U.S. patent application number 15/868892 was filed with the patent office on 2018-08-16 for subtypes of humanized antibody against interleukin-6 receptor.
The applicant listed for this patent is CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Katsuhiro KANO, Isamu TERASHIMA.
Application Number | 20180230222 15/868892 |
Document ID | / |
Family ID | 34993643 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180230222 |
Kind Code |
A1 |
KANO; Katsuhiro ; et
al. |
August 16, 2018 |
SUBTYPES OF HUMANIZED ANTIBODY AGAINST INTERLEUKIN-6 RECEPTOR
Abstract
An antibody subtype (1) which is a subtype of the humanized PM-1
antibody against interleukin-6 receptor (IL-6R) and in which one
C-terminal of the heavy chain is Pro-NH.sub.2 (447), and an
antibody subtype (2) which is a subtype of the humanized PM-1
antibody against interleukin-6 receptor (IL-6R) and in which both
C-terminals of the heavy chain are Pro-NH.sub.2 (447), and a
pharmaceutical composition comprising them.
Inventors: |
KANO; Katsuhiro; (Tokyo,
JP) ; TERASHIMA; Isamu; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHUGAI SEIYAKU KABUSHIKI KAISHA |
Tokyo |
|
JP |
|
|
Family ID: |
34993643 |
Appl. No.: |
15/868892 |
Filed: |
January 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14836813 |
Aug 26, 2015 |
9902777 |
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15868892 |
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14252648 |
Apr 14, 2014 |
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14836813 |
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13722919 |
Dec 20, 2012 |
8734800 |
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14252648 |
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10593786 |
Aug 26, 2008 |
8398980 |
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PCT/JP2005/006229 |
Mar 24, 2005 |
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13722919 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 13/12 20180101; C07K 2317/52 20130101; A61P 17/06 20180101;
A61P 31/04 20180101; C07K 2317/92 20130101; C07K 2317/24 20130101;
C07K 2317/14 20130101; A61P 19/02 20180101; C07K 2317/76 20130101;
C07K 2317/40 20130101; A61P 35/00 20180101; A61K 39/3955 20130101;
C07K 16/2866 20130101; A61P 1/18 20180101; A61P 37/02 20180101;
A61P 29/00 20180101; A61P 1/04 20180101; A61P 31/18 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 24, 2004 |
JP |
2004087578 |
Claims
1-6. (canceled)
7. An antibody subtype 1 against interleukin-6 receptor (IL-6R)
obtained by a process comprising the steps of: a) culturing CHO
cells comprising at least one nucleic acid encoding for a heavy
chain and at least one nucleic acid encoding for a light chain of a
humanized PM-1 antibody, said culturing in a medium supplemented
with a protein hydrolysate for expression of said humanized PM-1
antibody; and b) isolating said antibody subtype 1 expressed by
said CHO cells, wherein said antibody subtype 1 comprises a heavy
chain that has amino acids 1-448 of the amino acid sequence set
forth in SEQ ID NO: 1, a heavy chain that has amino acids 1-447 of
the amino acid sequence set forth in SEQ ID NO: 1 and a C-terminal
that is Pro-NH.sub.2, and a light chain that has the amino acid
sequence set forth in SEQ ID NO: 2.
8. An antibody subtype against interleukin-6 receptor (IL-6R)
obtained by a process comprising the steps of: a) culturing CHO
cells comprising at least one nucleic acid encoding for a heavy
chain and at least one nucleic acid encoding for a light chain of a
humanized PM-1 antibody, said culturing in a medium supplemented
with a protein hydrolysate for expression of said humanized PM-1
antibody; and b) isolating said antibody subtype expressed by said
CHO cells, wherein said antibody subtype comprises a heavy chain
that has amino acids 1-448 of the amino acid sequence set forth in
SEQ ID NO: 1 in which an N-terminal glutamine (Gln) is
pyroglutamylated, a heavy chain that has amino acids 1-447 of the
amino acid sequence set forth in SEQ ID NO: 1 in which an
N-terminal glutamine (Gln) is pyroglutamylated and a C-terminal
that is Pro-NH.sub.2, and a light chain that has the amino acid
sequence set forth in SEQ ID NO: 2.
