTCAGGACACAGC 281KBgl: (SEQ ID NO: 21)
atatagatctGAGCTGCTCAGTTAGGACCCAGAGGG 4D11KBgl: (SEQ ID NO: 22)
atatagatctTAAGCAAGTGTAACAACTCAGAGTAC 643KBgl: (SEQ ID NO: 23)
atatagatctGAGGAACTGCTCAGTTAGGACCCAGAGG Lamda 5': (SEQ ID NO: 24)
AACTCCAGATCTGCCTCAGGAAGCAGCATC F2-103 K: (SEQ ID NO: 25)
aactccagatctagggcaagcagtggtaac hk6 primer: (SEQ ID NO: 26)
5'-TGGCGGGAAGATGAAGACAGATGGTG-3'
[0124] DNAs of 341-1-19 encoding the full-length H-chain and
L-chain variable regions and the amino acid sequences of H-chain
and L-chain are respectively shown below.
[0125] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 50th adenine (A) from the 5' end of
SEQ ID NO: 27, and the termination codon is TGA beginning from the
1472nd thymine (T). The boundary of the antibody variable region
and the constant region is located between the 493rd adenine (A)
and the 494th guanine (G) from the 5' end. In the amino acid
sequence, the H-chain variable region ranges from the N-terminus to
the 148th serine (S) residue of SEQ ID NO: 28, and the constant
region is of the 149th alanine (A) and the following residues. It
was predicted by a gene sequence prediction software (Signal P
ver.2) that the H-chain signal sequence ranges from the N-terminus
to the 20th serine (S) of SEQ ID NO: 28. It is thought that the
N-terminus of the mature protein is the 21st glutamine (Q) of SEQ
ID NO: 28.
[0126] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 29th A from the 5' end of SEQ ID NO:
29, and the variable region ranges from the 5' end to the 400th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 124th lysine (K) of SEQ ID NO: 30.
Analysis of the N-terminus of the purified L-chain protein revealed
that the L-chain signal sequence ranges from the N-terminus to the
20th glycine (G) of SEQ ID NO: 30, and the N-terminus of the mature
protein is the 21st glutamic acid (E) of SEQ ID NO: 30.
TABLE-US-00014 341-1-19 H-chain (SEQ ID NO: 27):
GTCGACGCTGAATTCTGGCTGACCAGGGCAGCCACCAGAGCTCCAGACAA
TGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGT
GTCCTGTCACAGGTCCAACTGCAGCAGTCAGGTCCAGGACTGGTGAAGCC
CTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTA
GCAACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGAGAGACCTT
GAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATCGTGATTATGT
AGGATCTGTGAAAAGTCGAATAATCATCAACCCAGACACATCCAACAACC
AGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTATATAT
TACTGTACAAGAGCACAGTGGCTGGGAGGGGATTACCCCTACTACTACAG
TATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCAGCCTCCA
CCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCC
GAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC
GGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCT
TCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGA
TCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTT
GTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC
TGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCC
AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCAC
CGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCT
CCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACACCTCCCATGCTGGACTCAGACGGCTCCTTCTTCCTCTA
CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT
CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC
CTCTCCCTGTCTCCGGGTAAATGAGGATCC 341-1-19 H-chain amino acid sequence
(SEQ ID NO: 28) MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSLTCAISGDSVS
SNSATWNWIRQSPSRDLEWLGRTYYRSKWYRDYVGSVKSRIIINPDTSNN
QFSLQLNSVTPEDTAIYYCTRAQWLGGDYPYYYSMDVWGQGTTVTVSSAS
TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK 341-1-19 L-chain (SEQ ID NO: 29):
ACTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTC
TCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTG
ACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAAC
AGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGG
GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACT
GTCAGCAGCGTAGCAACACTTTCGGCCCTGGGACCAAAGTGGATATCAAA CGTACG 341-1-19
L-chain amino acid sequence (SEQ ID NO: 30)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVS
SYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP
EDFAVYYCQQRSNTFGPGTKVDIKRT
[0127] DNAs of 2105 encoding the H-chain variable region and
L-chain variable region and the amino acid sequences of the H-chain
and L-chain are shown below.
[0128] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 70th adenine (A) from the 5' end of
SEQ ID NO: 31. The boundary of the antibody variable region and the
constant region is located between the 495th adenine (A) and the
496th guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus to the 142nd
serine (S) residue of SEQ ID NO: 32, and the constant region is of
the 149th alanine (A) and the following residues. It was predicted
by a gene sequence prediction software (Signal P ver.2) that the
H-chain signal sequence ranges from the N-terminus to the 19th
cystein (C) of SEQ ID NO: 32. It is thought that the N-terminus of
the mature protein is the 20th glutamic acid (E) of SEQ ID NO:
32.
[0129] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 28th A from the 5' end of SEQ ID NO:
33, and the variable region ranges from the 5' end to the 405th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 126th lysine (K) of SEQ ID NO: 34. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
20th glycine (G) of SEQ ID NO: 34. It is thought that the
N-terminus of the mature protein is the 21st glutamic acid (E) of
SEQ ID NO: 34.
TABLE-US-00015 2105 H-chain (SEQ ID NO: 31)
CTGAACACAGACCCGTCGACTCCCAGGTGTTTCCATTCAGTGATCAGCAC
TGAACACAGAGGACTCACCATGGAGTTGGGACTGAGCTGGATTTTCCTTT
TGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGG
GGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAG
GGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCTTG
GTGCATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGG
CCTTGTATTACTGTGCAAGAGATAGGCTATTTCGGGGAGTTAGGTACTAC
GGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAG CACCAAGG 2105
H-chain amino acid sequence (SEQ ID NO: 32)
MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDD
YAMHWVRQAPGKGLEWVSGISWNSGSLVHADSVKGRFTISRDNAKNSLYL
QMNSLRAEDTALYYCARDRLFRGVRYYGMDVWGQGTTVTVSSASTK 2105 L-chain (SEQ ID
NO: 33) CTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCT
CTTCCTCCTGCTACTCTGGCTCCGAGATACCACCGGAGAAATTGTGTTGA
CACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC
TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACA
GAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGG
CCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC
ACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCACTTTATTACTG
TCAGCAGCGTAGCCACTGGCTCACTTTCGGCGGGGGGACCAAGGTGGAGA TCAAACGTACGGTG
2105 L-chain amino acid sequence (SEQ ID NO: 34)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVS
SYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP
EDFAVYYCQQRSHWLTFGGGTKVEIKRTV
[0130] DNAs of 110 encoding the H-chain variable region and L-chain
variable region and the amino acid sequences of the H-chain and
L-chain are respectively shown below.
[0131] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 60th adenine (A) from the 5' end of
SEQ ID NO: 35. The boundary of the antibody variable region and the
constant region is located between the 479th adenine (A) and the
480th guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 36
to the 140th serine (S) residue, and the constant region is of the
141st alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the H-chain
signal sequence ranges from the N-terminus to the 19th cystein (C)
of SEQ ID NO: 36. It is thought that the N-terminus of the mature
protein is the 20th glutamine (Q) of SEQ ID NO: 36.
[0132] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 35th A from the 5' end of SEQ ID NO:
37, and the variable region ranges from the 5' end to the 421st
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 129th lysine (K) of SEQ ID NO: 38. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
22nd cystein (C) of SEQ ID NO: 38. It is thought that the
N-terminus of the mature protein is the 23rd valine (V) of SEQ ID
NO: 38.
TABLE-US-00016 110 H-chain (SEQ ID NO: 35)
CTGAACACAGACCCGTCGACTTCCATTCGGTGATCAGCACTGAACACAGA
GGACTCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTT
AAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGG
TCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACC
TTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCT
GGAGTGGGTGGCAGTTATATGGTATGATGGAAGTATTAAATACTATGCAG
ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTA
CTGTGCGAGAGAGGGCTACAATATTTTGACTGGTTATTTTGGCTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGG 110 H-chain amino acid
sequence (SEQ ID NO: 36)
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSS
YGMHWVRQAPGKGLEWVAVIWYDGSIKYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAREGYNILTGYFGYWGQGTLVTVSSASTK 110 L-chain (SEQ ID
NO: 37) TCACAGATCTAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCC
CCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGT
GTCATCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGA
CAGAGTCACCATCAGTTGTCGGATGAGTCAGGGCATTAGCAGTGATTTAG
CCTGGTATCAGCAAAAACCAGGGAAAGCCCCTGAGCTCCTGATCTCTGCT
GCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCTGCCTGCAGTCTGAAGATTTTG
CAACTTATTACTGTCAACAGTATTATAGTTTTCCGTGGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGTACG 110 L-chain amino acid sequence (SEQ ID
NO: 38) MDMRVPAQLLGLLLLWLPGARCVIWMTQSPSLLSASTGDRVTISCRMSQG
ISSDLAWYQQKPGKAPELLISAASTLQSGVPSRFSGSGSGTDFTLTISCL
QSEDFATYYCQQYYSFPWTFGQGTKVEIKRT
[0133] DNAs of 115 encoding the H-chain variable region and L-chain
variable region and the amino acid sequences of the H-chain and
L-chain are respectively shown below.
