U.S. patent application number 10/485466 was filed with the patent office on 2004-12-02 for substance specific to pd-1.
Invention is credited to Honjo, Tasuku, Matsuo, Masayoshi, Shibayama, Shiro, Yoshida, Takao.
Application Number | 20040241745 10/485466 |
Document ID | / |
Family ID | 19064242 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241745 |
Kind Code |
A1 |
Honjo, Tasuku ; et
al. |
December 2, 2004 |
Substance specific to pd-1
Abstract
Substances comprising a substance that recognizes PD-1, a
substance that recognizes a membrane protein co-existing with PD-1
on a cell membrane, and a linker. As the substances that
specifically recognizes PD-1 by the present invention can recognize
selectively both PD-1 and a membrane protein co-existing with PD-1
on a cell membrane, and can transmit the suppressive signal of
PD-1, they are useful for the medical treatment and/or prevention
of the diseases caused by immune abnormality.
Inventors: |
Honjo, Tasuku; (Kyoto-shi,
JP) ; Shibayama, Shiro; (Mishima-gun Osaka, JP)
; Matsuo, Masayoshi; (Mishima-gun Osaka, JP) ;
Yoshida, Takao; (Mishima-gun, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Family ID: |
19064242 |
Appl. No.: |
10/485466 |
Filed: |
February 2, 2004 |
PCT Filed: |
July 30, 2002 |
PCT NO: |
PCT/JP02/07735 |
Current U.S.
Class: |
435/7.1 ;
530/388.26 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 21/00 20180101; A61P 35/00 20180101; C07K 2317/626 20130101;
A61P 25/14 20180101; A61P 9/00 20180101; A61P 37/02 20180101; C07K
2319/00 20130101; A61P 31/02 20180101; A61P 37/06 20180101; C07K
16/2809 20130101; A61P 17/00 20180101; C07K 16/18 20130101; A61P
19/02 20180101; A61K 2039/505 20130101; A61P 13/12 20180101; A61P
1/04 20180101; A61P 25/16 20180101; A61P 17/06 20180101; A61P 25/28
20180101; A61P 29/00 20180101; A61P 3/10 20180101; A61P 25/02
20180101; A61P 37/08 20180101; C07K 16/2803 20130101; C07K 2317/55
20130101; A61P 1/00 20180101; A61P 37/00 20180101 |
Class at
Publication: |
435/007.1 ;
530/388.26 |
International
Class: |
G01N 033/53; C07K
016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2001 |
JP |
2001-232303 |
Claims
1. A substance comprising a substance that recognizes PD-1, a
substance that recognizes a membrane protein co-existing with PD-1
on a cell membrane, and a linker.
2. The substance according to claim 1, which is a divalent
substance comprising a substance that recognizes PD-1, a substance
that recognizes a membrane protein co-existing with PD-1 on a cell
membrane, and a linker.
3. The substance according to claim 1, in which a membrane protein
is a protein existing on a T cell membrane or B cell membrane.
4. The substance according to claim 1, which comprises a substance
that recognizes PD-1, a substance that recognizes a protein
constituting T cell receptor complex or a substance that recognizes
a protein constituting B cell receptor complex, and a linker.
5. The substance according to claim 1, in which a substance that
recognizes PD-1 and in which a substance that recognizes a protein
is dimer to pentamer, respectively.
6. The substance according to claim 1, in which one of or both
substance that recognizes PD-1 and substance that recognizes a
protein are an antibody.
7. The substance according to claim 1, in which one of the two or
both substance that recognizes PD-1 and substance that recognizes a
protein are a Fab portion of antibody.
8. The substance according to claim 1, in which a linker comprises
an organic compound.
9. The substance according to claim 1, in which a linker comprises
a peptide.
10. The substance according to claim 1, in which a substance that
recognizes PD-1 and in which a substance that recognizes a protein
is a peptide, respectively.
11. The substance according to claim 1, in which a substance that
recognizes PD-1 and in which a substance that recognizes a protein
comprises two or more peptides including a heavy chain variable
region and a light chain variable region of antibody,
respectively.
12. The substance according to claim 1, in which a substance that
recognizes PD-1, in which a substance that recognizes a protein,
and in which a linker is a peptide, respectively.
13. A pharmaceutical composition containing an effective dose of
the substance according to claim 1 for the medical treatment and/or
prevention of a disease in which PD-1 participates.
14. The pharmaceutical composition according to claim 13, in which
disease is selected from the group consisting of neurodegenerative
disease, autoimmune disease, organ transplant rejection, neoplasm
and infection.
15. The pharmaceutical composition according to claim 14, in which
neulodegenerative disease is selected from the group consisting of
Parkinson's disease, parkinsonian syndrome, Huntington's disease,
Machado-Joseph disease, amyotrophic lateral sclerosis and
Creutzfeldt-Jakob disease.
16. The pharmaceutical composition according to claim 14, in which
autoimmune disease is selected from the group consisting of
glomerular nephritis, arthritis, dilated cardiomyopathy-like
disease, ulcerative colitis, Sjogren's syndrome, Crohn's disease,
systemic lupus erythematosus, chronic rheumatoid arthritis,
multiple sclerosis, Psoriasis, allergic contact dermatitis,
polymyositis, scleroderma, periarteritis nodosa, rheumatic fever,
vitiligo vulgaris, insulin-dependent diabetes mellitus, Behcet's
Syndrome and chronic thyroiditis.
17. A method for the medical treatment and/or prevention of a
disease in which PD-1 participates which comprises administering to
a subject in need thereof an effective amount of the substance
according to claim 1.
18. The method according to claim 17, in which disease is selected
from the group consisting of neurodegenerative disease, autoimmune
disease, organ transplant rejection, neoplasm and infection.
19. The method according to claim 18, in which neurodegenerative
disease is selected from the group consisting of Parkinson's
disease, parkinsonian syndrome, Huntington's disease,
Machado-Joseph disease, amyotrophic lateral sclerosis and
Creutzfeldt-Jakob disease.
20. The method according to claim 18, in which autoimmune disease
is selected from the group consisting of glomerular nephritis,
arthritis, dilated cardiomyopathy-like disease, ulcerative colitis,
Sjogren's syndrome, Crohn's disease, systemic lupus erythematosus,
chronic rheumatoid arthritis, multiple sclerosis, Psoriasis,
allergic contact dermatitis, polymyositis, scleroderma,
periarteritis nodosa, rheumatic fever, vitiligo vulgaris,
insulin-dependent diabetes mellitus, Behcet's Syndrome and chronic
thyroiditis.
Description
TECHNICAL FIELD
[0001] The invention is related to a substance comprising a
substance that recognizes PD-1, a substance that recognizes a
membrane protein co-existing with PD-1 on a cell membrane, and a
linker.
BACKGROUND
[0002] The immune system acquired the mechanism that can respond to
various foreign antigens. The mechanism brings about the diversity
of an antigen receptor by recombination of V, (D) and J fragment in
T cells and B cells. Although this mechanism brought a result that
produces self-reactive lymphocytes simultaneously, these
self-reactive lymphocytes are removed by the negative selection in
the thymus or bone marrow, and are further controlled by the
self-tolerance mechanism of clone removal or anergy in the
periphery.
[0003] Although it is thought that an autoimmune disease is
developed by the breakdown of self-tolerance, the researches using
various disease model mice have been made towards the elucidation
of the mechanism of pathogenesis of the disease. However, there are
many still unknown matters about the etiology of an autoimmune
disease and the molecular mechanism of self-tolerance. In such a
situation, existence of the mouse which shows the symptoms of an
autoimmune disease with a single gene deficit is very important,
when trying to study the etiology of an autoimmune disease from a
molecular biological viewpoint. CTLA4-/-mouse which causes lethal
systemic lymphocytes infiltration (Waterhouse P. et. al., Science
270:985.about.988, 1995, TivolE.A.et.al., Immunity3: 541.about.547,
1995) SHP-1 deficit mothaten mice (Shulltz L. D. et. al., Cell 73:
1445.about.1454, 1993) , TGF-beta-1 knockout mouse (Shull M. M. et.
al., Nature 359:693-699, 1992), lyn-/-mouse which shows the
symptoms of glomerular nephritis (Hibbs M. L. et. al., Cell
83:301-311, 1995), and FCRIIB-/-mouse (Bolland S. & Ravetch J.
V., Immunity 13:277-285, 2000) are the representation, and the
relation of these molecules and self-tolerance is studied.
[0004] PD-1 is type I membrane protein of 55 kD belonging to an
immunoglobulin family. Both mouse PD-1 and human PD-1 consist of
288 amino acids, and have signal peptide at N terminal (20 amino
acid) and hydrophobic region in the middle part, which is a
transmembrane region (The EMBO J. 11 (11):3887-3895, 1992);
Japanese patent Publication No. 5-336973; EMBL/GenBank/DDJB Acc.
No. X67914, Genomics 23:704, 1994; Japanese patent Publication No.
7-291996 (U.S. Pat. No. 5,629,204).
[0005] In a thymocyte, PD-1 is expressed during a CD4-CD8- cell
differentiation to a CD4+CD8+ cell (Nishimura H. et. al.,
Int.Immunol. 8:773-780, 1996, Nishimura H. et. al., J. Exp. Med.
191:891-898, 2000). Moreover, in the periphery, PD-1 is expressed
in T cells and B cells which were activated by the stimulus from an
antigen receptor (Agata Y. et. al., Int. Immunol. 8:765-772, 1996),
and in bone marrow cells including activated macrophage.
[0006] PD-1 has ITIM (Immunoreceptor tyrosine-based inhibitory
motif) in its intracellular region, therefore it is considered to
be a negative regulator in immune reaction. PD-1 deficit mice
develop a lupus-like autoimmune disease such as glomerular
nephritis and arthritis (C57BL/6 genetic background) (Nishimura H.
et. al., Int. Imuunol. 10:1563-1572, 1998, Nishimura H. et. al.,
Immunity 11:141-151, 1999) and dilated cardiomyopathy-like disease
(BALB/c genetic background) (Nishimura H. et. al., Science
291:319-322, 2001), it became clear that PD-1 is a regulator of the
development of autoimmune disease, especially one of self-tolerance
in the periphery.
DISCLOSURE OF THE INVENTION
[0007] It is thought that PD-1 is the regulator of various
autoimmune diseases, and that it is one of the genes that cause an
autoimmune disease. By controlling the function of PD-1, it thought
that the medical treatment and diagnosis of suppression or
enhancement of the immune function, infection, the rejection at the
time of a transplant, a neoplasm, etc. could be performed, and as a
result of repeating examination wholeheartedly, the inventors
reached this invention concerning the substance which controls the
function of PD-1.
[0008] The stimulus to lymphocytes which control immunity is
transmitted mainly through T cell receptor (TCR) in the case of T
cells, and B cell receptor (BCR) in the case of B cells, and the
intracellular phosphorylation play an important role in its
molecular mechanism.
[0009] Since it became clear that PD-1 is controlling negatively
various cells responsible for immunity, such as lymphocytes and
myeloid cells etc., and PD-1 has ITIM (Immunoreceptor
tyrosine-based inhibitory motif) in its intracellular region, the
inventors considered the molecular mechanism involved in the
inhibitory signal transduction of PD-1 to be the recruit of
de-phosphorylation enzymes (phosphatases). Therefore, it came to be
considered by locating PD-1 near TCR or BCR that it can display the
function of PD-1. The inventors confirmed that the inhibitory
signal of PD-1 was transmitted with the substance in which PD-1 was
cross-linked to TCR or BCR, and completed this invention.
[0010] The inventors confirmed first that the above-mentioned idea
was right using anti-PD-1 antibody, and anti-BCR antibody or
anti-CD3 antibody. CD3 is the membrane protein expressed on a T
cell, and is one component of the complexes that constitute TCR.
The divalent antibody was constructed by bridging anti-PD-1
antibody and anti-BCR or anti-CD3 antibody. In the present
invention, this divalent antibody is called hybrid antibody. The
inventors produced this hybrid antibody for the first time.
[0011] Moreover, the knowledge that a signal transmits by
cross-linking two kind of receptors using this hybrid antibody is
also acquired for the first time.