9. An antibody subtype 2 against interleukin-6 receptor (IL-6R)
obtained by a process comprising the steps of: a) culturing CHO
cells comprising at least one nucleic acid encoding for a heavy
chain and at least one nucleic acid encoding for a light chain of a
humanized PM-1 antibody, said culturing in a medium supplemented
with a protein hydrolysate for expression of said humanized PM-1
antibody; and b) isolating said antibody subtype 2 expressed by
said CHO cells, wherein said antibody subtype 2 comprises two heavy
chains that have amino acids 1-447 of the amino acid sequence set
forth in SEQ ID NO: 1 and a C-terminal that is Pro-NH.sub.2, and a
light chain that has the amino acid sequence set forth in SEQ ID
NO: 2.
10. An antibody subtype against interleukin-6 receptor (IL-6R)
obtained by a process comprising the steps of: a) culturing CHO
cells comprising at least one nucleic acid encoding for a heavy
chain and at least one nucleic acid encoding for a light chain of a
humanized PM-1 antibody, said culturing in a medium supplemented
with a protein hydrolysate for expression of said humanized PM-1
antibody; and b) isolating said antibody subtype expressed by said
CHO cells, wherein said antibody subtype comprises two heavy chains
that have amino acids 1-447 of the amino acid sequence set forth in
SEQ ID NO: 1 in which an N-terminal glutamine (Gln) is
pyroglutamylated and a C-terminal that is Pro-NH.sub.2, and a light
chain that has the amino acid sequence set forth in SEQ ID NO:
2.
11. An antibody subtype according to claim 7, wherein said medium
is a serum free medium.
12. An antibody subtype according to claim 7, wherein said protein
hydrolysate is derived from beef, pork, soy beans, rice, vegetables
or fish meat.
13. An antibody subtype according to claim 7, wherein said protein
hydrolysate is a fish meat-derived protein hydrolysate.
14. An antibody subtype according to claim 7, wherein said protein
hydrolysate is a vegetable-derived protein hydrolysate.
15. A pharmaceutical composition comprising the antibody subtype of
claim 7, and a pharmaceutical acceptable carrier.
16. A pharmaceutical composition comprising the antibody subtype of
claim 24, and a pharmaceutical acceptable carrier.
17. A pharmaceutical composition comprising the antibody subtype of
claim 9, and a pharmaceutical acceptable carrier.
18. A pharmaceutical composition comprising the antibody subtype of
claim 10, and a pharmaceutical acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/836,813, filed Aug. 26, 2015, now U.S. Pat. No. 9,902,777,
which is a continuation of U.S. application Ser. No. 14/252,648,
filed Apr. 14, 2014, which is a continuation of U.S. application
Ser. No. 13/722,919, filed Dec. 20, 2012, now U.S. Pat. No.
8,734,800, which is a continuation of U.S. application Ser. No.
10/593,786, filed Aug. 26, 2008, now U.S. Pat. No. 8,398,980, which
is a U.S. National Phase Application filed under 35 U.S.C. .sctn.
371 of International Application No. PCT/JP2005/006229 filed Mar.
24, 2005, which claims priority to Japanese application
2004-087578, filed Mar. 24, 2004. The disclosures of these
applications are incorporated herein by reference in their
entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
350292003103SEQLISTING.TXT, date recorded: Nov. 13, 2015, size: 7
KB)
TECHNICAL FIELD
[0003] The present invention relates to novel subtypes of the
humanized. PM-1 antibody which is an antibody against interleukin-6
receptor (IL-6R).
BACKGROUND ART
[0004] Though proteins produced by gene recombinant technology
should have an amino acid sequence predicted from the gene
sequence, a number of variants may actually be produced. This is
due to known or novel in vivo (post-transcription) modification or
naturally occurring (non-enzymatic) proteolysis (R. J. Harris, J.
Chromatogr. A 705 (1995) 129-134). Since proteins for use as
ingredients of pharmaceutical drugs are produced by gene
recombinant technology utilizing in vivo biosynthetic processes,
there is a possibility that subtypes having different molecular
structures may be produced. The kinds and contents of subtypes
define the quality of pharmaceutical drugs, and therefore it is
important to characterize the subtype profiles and assure their
usefulness as pharmaceutical compositions.