[0134] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 60th adenine (A) from the 5' end of
SEQ ID NO: 39. The boundary of the antibody variable region and the
constant region is located between the 479th adenine (A) and the
480th guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 40
to the 140th serine (S) residue, and the constant region is of the
141st alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the H-chain
signal sequence ranges from the N-terminus to the 19th cystein (C)
of SEQ ID NO: 40. It is thought that the N-terminus of the mature
protein is the 20th glutamine (Q) of SEQ ID NO: 40.
[0135] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 35th A from the 5' end of SEQ ID NO:
41, and the variable region ranges from the 5' end to the 421st
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 129th lysine (K) of SEQ ID NO: 42. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
22nd cystein (C) of SEQ ID NO: 42. It is thought that the
N-terminus of the mature protein is the 23rd valine (V) of SEQ ID
NO: 42.
TABLE-US-00017 115 H-chain (SEQ ID NO: 39)
CTGAACACAGACCCGTCGACTTCCATTCGGTGATCAGCACTGAACACAGA
GGACTCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTT
AAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGG
TCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACC
TTCAGTAGCTATGGCATGCACTGGGTCCGGCAGGCTCCAGGCAAGGGGCT
GGAGTGGGTGGCAGTTATATGGAATGATGGAAGTATTAAATACTATGCAG
ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTA
CTGTGCGAGAGAGGGCTACAATATTTTGACTGGTTATTTTGGCTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGG 115 H-chain amino acid
sequence (SEQ ID NO: 40)
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSS
YGMHWVRQAPGKGLEWVAVIWNDGSIKYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAREGYNILTGYFGYWGQGTLVTVSSASTK 115 L-chain (SEQ ID
NO: 41) TCACAGATCTAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCC
CCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGT
GTCATCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGA
CAGAGTCACCATCAGTTGTCGGATGAGTCAGGGCATTAGCAGTGATTTAG
CCTGGTATCAGCAAAAACCAGGGAAAGCCCCTGAGCTCCTGATCTCTGCT
GCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCTGCCTGCAGTCTGAAGATTTTG
CAACTTATTACTGTCAACAGTATTATAGTTTTCCGTGGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGTACG 115 L-chain amino acid sequence (SEQ ID
NO: 42) MDMRVPAQLLGLLLLWLPGARCVIWMTQSPSLLSASTGDRVTISCRMSQG
ISSDLAWYQQKPGKAPELLISAASTLQSGVPSRFSGSGSGTDFTLTISCL
QSEDFATYYCQQYYSFPWTFGQGTKVEIKRT
[0136] DNAs of 281-1-10 encoding the H-chain variable region and
L-chain variable region and the amino acid sequences of the H-chain
and L-chain are respectively shown below.
[0137] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 52nd adenine (A) from the 5' end of
SEQ ID NO: 43. The boundary of the antibody variable region and the
constant region is located between the 468th adenine (A) and the
469th guanine (G) from the Send. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 44
to the 139th serine (S) residue, and the constant region is of the
140th alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the H-chain
signal sequence ranges from the N-terminus to the 19th serine (S)
of SEQ ID NO: 44. It is thought that the N-terminus of the mature
protein is the 20th glutamine (Q) of SEQ ID NO: 44.
[0138] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 41 st A from the 5' end of SEQ ID
NO: 45, and the variable region ranges from the 5' end to the 424th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 128th lysine (K) of SEQ ID NO: 46. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
20th glycine (G) of SEQ ID NO: 46. It is thought that the
N-terminus of the mature protein is the 21st glutamic acid (E) of
SEQ ID NO: 46.
TABLE-US-00018 281-1-10 H-chain (SEQ ID NO: 43)
CTGAACACAGACCCGTCGACTTTGAGAGTCCTGGACCTCCTGTGCAAGAA
CATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGG
TCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCT
TCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTGG
TTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGA
TTGGGTATATCTATTACAGTGGGAGCACCAACTACAATCCCTCCCTCAAG
AGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAA
GCTGAATTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAG
CCCCCTTGCACGGTGACTACAAATGGTTCCACCCCTGGGGCCAGGGAACC
CTGGTCACCGTCTCCTCAGCTAGCACCAAGG 281-1-10 H-chain amino acid
sequence (SEQ ID NO: 44)
MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSISG
YYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLK
LNSVTAADTAVYYCARAPLHGDYKWFHPWGQGTLVTVSSASTK 281-1-10 L-chain (SEQ
ID NO: 45) TCACAGATCTGAGCTGCTCAGTTAGGACCCAGAGGGAACCATGGAAACCC
CAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT
TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT
TTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGATCACCTTCGGC
CAAGGGACACGACTGGAGATCAAACGTACG 281-1-10 L-chain amino acid sequence
(SEQ ID NO: 46) METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVS
SSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGSSPITFGQGTRLEIKRT
[0139] DNAs of 4D11 encoding the H-chain variable region and
L-chain variable region and the amino acid sequences of the H-chain
and L-chain are respectively shown below.
[0140] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 16th adenine (A) from the 5' end of
SEQ ID NO: 47. The boundary of the antibody variable region and the
constant region is located between the 456th adenine (A) and the
457th guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 48
to the 147th serine (S) residue, and the constant region is of the
148th alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the H-chain
signal sequence ranges from the N-terminus to the 26th serine (S)
of SEQ ID NO: 48. It is thought that the N-terminus of the mature
protein is the 27th glutamine (Q) of SEQ ID NO: 48.
[0141] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 59th A from the 5' end of SEQ ID NO:
49, and the variable region ranges from the 5' end to the 442nd
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 128th lysine (K) of SEQ ID NO: 50. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
22nd cystein (C) of SEQ ID NO: 50. It is thought that the
N-terminus of the mature protein is the 21st alanine (A) of SEQ ID
NO: 50.
TABLE-US-00019 4D11 H-chain (SEQ ID NO: 47)
ATATGTCGACGAGTCATGGATCTCATGTGCAAGAAAATGAAGCACCTGTG
GTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGC
AGCTGCAGGAGTCGGGCCCAGGACTACTGAAGCCTTCGGAGACCCTGTCC
CTCACCTGCACTGTCTCTGGCGGCTCCATCAGCAGTCCTGGTTACTACGG
GGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTA
TCTATAAAAGTGGGAGCACCTACCACAACCCGTCCCTCAAGAGTCGAGTC
ACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTC
TGTGACCGCCGCAGACACGGCTGTGTATTACTGTACGAGACCTGTAGTAC
GATATTTTGGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCAGCTAGC
4D11 H-chain amino acid sequence (SEQ ID NO: 48)
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLLKPSETLSLTCTV
SGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYHNPSLKSRVTISVD
TSKNQFSLKLSSVTAADTAVYYCTRPVVRYFGWFDPWGQGTLVTVSSAS 4D11 L-chain (SEQ
ID NO: 49) AGATCTTAAGCAAGTGTAACAACTCAGAGTACGCGGGGAGACCCACTCAG
GACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTG
CTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATC
CTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAA
GTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAA
GCTCCTAAGCTCCTGATCTATGATGCCTCCAATTTGGAAAGTGGGGTCCC
ATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCA
GCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAAT
AGTTACCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACG 4D11 L-chain amino
acid sequence (SEQ ID NO: 50)
MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQG
ISSALAWYQQKPGKAPKLLIYDASNLESGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQFNSYPTFGQGTKVEIKRT
[0142] DNAs of KM643-4-11 encoding the H-chain variable region and
L-chain variable region and the amino acid sequences of the H-chain
and L-chain are respectively shown below.
[0143] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 1st adenine (A) from the 5' end of
SEQ ID NO: 51. The boundary of the antibody variable region and the
constant region is located between the 447th adenine (A) and the
448th guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 52
to the 149th serine (S) residue, and the constant region is of the
150th alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the H-chain
signal sequence ranges from the N-terminus to the 20th serine (S)
of SEQ ID NO: 52. It is thought that the N-terminus of the mature
protein is the 21st glutamine (Q) of SEQ ID NO: 52.
[0144] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 38th A from the 5' end of SEQ ID NO:
53, and the variable region ranges from the 5' end to the 409th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 124th lysine (K) of SEQ ID NO: 54. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
20th glycine (G) of SEQ ID NO: 54. It is thought that the
N-terminus of the mature protein is the 21st Glutamic acid (E) of
SEQ ID NO: 54.