[0012] Namely, the present invention relates to,
[0013] 1. A substance comprising a substance that recognizes PD-1,
a substance that recognizes a membrane protein co-existing with
PD-1 on a cell membrane, and a linker,
[0014] 2. The substance according to (1), which is a divalent
substance comprising a substance that recognizes PD-1, a substance
that recognizes a membrane protein co-existing with PD-1 on a cell
membrane, and a linker,
[0015] 3. The substance according to (1) or (2), in which a
membrane protein is a protein existing on a T cell membrane or B
cell membrane,
[0016] 4. The substance according to (1) or (2), which comprises a
substance that recognizes PD-1, a substance that recognizes a
protein constituting T cell receptor complex or a substance that
recognizes a protein constituting B cell receptor complex, and a
linker,
[0017] 5. The substance according to any one of (1) to (4), in
which a substance that recognizes PD-1 and in which a substance
that recognizes a protein is dimer to pentamer, respectively,
[0018] 6. The substance according to any one of (1) to (5), in
which one of or both substance that recognizes PD-1 and substance
that recognizes a protein are an antibody,
[0019] 7. The substance according to any one of (1) to (6), in
which one of the two or both substance that recognizes PD-1 and
substance that recognizes a protein are a Fab portion of
antibody,
[0020] 8. The substance according to any one of (1) to (5), in
which a linker comprises an organic compound,
[0021] 9. The substance according to any one of (1) to (5), in
which a linker comprises a peptide,
[0022] 10. The substance according to any one of (1) to (5), in
which a substance that recognizes PD-1 and in which a substance
that recognizes a protein is a peptide, respectively,
[0023] 11. The substance according to any one of (1) to (5), in
which a substance that recognizes PD-1 and in which a substance
that recognizes a protein comprises two or more peptides including
a heavy chain variable region and a light chain variable region of
antibody, respectively,
[0024] 12. The substance according to any one of (1) to (5), in
which a substance that recognizes PD-1, in which a substance that
recognizes a protein, and in which a linker is a peptide,
respectively,
[0025] 13. A pharmaceutical composition containing an effective
dose of the substance according to (1) for the medical treatment
and/or prevention of a disease in which PD-1 participates,
[0026] 14. The pharmaceutical composition according to (13), in
which disease is selected from the group consisting of
neurodegenerative disease, autoimmune disease, organ transplant
rejection, neoplasm and infection,
[0027] 15. The pharmaceutical composition according to (14), in
which neurodegenerative disease is selected from the group
consisting of Parkinson's disease, parkinsonian syndrome,
Huntington's disease, Machado-Joseph disease, amyotrophic lateral
sclerosis and Creutzfeldt-Jakob disease,
[0028] 16. The pharmaceutical composition according to (14), in
which autoimmune disease is selected from the group consisting of
glomerular nephritis, arthritis, dilated cardiomyopathy-like
disease, ulcerative colitis, Sjogren's syndrome, Crohn's disease,
systemic lupus erythematosus, chronic rheumatoid arthritis,
multiple sclerosis, Psoriasis, allergic contact dermatitis,
polymyositis, scleroderma, periarteritis nodosa, rheumatic fever,
vitiligo vulgaris, insulin-dependent diabetes mellitus, Behcet's
Syndrome and chronic thyroiditis.
[0029] A substance that recognizes PD-1 said by the present
invention maybe a substance which recognizes PD-1 specifically, for
example, anti-PD-1 antibody, the fragment of anti-PD-1 antibody,
PD-1 in itself, the fragment of PD-1, ligand of PD-1 (PD-L1
(Freeman G. J. et. al., J. Exp. Med. 192:1027-1034, 2000), PD-L2,
PD-H3), the fragment thereof and a low molecule organic compound,
etc.
[0030] In more concretely, it is anti-PD-1 antibody produced by the
hybridoma strain "J43", which was deposited on May 30, 2001 in
International Patent Organism Depositary, National Institute of
Advanced Industrial Science and Technology, AIST Tsukuba Central 6,
1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566 Japan as
Accession number FERM P-18356, and transferred to international
deposition on Jul. 16, 2002 as international deposition Accession
number FERM BP-8118.
[0031] Preferably, although it is the Fab portion of the antibody,
it is not limited to this.
[0032] A substance that recognizes a membrane protein co-existing
with PD-1 on a cell membrane said by the present invention may be a
substance which recognizes a membrane protein specifically, for
example, a substance which specifically recognizes a complex that
constitute T cell receptor (TCR) (TCR complex) etc. expressed on T
cells, or a substance which specifically recognizes a complex that
constitute B cell receptor (BCR) (BCR complex) etc. expressed on B
cells, such as the fragment of a protein constituting TCR complex,
anti-TCR antibody, the fragment of anti-TCR antibody, the fragment
of a protein constituting BCR complex, anti-BCR antibody and the
fragment of anti-BCR antibody.
[0033] Preferably, although it is the Fab portion of an anti-TCR
antibody or the Fab portion of an anti-BCR antibody, it is not
limited to these.
[0034] Both an anti-TCR antibody and an anti-BCR antibody are
available commercially. For example, anti-CD3 antibody
(.alpha.-CD3.epsilon. mAb, manufactured by Pharmingen) as an
anti-TCR antibody, and anti-IgG (H+L) polyclonal antibody
(manufactured by Zymed) as an anti-BCR antibody are available,
respectively.
[0035] A linker said by the present invention may be a substance
which can connect the above mentioned substance recognizing PD-1
and the substance recognizing a membrane protein co-existing with
PD-1 on a cell membrane if a suitable distance can be maintained.
More specifically, it may be a peptide, amide, etc.
[0036] A linker can be used what is marketed, for example,
Phenylenedimaleimide (manufactured by Aldrich) is available.
[0037] A substance that specifically recognizes PD-1, which is the
subject of the present invention, can be produced as follows, for
example.
[0038] In the case that an antibody is chosen as a substance which
recognizes PD-1 specifically, and that an antibody is also chosen
as a substance which recognizes specifically a membrane protein
co-existing with PD-1 on a cell membrane (hereinafter, simply
referred to as "the membrane protein"), a substance is called
hybrid antibody by this invention. The method of producing this
hybrid antibody is explained.
[0039] (1) Animals are immunized with PD-1 or the membrane protein
as an antigen,
[0040] (2) Spleen cells from the immunized animal and myeloma cells
are fused,
[0041] (3) Cells producing monoclonal antibody against the antigen
(PD-1 or the membrane protein) are screened among thus obtained
hybridoma cells,
[0042] (4) Aimed hybridoma cells are cloned,
[0043] (5) Cloned hybridoma cells are grown,
[0044] (6) Antibodies produced are isolated and purified,
[0045] (7) A hybrid antibody can be produced by bridging thus
obtained anti-PD-1 antibody and anti-membrane antibody with a
linker.
[0046] Alternatively,
[0047] (8) To obtain F(ab').sub.2, the antibodies produced are
treated with pepsin, isolated and purified,
[0048] (9) Each F(ab').sub.2 thus prepared is reduced, isolated and
purified,
[0049] (10)A hybrid antibody can be produced by bridging each
F(ab').sub.SH thus prepared with a linker.
[0050] In the case that both or one of the two of a substance which
specifically recognizes PD-1 and a substance which specifically
recognizes a membrane protein co-existing with PD-1 on a cell
membrane (the membrane protein) are a low molecule organic
compound,
[0051] (11) A low molecule compound which inhibits the binding of
each antibody to PD-1 or the membrane protein which is a
corresponding antigen, respectively, is found out by using antibody
produced by the above-mentioned technique and suitable detection
equipment,
[0052] (12) A substance can be produced by bridging the
compound-to-compound, the compound to antibody, or the compound to
Fab with a linker.
[0053] It is as follows when each step is explained more
concretely.
[0054] In an immunization step (1), it is desirable to administer
PD-1 or the membrane protein to an animal into the peritoneal
cavity or foot pad. Moreover, the animal to be immunized will not
be limited especially if it is the one from which the monoclonal
antibody is obtained generally such as mouse, rat or the like. In
the case of a mouse, the amount of an antigen is enough if 10-200
micrograms is administered per time.
[0055] The cell fusion of (2) is carried out by excising spleen
from an immunized animal with which the antibody titer has fully
risen among the animals immunized in the step (1), preparing spleen
cell suspension according to a usual method, and adding
polyethylene glycol (preferably PEG4000) to a mixture of the spleen
cells thus obtained and myeloma cells at 37.degree. C. Some kinds,
such as P3X63Ag8, P3/NS1/1-Ag4-1, and SP-2/0-Ag-14, are known as
the mouse myeloma cell, and all of them are easily available.
[0056] As the myeloma cell, HGPRT (hypoxanthine-guanine
phosphoribosyl transferase)-defective cell line which cannot
survive in HAT medium (a medium containing hypoxanthine,
aminopterin and thymidine) is useful, and it is further preferred
that it is a cell line in which the myeloma cells themselves do not
secrete any antibody. Preferably, SP-2O--Ag-14 is used.
[0057] Next, the obtained cell fusion mixture is dispensed into a
96-well micro plate at a low density, and cultivated in the HAT
medium. By culturing them for 1 to 2 weeks, un-fused myeloma cells,
hybridomas of myeloma cells themselves, un-fused spleen cells and
dybridomas of spleen cells themselves die because their surviving
conditions are not satisfied, and only the hybridomas of spleen
cell with myeloma cell are propagated.
[0058] In the screening of (3), whether or not a hybridoma is the
one which produces an antibody against PD-1 or the membrane protein
is judged by allowing each hybridoma culture supernatant to react
with the immobilized antigen, and then determining amount of the
antibody in the supernatant specifically adsorbed to the antigen
using a labeled second antibody.
[0059] The step (4) is carried out by cloning the
antibody-producing hybridoma in accordance with the soft agar
culture method (Monoclonal Antibodies, 372 (1980)). In this case,
it is also possible to use the limiting dilution method.
[0060] The step (5) is carried out by culturing the cloned
hybridoma in usual medium and then separating and purifying fromthe
culture supernatant, however, to obtain a larger amount of the
antibody efficiently a method in which the hybridoma is
administered into the abdominal cavity of mouse, allowed to
propagate therein and then separated and purified from the ascites
is used.
[0061] In the step (6), the purification can be carried out by
usual methods such as salting out, ion exchange chromatography, gel
filtration, hydrophobic chromatography and affinity chromatography,
however, affinity chromatography using protein A-sepharose CL-4B
(manufactured by Amersham Bioscience) is used more effectively.
[0062] Since a hybrid antibody of the present invention recognizes
PD-1 specifically, it can be used for the purification and
concentration of PD-1, for example, for affinity chromatography
etc.
[0063] The step (7) can be carried out by bridging a linker such as
sulfo-EMCS (N-(6-maleimidcaproxy) sulfo-succinimide sodium salt) to
amide groups or SH (mercapto) groups of an antibody. First, one
antibody is combined to sulfo-EMCS by amide coupling, un-reacted
sulfo-EMCS is discarded by gel filtration, the maleimide groups of
the sulfo-EMCS that is bound to the first antibody is reacted with
SH (mercapto) groups of the other antibody that is reduced with
2-mercaptoethylamine etc., and then a substance bridged over two
kinds of antibodies is size-fractionated using gel filtration.
[0064] The step (8) is carried out by digesting each antibody
obtained in the step (6) with pepsin at 37.degree. C. for 48 hours.
The separation and purification of F(ab') .sub.2 digested with
pepsin can be carried out by usual methods such as salting out, ion
exchange chromatography, gel filtration, hydrophobic chromatography
and affinity chromatography, however, gel filtration using
Sephacryl S-200 (manufactured by Amersham Bioscience) is used more
effectively.
[0065] The step (9) is carried out by reducing F(ab').sub.2 with
2-mercaptoethanol at 30.degree. C. for 30 minutes. The separation
and purification of the reduced Fab.sub.SH can be carried out by
usual methods such as salting out, ion exchange chromatography, gel
filtration, hydrophobic chromatography and affinity chromatography,
however, gel filtration using Sephacryl S-200 is used more
effectively.
[0066] In the step (10), Fab.sub.SH fraction of one antibody is
combined with a linker. As a linker, a substance that is combinable
with mercapto (SH) groups of Fab.sub.SH may be used, for example, a
reaction is performed by adding phenylene dimaleimide for 30
minutes at room temperature. Next, the reaction is followed by
adding the other Fab.sub.SH multiplied by 1.3 at room temperature
for 4 hours. The separation and purification of bispecific
substance can be carried out by usual methods such as salting out,
ion exchange chromatography, gel filtration, hydrophobic
chromatography and affinity chromatography, however, gel filtration
using Sephacryl S-200 is used more effectively.
[0067] The step (11) can be carried out by using the antibody
obtained at the step (6) without modification, or by using the
antibody with appropriate labeling (for example, biotin-conjugated
or FITC-conjugated etc.) in accordance with a usual method. In the
case that ELISA method is used, an antigen is immobilized by a
usual method, and then an antibody is added. Next, when
enzyme-conjugated second antibody and biotin-conjugated antibody
are used, enzyme-conjugated streptavidin is added, then the
specific binding between antigen and antibody is measured in the
presence of chromophore-producing substance by using
absorptiometer. By using this assay system a low molecule that
specifically recognizes PD-1 or the membrane protein can be
obtained.
[0068] In the step (12), when one of the two is an antibody or Fab,
the obtained low molecule can be combined with the antibody or Fab
by introducing the suitable functional group in it. For example,
when maleimide groups is introduced, it is possible to make it
combine with mercapto groups of an antibody or Fab. Moreover, when
both substances are low molecules, it is possible to synthesize a
molecule containing both ones.
[0069] In the case that an antibody is chosen as a substance which
recognizes PD-1 specifically, that an antibody is also chosen as a
substance which recognizes specifically a membrane protein
co-existing with PD-1 on a cell membrane, and that both antibodies
are included in the same peptide, the substance is called
bispecific antibody in the present invention. The method of
producing this bispecific antibody is explained.
[0070] (1) An antibody gene is isolated from hybridoma cells
producing antibodies against PD-1 and the membrane protein,
respectively,
[0071] (2) A variable domain of an antibody gene against PD-1 and a
variable domain of an antibody gene against the membrane protein
are connected using a linker DNA, the connected DNA fragment is
inserted to an expression vector, and cells are transfected with
the expression vector and propagated,
[0072] (3) Bispecific antibody can be prepared by separating and
purifying the protein thus produced.
[0073] It is as follows when each step is explained more
concretely.
[0074] The step (1) consists of the processes, which isolates RNA
from hybridoma cells, and which isolates cDNA encoding an antibody
or its partial peptide.