[0005] IL-6 is a cytokine which is also called B cell stimulating
factor 2 (BSF2) or interferon .beta.2. IL6 was discovered as a
differentiation factor involved in the activation of B-lymphatic
cells (Hirano, T. et al., Nature (1986) 324, 73-76). Thereafter, it
was found to be a multifunctional cytokine that influences various
functions of the cell (Akira, S. et al., Adv. in Immunology (1993)
54, 1-78). IL-6 has been reported to induce the maturation of
T-lymphatic cells (Lott, M. et al., J. Exp. Med. (1988) 167,
1253-1258).
[0006] IL-6 transmits its biological activity through two types of
proteins on the cell. One type is interleukin-6 receptor (IL-6R), a
ligand-biding protein with a molecular weight of about 80 kD, to
which IL-6 binds (Taga, T. et al., J. Exp. Med. (1987) 166,
967-981; Yamasaki, K. et al., Science (1987) 241, 825-828). IL-6R
occurs not only in the membrane-bound form that penetrates through
and is expressed on the cell membrane but also as a soluble IL-6R
consisting mainly of the extracellular region.
[0007] Anti-IL-6R antibody has been described in several reports
(Novick D. et. al., Hybridoma (1991) 10, 137-146, Huang; Y. W. et
al., Hybridoma (1993) 12, 621-630; International Patent Publication
WO 95-09873; French Patent Application FR 2694767; United States
Patent U.S. Ser. No. 521,628). A known Humanized PM-1 antibody was
obtained by transplanting the complementarity determining region
(CDR) of a mouse antibody PM-1 (Hirata, Y. et al., J. Immunol.
(1989) 143, 2900-2906), to a human antibody (International Patent
Publication WO 92-19759).
[0008] However, subtypes of the humanized PM-1 antibody are not
known.
[0009] Patent document 1: WO 92/19759
[0010] Patent document 2: Japanese Unexamined Patent Publication
(Kokai) No. 8-99902
[0011] Patent document 3: French Patent Publication FR 2694767
[0012] Patent document 4: U.S. Pat. No. U.S. Ser. No. 521,628
[0013] Non-patent document 1: R. J. Harris, J. Chromatogr. A 705
(1995) 129-134
[0014] Non-patent document 2: Hirano, T. et al., Nature (1986) 324,
73-76
[0015] Non-patent document 3: Akira, S. et al., Adv. in Immunology
(1993) 54, 1-78
[0016] Non-patent document Lotz, M. et al., J. Exp. Med. (1988)
167, 1253-1258
[0017] Non-patent document 5: Taga, J. Exp. Med. (1987) 166,
967-981
[0018] Non-patent document 6: Yamasaki, K. et al., Science (1987)
241, 825-828
[0019] Non-patent document 7: Novick, D. et al., Hybridoma (1991)
10, 137-146
[0020] Non-patent document 8: Huang, Y. W. et al., Hybridoma (1993)
12, 621-630
[0021] Non-patent document 9: Hirata, Y. et al., J. Immunol. (1989)
143, 2900-2906
DISCLOSURE OF THE INVENTION
[0022] Thus, the present invention relates to novel subtypes of the
humanized PM-1 antibody and pharmaceutical composition comprising
said subtypes.
[0023] After careful separation of the recombinantly produced
humanized PM-1 antibody, the present inventors have found that
there is a molecular species in which Gly at the C-terminal
(position 448) of the constant region constituting the heavy chain
of the humanized PM-1 antibody is lost and Pro at position 447 has
been amidated, as well as an antibody subtype (referred to as
subtype 1) in which only one of the two heavy chains constituting
the antibody has been amidated and an antibody subtype (referred to
as subtype 2) in which both have been amidated. Furthermore, the
present inventors have found that both of the above subtypes retain
the same antigen-binding activity and cell growth-inhibiting
activity as the native antibody in which the C-terminal is Gly
(448), and have thus completed the present invention.
[0024] Thus, the present invention provides an antibody subtype (1)
which is a subtype of the humanized PM-1 antibody against
interleukin-6 receptor (IL-6R) and in which one C-terminal of the
heavy chain is Pro-NH.sub.2 (447), and an antibody subtype (2)
which is a subtype of the humanized PM-1 antibody against
interleukin-6 receptor (IL-6R) and in which both C-terminals of the
heavy chain are Pro-NH.sub.2 (447). The native heavy chain
C-terminal of the humanized PM-1 antibody corresponding to both of
the above subtypes is Gly (448). In a preferred embodiment, the
native heavy chain corresponding to the amidated heavy chain
subtype has the amino acid sequence set forth in SEQ ID NO: 1. In a
preferred embodiment, glutamine (Gln) at the heavy chain N-terminal
has been replaced with pyroglutamic acid (pGlu). Also, in a
preferred embodiment, the light chain constituting the antibody
subtype of the present invention has the amino acid sequence set
forth in SEQ ID NO: 2.