TABLE-US-00020 KM643-4-11 H-chain (SEQ ID NO: 51)
ATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGG
TGTCCTGTCACAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGC
CCTCGCAGACCCTCTCATTCACCTGTGCCATCTCCGGGGACAGTGTCTCT
AGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCT
TGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAAAGATTATG
CAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAAC
CAGTTCTCCCTGCAGCTGAACTCTGTGACCCCCGAGGACACGGCTGTGTA
TTACTGTGCAAGAGGGTATTACTATGGTTCGGGGAGCTATCCCTACTACT
ACCAAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCT AGC KM643-4-11
H-chain amino acid sequence (SEQ ID NO: 52)
MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSFTCAISGDSVS
SNSAAWNWIRQSPSRGLEWLGRTYYRSKWYKDYAVSVKSRITINPDTSKN
QFSLQLNSVTPEDTAVYYCARGYYYGSGSYPYYYQMDVWGQGTTVTVSSA S KM643-4-11
L-chain (SEQ ID NO: 53)
AATTGAGGAACTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAG
CTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAA
ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
TGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCT
GGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCA
TCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGG
GACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAG
TTTATTACTGTCAGCAGCGTAGCAACACTTTCGGCGGAGGGACCAAGGTG GAGATCAAACGAAC
KM643-4-11 L-chain amino acid sequence (SEQ ID NO: 54)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGESATLSCRASQSVS
SYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP
EDFAVYYCQQRSNTFGGGTKVEIKR
[0145] DNAs of F4-465 encoding the H-chain variable region and
L-chain variable region and the amino acid sequences of the H-chain
and L-chain are respectively shown below.
[0146] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 47th adenine (A) from the 5' end of
SEQ ID NO: 55. The boundary of the antibody variable region and the
constant region is located between the 484th adenine (A) and the
445th guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 56
to the 146th serine (S) residue, and the constant region is of the
147th alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the H-chain
signal sequence ranges from the N-terminus to the 19th serine (S)
of SEQ ID NO: 56. It is thought that the N-terminus of the mature
protein is the 20th glutamine (Q) of SEQ ID NO: 56.
[0147] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 81st A from the 5' end of SEQ ID NO:
57, and the variable region ranges from the 5' end to the 440th
(C). In the amino acid sequence, the variable region ranges from
the N-terminus to the 120th Threonine (T) of SEQ ID NO: 58. It was
predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
19th alanine (G) of SEQ ID NO: 58. It is thought that the
N-terminus of the mature protein is the 20th serine (S) of SEQ ID
NO: 58.
TABLE-US-00021 F4-465 H-chain (SEQ ID NO: 55)
CTGAACACAGACCCGTCGACTACGCGGGAGACCACAGCTCCACACCATGG
ACTGGACCTGGAGGATCCTATTCTTGGTGGCAGCAGCAACAGGTGCCCAC
TCCCAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGC
CTCAGTGAAGGTCCCCTGCAAGGCTTCTGGATACACCTTCACTAGCTATG
CTATGAATTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGA
TGGATCAACACCAACACTGGGAACCCAACGTATGCCCAGGGCTTCACAGG
ACGGTTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGA
TCAGCAGCCTAAAGGCTGAGGACACTGCCGTGTATTACTGTGCGAGAGAG
GTAGTACCAGTTGCTATGAGGGTAACTCACTACTACTACGGTATGGACGT
CTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAA F4-465 H-chain amino
acid sequence (SEQ ID NO: 56)
MDWTWRILFLVAAATGAHSQVQLVQSGSELKKPGASVKVPCKASGYTFTS
YAMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYL
QISSLKAEDTAVYYCAREVVPVAMRVTHYYYGMDVWGQGTTVTVSSAST F4-465 L-chain
(SEQ ID NO: 57) CTGGGTACGGTAACCGTCAGATCGCCTGGAGACGCCATCACAGATCTGCC
TCAGGAAGCAGCATCGGAGGTGCCTCAGCCATGGCATGGATCCCTCTCTT
CCTCGGCGTCCTTGTTTACTGCACAGGATCCGTGGCCTCCTATGAGCTGA
CTCAGCCACCCTCAGTGTCCGTGGCCCCAGGACAGACAGCCAGCATCACC
TGTTCTGGAGATAAATTGGGGGATAATTTTACTTGCTGGTATCAGCAGAA
GCCAGGCCAGTCCCCTGTGCTGGTCATCTTTCAGGATTGGAAGCGGCGCC
CAGGGATCCCTGCGCGATTCTCTGGCTCCAAGTCTGGGAACACAGCCACT
CTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTCA
GGCGTGGGACATCAGCACTGTGGTATTCGGCGGAGGGACCAAGCTGACCG
TCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCC
TCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGA
CTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCG
TCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAG
TACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCA
CAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGA
CAGTGGCCCCTACAGAATGTTCATGAATTCAGATCCGTTAACGGTTACCA
ACTACCTAGACTGGATTCGTGACCAACATA F4-465 L-chain amino acid sequence
(SEQ ID NO: 58) MAWIPLFLGVLVYCTGSVASYELTQPPSVSVAPGQTASITCSGDKLGDNF
TCWYQQKPGQSPVLVIFQDWKRRPGIPARFSGSKSGNTATLTISGTQAMD
EADYYCQAWDISTVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKAT
LVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT
PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
[0148] DNAs of F2-103 encoding the H-chain variable region and
L-chain variable region and the amino acid sequences of the H-chain
and L-chain are respectively shown below.
[0149] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 32nd adenine (A) from the 5' end of
SEQ ID NO: 59. The boundary of the antibody variable region and the
constant region is located between the 463rd adenine (A) and the
464th Guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 60
to the 144th Serine (S) residue, and the constant region is of the
145th Alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the H-chain
signal sequence ranges from the N-terminus to the 19th Cystein (C)
of SEQ ID NO: 60. It is thought that the N-terminus of the mature
protein is the 20th Glutamic acid (E) of SEQ ID NO: 60.
[0150] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 29th A from the 5' end of SEQ ID NO:
61, and the variable region ranges from the 5' end to the 415th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 129th Lysine (K) of SEQ ID NO: 62. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
22nd Cystein (C) of SEQ ID NO: 62. It is thought that the
N-terminus of the mature protein is the 23rd Asp (D) of SEQ ID NO:
62.
TABLE-US-00022 F2-103 H-chain (SEQ ID NO: 59)
GCTGATCAGGACTGCACACAGAGAACTCACCATGGAGTTTGGGCTGAGCT
GGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTG
GTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTC
CTGTGCAGTCTCTGGATTCACCTTCAGTACCTACTGGATGCACTGGGTCC
GCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTATTAATAGTGAT
GGGAGTAGCACAACCTACGCGGACTCCGTGAAGGGCCGATTCACCATCTC
CAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAG
CCGAGGACACGGCTGTGTATTACTGTGCAAGAGATAGAGTACTATGGATC
GGGGAGTTATCCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGT
CACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC
CCTCCTCCAAGAGCACCTCT F2-1103 H-chain amino acid sequence (SEQ ID
NO: 60) MEFGLSWVFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAVSGFTFST
YWMHWVRQAPGKGLVWVSRINSDGSSTTYADSVKGRFTISRDNAKNTLYL
QMNSLRAEDTAVYYCARDRVLWIGELSYYGMDVWGQGTTVTVSSASTKGP SVFPLAPSSKSTS
F2-103 L-chain (SEQ ID NO: 61)
GGGGAGTCAGACCCAGTCAGGACACAGCATGGACATGACGGTCCCCGCTC
AGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAAATGTGACATC
CAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGT
CACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAACTGGTTGGCCTGGT
ATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGCTCTATAAGGCATCT
GGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGAC
AGAATTCACTCTCACCATCAACAGCCTGCAGCCTGATGATTTTGCAACTT
ATTACTGCCAACAGTCTAATAGTTATTCGTGGACGTTCGGCCACGGGACC
AAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC
GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC
TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT
ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA F2-103 L-chain
amino acid sequence (SEQ ID NO: 62)
MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQS
ISNWLAWYQQKPGKAPKLLLYKASGLESGVPSRFSGSGSGTEFTLTINSL
QPDDFATYYCQQSNSYSWTFGHGTKVEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGL
[0151] DNAs of F5-77 encoding the H-chain variable region and
L-chain variable region and the amino acid sequences of the H-chain
and L-chain are respectively shown below.