[0075] The process which isolates total RNA or mRNA from hybridoma
cells can be carried out in accordance with known methods
(hereinafter, if unstated especially, a known method is the method
described in Molecular Cloning (Sambrook J., Fritsch E. F. and
Maniatis T., Cold Spring Harbor Laboratory Press, 1989) or Current
Protocol in Molecular Biology (Ausubel F. M. et al., John Wiley
& Sons and Inc.).
[0076] The cloning of a cDNA encoding an antibody gene or its
partial peptide of the present invention can be performed by an
amplification using Polymerase Chain Reaction (hereinafter simply
referred to as "PCR method") with synthesized DNA primers having
partial nucleotide sequence encoding an antibody of the present
invention, or by a selection using hybridization of cDNAs inserted
into a suitable vector with a labeled DNA fragment or synthetic DNA
encoding an antibody of the present invention partially or
entirely. Hybridization can be carried out in accordance with known
methods. An antibody gene may be amplified directly by using
Reverse Transcriptase Polymerase Chain Reaction (hereinafter simply
referred to as "RT-PCR method") from total RNA or mRNA.
[0077] (2) Bispecific antibody of the present invention may be
prepared by:
[0078] i) A method of peptide synthesis, or
[0079] ii) A method of production using recombinant DNA technology,
preferably, by the method described in ii) in an industrial
production.
[0080] Examples of expression system (host-vector system) for the
production of peptide by using recombinant DNA technology are the
expression systems using bacteria, yeast, insect cells and
mammalian cells.
[0081] For example, in an E. Coli expression system, the initiation
codon (ATG) is added to 5'-end of the nucleotide sequence, for
example, shown in SEQ ID NO:28, then the expression vector is
prepared by connecting the obtained DNA to the downstream of a
suitable promoter (e.g., trp promoter, lac promoter, .lambda. PL
promoter, and T7 promoter), and by inserting it into a vector
(e.g., pBR322, pUC18and pUC19) which functions in an E. coli
strain.
[0082] Then, an E. coli strain (e.g., E. coli strain DH1, E. coli
strain JM 109 and E. coli strain HB101) which is transformed with
the expression vector described above may be cultured in an
appropriate medium to obtain the desired peptide. When a signal
peptide of bacteria (e.g., signal peptide of pel B) is utilized,
the desired peptide maybe released in periplasm. Furthermore, a
fusion protein with other peptide may be produced easily.
[0083] In a mammalian cell expression system, an expression vector
is prepared by inserting a DNA, for example, having the nucleotide
sequence shown in SEQ ID NO: 28, into the downstream of a proper
promoter (e.g., SV40 promoter, LTR promoter and metallothione in
promoter) in a proper vector (e.g., retro virus vector, papilloma
virus vector, vaccinia virus vector and SV40vector). Next, suitable
mammalian cells (e.g., monkey COS-1 cells, COS-7 cells, Chinese
hamster CHO cells, mouse L cells, 293 cells etc.) are transfected
with the expression vector thus obtained, and then the transfected
cells are cultured in an appropriate medium, the aimed peptide can
be secreted into the culture medium.
[0084] A transformation of E. coli can be carried out in accordance
with the method, for example, described in Proc. Natl. Acad. Sci.
(USA) 69:2110, 1972 and Gene 17: 107, 1982.
[0085] A transfection of mammalian cells can be carried out in
accordance with the method, for example, described in Saiboukougaku
supl.8:263 (New experimental protocol for cell technology),
Shujun-sha, 1995 and Virology 52:456, 1973.
[0086] It is known that a bispecific antibody can be prepared
directly by using recombinant DNA technology. For example, Alt et
al. (FEBS Letter 454:90, 1999) reported the generation of a
bispecific antibody (it is referred to a single-chain diabody)
directed against carcinoembryonic antigen and E. coli
beta-galactosidase. In said fragment, a heavy chain variable domain
(VH) of one antibody is connected to a light chain variable domain
(VL) of the other antibody with a short linker, which prevent the
pairing of these two continuous domains on the same chain.
Therefore, the VH and VL domains of this fragment are obliged to
pair with each complementary VL and VH domain on other chain, and
thereby form two antigen binding sites.
[0087] It is preferred that a peptide linker contains 3 to 12 amino
acid residues, but it is not limited particularly to its amino acid
sequence (Hudson et al., J. Immunol. Met. 231:177, 1999).
[0088] (3) The peptide thus obtained can be purified by usual
methods such as salting out, ion exchange chromatography, gel
filtration, hydrophobic chromatography and affinity
chromatography.
[0089] Since bispecific antibody of the present invention also
recognizes PD-1 specifically, it can be used for the purification
and concentration of PD-1, for example, for affinity chromatography
etc.
Industrial Applicability
[0090] Application for Pharmaceuticals:
[0091] The greatest and important usage of a substance that
specifically recognizes PD-1 by the present invention is for the
medical treatment of the following disease.
[0092] A substance that specifically recognizes PD-1 by the present
invention is useful for the medical treatment and/or prevention of
diseases such as neurodegenerative disease (Parkinson's disease,
parkinsonian syndrome, Huntington's disease, Machado-Joseph
disease, amyotrophic lateral sclerosis and Creutzfeldt-Jakob
disease etc.).
[0093] A substance that specifically recognizes PD-1 by the present
invention is also useful for the medical treatment and/or
prevention of diseases, in which PD-1 is involved and immune
responses are enhanced, such as autoimmune diseases (glomerular
nephritis, arthritis, dilated cardiomyopathy-like disease,
ulcerative colitis, Sjogren's syndrome, Crohn's disease, systemic
lupus erythematosus, chronic rheumatoid arthritis, multiple
sclerosis, Psoriasis, allergic contact dermatitis, polymyositis,
scleroderma, periarteritis nodosa, rheumatic fever, vitiligo
vulgaris, insulin-dependent diabetes mellitus, Behcet's Syndrome
and chronic thyroiditis etc.), organ transplant rejection, and
allergy.
[0094] A substance that specifically recognizes PD-1 by the present
invention is also useful for the medical treatment and/or
prevention of diseases, in which PD-1 is involved and immune
responses are reduced, such as neoplasm and infections.
[0095] For the above mentioned usage, administration of the
substance that specifically recognizes PD-1 by the present
invention can be carried out in systemic or local, generally
peroral or parenteral ways.
[0096] The dosage to be administered depends upon age, body weight,
symptom, desired therapeutic effect, route of administration, and
duration of the treatment etc. In human adults, one dose per person
is generally between 0.1 mg and 100 mg by oral administration up to
several times per day, or between 0.01 mg and 30 mg by parenteral
administration (preferably intravenous administration) up to
several times per day, or continuous administration between 1 and
24 hrs. per day into vein.
[0097] As mentioned above, the doses to be used depend upon various
conditions. Therefore, there are cases in which doses lower than or
greater than the ranges specified above may be used.
[0098] The compounds of the present invention may be administered
as inner solid compositions or inner liquid compositions for oral
administration, or as injections, liniments or suppositories etc.
for parenteral administration.
[0099] Examples of Inner solid compositions for oral administration
include compressed tablets, pills, capsules, dispersible powders
and granules etc. Examples of capsules include hard capsules and
soft capsules.
[0100] In such inner solid compositions, one or more of the active
compound(s) remains intact, or is/are admixed with excipients
(lactose, mannitol, glucose, microcrystalline cellulose and starch
etc.), connecting agents (hydroxypropyl cellulose,
polyvinylpyrrolidone, magnesium metasilicate aluminate, etc.),
disintegrating agents (cellulose calcium glycolate etc.),
lubricating agents (magnesium stearate etc.), stabilizing agents,
assisting agents for dissolving (glutamic acid, asparaginic acid
etc.) etc. to prepare pharmaceuticals by known methods. The
pharmaceuticals may, if desired, be coated with coating agent
(sugar, gelatin, hydroxypropyl cellulose or hydroxypropylmethyl
cellulose phthalate etc.), or be coated with two or more films.
Further, coating may include capsules of absorbable materials such
as gelatin.
[0101] Inner liquid compositions for oral administration may
contain pharmaceutically acceptable water-agents, suspensions,
emulsions, syrups and elixirs etc. In such liquid compositions, one
or more of the active compound(s) is/are resolved, suspended or
emulsified in inert diluent (s) commonly used in the art (purified
water, ethanol or mixture thereof etc.). Such liquid compositions
may also comprise wetting agents, suspending agents, emulsifying
agent, sweetening agents, flavoring agents, perfuming agents,
preserving agents and buffer agents etc.
[0102] Injections for parenteral administration include solutions,
suspensions and emulsions and solid injections, which are dissolved
or suspended in solvent when it is used. In such compositions, one
or more active compound(s) is/are dissolved, suspended or
emulsified in a solvent. Solvents include distilled water for
injection, physiological salt solution, plant oil, propylene
glycol, polyethylene glycol, alcohol such as ethanol, and mixture
thereof etc. Such compositions may comprise additional stabilizing
agents, assisting agents for dissolving (glutamic acid, asparaginic
acid, POLYSOLBATE80 (resistered trade mark) etc.), suspending
agents, emulsifying agents, soothing agent, buffer agents,
preserving agents etc. They may be manufactured or prepared by
sterilization or by aseptic manipulation in a final process. They
may also be manufactured in the form of sterile solid compositions
such as freeze-dried compositions, and can be dissolved in sterile
water or some other sterile solvent for injection immediately
before use.
[0103] Other compositions for parenteral administration include
liquids for external use, ointments, endermic liniments, aerosols,
spray compositions, suppositories and pessaries for vaginal
administration etc., which comprise one or more of the active
compound(s) and may be prepared by known methods.
[0104] Spray compositions may comprise additional substances other
than inert diluents generally used: e.g. stabilizing agents such as
sodium hydrogen sulfate, buffer agents to give isotonicity,
isotonic buffer such as sodium chloride, sodium citrate and citric
acid. For preparation of such spray compositions, for example, the
methods described in the U.S. Pat. No. 2,868,691 and 3,095,355 may
be used.
[0105] Since PD-1 is involved in immune responses, the substance
that specifically recognizes PD-1 by the present invention can also
be used for the screening of substances, which are involved in
immune responses, by measuring the expression of PD-1.
Effect of the Invention
[0106] A substance that specifically recognizes PD-1 by the present
invention comprises a substance that recognizes PD-1, a substance
that recognizes a membrane protein co-existing with PD-1 on a cell
membrane, and a linker, is a superior substance recognizable both
PD-1 and the membrane protein specifically and transmittable the
signal of PD-1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0107] FIG. 1 shows the results of FACS analysis using
anti-PD-1/anti-TCR hybrid Fab antibodies;
[0108] FIG. 2 shows the effect of anti-PD-1/anti-TCR hybrid Fab
antibodies on activated T cells;
[0109] FIG. 3 shows the inhibitory effect of anti-PD-1/anti-BCR
hybrid Fab antibodies on the production of IL-2 by anti-BCR
antibody stimulation;
[0110] FIG. 4 shows the effect of anti-PD-1/anti-BCR hybrid Fab
antibodies on SHP-2 recruitment after anti-BCR antibody
stimulation;
[0111] FIG. 5 shows the plasmid J43-2C11scDb-pSec/hygro B for the
expression of J43-2C11 bispecific antibody; and
[0112] FIG. 6 shows the effect of J43-2C11 bispecific antibodies on
the production of IFN-r by activated mouse spleen T cells.
BEST MODE FOR CARRYING OUT THE INVENTION
[0113] The present invention is more specifically explained by
means of the following examples, but is not limited only to these
examples.
[0114] Hereinafter, a substance in which anti-PD-1 antibody and
anti-T cell receptor antibody are linked by a linker simply
referred to as "anti-PD-1/anti-TCR hybrid antibody", and a
substance in which anti-PD-1 antibody and anti-B cell receptor
antibody are linked by a linker "anti-PD-1/anti-BCR hybrid
antibody", respectively. A substance in which the Fab.sub.SH
portion of anti-PD-1 antibody and the Fab.sub.SH portion of anti-T
cell receptor antibody are linked by a linker hereinafter simply
referred to as "anti-PD-1/anti-TCR hybrid Fab antibody", and a
substance in which the Fab.sub.SH portion of anti-PD-1 antibody and
the Fab.sub.SH portion of anti-B cell receptor antibody are linked
by a linker "anti-PD-1/anti-BCR hybrid Fab antibody",
respectively.
EXAMPLE 1
(1) Preparation of Anti-PD-1/Anti-TCR Hybrid Antibodies
[0115] (1-A) Introduction of Maleimide to Anti-Mouse CD3.epsilon.
Monoclonal Antibodies
[0116] Anti-mouse CD3.epsilon. monoclonal antibodies were
substituted with Sodium phosphate (0.1 M, pH7.0) and Nacl (50 mM),
then 200 times quantity of sulfo-EMCS (manufactured by Dojin
Chemical) were added and incubated at 20.degree. C. for 1 hour.
Then the reaction mixture was size-fractionated by gel filtration
using Sephacril S-300 [Sodium phosphate (0.1 M, pH7.0)] and major
peak fractions were collected by monitoring the absorbency at 280
nm. The protein content was calculated from the absorbency at 280
nm simultaneously.