[0025] The present invention also provides a pharmaceutical
composition comprising either subtype (1) or subtype (2) described
above, or both subtypes (1) and (2).
BRIEF EXPLANATION OF THE DRAWINGS
[0026] FIG. 1 shows the result of liquid chromatography in the
liquid chromatography (LC)-mass spectrometry (MS) of the peptide
fragment SLSLSP (SEQ ID NO: 3), in which the top graph is a
chromatogram detected by a UV at 215 nm and the bottom graph is a
base peak chromatogram.
[0027] FIG. 2 shows a mass spectrum in the liquid chromatography
(LC)-mass spectrometry (MS) of the peptide fragment SLSLSP (SEQ ID
NO: 3).
[0028] FIG. 3 shows a zoom scan spectrum in the liquid
chromatography (LC)-mass spectrometry (MS) of the peptide fragment
SLSLSP (SEQ ID NO: 3).
[0029] FIG. 4 shows the result of liquid chromatography in the
liquid chromatography (LC)-mass spectrometry (MS) of the peptide
fragment SLSLSP-NH.sub.2 (SEQ ID NO: 4), in which the top graph is
a chromatogram detected by a UV at 215 nm and the bottom graph is a
base peak chromatogram.
[0030] FIG. 5 shows a mass spectrum in the liquid chromatography
(LC)-mass spectrometry (MS) of the peptide fragment SLSLSP-NH.sub.2
(SEQ ID NO: 4).
[0031] FIG. 6 shows a zoom scan spectrum in the liquid
chromatography (LC)-mass spectrometry (MS) of the peptide fragment
SLSLSP-NH.sub.2 (SEQ ID NO: 4).
[0032] FIG. 7 shows the result of liquid chromatography in the
liquid chromatography (LC)-mass spectrometry (MS) of the mixture of
the peptide fragments SLSLSP(SEQ ID NO: 3) and SLSLSP-NH.sub.2 (SEQ
ID NO: 4), in which the top graph is a chromatogram detected by
a UV at 215 nm and the bottom graph is a base peak
chromatogram.
[0033] FIG. 8 shows a mass spectrum of the peak at a retention time
of 44 minutes in FIG. 7.
[0034] FIG. 9 shows a mass spectrum of the peak at a retention time
of 51 minutes in FIG. 7.
[0035] FIG. 10 A shows a peptide map of peptides obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody (Main)
followed by trypsin digestion; FIG. 10 B shows the MS chromatogram
of molecular weight of SLSLSPG (SEQ ID NO: 5) (selective monitoring
at m/z 660.3.+-.0.5), FIG. 10 C shows that of SLSLSP-NH.sub.2 (SEQ
ID NO: 4) (selective monitoring at m/z 602.3.+-.0.5), and FIG. 10 D
shows that of SLSLSP (SEQ ID NO: 3) (selective monitoring at m/z
603.3.+-.0.5).
[0036] FIG. 11 shows the result of LC-MS/MS analysis of a peptide
obtained by the reduction/carboxymethylation of the humanized PM-1
antibody (Main) followed by trypsin digestion, in which the top is
a chromatogram detected by a UV at 215 nm and the bottom is a base
peak chromatogram.
[0037] FIG. 12 shows a mass spectrum of the peak at a retention
time of 50 minutes in FIG. 11.
[0038] FIG. 13 shows a zoom scan spectrum of the same peak as in
FIG. 11.
[0039] FIG. 14 A shows a peptide map of peptides obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody subtype
1 followed by trypsin digestion; FIG. 14 B shows the MS
chromatogram of molecular weight of SLSLSPG (SEQ ID NO: 5)
(selective monitoring at m/z 660.3.+-.0.5), FIG. 14 C shows that of
SLSLSP-NH.sub.2 (SEQ ID NO: 4) (selective monitoring at m/z
602.3.+-.0.5), and FIG. 14 D shows that of SLSLSP (SEQ ID NO: 3)
(selective monitoring at m/z 603.3.+-.0.5).