[0152] The translation initiation point of the H-chain DNA is an
ATG codon that begins from the 100th adenine (A) from the 5' end of
SEQ ID NO: 63. The boundary of the antibody variable region and the
constant region is located between the 528th adenine (A) and the
529th Guanine (G) from the 5' end. In the amino acid sequence, the
H-chain variable region ranges from the N-terminus of SEQ ID NO: 64
to the 143rd Serine (S) residue, and the constant region is of the
144th Alanine (A) and the following residues. It was predicted by
gene sequence prediction software (Signal P ver.2) that the U-chain
signal sequence ranges from the N-terminus to the 19th Cystein (C)
of SEQ ID NO: 64. It is thought that the N-terminus of the mature
protein is the 20th Glutamic acid (E) of SEQ ID NO: 64.
[0153] The translation initiation point of the L-chain DNA is an
ATG codon that begins from the 59th A from the 5' end of SEQ ID NO:
65, and the variable region ranges from the 5' end to the 445th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 129th Lysine (K) of SEQ ID NO: 66. It
was predicted by gene sequence prediction software (Signal P ver.2)
that the L-chain signal sequence ranges from the N-terminus to the
22nd Cystein (C) of SEQ ID NO: 66. It is thought that the
N-terminus of the mature protein is the 23rd Asp (D) of SEQ ID NO:
66.
TABLE-US-00023 F5-77 H-chain (SEQ ID NO: 63)
GGTCTATATAAGCAGAGCTGGGTACGTCCTCACATTCAGTGATCAGCACT
GAACACAGACCCGTCGACGGTGATCAGGACTGAACAGAGAGAACTCACCA
TGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTC
CAGTGTGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
ATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
GATGGGGGGTACTATGGTTCGGGGAGTTATGGGTACTTTGACTACTGGGG
CCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC
CTGGGCTGCCTGGTCAAGGACTACTTCCCC F5-77 H-chain amino acid sequence
(SEQ ID NO: 64) MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSS
YAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAKDGGYYGSGSYGYFDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFP F5-77 L-chain (SEQ ID NO: 65)
CAAGCAGTGGTAACAACGCAGAGTACGCGGGGGGAGTCAGACCCAGTCAG
GACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTG
CTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATC
TTCCGTGTCTGGATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGA
GTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATGCTGGATCCAGTTTGCAAAGTGGGGTCCC
ATCAAGGTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATCA
GCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAGC
AGTTTCCCTCGGACATTCGGCCAAGGGACCAAGGTGGAGATCAAACGTAC
GGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA
AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA
GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC
CCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGA F5-77 L-chain amino acid sequence (SEQ
ID NO: 66) MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSGSVGDRVTITCRASQG
ISSWLAWYQQKPGKAPKLLIYAGSSLQSGVPSRFSGSGFGTDFTLTISSL
QPEDFATYYCQQASSFPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGL
Example 18
Expression of Antibody Protein in Animal Cell
[0154] The above obtained DNA fragment containing the variable
region of the antibody was incorporated into an appropriate vector
such as N5KG1 (IDEC Pharmaceuticals, U.S. Pat. No. 6,001,358),
thereby preparing an antibody expression vector. As a host cell for
expression, for example, CHO-Ras (Katakura Y., et al.,
Cytotechnology, 31: 103-109, 1999) is appropriately used. The
vector can be introduced into the host cell by, for example,
electroporation. Approximately 2 .mu.g of the antibody expression
vector was linearized with a restriction enzyme. The gene was
introduced into 4.times.10.sup.7 CHO-Ras cells under conditions of
350V and 500 .mu.F using a Bio-Rad electrophoreter, and then
inoculated to a 96-well culture plate. A drug corresponding to the
selection marker of the expression vector was added, and culturing
was continued. When colonies were observed, antibody-expressing
lines were selected by the method described in Example 4.
Antibodies can be purified from the selected cells according to
Example 5.
Example 19
Antigen-Specific Antibody Production Suppressive Action of CD40
Antagonistic Antibody
[0155] Mice having a genetic background whereby they were
homozygotes for mouse endogenous disrupted CD40 and harboring a
transgene of a human CD40 gene (Yasui. et al. Int. Immunol. 2002
Vol 14: 319) were sensitized by intraperitoneally injecting 100
.mu.g (in an amount of NP-CGG) of a complex of
4-hydroxy-3-nitrophenylacetyl-chicken .gamma.-globulin conjugates
(NP-CGG: distributed by Hitoshi KIKUTANI, Professor, Research
Institute for Microbial Diseases, Osaka University) and ARAM (ARAM:
Antigen Recognition Activation Motif). 30 or 100 .mu.g of each
monoclonal antibody was administered via caudate vein immediately
before antigen sensitization. 100 .mu.g of anti-human albumin human
IgG4 antibody was administered as a negative control. 7 days after
sensitization, blood was collected from the orbital venous plexus,
and then the amounts of NP specific IgG1 and IgM antibodies in sera
were measured by the ELISA method. The ELISA method was performed
by adding NP-bound bovine serum albumin (NP-BSA: 2.5 .mu.g/ml) (50
.mu.l/well) to each well of a 96-well micro plate for ELISA
(Maxisorp, Nunc), incubating at 4.degree. C. and then absorbing
NP-BSA. Next, the supernatant was discarded, a blocking reagent
(Super Block, Pierce) was added to each well, and then incubation
was performed at room temperature for blocking. Each well was then
washed 3 times with a 0.1% Tween20-containing phosphate buffer
(PBS-T). Subsequently, each serum diluted with 10%
BlockAce-containing PBS-T was added (50 Ill/well) to each well,
followed by incubation at 37.degree. C. for 2 hours for reaction.
The microplate was washed 3 times with PBS-T. A solution prepared
by diluting 1.000-fold alkaline phosphatase-labeled goat anti-mouse
IgG1 or IgM antibody (COSMO BIO, 1070-04 or 1020-04) with 10%
BlockAce-containing PBS-T was added (50 .mu.l/well) to each well,
followed by 2 hours of incubation at 37.degree. C. Next, the
microplate was washed 3 times with PBS-T, a chromogenic substrate
solution (50 .mu.l/well, Sigma104, phosphatase substrate) was added
to each well, and then absorbance at a wavelength of 405 nm was
measured with a microplate reader. FIGS. 18 and 19 show the
results. In the figures, longitudinal axes indicate values obtained
by conversion using as a unit the serum diluted 10,000-fold in the
case of IgG1 antibody, and the serum diluted 100-fold in the case
of IgM antibody. Here, the serum was prepared by injecting NP-CGG
twice into C57BL/6 mice, collecting blood from the mice, and
pooling the serum. Administration of 100 .mu.g each of F4-465, 4D11
and KM281-1-10 antibodies strongly suppressed NP-specific IgG1 and
IgM antibody production.
Example 20
Proliferation Suppression of Tonsillar B Cells by CD40 Antagonistic
Antibody
[0156] Human tonsils were obtained from Children's Hospital (San
Diego, Calif., U.S.A.). Tonsils were cut into small pieces, minced,
and then passed through a 70-micrometer nylon mesh cell strainer,
thereby preparing a cell suspension. The cell suspension was washed
several times with PBS, the cell number was counted, and then the
suspension was cryopreserved with 90% human serum (ICN) and 10%
DMSO. After thawing, the cells were re-suspended in a standard RPMI
medium supplemented with 10% human serum and 2.5 .mu.g/ml
amphotericin (fangizon, Gibco/BRL), and then used.
[0157] 1.times.10.sup.5 cells were added to a 96-well plate, and
then anti-human CD40 antibodies were added at concentrations of
0.01, 0.1, 1.0 and 10 .mu.g/ml. The test was conducted in
triplicate at each concentration. 1 .mu.g/ml flag-labeled CD40L
(Alexis) and 1 .mu.g/ml CD40L enhancer antibodies (Alexis) were
added to each well, followed by 3 days of culturing. Then, 1 .mu.Ci
[.sup.3H] thymidine was added to each well. 12 to 15 hours later,
the cells were collected, and then the proliferation of tonsillar B
cells was measured using a liquid scintillation counter. The count
obtained when B cells had proliferated due to the stimulation of
CD40L and no antibody had been added was determined as 100, and the
count obtained when no CD40L had been added and B cells had not
been not stimulated was determined as 0. For example, when the
relative count measured was 30, this was expressed in this
experiment as that 70% proliferation inhibition had occurred.
[0158] 5D12, which is the known antagonistic antibody, did not show
more than 50% proliferation inhibition even with the antibody
concentration of 100 .mu.g/ml. F4-465 showed approximately 80%
proliferation suppression even with the antibody concentration as
low as 0.01 .mu.g/ml, and showed approximately 95% proliferation
suppression with an antibody concentration of 0.1 to 10 .mu.g/ml
(FIG. 20).