[0117] (1-B) Reduction of Anti-PD-1 Antibodies
[0118] Antibodies against mouse PD-1 (produced by the hybridoma
cells named (J43) and deposited as international deposition
Accession number FERM BP-8118) were substituted with Sodium
phosphate (0.1 M, pH6.0), added with 2-mercaptoethylamine (final
concentration of 10 mM) and EDTA (final concentration of 1 mM), and
reduced at 37.degree. C. for 90 minutes. Then the reaction mixture
was size-fractionated by gel filtration using Sephacryl S-300
[Sodium phosphate (0.1 M, pH6.0)] and major peak fractions (single
chain fractions) were collected by monitoring the absorbency at 280
nm. The protein content was calculated from the absorbency at 280
nm simultaneously. (1-C) Bridging of Maleimide-conjugated
anti-mouse CD3.epsilon. monoclonal antibodies and Reduced anti-PD-1
antibodies
[0119] Maleimide-conjugated anti-mouse CD3.epsilon. monoclonal
antibodies and Reduced anti-PD-1 antibodies were mixed at the rate
of 1:4 and incubated at 15.degree. C. for 18 hours. Then the
reaction mixture was size-fractionated by gel filtration using
Sephacryl S-300 [Sodium phosphate (0.1 M, pH7.0)] and major peak
fractions were collected by monitoring the absorbency at 280 nm.
The protein content was calculated from the absorbency at 280 nm
simultaneously.
(2) Preparation of Anti-PD-1/Anti-TCR Hybrid Fab Antibodies
[0120] (2-A) Preparation of F(ab').sub.2 Fraction of Anti-PD-1
Antibodies
[0121] Antibodies against mouse PD-1 (produced by the hybridoma
cells named (J43) and deposited as international deposition
Accession number FERM BP-8118) were substituted with pepsin-buffer
[(sodium acetate 0.1 M, pH4.5), NaCl (0.1 M)], added with pepsin
(final concentration of 0.2 mg/ml), and digested for 48 hours at
37.degree. C. Then the reaction mixture was size-fractionated by
gel filtration using Sephacryl S-200 [Tris-HCl (0.2 M, pH8.0), EDTA
(10 mM)] and major peak fractions (F(ab').sub.2 fraction) were
collected by monitoring the absorbency at 280 nm. The protein
content was calculated from the absorbency at 280 nm
simultaneously.
[0122] (2-B) Preparation of Fab.sub.SH Fraction of Anti-PD-1
Antibodies
[0123] 2-mercaptoethanol (final concentration of 20 mM) was added
to reduce the F(ab').sub.2 fraction at 30.degree. C. for 30
minutes. After cooling the reaction mixture on ice, it was
size-fractionated by gel filtration using Sephacryl S-200 [sodium
acetate (50 mM, pH6.3), EDTA (1 mM)] and major peak fractions
(Fab.sub.SH fraction) were collected by monitoring the absorbency
at 280 nm. The protein content was calculated from the absorbency
at 280 nm simultaneously.
[0124] (2-C) Preparation of F(ab').sub.2 Fraction of Anti-Mouse
CD3.epsilon. Monoclonal Antibodies
[0125] Anti-mouse CD3.epsilon. monoclonal antibodies (manufactured
by Pharmingen) were substituted with pepsin-buffer [(sodium acetate
0.1 M, pH4.5), NaCl (0.1 M)], added with pepsin (final
concentration of 0.2 mg/ml), and digested for 48 hours at
37.degree. C. Then the reaction mixture was size-fractionated by
gel filtration using Sephacryl S-200 [Tris-HCl (0.2 M, pH8.0), EDTA
(10 mM)] and major peak fractions (F(ab').sub.2 fraction) were
collected by monitoring the absorbency at 280 nm. The protein
content was calculated from the absorbency at 280 nm
simultaneously.
[0126] (2-D) Preparation of Fab.sub.SH Fraction of Anti-Mouse
CD3.epsilon. Monoclonal Antibodies
[0127] 2-mercaptoethanol (final concentration of 20 mM) was added
to reduce the F(ab').sub.2 fraction at 30.degree. C. for 30
minutes. After cooling the reaction mixture on ice, it was
size-fractionated by gel filtration using Sephacryl S-200 [sodium
acetate (50 mM, pH6.3), EDTA (1 mM)] and major peak fractions
(Fab.sub.SH fraction) were collected by monitoring the absorbency
at 280 nm. The protein content was calculated from the absorbency
at 280 nm simultaneously.
[0128] (2-E) Bridging of Fab.sub.SH Fraction of Anti-PD-1
Antibodies and Fab.sub.SH Fraction of Anti-Mouse CD3.epsilon.
Monoclonal Antibodies
[0129] Phenylenedimaleimide (manufactured by Aldrich) (final
concentration of 4 mM) was added to the Fab.sub.SH fraction of
anti-PD-1 antibodies prepared in step (2-A), and incubated
for30minutes at room temperature to prepare J43 Fab.sub.mal
fraction. The J43 Fab.sub.mal fraction and the Fab.sub.SH fraction
of anti-mouse CD3.epsilon. monoclonal antibodies were mixed at the
rate of 1:1.3 and incubated for 4 hours at room temperature. Next,
an appropriate amount of Tris-HCl (1 M, pH8.0) were added to make
the pH of the reaction mixture 8.0, 2-mercaptoethanol (final
concentration of 20 mM) was added and incubated at 30.degree. C.
for 30 minutes. Then iodoacetoamide (manufactured by Sigma) was
added (final concentration of 25 mM) and incubated for additional
10 minutes at room temperature under light shielding.
[0130] Finally, the reaction mixture was size-fractionated by gel
filtration using Sephacryl S-200 [sodium acetate (50 mM, pH6.3),
EDTA (1 mM)] and major peak fractions (BsAb fraction) were
collected by monitoring the absorbency at 280 nm. The protein
content was calculated from the absorbency at 280 nm
simultaneously.
EXAMPLE 2
Confirmation of Reactivity of Anti-PD-1/Anti-TCR Hybrid Fab
Antibodies on Cell Surface Antigen (PD-1 and CD3)
[0131] 1.times.10.sup.6 cells were recovered from mPD-1/A 20IIA1.6
cells as a PD-1 positive and CD3 negative cell, and naive T cells
prepared from mouse spleen cells as a PD-1 negative and CD3
positive cell, respectively. The cells were added with 91 .mu.l of
FACS buffer (PBS(-) containing 0.5% BSA, EDTA (2 mM) and 0.01%
NaN.sub.3), 5 .mu.l of mouse serum and 4 .mu.l of hybrid antibodies
(2 .mu.g) and incubated for 30 minutes on ice. After washing with
PBS(-) once, the cells were added with each 2 .mu.l (1 .mu.g) of
second antibodies, fill upped to final 100 .mu.l with FACS buffer,
and incubated for 30 minutes on ice. Then they were analyzed by
using FACS can. The results were shown in FIG. 1 (in the following
figures, hybrid Fab antibody simply referred to as "HFAb").
[0132] Analysis whether the prepared anti-PD-1/anti-TCR hybrid Fab
antibodies actually react with the cell surface antigens by using
FACS.sub.sort (manufactured by Becton Dickinson) showed the results
that the antibodies reacted with both surface antigens.
EXAMPLE 3
Effect of Anti-PD-1/Anti-TCR Hybrid Fab Antibodies on Activated T
cells
[0133] (A) Preparation of Spleen T Cells
[0134] Spleen was excised from BALB/c mouse. After red blood cells
were hemolyzed, the spleen cells were washed once with PBS(-) and
suspended in medium RPMI1640 (10% FCS, antibiotics)
(1.times.10.sup.8 cells/ml). Next, T cells were prepared by using
nylon fiber column (manufactured by WAKO) for T cell separation
equilibrated with the medium.
[0135] (B) Effect of Anti-PD-1/Anti-TCR Hybrid Fab Antibodies on
Activated Spleen T Cells
[0136] Ninety six-well plates were coated with 0.5 .mu.g/ml and 5
.mu.g/ml of anti-CD3 antibodies (clone KT3, manufactured by
Immuntech) at 37.degree. C. for 3 hours. T cells (2.times.10.sup.5
cells/well/200 .mu.l ) were seeded on the plates,
anti-PD-1/anti-TCR hybrid antibodies (0.03, 0.1, 0.3, 1, 3
.mu.g/100 ml) were added, and incubated in a CO.sub.2 incubator (at
37.degree. C.). After 72 hours, cytokine (IFN-r, IL-2, IL-4 and
IL-10) concentrations in the recovered culture supernatants were
measured by using assay kit (manufactured by R & D System).
[0137] As the results shown in FIG. 2, after 72 hours when 3 .mu.g
of anti-PD-1/anti-TCR hybrid Fab antibodies were used, the results
were obtained that the stimulation by the antibodies both in 0.5
.mu.g/ml and 5 .mu.g/ml suppressed the production of IFN-r, IL-4
and IL-10 dominantly.
EXAMPLE 4
Preparation of Anti-PD-1/Anti-BCR Hybrid Fab Antibodies
[0138] (A) Preparation of F(ab').sub.2 Fraction of Anti-PD-1
Antibodies
[0139] Antibodies against mouse PD-1 (the same antibodies as used
in Example 1) were substituted with pepsin-buffer [(sodium acetate
0.1 M, pH4.5), NaCl (0.1 M)], added with pepsin (manufactured by
SIGMA) (final concentration of 0.2 mg/ml), and digested at
37.degree. C. for 48 hours. Then the reaction mixture was
size-fractionated by gel filtration using Sephacryl S-200
(manufactured by AmarshamParmacia) [Tris-HCl (0.2 M, pH8.0), EDTA
(10 mM)] and major peak fractions (F(ab').sub.2 fraction) were
collected by monitoring the absorbency at 280 nm. The protein
content was calculated from the absorbency at 280 nm
simultaneously.
[0140] (B) Preparation of Fab.sub.SH Fraction of Anti-PD-1
Antibodies
[0141] 2-mercaptoethanol (final concentration of 20 mM) was added
to reduce the F(ab').sub.2 fraction at 30.degree. C. for 30
minutes. After cooling the reaction mixture on ice, it was
size-fractionated by gel filtration using Sephacryl S-200 [sodium
acetate (50 mM, pH6.3), EDTA (1 mM)] and major peak fractions
(Fab.sub.SH fraction) were collected by monitoring the absorbency
at 280 nm. The protein content was calculated from the absorbency
at 280 nm simultaneously.
[0142] (C) Preparation of F(ab').sub.2 Fraction of Anti-IgG (H+L)
Polyclonal Antibodies
[0143] Rabbit anti-mouse IgG (H+L) polyclonal antibodies
(manufactured by Zymed) were substituted with pepsin-buffer
[(sodium acetate 0.1 M, pH4.5), NaCl (0.1 M)], added with pepsin
(final concentration of 0.2 mg/ml), and digested at 37.degree. C.
for 48 hours. Then the reaction mixture was size-fractionated by
gel filtration using Sephacryl S-200 [Tris-HCl (0.2 M, pH8.0), EDTA
(10 mM)] and major peak fractions (F(ab').sub.2 fraction) were
collected by monitoring the absorbency at 280 nm. The protein
content was calculated from the absorbency at 280 nm
simultaneously.
[0144] (D) Preparation of Fab.sub.SH Fraction of Anti-IgG (H+L)
Polyclonal Antibodies
[0145] 2-mercaptoethanol (final concentration of 20 mM) was added
to reduce the F(ab').sub.2 fraction at 30.degree. C. for 30
minutes. After cooling the reaction mixture on ice, it was
size-fractionated by gel filtration using Sephacryl S-200 [sodium
acetate (50 mM, pH6.3), EDTA (1 mM)] and major peak fractions
(Fab.sub.SH fraction) were collected by monitoring the absorbency
at 280 nm. The protein content was calculated from the absorbency
at 280 nm simultaneously.
[0146] (E) Bridging of Fab.sub.SH Fraction of Anti-PD-1 Antibodies
and Fab.sub.SH Fraction of Anti-IgG (H+L) Polyclonal Antibodies
[0147] Phenylenedimaleimide (manufactured by Aldrich) (final
concentration of 4 mM) was added to the Fab.sub.SH fraction of J43
anti-PD-1 antibodies, and incubated for 30 minutes at room
temperature to prepare J43 Fab.sub.malfraction. The J43 Fab.sub.mal
fraction and the Fab.sub.SH fraction of anti-IgG (H+L) polyclonal
antibodies were mixed at the rate of 1:1.3 and incubated for 4
hours at room temperature. Next, an appropriate amount of Tris-HCl
(1 M, pH8.0) were added to make the pH of the reaction mixture 8.0,
2-mercaptoethanol (final concentration of 20 mM) was added and
incubated at 30.degree. C. for 30 minutes. Then iodoacetoamide
(manufactured by SIGMA) was added (final concentration of 25 mM)
and incubated for additional 10 minutes at room temperature under
light shielding. Finally, the reaction mixture was
size-fractionated by gel filtration using Sephacryl S-200 [sodium
acetate (50 mM, pH6.3), EDTA (1 mM)] and major peak fractions (BsAb
fraction) were collected by monitoring the absorbency at 280 nm.
The protein content was calculated from the absorbency at 280 nm
simultaneously.
EXAMPLE 5
Effect of Anti-PD-1/Anti-BCR Hybrid Fab Antibodies on B Cell
Line
[0148] (A) Generation of A20IIA1.6 (B cell line) that was Forced to
Express Mouse PD-1
[0149] (1) Construction of expression plasmid for mouse PD-1
[0150] DNA fragments of mPD1-flag digested with EcoRI were inserted
into the EcoRI site of a commercially available expression vector
to construct the expression plasmid mPD1-pA.