[0040] FIG. 15 shows the result (on the peak in FIG. 17 B) of
liquid chromatography in the LC-MS/MS analysis of a peptide
obtained by the reduction/carboxymethylation of the humanized PM-1
antibody subtype 1 followed by trypsin digestion, in which the top
is a chromatogram detected by a UV at 215 nm and the bottom is a
base peak chromatogram.
[0041] FIG. 16 shows a mass spectrum of the peak at a retention
time of 48 minutes in FIG. 15.
[0042] FIG. 17 shows a zoom scan spectrum of the same peak as in
FIG. 16.
[0043] FIG. 18 shows the result (on the peak in FIG. 17 C) of
liquid chromatography in the LC-MS/MS analysis of a peptide
obtained by the reduction/carboxymethylation of the humanized PM-1
antibody subtype 1 followed by trypsin digestion, in which the top
is a chromatogram detected by a UV at 215 nm and the bottom is a
base peak chromatogram.
[0044] FIG. 19 shows a mass spectrum of the peak at a retention
time of 46 minutes in FIG. 18.
[0045] FIG. 20 shows a zoom scan spectrum of the same peak as in
FIG. 19.
[0046] FIG. 21 A shows a peptide map of peptides obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody subtype
2 followed by trypsin digestion; FIG. 21 B shows the MS
chromatogram of molecular weight of SLSLSPG (SEQ ID NO: 5)
(selective monitoring at m/z 660.3.+-.0.5), FIG. 21 C shows that of
SLSLSP-NH2 (SEQ ID NO: 4) (selective monitoring at m/z
602.3.+-.0.5), and FIG. 21 D shows that of SLSLSP (SEQ ID NO: 3)
(selective monitoring at m/z 603.3.+-.0.5).
[0047] FIG. 22 shows the result of liquid chromatography in the
LC-MS/MS analysis of a peptide obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody subtype
2 followed by trypsin digestion, in which the top is a chromatogram
detected by a UV at 215 nm and the bottom is a base peak
chromatogram.
[0048] FIG. 23 shows a mass spectrum of the peak at a retention
time of 45 minutes in FIG. 22.
[0049] FIG. 24 shows a zoom scan spectrum of the same peak as in
FIG. 23.
BEST MODE FOR CARRYING OUT THE INVENTION
[0050] The native humanized PM-1 antibody corresponding to the
antibody subtype of the present invention is an antibody in which
the complementarity determining region (CDR) in the variable region
(V region) constituting the heavy chain (H chain) and the light
chain (L chain) of a mouse monoclonal antibody termed PM-1 against
IL-6R has been replaced with the corresponding CDR region of the
human antibody V region. The amino acid sequence of the CDR of the
L chain V region of the above mouse anti-IL-6R antibody is
described in CDR 1, CDR 2 and CDR 3 on the line of L.sub.VPM-1 in
Table 2 of International Patent Application WO 92/19759, and the
amino acid sequence of the CDR of the H chain V region of the above
mouse anti-IL-6R antibody is described in CDR I, CDR 2 and CDR 3 on
the line of H.sub.VPM-1 in Table 3 of. International Patent
Application WO 92/19759.
[0051] The framework region (FR) of the L chain V region of the
above humanized PM-1 antibody is preferably derived from a human
antibody REI, and the amino acid sequence is described in the FR1,
FR2, FR3 and FR4 on the line of REI in Table 2 of International
Patent Application WO 92/19759. Also, the framework region (FR) of
the H chain V region of the above humanized PM-1 antibody is
preferably derived from a human antibody NEW, and the amino acid
sequence is described in the FR1, FR2, FR3 and FR4 on the line of
NEW in Table 3 of International Patent Application WO 92/19759.
[0052] Furthermore, among the above V chains composed of the FR of
a human antibody and the CDR of the mouse PM-1 antibody, the FR
region may be modified in various manners to improve
antigen-binding activity and neutralization activity. For example,
the L chain V region is described in the FR1, FR2, FR3 and FR4 on
the line of RV.sub.La and RV.sub.Lb in Table 2 of International
Patent Application WO 92/19759 (termed version a to version b), and
the H chain V region is described in the FR1, FR2, FR3 and FR4 on
the line of RV.sub.Ha to RV.sub.Hf in Table 3 of International
Patent Application WO 92/19759 (termed version a to version f).