[0159] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
[0160] The present invention provides an antibody against CD40. The
antibody of the present invention includes both an antibody that
acts agonistically on CD40 and an antibody that acts
antagonistically on CD40. Thus, these antibodies are useful as, for
example, an immunopotentiating agent and immunosuppressive agent,
respectively.
[0161] Sequence Listing Free Text
SEQ ID NO: 1: Synthetic DNA
SEQ ID NO: 2: Synthetic DNA
SEQ ID NO: 3: Synthetic DNA
SEQ ID NO: 4: Synthetic DNA
SEQ ID NO: 5: Synthetic DNA
SEQ ID NO: 6: Synthetic DNA
SEQ ID NO: 7: Synthetic DNA
SEQ ID NO: 8: Synthetic DNA
SEQ ID NO: 9: Synthetic DNA
SEQ ID NO: 10: Synthetic DNA
SEQ ID NO: 11: Synthetic DNA
SEQ ID NO: 12: Synthetic DNA
SEQ ID NO: 13: Synthetic DNA
SEQ ID NO: 14: Synthetic DNA
SEQ ID NO: 15: Synthetic DNA
SEQ ID NO: 16: Synthetic DNA
SEQ ID NO: 17: Synthetic DNA
SEQ ID NO: 18: Synthetic DNA
SEQ ID NO: 19: Synthetic DNA
SEQ ID NO: 20: Synthetic DNA
SEQ ID NO: 21: Synthetic DNA
SEQ ID NO: 22: Synthetic DNA
SEQ ID NO: 23: Synthetic DNA
SEQ ID NO: 24: Synthetic DNA
SEQ ID NO: 25: Synthetic DNA
[0162] SEQ ID NO: 26: Synthetic DNA
Sequence CWU 1
1
66140DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 1cccagatctg tccatccaga accacccact gcatgcagag
40241DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 2acaagatctg ggctctacgt atctcagccg atcctgggga
c 41327DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 3ggtccgggag atcatgaggg tgtcctt
27426DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 4gtgcacgccg ctggtcaggg cgcctg
26525DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 5gctggagggc acggtcacca cgctg
25623DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 6ggtgccaggg ggaagaccga tgg 23735DNAArtificial
SequenceDescription of Artificial SequenceSynthetic DNA 7atatgtcgac
gctgaattct ggctgaccag ggcag 35837DNAArtificial SequenceDescription
of Artificial SequenceSynthetic DNA 8atatgtcgac tcccaggtgt
ttccattcag tgatcag 37937DNAArtificial SequenceDescription of
Artificial SequenceSynthetic DNA 9atatgtcgac ttccattcgg tgatcagcac
tgaacac 371034DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 10atatgtcgac tttgagagtc ctggacctcc tgtg
341133DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 11atatgtcgac gagtcatgga tctcatgtgc aag
331236DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 12atatgtcgac ccagggcagt caccagagct ccagac
361326DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 13accgtgtcga ctacgcggga gtgact
261429DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 14accgtgtcga cgctgatcag gactgcaca
291530DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 15accgtgtcga cggtgatcag gactgaacag
301626DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 16gttgaagctc tttgtgacgg gcgagc
261724DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 17tcttctccac ggtgctccct tcat
241835DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 18atatagatct gaactgctca gttaggaccc agagg
351934DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 19atatagatct cgcggggaag gagactgctc agtt
342034DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 20atatagatct agtcagaccc agtcaggaca cagc
342136DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 21atatagatct gagctgctca gttaggaccc agaggg
362236DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 22atatagatct taagcaagtg taacaactca gagtac
362338DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 23atatagatct gaggaactgc tcagttagga cccagagg
382430DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 24aactccagat ctgcctcagg aagcagcatc
302530DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 25aactccagat ctagggcaag cagtggtaac
302626DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 26tggcgggaag atgaagacag atggtg
26271480DNAHomo sapiens 27gtcgacgctg aattctggct gaccagggca
gccaccagag ctccagacaa tgtctgtctc 60cttcctcatc ttcctgcccg tgctgggcct
cccatggggt gtcctgtcac aggtccaact 120gcagcagtca ggtccaggac
tggtgaagcc ctcgcagacc ctctcactca cctgtgccat 180ctccggggac
agtgtctcta gcaacagtgc tacttggaac tggatcaggc agtccccatc
240gagagacctt gagtggctgg gaaggacata ctacaggtcc aagtggtatc
gtgattatgt 300aggatctgtg aaaagtcgaa taatcatcaa cccagacaca
tccaacaacc agttctccct 360gcagctgaac tctgtgactc ccgaggacac
ggctatatat tactgtacaa gagcacagtg 420gctgggaggg gattacccct
actactacag tatggacgtc tggggccaag ggaccacggt 480caccgtctct
tcagcctcca ccaagggccc atcggtcttc cccctggcgc cctgctccag
540gagcacctcc gagagcacag cggccctggg ctgcctggtc aaggactact
tccccgaacc 600ggtgacggtg tcgtggaact caggcgctct gaccagcggc
gtgcacacct tcccagctgt 660cctacagtcc tcaggactct actccctcag
cagcgtggtg accgtgccct ccagcaactt 720cggcacccag acctacacct
gcaacgtaga tcacaagccc agcaacacca aggtggacaa 780gacagttgag
cgcaaatgtt gtgtcgagtg cccaccgtgc ccagcaccac ctgtggcagg
840accgtcagtc ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc 900tgaggtcacg tgcgtggtgg tggacgtgag ccacgaagac
cccgaggtcc agttcaactg 960gtacgtggac ggcgtggagg tgcataatgc
caagacaaag ccacgggagg agcagttcaa 1020cagcacgttc cgtgtggtca
gcgtcctcac cgttgtgcac caggactggc tgaacggcaa 1080ggagtacaag
tgcaaggtct ccaacaaagg cctcccagcc cccatcgaga aaaccatctc
1140caaaaccaaa gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga 1200gatgaccaag aaccaggtca gcctgacctg cctggtcaaa
ggcttctacc ccagcgacat 1260cgccgtggag tgggagagca atgggcagcc
ggagaacaac tacaagacca cacctcccat 1320gctggactca gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg 1380gcagcagggg
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
1440gcagaagagc ctctccctgt ctccgggtaa atgaggatcc 148028474PRTHomo
sapiens 28Met Ser Val Ser Phe Leu Ile Phe Leu Pro Val Leu Gly Leu
Pro Trp1 5 10 15Gly Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro
Gly Leu Val20 25 30Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser35 40 45Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile
Arg Gln Ser Pro Ser50 55 60Arg Asp Leu Glu Trp Leu Gly Arg Thr Tyr
Tyr Arg Ser Lys Trp Tyr65 70 75 80Arg Asp Tyr Val Gly Ser Val Lys
Ser Arg Ile Ile Ile Asn Pro Asp85 90 95Thr Ser Asn Asn Gln Phe Ser
Leu Gln Leu Asn Ser Val Thr Pro Glu100 105 110Asp Thr Ala Ile Tyr
Tyr Cys Thr Arg Ala Gln Trp Leu Gly Gly Asp115 120 125Tyr Pro Tyr
Tyr Tyr Ser Met Asp Val Trp Gly Gln Gly Thr Thr Val130 135 140Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala145 150
155 160Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu165 170 175Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly180 185 190Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser195 200 205Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe210 215 220Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr225 230 235 240Lys Val Asp Lys
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro245 250 255Cys Pro
Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro260 265
270Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys275 280 285Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp290 295 300Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu305 310 315 320Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val325 330 335His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn340 345 350Lys Gly Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly355 360 365Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu370 375
380Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr385 390 395 400Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn405 410 415Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe420 425 430Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn435 440 445Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr450 455 460Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys465 47029406DNAHomo sapiens 29actgctcagt
taggacccag agggaaccat ggaagcccca gctcagcttc tcttcctcct 60gctactctgg
ctcccagata ccaccggaga aattgtgttg acacagtctc cagccaccct
120gtctttgtct ccaggggaaa gagccaccct ctcctgcagg gccagtcaga
gtgttagcag 180ctacttagcc tggtaccaac agaaacctgg ccaggctccc
aggctcctca tctatgatgc 240atccaacagg gccactggca tcccagccag