[0151] (2) Transfection
[0152] A20IIA1.6 cells (1.times.10.sup.7) in 325 .mu.l of ice-cold
RPMI1640 medium containing 15% FCS and the PD-1 expression plasmid
linearized with ScaI in 10 .mu.l of distilled water were
incorporated into a Cuvette for electroporation (Gene Pulser
Cuvette 0.4 cm electrode gap, 50, BIO RAD), and pulsed under the
condition of 250V/960 .mu.F (Gene Pulser, BIO RAD). After leaving
at rest for 10 minutes at room temperature, the cells were
suspended in 30 ml of medium (RPMI1640 containing 10% FBS, 50 .mu.l
of 2-mercaptoethanol, penicillin and streptomycin), diluted
thirtyfold further, and dispensed onto 96-well plates (10.sup.3/100
.mu.l/well). After 48 hours, the selection was initiated using
final 3 .mu.M of Puromycin to establish the cell line expressing
mouse PD-1.
[0153] (B) Effect of Anti-PD-1/Anti-BCR Hybrid Fab Antibodies on
A20IIA1.6 Cells that was Forced to Express Mouse PD-1
[0154] A20IIA1.6 cells that was forced to express mouse PD-1 were
seeded on 96-well plates (5.times.10.sup.5 cells/100 .mu.l).
Anti-PD-1/anti-BCR hybrid Fab antibodies (0, 1, 3, 10 .mu.g/100
.mu.l) were added, 10 minutes later 100 .mu.l of anti-mouse IgG
(H+L) F(ab').sub.2 (manufactured by Zymed) (final concentration of
0.3, 1, 3 .mu.g/ml) were dispensed and cultivated for 12 hours in a
CO.sub.2 incubator (at 37.degree. C.). The culture supernatants
were recovered and the concentrations of IL-2 in the culture
supernatants were measured by using mouse IL-2 assay kit
(manufactured by R & G System). The results were shown in FIG.
3.
[0155] The studies using various doses of anti-PD-1/anti-BCR hybrid
Fab antibodies and anti-BCR antibodies F(ab').sub.2 showed the
suppressive effects of the hybrid Fab antibodies in all cases,
regardless of the concentration of anti-BCR antibodies
F(ab').sub.2.
[0156] (C) Confirmation of SHP-2 Recruitment
[0157] Anti-PD-1/anti-BCR hybrid Fab antibodies (0, 1, 3, 10
.mu.g/100 .mu.l) were added to 3.times.10.sup.6 of A20IIA1.6 cells
(B cell line) that was forced to express mouse PD-1. After 10
minutes, 100 .mu.l of anti-mouse IgG (H+L) F(ab').sub.2 were added
and incubated for 5 minutes at room temperature. After discarding
the supernatants by centrifugation, the cells were suspended in 200
.mu.l of lysis buffer (composition: Tris-HCl (20 mM, pH7.4), NaCl
(150 mM), Na.sub.2EDTA (1 mM), EGTA (1 mM), 1% Triton-X100, sodium
pyrophosphate (2.5 mM), .beta.-sodium glycerophosphate (1 mM),
Na.sub.3VO.sub.4 (1 mM), leupeptin (1 .mu.g/ml) and PMSF (1 mM) and
left at rest on ice. After 30 minutes, the supernatants were
recovered by centrifugation, 20 .mu.l of protein G-cepharose beads
(manufactured by Amersham Bioscience) added, and incubated at
4.degree. C. for 30 minutes. After recovering the supernatants by
centrifugation, 20 .mu.l of protein G-cepharose beads bound
beforehand with anti-FLAG antibodies (manufactured by SIGMA) were
added and mixed over night at 4.degree. C.
[0158] The beads were washed five times with 400 .mu.l of lysis
buffer, added with 20 .mu.l of lysis buffer and 20 .mu.l of
2.times. SDS sample buffer, and boiled at 100.degree. C. for 5
minutes. After discarding the beads by centrifugation, 15 .mu.l of
supernatants were subjected to 4-20% SDS-PAGE. After
electrophoresis the gels were substituted with blotting buffer and
transferred onto PVDF membrane (manufactured by BIO RAD). Then the
membrane was blocked with Block Ace (manufactured by Dainippon
Pharmaceuticals) for 1 hour at room temperature.
[0159] The membrane was incubated with anti-SHP-2 antibody
(manufactured by SANTA CRUZ) diluted 1/200 for 1 hour at room
temperature, then washed three times with TBS-T for 10 minutes.
Next, the membrane was incubated with HRP-conjugated anti-rabbit Ig
antibody (manufactured by Amersham Bioscience) diluted 1/2000 for 1
hour at room temperature, then washed three times with TBS-T for 10
minutes. Finally the membrane was emitted light by using ECT plus
detection kit (manufactured by Amersham Bioscience) and analyzed
using luminoimager LAS1000 plus (manufactured by FUJI Film).
[0160] Since anti-PD-1/anti-BCR hybrid Fab antibodies suppressed
the production of IL-2 from anti-BCR antibody stimulated B cells,
the evaluation whether the effects were caused by the recruitment
of phosphatase SHP-2 to ITIM of PD-1was carried out. As shown in
FIG. 4, by adjusting the amount of samples with that of PD-1 as a
control, the determination of the quantity of SHP-2 recruitment
resulted in the obvious recruitment of SHP-2, in 1 and 10 .mu.g, as
compared with control hybrid antibodies.
EXAMPLE 6
cDNA Cloning of Anti-Mouse PD-1 Antibody J43
[0161] (1) Preparation of Anti-Mouse PD-1 Antibody J43
[0162] Anti-mouse PD-1 antibody producing hybridoma (J43) cells
were cultured in Hybridoma SFM medium (manufactured by Invitrogen)
at 37.degree. C. under 5% CO.sub.2, a few days after the culture
supernatants of hybridoma cells were recovered. IgG fraction was
purified from the culture supernatants recovered by using HiTrap
Protein G (manufactured by Amersham Bioscience).
[0163] (2) Peptide Sequencing
[0164] J43 IgG was subjected to 10-20% SDS-PAGE. After
electrophoresis the IgG was electrically transferred from the gel
onto PVDF membrane (manufactured by BIO RAD). The membrane
transferred was stained with coomassie, the membrane fraction
containing the light chain of J43 IgG was removed, and amino
terminal 15 residues of the light chain were determined by using
peptide sequencer Procise492 (manufactured by Applied
Biosystems)(SEQ ID NO:1).
[0165] (3) Extraction of RNA
[0166] 5.times.10.sup.6 hybridoma cells were lysed with 1 ml of
TRIzol (manufactured by Invitrogen). Total RNA was prepared
according to the direction of attached document. mRNA was purified
from total RNA thus prepared by using Oligotex-MAG mRNA
Purification Kit (manufactured by Takara Shuzo).
[0167] (4) Cloning of Light Chain cDNA (3'RACE) Degenerated primer
(primer No.1) was designed based on the amino terminal sequence
(YELTQPPSASVNVGE) of the light chain determined by peptide
sequencing. 3'RACE was carried out by using 3'-Full RACE Core Set
(manufactured by Takara Shuzo) under the following conditions.
1 Primer No. 1 5'-ta(c/t) ga(a/g) ct(g/a/t/c) ac(g/a/t/c) ca(a/g)
(SEQ ID NO:2) cc(g/a/t/c) cc-3' 1) Synthesis of first strand cDNA
10 .times. RNA polymerase chain reaction (PCR) buffer 2 J43 mRNA
(50 ng/.mu.l) 2 Magnesium chloride (25 mM) 4 dNTP mixture (each 10
mM) 2 AMV Reverse Transcriptase XL (5 U/.mu.l) 1 Oligo dT-3sites
Adapter primer (2.5 pmol/.mu.l) 1 Ribonuclease (RNase) inhibitor
(40 U/.mu.l) 0.5 dH.sub.2O 7.5 15 .mu.l 30.degree. C. 10 min.
.fwdarw. 50.degree. C. .fwdarw. 30 min. .fwdarw. 95.degree. C. 5
min. .fwdarw. 4.degree. C. 5 min. 2) Polymerase chain reaction
(PCR) First strand cDNA 1 Primer No. 1 (20 pmol/.mu.l) 1
Anchorprimer 1 dH.sub.2O 22 One shot LA PCR Mix.TM. 25 50 .mu.l
95.degree. C. 5 min. .fwdarw. (94.degree. C. 29 sec., 50.degree. C.
20 sec., 72.degree. C. 60 sec.) .times. 30 cycles
[0168] The PCR products were subjected to electrophoresis using 1%
Agarose gel, then the gel was stained by EtBr. DNA fragment was
recovered from the gel by using MinElute Gel Extraction Kit
(manufactured by Quiagen), the DNA fragment recovered was ligated
to pGEM-T Easy Vector (manufactured by Promega) by using DNA
Ligation Kit ver.2 (manufactured by Takara Shuzo). E. coli
DH5.alpha. was transformed with the ligated plasmid. Finally, the
plasmid was purified from E. coli and J43 IgG light chain cDNA was
sequenced (SEQ ID NO:3). The deduced amino acid sequence of the
cDNA is shown in sequence listing (SEQ ID NO:4).
[0169] (5) Cloning of Heavy Chain cDNA (5'RACE)
[0170] To perform 5'RACE, primers for constant region were designed
based upon the reported information of hamster IgG heavy chain cDNA
sequence (GenBank Accession No.U17166). 5'RACE was carried out by
using 5'-Full RACE Core Set (manufactured by Takara Shuzo) under
the following conditions.
2 Primer No. 2 5'-ccc aag agg tca gga gtt gga-3' (5'
phosphorylated) (SEQ ID NO:5) Primer No. 3 5'-ttg acc agg cat ccc
agg gtc-3' (SEQ ID NO:6) Primer No. 4 5'-cgt aag ctg gaa ctc tgg
agc-3' (SEQ ID NO:7) Primer No. 5 5'-tgg ttg tgc tgt cac agg cag-3'
(SEQ ID NO:8) Primer No. 6 5'-tgc aca cct tcc cat ctg tcc t-3' (SEQ
ID NO:9) 1) Synthesis of first strand cDNA J43 total RNA (2
.mu.g/.mu.l) 2 10 .times. RNA polymerase chain reaction (PCR)
buffer 1.5 Ribonuclease (RNase) inhibitor (40 U/.mu.l) 0.5 AMV
Reverse Transcriptase XL (5 U/.mu.l) 1 Primer No. 2 (100
pmol/.mu.l) 2 dH.sub.2O 8 15 .mu.l 30.degree. C. 10 min. .fwdarw.
50.degree. C. .fwdarw. 40 min. .fwdarw. 30.degree. C. 2 min. 2)
Degeneration of hybrid RNA First strand cDNA 15 5 .times. Hybrid
RNA Degeneration buffer 15 dH.sub.2O 45 Ribonuclease H (RNaseH) 1
76 .mu.l 30.degree. 1 hour After the reaction, ethanol
precipitation was carried out. 3) Cyclization of single chain cDNA
by ligation 5 .times. RNA (ssDNA) Ligation buffer 8 40%
Polyethylene glycol (PEG) #6000 20 dH.sub.2O 12 Pellet after
ethanol precipitation T4 RNA ligase 1 41 .mu.l 15.degree. C. 15
hours 4) First PCR Sample after ligation 1 Primer No. 3 (5
pmol/.mu.l) 2 Primer No. 4 (5 pmol/.mu.l) 2 dH.sub.2O 20 One shot
LA PCR Mix.TM. 25 50 .mu.l 94.degree. C. 3 min. .fwdarw.
(94.degree. C. .fwdarw. 30 sec., 52.degree. C. 30 sec, 72.degree.
C. 120 sec.) .times. 25 cycles 5) Second PCR Product of first PCR 2
Primer No. 5 (5 pmol/.mu.l) 2 Primer No. 6 (5 pmol/.mu.l) 2
dH.sub.2O 20 One shot LA PCR Mix.TM. 25 50 .mu.l 94.degree. C. 3
min. .fwdarw. (94.degree. C. 30 sec., 52.degree. C. 30 sec,
72.degree. C. 120 sec.) .times. 30 cycles
[0171] The PCR products were subjected to electrophoresis using 1%
Agarose gel, then the gel was stained by EtBr. DNA fragment was
recovered from the gel by using MinElute Gel Extraction Kit
(manufactured by Quiagen), the DNA fragment recovered was ligated
to pGEM-T Easy Vector (manufactured by Promega) by using DNA
Ligation Kit ver.2 (manufactured by Takara Shuzo). E. coli
DH5.alpha. was transformed with the ligated plasmid. Finally, the
plasmid was purified from E. coli and J43 IgG heavy chain cDNA was
sequenced (SEQ ID NO:10). The deduced amino acid sequence of the
cDNA is shown in sequence listing (SEQ ID NO:11)
EXAMPLE 7
cDNA Cloning of Anti-Mouse CD3.epsilon. Antibody
[0172] (1) Preparation of RNA
[0173] Anti-mouse CD3.epsilon. antibody producing hybridoma
(145-2C11: manufactured by Pharmingen) cells were cultured in
Hybridoma SFM medium (manufactured by Invitrogen) at 37.degree. C.
under 5% CO.sub.2, a few days after 5.times.10.sup.6 hybrisoma
cells were lysed with 1 ml of TRIzol (manufactured by Invitrogen).
Total RNA was prepared according to the direction of attached
document.
[0174] (1) Preparation of cDNA Library
[0175] cDNA was synthesized from 2.5 .mu.g of total RNA extracted
from hybridoma (145-2C11) cells by oligo-dT prime method using
Ready-To-Go You-Prime First-Strand Beads (manufactured by Amersham
Pharmacia). Operations and procedures were followed by the
instructions of attached document.
[0176] (3) cDNA Cloning of Heavy and Light Chains
[0177] Based upon the reported cDNA sequence information of heavy
chain variable region of hybridoma 145-2C11 (GenBank Accession
No.AF000357), primers No.7 and No.8 were designed. Also, based upon
the cDNA sequence information of light chain variable region of
145-2C11 (GenBank Accession No.AF000356), primers No.9 and No.10
were designed. PCR was carried out using these primers and the cDNA
library from hybridoma 145-2C11 as a template.
3 Primer No. 7 5'-gag gtg cag ctg gtg gag tct-3' (SEQ ID NO:12)
Primer No. 8 5'-tga gga gac ggt gac cat ggt t-3' (SEQ ID NO:13)
Primer No. 9 5'-gac atc cag atg acc cag tct c-3' (SEQ ID NO:14)
Primer No. 10 5'-ttt gat ttc cag ctt ggt gcc ag-3' (SEQ ID NO:15)
cDNA library 2 Primer No. 7 or No. 9 (5 pmol/.mu.l) 2 Primer No. 8
or No. 10 (5 pmol/.mu.l) 2 dH.sub.2O 20 One shot LA PCR Mix.TM. 25
50 .mu.l (94.degree. C. 30 sec., 52.degree. C. 30 sec., 72.degree.
C. 120 sec.) .times. 30 cycles
[0178] The PCR products were subjected to electrophoresis using 1%
Agarose gel, then the gel was stained by EtBr. DNA fragments were
recovered from the gel by using MinElute Gel Extraction Kit
(manufactured by Quiagen), the DNA fragments recovered was ligated
to pGEM-T Easy Vector (manufactured by Promega) by using DNA
Ligation Kit ver.2 (manufactured by Takara Shuzo). E. coli
DH5.alpha. were transformed with the ligated plasmids. Finally, the
plasmids were purified from E. coli, and both DNA were sequenced.
It was confirmed that their sequences were identical to those of
GenBank Accession No.AF000357and GenBank Accession No.AF000356,
respectively.
EXAMPLE 8
Constraction of Expression Plasmid for J43-2C11 Bispecific
Antibody
[0179] J43 IgG heavy chain cDNA and 145-2C11 IgG light chain cDNA
were connected by PCR using linker No.1, No.2, primer No.11 and
No.12 to prepare fragment 1 (see FIG. 5). Next, 145-2C11 IgG light
chain cDNA and 145-2C11 IgG heavy chain cDNA were connected by PCR
using linker No.3, No.4, primer No.13 and No.14 to prepare fragment
2 (see FIG. 5). 145-2C11 IgG heavy chain cDNA and J43 IgG light
chain cDNA were connected by PCR using linker No.5, No.6, primer
No.15and No.16 to prepare fragment 3 (see FIG. 5).
4 Primer No. 11 5'-ttt gaa ttc aga ggt gcg gct tct gga gtc t-3'
(SEQ ID NO:16) Primer No. 12 5'-gat cag gag ctt agg agc ttt cc-3'
(SEQ ID NO:17) Primer No. 13 5'-cag gcc agt cag gac att agc aa-3'
(SEQ ID NO:18) Primer No. 14 5'-taa tgt atg cga ccg act cca gc-3'
(SEQ ID NO:19) Primer No. 15 5'-tga ggc ctc tgg att cac ctt ca-3'
(SEQ ID NO:20) Primer No. 16 5'-aaa aaa aaa ctc gag gac cta gga cgg
tga gct ggg t-3' (SEQ ID NO:21) Linker No. 1 5'-agg gac cca agt cac
tgt ctc ctc agg tgg agg cgg ttc aga (SEQ ID NO:22) cat cca gat gac
cca gtc tcc at-3' Linker No. 2 5'-tcc ctg ggt tca gtg aca gag gag
tcc acc tcc gcc aag tct (SEQ ID NO:23) gta ggt cta ctg ggt cag agg
ta-3' Linker No. 3 5'-acc tgg cac caa gct gga aat caa agg tgg agg
cgg ttc agg (SEQ ID NO:24) cgg agg tgg ctc tgg cgg tgg cgg atc gga
ggt gca gct ggt gga gtc tgg gg-3' Linker No. 4 5'-tgg acc gtg gtt
cga cct tta gtt tcc acc tcc gcc aag tcc (SEQ ID NO:25) gcc tcc acc
gag acc gcc acc gcc tag cct cca cgt cga cca cct cag acc cc-3'
Linker No. 5 5'-agg aac cat ggt cac cgt ctc ctc agg tgg agg cgg ttc
ata (SEQ ID NO:26) tga gct gac tca gcc acc ttc ag-3' Linker No. 6
5'-tcc ttg gta cca gtg gca gag gag tcc acc tcc gcc aag tat (SEQ ID
NO:27) act cga ctg agt cgg tgg aag tc-3' PCR condition for
fragments 1, 2 and 3 First PCR Template 1 2 Template 2 2 Linker
(100 ng/.mu.l) 2 Linker (100 ng/.mu.l) 2 dH.sub.2O 17 One shot LA
PCR Mix.TM. 25 50 .mu.l 95.degree. C. 5 min., .fwdarw. (94.degree.
C. 30 sec., 40.degree. C. 30 sec., 72.degree. C. 60 sec.) .times.
20 cycles Second PCR Product of first PCR 5 Primer (5 pmol/.mu.l) 2
Primer (5 pmol/.mu.l) 2 dH.sub.2O 16 One shot LA PCR Mix.TM. 25 50
.mu.l 95.degree. C. 5 min., .fwdarw. (94.degree. C. 30 sec.,
50.degree. C. 30 sec., 72.degree. C. 60 sec.) .times. 30 cycles
[0180] The DNA fragments 1, 2 and 3 and plasmid pBluescriptII SK(+)
(manufactured by StrateGene) were digested with restriction enzymes
EcoRI/KpnI, KpnI/SphI, SphI/XhoI, EcoRI/XhoI, respectively. After
electrophoresis on 1% agarose gel, the DNA fragments were purified
from the gel using MinElute Gel Extraction Kit. Next, these three
fragments and plasmid pBluescriptII SK(+) (manufactured by
StrateGene) were connected using DNA Ligation Kit ver.2
(manufactured by Takara Shuzo), then E. coli DH5.alpha. were
transformed with the connected plasmid. Plasmid
J43-2C11scDb-pBluescriptII SK(+) was prepared from E. coli, then
the nucleotide sequence of the insert was determined (SEQ ID
NO:28). The deduced amino acid sequence is shown in sequence
listing (SEQ ID NO:29).
[0181] J43-2C11scDb-pBluescriptII SK(+) was digested with
restriction enzymes BamHI and XhoI, the BamHI-XhoI fragment out of
BamHI-XhoI and BamHI-BamHI fragments generated by the digestion was
connected with BamHI/XhoI digested mammalian expression vector
pSecTag2/Hygro B (manufactured by Invitrogen). Next, the
pSecTag2/Hygro B connected with BamHI-XhoI fragment was digested
again with restriction enzyme BamHI, and connected with the other
BamHI-BamHI fragment. E. coli DH5.alpha. were transformed with the
connected plasmid. Finally, Plasmid J43-2C11scDb-pSec/Hygro B was
prepared from E. coli, then the nucleotide sequence of thus
prepared J43-2C11bispecific antibody was determined (SEQ ID NO:30).
The deduced amino acid sequence is shown in sequence listing (SEQ
ID NO:31)
EXAMPLE 9
Expression of J43-2C11 Bispecific Antibody
[0182] 6.times.10.sup.6 293 T cells were suspended in 20 ml of
medium (DMEM containing 10% FBS) and seeded into 150 mm dish coated
with Type Ic collagen. Next day, the cells were washed with 10 ml
of DMEM and transfected with J43-2C11scDb-pSec/Hygro B by using
LipofectAMINE-plus. After 3 hours, 5 ml of DMEM containing 40% FBS
were added to the cells. At day 2, the cells were washed with 10 ml
of DMEM, added 20 ml of new DMEM, and the culture supernatant was
recovered at day 4.
[0183] The cells were discarded by centrifugation, then the
supernatant was filtrated using 0.22 .mu.m PVDF filter. The
supernatant was enclosed in dialysis tube, dialyzed against PBS
containing 40% PEG20000, and concentrated. The concentrated
supernatant was purified using HiTrap chelating HP column
(manufactured by Amersham Farmacia). To purify further, the
antibodies were purified by gel-filtration using Hiprep 16/60
Sephacryl S-200 High Resolution (manufactured by Amersham
Farmacia).
EXAMPLE 10
Suppression of T Cell Activation
[0184] Spleen was removed from BALB/c mouse, and cells were
prepared using CellStrainer (70 .mu.m Nyron). The cells were
recovered by centrifugation, and red blood cells were hemolyzed by
the addition of hemolysis buffer [NH.sub.4Cl (0.8%), KCO.sub.3
(0.1%) and EDTA (1 mM)]. The cells were washed with PBS (-) once, T
cells were enriched by using mouse CD3.sup.+T cell enrichment
column kit (manufactured by R&D), and suspended in medium
(RPMI1640 containing 10% FBS) in the proportion of 5.times.10.sup.6
cells/ml. The T cells thus prepared were seeded on 96-well plates
(2.times.10.sup.5 cells/100 .mu.l /well), which were coated
preliminarily with 5 .mu.g/ml of anti-CD3 antibodies (clone KT3) at
37.degree. C. for 3 hours, added with J43-2C11 bispecific antibody
diluted in the medium (0.01, 0.03, 0.1, 0.3, 1 and 3 .mu.g/100
.mu.l ), and cultivated for 72 hours at 37.degree. C. under 5%
CO.sub.2. After 72 hours, the culture supernatants were recovered,
and the concentrations of IFN-r in the supernatants were determined
by using Quantikine Immunoassay Kit (manufactured by R&D).
[0185] As shown in FIG. 6, J43-2C11 bispecific antibody suppressed
dose dependently the production of IFN-r from activated mouse
spleen T cells in vitro.