gttcagtggc agtgggtctg ggacagactt 300cactctcacc atcagcagcc
tagagcctga agattttgca gtttattact gtcagcagcg 360tagcaacact
ttcggccctg ggaccaaagt ggatatcaaa cgtacg 40630126PRTHomo sapiens
30Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1
5 10 15Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser20 25 30Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser35 40 45Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro50 55 60Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser85 90 95Ser Leu Glu Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser100 105 110Asn Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Lys Arg Thr115 120 12531508DNAHomo sapiens
31ctgaacacag acccgtcgac tcccaggtgt ttccattcag tgatcagcac tgaacacaga
60ggactcacca tggagttggg actgagctgg attttccttt tggctatttt aaaaggtgtc
120cagtgtgaag tgcagctggt ggagtctggg ggaggcttgg tacagcctgg
caggtccctg 180agactctcct gtgcagcctc tggattcacc tttgatgatt
atgccatgca ctgggtccgg 240caagctccag ggaagggcct ggagtgggtc
tcaggtatta gttggaatag tggtagcttg 300gtgcatgcgg actctgtgaa
gggccgattc accatctcca gagacaacgc caagaactcc 360ctgtatctgc
aaatgaacag tctgagagct gaggacacgg ccttgtatta ctgtgcaaga
420gataggctat ttcggggagt taggtactac ggtatggacg tctggggcca
agggaccacg 480gtcaccgtct cctcagctag caccaagg 50832146PRTHomo
sapiens 32Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu
Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe35 40 45Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu50 55 60Glu Trp Val Ser Gly Ile Ser Trp Asn Ser
Gly Ser Leu Val His Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn85 90 95Ser Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Leu100 105 110Tyr Tyr Cys Ala Arg
Asp Arg Leu Phe Arg Gly Val Arg Tyr Tyr Gly115 120 125Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser130 135 140Thr
Lys14533414DNAHomo sapiens 33ctgctcagtt aggacccaga gggaaccatg
gaagccccag ctcagcttct cttcctcctg 60ctactctggc tcccagatac caccggagaa
attgtgttga cacagtctcc agccaccctg 120tctttgtctc caggggaaag
agccaccctc tcctgcaggg ccagtcagag tgttagcagc 180tacttagcct
ggtaccaaca gaaacctggc caggctccca ggctcctcat ctatgatgca
240tccaacaggg ccactggcat cccagccagg ttcagtggca gtgggtctgg
gacagacttc 300actctcacca tcagcagcct agagcctgaa gattttgcag
tttattactg tcagcagcgt 360agccactggc tcactttcgg cggggggacc
aaggtggaga tcaaacgtac ggtg 41434129PRTHomo sapiens 34Met Glu Ala
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr
Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser20 25 30Leu
Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser35 40
45Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro50
55 60Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser85 90 95Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser100 105 110His Trp Leu Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg Thr115 120 125Val35493DNAHomo sapiens
35ctgaacacag acccgtcgac ttccattcgg tgatcagcac tgaacacaga ggactcacca
60tggagtttgg gctgagctgg gttttcctcg ttgctctttt aagaggtgtc cagtgtcagg
120tgcagctggt ggagtctggg ggaggcgtgg tccagcctgg gaggtccctg
agactctcct 180gtgcagcgtc tggattcacc ttcagtagct atggcatgca
ctgggtccgc caggctccag 240gcaaggggct ggagtgggtg gcagttatat
ggtatgatgg aagtattaaa tactatgcag 300actccgtgaa gggccgattc
accatctcca gagacaattc caagaacacg ctgtatctgc 360aaatgaacag
cctgagagcc gaggacacgg ctgtgtatta ctgtgcgaga gagggctaca
420atattttgac tggttatttt ggctactggg gccagggaac cctggtcacc
gtctcctcag 480ctagcaccaa ggg 49336144PRTHomo sapiens 36Met Glu Phe
Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln
Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln20 25 30Pro
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe35 40
45Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu50
55 60Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Ile Lys Tyr Tyr
Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn85 90 95Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val100 105 110Tyr Tyr Cys Ala Arg Glu Gly Tyr Asn Ile
Leu Thr Gly Tyr Phe Gly115 120 125Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys130 135 14037427DNAHomo sapiens
37tcacagatct agtcagaccc agtcaggaca cagcatggac atgagggtcc ccgctcagct
60cctggggctc ctgctgctct ggctcccagg tgccagatgt gtcatctgga tgacccagtc
120tccatcctta ctctctgcat ctacaggaga cagagtcacc atcagttgtc
ggatgagtca 180gggcattagc agtgatttag cctggtatca gcaaaaacca
gggaaagccc ctgagctcct 240gatctctgct gcatccactt tgcaaagtgg
ggtcccatca aggttcagtg gcagtggatc 300tgggacagat ttcactctca
ccatcagctg cctgcagtct gaagattttg caacttatta 360ctgtcaacag
tattatagtt ttccgtggac gttcggccaa gggaccaagg tggaaatcaa 420acgtacg
42738131PRTHomo sapiens 38Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Val Ile Trp
Met Thr Gln Ser Pro Ser Leu20 25 30Leu Ser Ala Ser Thr Gly Asp Arg
Val Thr Ile Ser Cys Arg Met Ser35 40 45Gln Gly Ile Ser Ser Asp Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys50 55 60Ala Pro Glu Leu Leu Ile
Ser Ala Ala Ser Thr Leu Gln Ser Gly Val65 70 75 80Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr85 90 95Ile Ser Cys
Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln100 105 110Tyr
Tyr Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile115 120
125Lys Arg Thr13039492DNAHomo sapiens 39ctgaacacag acccgtcgac
ttccattcgg tgatcagcac tgaacacaga ggactcacca 60tggagtttgg gctgagctgg
gttttcctcg ttgctctttt aagaggtgtc cagtgtcagg 120tgcagctggt
ggagtctggg ggaggcgtgg tccagcctgg gaggtccctg agactctcct
180gtgcagcgtc tggattcacc ttcagtagct atggcatgca ctgggtccgc
caggctccag 240gcaaggggct ggagtgggtg gcagttatat ggaatgatgg
aagtattaaa tactatgcag 300actccgtgaa gggccgattc accatctcca
gagacaattc caagaacacg ctgtatctgc 360aaatgaacag cctgagagcc
gaggacacgg ctgtgtatta ctgtgcgaga gagggctaca 420atattttgac
tggttatttt ggctactggg gccagggaac cctggtcacc gtctcctcag
480ctagcaccaa gg 49240144PRTHomo sapiens 40Met Glu Phe Gly Leu Ser
Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln20 25 30Pro Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe35 40 45Ser Ser Tyr
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp
Val Ala Val Ile Trp
Asn Asp Gly Ser Ile Lys Tyr Tyr Ala65 70 75 80Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn85 90 95Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val100 105 110Tyr Tyr
Cys Ala Arg Glu Gly Tyr Asn Ile Leu Thr Gly Tyr Phe Gly115 120
125Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys130 135 14041427DNAHomo sapiens 41tcacagatct agtcagaccc
agtcaggaca cagcatggac atgagggtcc ccgctcagct 60cctggggctc ctgctgctct
ggctcccagg tgccagatgt gtcatctgga tgacccagtc 120tccatcctta
ctctctgcat ctacaggaga cagagtcacc atcagttgtc ggatgagtca
180gggcattagc agtgatttag cctggtatca gcaaaaacca gggaaagccc
ctgagctcct 240gatctctgct gcatccactt tgcaaagtgg ggtcccatca
aggttcagtg gcagtggatc 300tgggacagat ttcactctca ccatcagctg
cctgcagtct gaagattttg caacttatta 360ctgtcaacag tattatagtt
ttccgtggac gttcggccaa gggaccaagg tggaaatcaa 420acgtacg
42742131PRTHomo sapiens 42Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Val Ile Trp
Met Thr Gln Ser Pro Ser Leu20 25 30Leu Ser Ala Ser Thr Gly Asp Arg
Val Thr Ile Ser Cys Arg Met Ser35 40 45Gln Gly Ile Ser Ser Asp Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys50 55 60Ala Pro Glu Leu Leu Ile
Ser Ala Ala Ser Thr Leu Gln Ser Gly Val65 70 75 80Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr85 90 95Ile Ser Cys
Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln100 105 110Tyr
Tyr Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile115 120
125Lys Arg Thr13043481DNAHomo sapiens 43ctgaacacag acccgtcgac
tttgagagtc ctggacctcc tgtgcaagaa catgaaacat 60ctgtggttct tccttctcct
ggtggcagct cccagatggg tcctgtccca ggtgcagctg 120caggagtcgg
gcccaggact ggtgaagcct tcggagaccc tgtccctcac ctgcactgtc
180tctggtggct ccatcagtgg ttactactgg agctggatcc ggcagccccc
agggaaggga 240ctggagtgga ttgggtatat ctattacagt gggagcacca
actacaatcc ctccctcaag 300agtcgagtca ccatatcagt agacacgtcc
aagaaccagt tctccctgaa gctgaattct 360gtgaccgctg cggacacggc
cgtgtattac tgtgcgagag cccccttgca cggtgactac 420aaatggttcc
acccctgggg ccagggaacc ctggtcaccg tctcctcagc tagcaccaag 480g