Sequence CWU 1
1
31 1 15 PRT Mus musculus 1 Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser
Val Asn Val Gly Glu 1 5 10 15 2 20 DNA Artificial Sequence Designed
DNA based on amino terminal sequence of monoclonal antibody J43
light chain to act as a degenerated primer 2 taygarctna cncarccncc
20 3 798 DNA Mus musculus CDS (1)..(642) mat_peptide (1)..(642) 3
tat gag ctg act cag cca cct tca gca tca gtc aat gta gga gag act 48
Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Val Asn Val Gly Glu Thr 1 5
10 15 gtc aaa atc acc tgc tct ggg gac caa ttg ccg aaa tat ttt gca
gat 96 Val Lys Ile Thr Cys Ser Gly Asp Gln Leu Pro Lys Tyr Phe Ala
Asp 20 25 30 tgg ttt cat caa agg tca gac cag acc att ttg caa gtg
ata tat gat 144 Trp Phe His Gln Arg Ser Asp Gln Thr Ile Leu Gln Val
Ile Tyr Asp 35 40 45 gat aat aag cgc ccc tcg ggg atc cct gaa aga
atc tct ggg tcc agc 192 Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg
Ile Ser Gly Ser Ser 50 55 60 tca ggg aca aca gcc acc ttg acc atc
aga gat gtc cgg gct gag gat 240 Ser Gly Thr Thr Ala Thr Leu Thr Ile
Arg Asp Val Arg Ala Glu Asp 65 70 75 80 gaa ggt gac tat tac tgt ttc
tca gga tat gtt gat agt gat agc aaa 288 Glu Gly Asp Tyr Tyr Cys Phe
Ser Gly Tyr Val Asp Ser Asp Ser Lys 85 90 95 ttg tat gtt ttt ggc
agc gga acc cag ctc acc gtc cta ggt gga ccc 336 Leu Tyr Val Phe Gly
Ser Gly Thr Gln Leu Thr Val Leu Gly Gly Pro 100 105 110 aag tct tct
ccc aaa gtc aca gtg ttt cca cct tca cct gag gag ctc 384 Lys Ser Ser
Pro Lys Val Thr Val Phe Pro Pro Ser Pro Glu Glu Leu 115 120 125 cgg
aca aac aaa gcc aca ctg gtg tgt ctg gtt aat gac ttc tac ccg 432 Arg
Thr Asn Lys Ala Thr Leu Val Cys Leu Val Asn Asp Phe Tyr Pro 130 135
140 ggt tct gca aca gtg acc tgg aag gca aat gga gca act atc aat gat
480 Gly Ser Ala Thr Val Thr Trp Lys Ala Asn Gly Ala Thr Ile Asn Asp
145 150 155 160 ggg gtg aag act aca aag cct tcc aaa cag ggc caa aac
tac atg acc 528 Gly Val Lys Thr Thr Lys Pro Ser Lys Gln Gly Gln Asn
Tyr Met Thr 165 170 175 agc agc tac cta agt ttg aca gca gac cag tgg
aaa tct cac aac agg 576 Ser Ser Tyr Leu Ser Leu Thr Ala Asp Gln Trp
Lys Ser His Asn Arg 180 185 190 gtt tcc tgc caa gtt acc cat gaa ggg
gaa act gtg gag aag agt ttg 624 Val Ser Cys Gln Val Thr His Glu Gly
Glu Thr Val Glu Lys Ser Leu 195 200 205 tcc cct gca gaa tgt ctc
taggagccca gtctttttct tagcccagga 672 Ser Pro Ala Glu Cys Leu 210
agcctggagc tacgggaccc agaatgtggt cttctctcta ttctatcaat ctcaaacctt
732 ctgctcttac ccactgagta ttcaataaag tatcattagt taatcaaaaa
aaaaaaaaaa 792 acaaaa 798 4 214 PRT Mus musculus 4 Tyr Glu Leu Thr
Gln Pro Pro Ser Ala Ser Val Asn Val Gly Glu Thr 1 5 10 15 Val Lys
Ile Thr Cys Ser Gly Asp Gln Leu Pro Lys Tyr Phe Ala Asp 20 25 30
Trp Phe His Gln Arg Ser Asp Gln Thr Ile Leu Gln Val Ile Tyr Asp 35
40 45 Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Ile Ser Gly Ser
Ser 50 55 60 Ser Gly Thr Thr Ala Thr Leu Thr Ile Arg Asp Val Arg
Ala Glu Asp 65 70 75 80 Glu Gly Asp Tyr Tyr Cys Phe Ser Gly Tyr Val
Asp Ser Asp Ser Lys 85 90 95 Leu Tyr Val Phe Gly Ser Gly Thr Gln
Leu Thr Val Leu Gly Gly Pro 100 105 110 Lys Ser Ser Pro Lys Val Thr
Val Phe Pro Pro Ser Pro Glu Glu Leu 115 120 125 Arg Thr Asn Lys Ala
Thr Leu Val Cys Leu Val Asn Asp Phe Tyr Pro 130 135 140 Gly Ser Ala
Thr Val Thr Trp Lys Ala Asn Gly Ala Thr Ile Asn Asp 145 150 155 160
Gly Val Lys Thr Thr Lys Pro Ser Lys Gln Gly Gln Asn Tyr Met Thr 165
170 175 Ser Ser Tyr Leu Ser Leu Thr Ala Asp Gln Trp Lys Ser His Asn
Arg 180 185 190 Val Ser Cys Gln Val Thr His Glu Gly Glu Thr Val Glu
Lys Ser Leu 195 200 205 Ser Pro Ala Glu Cys Leu 210 5 21 DNA
Cricetulus migratorius modified_base (1)..(1) phosphorylated 5
cccaagaggt caggagttgg a 21 6 21 DNA Cricetulus migratorius 6
ttgaccaggc atcccagggt c 21 7 21 DNA Cricetulus migratorius 7
cgtaagctgg aactctggag c 21 8 21 DNA Cricetulus migratorius 8
tggttgtgct gtcacaggca g 21 9 22 DNA Cricetulus migratorius 9
tgcacacctt cccatctgtc ct 22 10 548 DNA Mus musculus CDS (66)..(548)
sig_peptide (66)..(128) mat_peptide (129)..(548) 10 ggaggcagag
gactctagcc ctgtcttccc attcagtgag cagcactgaa aacaagacca 60 tcaac atg
gga ttg gga ctg cag tgg gtt ttc ttt gtt gct ctt tta aaa 110 Met Gly
Leu Gly Leu Gln Trp Val Phe Phe Val Ala Leu Leu Lys -20 -15 -10 ggt
gtc cac tgt gag gtg cgg ctt ctg gag tct ggt gga gga tta gtg 158 Gly
Val His Cys Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val -5 -1 1
5 10 aag cct gag ggg tca ctg aaa ctc tcc tgt gtg gcc tct gga ttc
acc 206 Lys Pro Glu Gly Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe
Thr 15 20 25 ttc agt gac tat ttc atg agc tgg gtc cgc cag gct cca
ggg aag ggg 254 Phe Ser Asp Tyr Phe Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly 30 35 40 ctg gag tgg gtt gct cac ata tac acg aaa agt
tat aat tat gca act 302 Leu Glu Trp Val Ala His Ile Tyr Thr Lys Ser
Tyr Asn Tyr Ala Thr 45 50 55 tat tac tcg ggt tcg gtg aaa ggc aga
ttc acc atc tcc aga gat gat 350 Tyr Tyr Ser Gly Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asp 60 65 70 tcc cga agc atg gtc tac ctg
caa atg aac aac ctg aga act gag gac 398 Ser Arg Ser Met Val Tyr Leu
Gln Met Asn Asn Leu Arg Thr Glu Asp 75 80 85 90 acg gcc act tat tac
tgt aca aga gat gga agc gga tat ccc tct ctg 446 Thr Ala Thr Tyr Tyr
Cys Thr Arg Asp Gly Ser Gly Tyr Pro Ser Leu 95 100 105 gat ttc tgg
ggt caa ggg acc caa gtc act gtc tcc tca gcc aca aca 494 Asp Phe Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Thr Thr 110 115 120 aca
gcc cca tct gtc tat ccc ttg gcc cct gcc tgt gac agc aca acc 542 Thr
Ala Pro Ser Val Tyr Pro Leu Ala Pro Ala Cys Asp Ser Thr Thr 125 130
135 aaa tcg 548 Lys Ser 140 11 161 PRT Mus musculus 11 Met Gly Leu
Gly Leu Gln Trp Val Phe Phe Val Ala Leu Leu Lys Gly -20 -15 -10 Val
His Cys Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Lys -5 -1 1
5 10 Pro Glu Gly Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr
Phe 15 20 25 Ser Asp Tyr Phe Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 30 35 40 Glu Trp Val Ala His Ile Tyr Thr Lys Ser Tyr
Asn Tyr Ala Thr Tyr 45 50 55 Tyr Ser Gly Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser 60 65 70 75 Arg Ser Met Val Tyr Leu Gln
Met Asn Asn Leu Arg Thr Glu Asp Thr 80 85 90 Ala Thr Tyr Tyr Cys
Thr Arg Asp Gly Ser Gly Tyr Pro Ser Leu Asp 95 100 105 Phe Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Thr Thr Thr 110 115 120 Ala
Pro Ser Val Tyr Pro Leu Ala Pro Ala Cys Asp Ser Thr Thr Lys 125 130
135 Ser 140 12 21 DNA Mus musculus 12 gaggtgcagc tggtggagtc t 21 13
22 DNA Mus musculus 13 tgaggagacg gtgaccatgg tt 22 14 22 DNA Mus
musculus 14 gacatccaga tgacccagtc tc 22 15 23 DNA Mus musculus 15
tttgatttcc agcttggtgc cag 23 16 31 DNA Mus musculus 16 tttgaattca
gaggtgcggc ttctggagtc t 31 17 23 DNA Mus musculus 17 gatcaggagc
ttaggagctt tcc 23 18 23 DNA Mus musculus 18 caggccagtc aggacattag
caa 23 19 23 DNA Mus musculus 19 taatgtatgc gaccgactcc agc 23 20 23
DNA Mus musculus 20 tgaggcctct ggattcacct tca 23 21 37 DNA Mus
musculus 21 aaaaaaaaac tcgaggacct aggacggtga gctgggt 37 22 65 DNA
Artificial Sequence Designed DNA to act as a linker between the
heavy chain of J43 and the light chain of 145-2C11 22 agggacccaa
gtcactgtct cctcaggtgg aggcggttca gacatccaga tgacccagtc 60 tccat 65
23 65 DNA Artificial Sequence Designed DNA to act as a linker
between the heavy chain of J43 and the light chain of 145-2C11 23
tccctgggtt cagtgacaga ggagtccacc tccgccaagt ctgtaggtct actgggtcag
60 aggta 65 24 95 DNA Artificial Sequence Designed DNA to act as a
linker between the heavy and light chains of 145-2C11 24 acctggcacc
aagctggaaa tcaaaggtgg aggcggttca ggcggaggtg gctctggcgg 60
tggcggatcg gaggtgcagc tggtggagtc tgggg 95 25 95 DNA Artificial
Sequence Designed DNA to act as a linker between the heavy and
light chains of 145-2C11 25 tggaccgtgg ttcgaccttt agtttccacc
tccgccaagt ccgcctccac cgagaccgcc 60 accgcctagc ctccacgtcg
accacctcag acccc 95 26 65 DNA Artificial Sequence Designed DNA to
act as a linker between the heavy chain of 145-2C11 and the light
chain of J43 26 aggaaccatg gtcaccgtct cctcaggtgg aggcggttca
tatgagctga ctcagccacc 60 ttcag 65 27 65 DNA Artificial Sequence
Designed DNA to act as a linker between the heavy chain of 145-2C11
and the light chain of J43 27 tccttggtac cagtggcaga ggagtccacc
tccgccaagt atactcgact gagtcggtgg 60 aagtc 65 28 1437 DNA Artificial
Sequence Designed DNA to produce the bispecific antibody 28 gag gtg
cgg ctt ctg gag tct ggt gga gga tta gtg aag cct gag ggg 48 Glu Val
Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly 1 5 10 15
tca ctg aaa ctc tcc tgt gtg gcc tct gga ttc acc ttc agt gac tat 96
Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30 ttc atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg
gtt 144 Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 gct cac ata tac acg aaa agt tat aat tat gca act tat
tac tcg ggt 192 Ala His Ile Tyr Thr Lys Ser Tyr Asn Tyr Ala Thr Tyr
Tyr Ser Gly 50 55 60 tcg gtg aaa ggc aga ttc acc atc tcc aga gat
gat tcc cga agc atg 240 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asp Ser Arg Ser Met 65 70 75 80 gtc tac ctg caa atg aac aac ctg aga
act gag gac acg gcc act tat 288 Val Tyr Leu Gln Met Asn Asn Leu Arg
Thr Glu Asp Thr Ala Thr Tyr 85 90 95 tac tgt aca aga gat gga agc
gga tat ccc tct ctg gat ttc tgg ggt 336 Tyr Cys Thr Arg Asp Gly Ser
Gly Tyr Pro Ser Leu Asp Phe Trp Gly 100 105 110 caa ggg acc caa gtc
act gtc tcc tca ggt gga ggc ggt tca gac atc 384 Gln Gly Thr Gln Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile 115 120 125 cag atg acc
cag tct cca tca tca ctg cct gcc tcc ctg gga gac aga 432 Gln Met Thr
Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly Asp Arg 130 135 140 gtc
act atc aat tgt cag gcc agt cag gac att agc aat tat tta aac 480 Val
Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 145 150
155 160 tgg tac cag cag aaa cca ggg aaa gct cct aag ctc ctg atc tat
tat 528 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Tyr 165 170 175 aca aat aaa ttg gca gat gga gtc cca tca agg ttc agt
ggc agt ggt 576 Thr Asn Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly 180 185 190 tct ggg aga gat tct tct ttc act atc agc agc
ctg gaa tcc gaa gat 624 Ser Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser
Leu Glu Ser Glu Asp 195 200 205 att gga tct tat tac tgt caa cag tat
tat aac tat ccg tgg acg ttc 672 Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr
Tyr Asn Tyr Pro Trp Thr Phe 210 215 220 gga cct ggc acc aag ctg gaa
atc aaa ggt gga ggc ggt tca ggc gga 720 Gly Pro Gly Thr Lys Leu Glu
Ile Lys Gly Gly Gly Gly Ser Gly Gly 225 230 235 240 ggt ggc tct ggc
ggt ggc gga tcg gag gtg cag ctg gtg gag tct ggg 768 Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 245 250 255 gga ggc
ttg gtg cag cct gga aag tcc ctg aaa ctc tcc tgt gag gcc 816 Gly Gly
Leu Val Gln Pro Gly Lys Ser Leu Lys Leu Ser Cys Glu Ala 260 265 270
tct gga ttc acc ttc agc ggc tat ggc atg cac tgg gtc cgc cag gct 864
Ser Gly Phe Thr Phe Ser Gly Tyr Gly Met His Trp Val Arg Gln Ala 275
280 285 cca ggg agg ggg ctg gag tcg gtc gca tac att act agt agt agt
att 912 Pro Gly Arg Gly Leu Glu Ser Val Ala Tyr Ile Thr Ser Ser Ser
Ile 290 295 300 aat atc aaa tat gct gac gct gtg aaa ggc cgg ttc acc
gtc tcc aga 960 Asn Ile Lys Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr
Val Ser Arg 305 310 315 320 gac aat gcc aag aac tta ctg ttt cta caa
atg aac att ctc aag tct 1008 Asp Asn Ala Lys Asn Leu Leu Phe Leu
Gln Met Asn Ile Leu Lys Ser 325 330 335 gag gac aca gcc atg tac tac
tgt gca aga ttc gac tgg gac aaa aat 1056 Glu Asp Thr Ala Met Tyr
Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn 340 345 350 tac tgg ggc caa
gga acc atg gtc acc gtc tcc tca ggt gga ggc ggt 1104 Tyr Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly 355 360 365 tca
tat gag ctg act cag cca cct tca gca tca gtc aat gta gga gag 1152
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Val Asn Val Gly Glu 370
375 380 act gtc aaa atc acc tgc tct ggg gac caa ttg ccg aaa tat ttt
gca 1200 Thr Val Lys Ile Thr Cys Ser Gly Asp Gln Leu Pro Lys Tyr
Phe Ala 385 390 395 400 gat tgg ttt cat caa agg tca gac cag acc att
ttg caa gtg ata tat 1248 Asp Trp Phe His Gln Arg Ser Asp Gln Thr
Ile Leu Gln Val Ile Tyr 405 410 415 gat gat aat aag cgc ccc tcg ggg
atc cct gaa aga atc tct ggg tcc 1296 Asp Asp Asn Lys Arg Pro Ser
Gly Ile Pro Glu Arg Ile Ser Gly Ser 420 425 430 agc tca ggg aca aca
gcc acc ttg acc atc aga gat gtc cgg gct gag 1344 Ser Ser Gly Thr
Thr Ala Thr Leu Thr Ile Arg Asp Val Arg Ala Glu 435 440 445 gat gaa
ggt gac tat tac tgt ttc tca gga tat gtt gat agt gat agc 1392 Asp
Glu Gly Asp Tyr Tyr Cys Phe Ser Gly Tyr Val Asp Ser Asp Ser 450 455
460 aaa ttg tat gtt ttt ggc agc gga acc cag ctc acc gtc cta ggt
1437 Lys Leu Tyr Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Gly
465 470 475 29 479 PRT Artificial Sequence Designed DNA to produce
the bispecific antibody 29 Glu Val Arg Leu Leu Glu Ser Gly Gly Gly
Leu Val Lys Pro Glu Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Phe Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala His Ile Tyr
Thr Lys Ser Tyr Asn Tyr Ala Thr Tyr Tyr Ser Gly 50 55 60 Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Ser Met 65 70 75 80
Val Tyr Leu Gln Met Asn Asn Leu Arg Thr Glu Asp Thr Ala Thr Tyr 85
90 95 Tyr Cys Thr Arg Asp Gly Ser Gly Tyr Pro Ser Leu Asp Phe Trp
Gly
100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Asp Ile 115 120 125 Gln Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser
Leu Gly Asp Arg 130 135 140 Val Thr Ile Asn Cys Gln Ala Ser Gln Asp
Ile Ser Asn Tyr Leu Asn 145 150 155 160 Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr Tyr 165 170 175 Thr Asn Lys Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 180 185 190 Ser Gly Arg
Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp 195 200 205 Ile
Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr Phe 210 215
220 Gly Pro Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
225 230 235 240 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
Glu Ser Gly 245 250 255 Gly Gly Leu Val Gln Pro Gly Lys Ser Leu Lys
Leu Ser Cys Glu Ala 260 265 270 Ser Gly Phe Thr Phe Ser Gly Tyr Gly
Met His Trp Val Arg Gln Ala 275 280 285 Pro Gly Arg Gly Leu Glu Ser
Val Ala Tyr Ile Thr Ser Ser Ser Ile 290 295 300 Asn Ile Lys Tyr Ala
Asp Ala Val Lys Gly Arg Phe Thr Val Ser Arg 305 310 315 320 Asp Asn
Ala Lys Asn Leu Leu Phe Leu Gln Met Asn Ile Leu Lys Ser 325 330 335
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn 340
345 350 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly
Gly 355 360 365 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Val Asn
Val Gly Glu 370 375 380 Thr Val Lys Ile Thr Cys Ser Gly Asp Gln Leu
Pro Lys Tyr Phe Ala 385 390 395 400 Asp Trp Phe His Gln Arg Ser Asp
Gln Thr Ile Leu Gln Val Ile Tyr 405 410 415 Asp Asp Asn Lys Arg Pro
Ser Gly Ile Pro Glu Arg Ile Ser Gly Ser 420 425 430 Ser Ser Gly Thr
Thr Ala Thr Leu Thr Ile Arg Asp Val Arg Ala Glu 435 440 445 Asp Glu
Gly Asp Tyr Tyr Cys Phe Ser Gly Tyr Val Asp Ser Asp Ser 450 455 460
Lys Leu Tyr Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Gly 465 470
475 30 1656 DNA Artificial Sequence Designed protein to act as the
bispecific antibody 30 atg gag aca gac aca ctc ctg cta tgg gta ctg
ctg ctc tgg gtt cca 48 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro -20 -15 -10 ggt tcc act ggt gac gcg gcc cag ccg
gcc agg cgc gcg cgc cgt acg 96 Gly Ser Thr Gly Asp Ala Ala Gln Pro
Ala Arg Arg Ala Arg Arg Thr -5 -1 1 5 10 aag ctt ggt acc gag ctc
gga tcc ccc ggg ctg cag gaa ttc gag gtg 144 Lys Leu Gly Thr Glu Leu
Gly Ser Pro Gly Leu Gln Glu Phe Glu Val 15 20 25 cgg ctt ctg gag
tct ggt gga gga tta gtg aag cct gag ggg tca ctg 192 Arg Leu Leu Glu
Ser Gly Gly Gly Leu Val Lys Pro Glu Gly Ser Leu 30 35 40 aaa ctc
tcc tgt gtg gcc tct gga ttc acc ttc agt gac tat ttc atg 240 Lys Leu
Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr Phe Met 45 50 55
agc tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtt gct cac 288
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His 60
65 70 75 ata tac acg aaa agt tat aat tat gca act tat tac tcg ggt
tcg gtg 336 Ile Tyr Thr Lys Ser Tyr Asn Tyr Ala Thr Tyr Tyr Ser Gly
Ser Val 80 85 90 aaa ggc aga ttc acc atc tcc aga gat gat tcc cga
agc atg gtc tac 384 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg
Ser Met Val Tyr 95 100 105 ctg caa atg aac aac ctg aga act gag gac
acg gcc act tat tac tgt 432 Leu Gln Met Asn Asn Leu Arg Thr Glu Asp
Thr Ala Thr Tyr Tyr Cys 110 115 120 aca aga gat gga agc gga tat ccc
tct ctg gat ttc tgg ggt caa ggg 480 Thr Arg Asp Gly Ser Gly Tyr Pro
Ser Leu Asp Phe Trp Gly Gln Gly 125 130 135 acc caa gtc act gtc tcc
tca ggt gga ggc ggt tca gac atc cag atg 528 Thr Gln Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 140 145 150 155 acc cag tct
cca tca tca ctg cct gcc tcc ctg gga gac aga gtc act 576 Thr Gln Ser
Pro Ser Ser Leu Pro Ala Ser Leu Gly Asp Arg Val Thr 160 165 170 atc
aat tgt cag gcc agt cag gac att agc aat tat tta aac tgg tac 624 Ile
Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr 175 180
185 cag cag aaa cca ggg aaa gct cct aag ctc ctg atc tat tat aca aat
672 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asn
190 195 200 aaa ttg gca gat gga gtc cca tca agg ttc agt ggc agt ggt
tct ggg 720 Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly 205 210 215 aga gat tct tct ttc act atc agc agc ctg gaa tcc
gaa gat att gga 768 Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser
Glu Asp Ile Gly 220 225 230 235 tct tat tac tgt caa cag tat tat aac
tat ccg tgg acg ttc gga cct 816 Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn
Tyr Pro Trp Thr Phe Gly Pro 240 245 250 ggc acc aag ctg gaa atc aaa
ggt gga ggc ggt tca ggc gga ggt ggc 864 Gly Thr Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser Gly Gly Gly Gly 255 260 265 tct ggc ggt ggc gga
tcg gag gtg cag ctg gtg gag tct ggg gga ggc 912 Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 270 275 280 ttg gtg cag
cct gga aag tcc ctg aaa ctc tcc tgt gag gcc tct gga 960 Leu Val Gln
Pro Gly Lys Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly 285 290 295 ttc
acc ttc agc ggc tat ggc atg cac tgg gtc cgc cag gct cca ggg 1008
Phe Thr Phe Ser Gly Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 300
305 310 315 agg ggg ctg gag tcg gtc gca tac att act agt agt agt att
aat atc 1056 Arg Gly Leu Glu Ser Val Ala Tyr Ile Thr Ser Ser Ser
Ile Asn Ile 320 325 330 aaa tat gct gac gct gtg aaa ggc cgg ttc acc
gtc tcc aga gac aat 1104 Lys Tyr Ala Asp Ala Val Lys Gly Arg Phe
Thr Val Ser Arg Asp Asn 335 340 345 gcc aag aac tta ctg ttt cta caa
atg aac att ctc aag tct gag gac 1152 Ala Lys Asn Leu Leu Phe Leu
Gln Met Asn Ile Leu Lys Ser Glu Asp 350 355 360 aca gcc atg tac tac
tgt gca aga ttc gac tgg gac aaa aat tac tgg 1200 Thr Ala Met Tyr
Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp 365 370 375 ggc caa
gga acc atg gtc acc gtc tcc tca ggt gga ggc ggt tca tat 1248 Gly
Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Tyr 380 385
390 395 gag ctg act cag cca cct tca gca tca gtc aat gta gga gag act
gtc 1296 Glu Leu Thr Gln Pro Pro Ser Ala Ser Val Asn Val Gly Glu
Thr Val 400 405 410 aaa atc acc tgc tct ggg gac caa ttg ccg aaa tat
ttt gca gat tgg 1344 Lys Ile Thr Cys Ser Gly Asp Gln Leu Pro Lys
Tyr Phe Ala Asp Trp 415 420 425 ttt cat caa agg tca gac cag acc att
ttg caa gtg ata tat gat gat 1392 Phe His Gln Arg Ser Asp Gln Thr
Ile Leu Gln Val Ile Tyr Asp Asp 430 435 440 aat aag cgc ccc tcg ggg
atc cct gaa aga atc tct ggg tcc agc tca 1440 Asn Lys Arg Pro Ser
Gly Ile Pro Glu Arg Ile Ser Gly Ser Ser Ser 445 450 455 ggg aca aca
gcc acc ttg acc atc aga gat gtc cgg gct gag gat gaa 1488 Gly Thr
Thr Ala Thr Leu Thr Ile Arg Asp Val Arg Ala Glu Asp Glu 460 465 470
475 ggt gac tat tac tgt ttc tca gga tat gtt gat agt gat agc aaa ttg
1536 Gly Asp Tyr Tyr Cys Phe Ser Gly Tyr Val Asp Ser Asp Ser Lys
Leu 480 485 490 tat gtt ttt ggc agc gga acc cag ctc acc gtc cta ggt
cct cga gga 1584 Tyr Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu
Gly Pro Arg Gly 495 500 505 ggg ccc gaa caa aaa ctc atc tca gaa gag
gat ctg aat agc gcc gtc 1632 Gly Pro Glu Gln Lys Leu Ile Ser Glu
Glu Asp Leu Asn Ser Ala Val 510 515 520 gac cat cat cat cat cat cat
tga 1656 Asp His His His His His His 525 530 31 551 PRT Artificial
Sequence Designed protein to act as the bispecific antibody 31 Met
Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20 -15
-10 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr
-5 -1 1 5 10 Lys Leu Gly Thr Glu Leu Gly Ser Pro Gly Leu Gln Glu
Phe Glu Val 15 20 25 Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Lys
Pro Glu Gly Ser Leu 30 35 40 Lys Leu Ser Cys Val Ala Ser Gly Phe
Thr Phe Ser Asp Tyr Phe Met 45 50 55 Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His 60 65 70 75 Ile Tyr Thr Lys Ser
Tyr Asn Tyr Ala Thr Tyr Tyr Ser Gly Ser Val 80 85 90 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asp Ser Arg Ser Met Val Tyr 95 100 105 Leu
Gln Met Asn Asn Leu Arg Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 110 115
120 Thr Arg Asp Gly Ser Gly Tyr Pro Ser Leu Asp Phe Trp Gly Gln Gly
125 130 135 Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile
Gln Met 140 145 150 155 Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu
Gly Asp Arg Val Thr 160 165 170 Ile Asn Cys Gln Ala Ser Gln Asp Ile
Ser Asn Tyr Leu Asn Trp Tyr 175 180 185 Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Asn 190 195 200 Lys Leu Ala Asp Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 205 210 215 Arg Asp Ser
Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp Ile Gly 220 225 230 235
Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Pro 240
245 250 Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly
Gly 255 260 265 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser
Gly Gly Gly 270 275 280 Leu Val Gln Pro Gly Lys Ser Leu Lys Leu Ser
Cys Glu Ala Ser Gly 285 290 295 Phe Thr Phe Ser Gly Tyr Gly Met His
Trp Val Arg Gln Ala Pro Gly 300 305 310 315 Arg Gly Leu Glu Ser Val
Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile 320 325 330 Lys Tyr Ala Asp
Ala Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn 335 340 345 Ala Lys
Asn Leu Leu Phe Leu Gln Met Asn Ile Leu Lys Ser Glu Asp 350 355 360
Thr Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp 365
370 375 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Tyr 380 385 390 395 Glu Leu Thr Gln Pro Pro Ser Ala Ser Val Asn Val
Gly Glu Thr Val 400 405 410 Lys Ile Thr Cys Ser Gly Asp Gln Leu Pro
Lys Tyr Phe Ala Asp Trp 415 420 425 Phe His Gln Arg Ser Asp Gln Thr
Ile Leu Gln Val Ile Tyr Asp Asp 430 435 440 Asn Lys Arg Pro Ser Gly
Ile Pro Glu Arg Ile Ser Gly Ser Ser Ser 445 450 455 Gly Thr Thr Ala
Thr Leu Thr Ile Arg Asp Val Arg Ala Glu Asp Glu 460 465 470 475 Gly
Asp Tyr Tyr Cys Phe Ser Gly Tyr Val Asp Ser Asp Ser Lys Leu 480 485
490 Tyr Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Gly Pro Arg Gly
495 500 505 Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser
Ala Val 510 515 520 Asp His His His His His His 525 530
* * * * *