48144143PRTHomo sapiens 44Met Lys His Leu Trp Phe Phe Leu Leu Leu
Val Ala Ala Pro Arg Trp1 5 10 15Val Leu Ser Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Gly Ser Ile35 40 45Ser Gly Tyr Tyr Trp Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile
Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro65 70 75 80Ser Leu Lys Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln85 90 95Phe Ser Leu
Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr100 105 110Tyr
Cys Ala Arg Ala Pro Leu His Gly Asp Tyr Lys Trp Phe His Pro115 120
125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys130
135 14045430DNAHomo sapiens 45tcacagatct gagctgctca gttaggaccc
agagggaacc atggaaaccc cagcgcagct 60tctcttcctc ctgctactct ggctcccaga
taccaccgga gaaattgtgt tgacgcagtc 120tccaggcacc ctgtctttgt
ctccagggga aagagccacc ctctcctgca gggccagtca 180gagtgttagc
agcagctact tagcctggta ccagcagaaa cctggccagg ctcccaggct
240cctcatctat ggtgcatcca gcagggccac tggcatccca gacaggttca
gtggcagtgg 300gtctgggaca gacttcactc tcaccatcag cagactggag
cctgaagatt ttgcagtgta 360ttactgtcag cagtatggta gctcaccgat
caccttcggc caagggacac gactggagat 420caaacgtacg 43046130PRTHomo
sapiens 46Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp
Leu Pro1 5 10 15Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly
Thr Leu Ser20 25 30Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser35 40 45Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala50 55 60Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Ile Pro65 70 75 80Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile85 90 95Ser Arg Leu Glu Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Tyr100 105 110Gly Ser Ser Pro Ile
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys115 120 125Arg
Thr13047462DNAHomo sapiens 47atatgtcgac gagtcatgga tctcatgtgc
aagaaaatga agcacctgtg gttcttcctc 60ctgctggtgg cggctcccag atgggtcctg
tcccagctgc agctgcagga gtcgggccca 120ggactactga agccttcgga
gaccctgtcc ctcacctgca ctgtctctgg cggctccatc 180agcagtcctg
gttactacgg gggctggatc cgccagcccc cagggaaggg gctggagtgg
240attgggagta tctataaaag tgggagcacc taccacaacc cgtccctcaa
gagtcgagtc 300accatatccg tagacacgtc caagaaccag ttctccctga
agctgagctc tgtgaccgcc 360gcagacacgg ctgtgtatta ctgtacgaga
cctgtagtac gatattttgg gtggttcgac 420ccctggggcc agggaaccct
ggtcaccgtc tcctcagcta gc 46248149PRTHomo sapiens 48Met Asp Leu Met
Cys Lys Lys Met Lys His Leu Trp Phe Phe Leu Leu1 5 10 15Leu Val Ala
Ala Pro Arg Trp Val Leu Ser Gln Leu Gln Leu Gln Glu20 25 30Ser Gly
Pro Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys35 40 45Thr
Val Ser Gly Gly Ser Ile Ser Ser Pro Gly Tyr Tyr Gly Gly Trp50 55
60Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr65
70 75 80Lys Ser Gly Ser Thr Tyr His Asn Pro Ser Leu Lys Ser Arg Val
Thr85 90 95Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu
Ser Ser100 105 110Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr
Arg Pro Val Val115 120 125Arg Tyr Phe Gly Trp Phe Asp Pro Trp Gly
Gln Gly Thr Leu Val Thr130 135 140Val Ser Ser Ala
Ser14549448DNAHomo sapiens 49agatcttaag caagtgtaac aactcagagt
acgcggggag acccactcag gacacagcat 60ggacatgagg gtccccgctc agctcctggg
gcttctgctg ctctggctcc caggtgccag 120atgtgccatc cagttgaccc
agtctccatc ctccctgtct gcatctgtag gagacagagt 180caccatcact
tgccgggcaa gtcagggcat tagcagtgct ttagcctggt atcagcagaa
240accagggaaa gctcctaagc tcctgatcta tgatgcctcc aatttggaaa
gtggggtccc 300atcaaggttc agcggcagtg gatctgggac agatttcact
ctcaccatca gcagcctgca 360gcctgaagat tttgcaactt attactgtca
acagtttaat agttacccga cgttcggcca 420agggaccaag gtggaaatca aacgtacg
44850130PRTHomo sapiens 50Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser20 25 30Leu Ser Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser35 40 45Gln Gly Ile Ser Ser Ala Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys50 55 60Ala Pro Lys Leu Leu Ile
Tyr Asp Ala Ser Asn Leu Glu Ser Gly Val65 70 75 80Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr85 90 95Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln100 105 110Phe
Asn Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys115 120
125Arg Thr13051453DNAHomo sapiens 51atgtctgtct ccttcctcat
cttcctgccc gtgctgggcc tcccatgggg tgtcctgtca 60caggtacagc tgcagcagtc
aggtccagga ctggtgaagc cctcgcagac cctctcattc 120acctgtgcca
tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg
180cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 240aaagattatg cagtatctgt gaaaagtcga ataaccatca
acccagacac atccaagaac 300cagttctccc tgcagctgaa ctctgtgacc
cccgaggaca cggctgtgta ttactgtgca 360agagggtatt actatggttc
ggggagctat ccctactact accaaatgga cgtctggggc 420caagggacca
cggtcaccgt ctcctcagct agc 45352151PRTHomo sapiens 52Met Ser Val Ser
Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp1 5 10 15Gly Val Leu
Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val20 25 30Lys Pro
Ser Gln Thr Leu Ser Phe Thr Cys Ala Ile Ser Gly Asp Ser35 40 45Val
Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser50 55
60Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr65
70 75 80Lys Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro
Asp85 90 95Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu100 105 110Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Tyr
Tyr Gly Ser Gly115 120 125Ser Tyr Pro Tyr Tyr Tyr Gln Met Asp Val
Trp Gly Gln Gly Thr Thr130 135 140Val Thr Val Ser Ser Ala Ser145
15053414DNAHomo sapiens 53aattgaggaa ctgctcagtt aggacccaga
gggaaccatg gaagccccag ctcagcttct 60cttcctcctg ctactctggc tcccagatac
caccggagaa attgtgttga cacagtctcc 120agccaccctg tctttgtctc
caggggaaag tgccaccctc tcctgcaggg ccagtcagag 180tgttagcagc
tacttagcct ggtaccaaca gaaacctggc caggctccca ggctcctcat
240ctatgatgca tccaacaggg ccactggcat cccagccagg ttcagtggca
gtgggtctgg 300gacagacttc actctcacca tcagcagcct agagcctgaa
gattttgcag tttattactg 360tcagcagcgt agcaacactt tcggcggagg
gaccaaggtg gagatcaaac gaac 41454125PRTHomo sapiens 54Met Glu Ala
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr
Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser20 25 30Leu
Ser Pro Gly Glu Ser Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser35 40
45Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro50
55 60Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser85 90 95Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser100 105 110Asn Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg115 120 12555495DNAHomo sapiens 55ctgaacacag acccgtcgac
tacgcgggag accacagctc cacaccatgg actggacctg 60gaggatccta ttcttggtgg
cagcagcaac aggtgcccac tcccaggtgc agctggtgca 120atctgggtct
gagttgaaga agcctggggc ctcagtgaag gtcccctgca aggcttctgg
180atacaccttc actagctatg ctatgaattg ggtgcgacag gcccctggac
aagggcttga 240gtggatggga tggatcaaca ccaacactgg gaacccaacg
tatgcccagg gcttcacagg 300acggtttgtc ttctccttgg acacctctgt
cagcacggca tatctgcaga tcagcagcct 360aaaggctgag gacactgccg
tgtattactg tgcgagagag gtagtaccag ttgctatgag 420ggtaactcac
tactactacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc
480ctcagctagc accaa 49556149PRTHomo sapiens 56Met Asp Trp Thr Trp
Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1 5 10 15Ala His Ser Gln
Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys20 25 30Pro Gly Ala
Ser Val Lys Val Pro Cys Lys Ala Ser Gly Tyr Thr Phe35 40 45Thr Ser
Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu50 55 60Glu
Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala65 70 75
80Gln Gly Phe Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser85
90 95Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala
Val100 105 110Tyr Tyr Cys Ala Arg Glu Val Val Pro Val Ala Met Arg
Val Thr His115 120 125Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly
Thr Thr Val Thr Val130 135 140Ser Ser Ala Ser Thr14557830DNAHomo
sapiens 57ctgggtacgg taaccgtcag atcgcctgga gacgccatca cagatctgcc
tcaggaagca 60gcatcggagg tgcctcagcc atggcatgga tccctctctt cctcggcgtc
cttgtttact 120gcacaggatc cgtggcctcc tatgagctga ctcagccacc
ctcagtgtcc gtggccccag 180gacagacagc cagcatcacc tgttctggag
ataaattggg ggataatttt acttgctggt 240atcagcagaa gccaggccag
tcccctgtgc tggtcatctt tcaggattgg aagcggcgcc 300cagggatccc
tgcgcgattc tctggctcca agtctgggaa cacagccact ctgaccatca
360gcgggaccca ggctatggat gaggctgact attactgtca ggcgtgggac
atcagcactg 420tggtattcgg cggagggacc aagctgaccg tcctaggtca
gcccaaggct gccccctcgg 480tcactctgtt cccgccctcc tctgaggagc
ttcaagccaa caaggccaca ctggtgtgtc 540tcataagtga cttctacccg
ggagccgtga cagtggcctg gaaggcagat agcagccccg 600tcaaggcggg
agtggagacc accacaccct ccaaacaaag caacaacaag tacgcggcca
660gcagctacct gagcctgacg cctgagcagt ggaagtccca cagaagctac
agctgccagg 720tcacgcatga agggagcacc gtggagaaga cagtggcccc
tacagaatgt tcatgaattc 780agatccgtta acggttacca actacctaga
ctggattcgt gaccaacata 83058231PRTHomo sapiens 58Met Ala Trp Ile Pro
Leu Phe Leu Gly Val Leu Val Tyr Cys Thr Gly1 5 10 15Ser Val Ala Ser
Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala20 25 30Pro Gly Gln
Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp35 40 45Asn Phe
Thr Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu50 55 60Val
Ile Phe Gln Asp Trp Lys Arg Arg Pro Gly Ile Pro Ala Arg Phe65 70 75
80Ser Gly Ser Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr85
90 95Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ile
Ser100 105 110Thr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln Pro115 120 125Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser Glu Glu Leu130 135 140Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe Tyr Pro145 150 155 160Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro Val Lys Ala165 170 175Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala180 185 190Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg195 200
205Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
Thr210 215 220Val Ala Pro Thr Glu Cys Ser225 23059520DNAHomo
sapiens 59gctgatcagg actgcacaca gagaactcac catggagttt gggctgagct
gggttttcct 60tgttgctatt ttaaaaggtg tccagtgtga ggtgcagctg gtggagtccg
ggggaggctt 120agttcagcct ggggggtccc tgagactctc ctgtgcagtc
tctggattca ccttcagtac 180ctactggatg cactgggtcc gccaagctcc
agggaagggg ctggtgtggg tctcacgtat 240taatagtgat gggagtagca
caacctacgc ggactccgtg aagggccgat tcaccatctc 300cagagacaac
gccaagaaca cgctgtatct gcaaatgaac agtctgagag ccgaggacac
360ggctgtgtat tactgtgcaa gagatagagt actatggatc ggggagttat
cctactacgg 420tatggacgtc tggggccaag ggaccacggt caccgtctcc
tcagctagca ccaagggccc 480atcggtcttc cccctggcac cctcctccaa
gagcacctct 52060163PRTHomo sapiens 60Met Glu Phe Gly Leu Ser Trp
Val Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln20 25 30Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe35 40 45Ser Thr Tyr Trp
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Val Trp Val
Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Thr Tyr Ala65 70 75 80Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn85 90
95Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val100 105 110Tyr Tyr Cys Ala Arg Asp Arg Val Leu Trp Ile Gly Glu
Leu Ser Tyr115 120 125Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser130 135 140Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys145 150 155 160Ser Thr Ser61698DNAHomo
sapiens 61ggggagtcag acccagtcag gacacagcat ggacatgagg gtccccgctc
agctcctggg 60gctcctgctg ctctggctcc caggtgccaa atgtgacatc cagatgaccc
agtctccttc 120caccctgtct gcatctgtag gagacagagt caccatcact
tgccgggcca gtcagagtat 180tagtaactgg ttggcctggt atcagcagaa
accagggaaa gcccctaagc tcctgctcta 240taaggcatct ggtttagaaa
gtggggtccc atcaaggttc agcggcagtg gatctgggac 300agaattcact
ctcaccatca acagcctgca gcctgatgat tttgcaactt attactgcca
360acagtctaat agttattcgt ggacgttcgg ccacgggacc aaggtggaaa
tcaaacgtac 420ggtggctgca ccatctgtct tcatcttccc gccatctgat
gagcagttga aatctggaac 480tgcctctgtt gtgtgcctgc tgaataactt
ctatcccaga gaggccaaag tacagtggaa 540ggtggataac gccctccaat
cgggtaactc ccaggagagt gtcacagagc aggacagcaa 600ggacagcacc
tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca
660caaagtctac gcctgcgaag tcacccatca gggcctga 69862223PRTHomo
sapiens 62Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu
Leu Trp1 5 10 15Leu Pro Gly Ala Lys Cys Asp Ile Gln Met Thr Gln Ser
Pro Ser Thr20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser35 40 45Gln Ser Ile Ser Asn Trp Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys50 55 60Ala Pro Lys Leu Leu Leu Tyr Lys Ala Ser
Gly Leu Glu Ser Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr85 90 95Ile Asn Ser Leu Gln Pro Asp
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln100 105 110Ser Asn Ser Tyr Ser
Trp Thr Phe Gly His Gly Thr Lys Val Glu Ile115 120 125Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp130 135 140Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150
155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu165 170 175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu210 215 22063630DNAHomo sapiens 63ggtctatata
agcagagctg ggtacgtcct cacattcagt gatcagcact gaacacagac 60ccgtcgacgg
tgatcaggac tgaacagaga gaactcacca tggagtttgg gctgagctgg
120ctttttcttg tggctatttt aaaaggtgtc cagtgtgagg tgcagctgtt
ggagtctggg 180ggaggcttgg tacagcctgg ggggtccctg agactctcct
gtgcagcctc tggattcacc 240tttagcagct atgccatgag ctgggtccgc
caggctccag ggaaggggct ggagtgggtc 300tcagctatta gtggtagtgg
tggtagcaca tactacgcag actccgtgaa gggccggttc 360accatctcca
gagacaattc caagaacacg ctgtatctgc aaatgaacag cctgagagcc
420gaggacacgg ccgtatatta ctgtgcgaaa gatggggggt actatggttc
ggggagttat 480gggtactttg actactgggg ccagggaacc ctggtcaccg
tctcctcagc tagcaccaag 540ggcccatcgg tcttccccct ggcaccctcc
tccaagagca cctctggggg cacagcggcc 600ctgggctgcc tggtcaagga
ctacttcccc 63064177PRTHomo sapiens 64Met Glu Phe Gly Leu Ser Trp
Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln20 25 30Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe35 40 45Ser Ser Tyr Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp Val
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala65 70 75 80Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn85 90
95Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val100 105 110Tyr Tyr Cys Ala Lys Asp Gly Gly Tyr Tyr Gly Ser Gly
Ser Tyr Gly115 120 125Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala130 135 140Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser145 150 155 160Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe165 170 175Pro65728DNAHomo
sapiens 65caagcagtgg taacaacgca gagtacgcgg ggggagtcag acccagtcag
gacacagcat 60ggacatgagg gtccccgctc agctcctggg gctcctgctg ctctggttcc
caggttccag 120atgcgacatc cagatgaccc agtctccatc ttccgtgtct
ggatctgtag gagacagagt 180caccatcact tgtcgggcga gtcagggtat
tagcagctgg ttagcctggt atcagcagaa 240accagggaaa gcccctaagc
tcctgatcta tgctggatcc agtttgcaaa gtggggtccc 300atcaaggttc
agcggcagtg gatttgggac agatttcact ctcaccatca gcagcctgca
360gcctgaagat tttgcaactt actattgtca acaggctagc agtttccctc
ggacattcgg 420ccaagggacc aaggtggaga tcaaacgtac ggtggctgca
ccatctgtct tcatcttccc 480gccatctgat gagcagttga aatctggaac
tgcctctgtt gtgtgcctgc tgaataactt 540ctatcccaga gaggccaaag
tacagtggaa ggtggataac gccctccaat cgggtaactc 600ccaggagagt
gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct
660gacgctgagc aaagcagact acgagaaaca caaagtctac gcctgcgaag
tcacccatca 720gggcctga 72866223PRTHomo sapiens 66Met Asp Met Arg
Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly
Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser20 25 30Val Ser
Gly Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser35 40 45Gln
Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys50 55
60Ala Pro Lys Leu Leu Ile Tyr Ala Gly Ser Ser Leu Gln Ser Gly Val65
70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu
Thr85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln100 105 110Ala Ser Ser Phe Pro Arg Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile115 120 125Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp130 135 140Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu165 170 175Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp180 185 190Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr195 200
205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu210
215 220
* * * * *