[0053] The L chain of the humanized PM-1 antibody is composed of
the L chain V region mentioned above and the constant region (C
region) of a human antibody L chain, and the H chain of the
humanized PM-1 antibody is composed of the H chain V region
mentioned above and the constant region (C region) of a human
antibody H chain. As the C region constituting the L chain, human
.gamma.-IC region is preferred, and as the C region constituting
the H chain, human .kappa.C region is preferred.
[0054] Glutamine, an N-terminal amino acid of a monoclonal
antibody, is known to be pyroglutamylated, and in the subtypes 1
and 2 of the humanized PM-1 antibody of the present invention the
N-terminal glutamine of the heavy chain may be pyroglutamylated.
Thus the subtypes 1 and 2 of the humanized. PM-1 antibody of the
present invention may be antibody subtypes in which the N-terminal
glutamine (Gln) of the heavy chain is pyroglutamic acid (pGlu).
[0055] As the L chain and the H chain of the thus constructed
humanized PM-1 antibody, as described above, there are various
versions present by the modification of the FR region, and as a
preferred example, there can be mentioned a H chain having the
amino acid sequence set forth in SEQ ID NO: 1 and a L chain having
the amino acid sequence set forth in SEQ ID NO: 2.
[0056] Incidentally, Escherichia coli (E. coli) DH5 .alpha. pPM-k3
containing a plasmid pPM-k3 comprising DNA encoding the L chain V
region of the monoclonal antibody PM-1 has been internationally
deposited as NCIMB 40366 and E. coli DH5 .alpha. pPM-h1 containing
a plasmid pPM-h1 comprising DNA encoding the H chain V region of
the monoclonal antibody PM-1 has been internationally deposited as
NCIMB 40362, under the provisions of the Budapest Treaty on Feb.
12, 1991, with the National Collections of Industrial and Marine
Bacteria Limited (Ferguson Building, Craibstone Estate, Bucksburn,
Aberdeen AB 21 9YA, United Kingdom). The hybridoma PM1 producing
the monoclonal antibody PM-1 has been internationally deposited as
FERM BP-2998 under the provisions of the Budapest Treaty on Jul.
12, 1989 with the Patent Microorganism Depository of the National
Institute of Industrial Science and Technology (Chuo 6, 1-1,
Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan).
[0057] DNA encoding the chain or the H chain having the amino acid
sequence as described above may be constructed according to a
standard method. Specifically, a DNA sequence which was designed to
ligate the CDR of a mouse antibody with the framework region (FR)
of a human antibody is synthesized by the PCR method from several
divided oligonucleotides having sections overlapping with one
another at the ends thereof. The DNA thus obtained is ligated to a
DNA encoding the C region of a human antibody and then is
integrated into an expression vector, which is introduced into a
host for antibody production (see European Patent Application EP
239400 and International Patent Publication WO 92-19759).
[0058] For the FR of a human antibody ligated through CDR, those in
which the complementarity determining region that forms a favorable
antigen binding site are selected. When desired, amino acids in the
framework region of the antibody variable region may be substituted
so that the complementarity determining region of a reshaped human
antibody may form an appropriate antigen biding site (Sato, K. et
al., Cancer Res. (1993) 53, 851-856).
[0059] For humanized antibody, the C region of a human antibody is
used. As the C region of a human antibody, there can be mentioned
C.gamma., and C.gamma.1, C.gamma.2, C.gamma.3, and C.gamma.4, for
example, can be used.
[0060] Antibody genes constructed as described above may be
expressed and obtained in a known method. In the case of mammalian
cells, expression may be accomplished using a vector containing a
commonly used useful promoter, the antibody gene to be expressed,
DNA in which the poly A signal has been operably linked at 3'
downstream thereof or a vector containing said DNA. Examples of the
promoter/enhancer include human cytomegalovirus immediate early
promoter/enhancer.
[0061] Additionally, as the promoter/enhancer which can be used for
expression of antibody for use in the present invention, there can
be used viral promoters/enhancers such as retrovirus, polyoma
virus, adenovirus, and simian virus 40 (SV40), and
promoters/enhancers derived from mammalian cells such as human
elongation factor 1.alpha. (HEF1.alpha.).
[0062] For example, expression may be readily accomplished by the
method of Mulligan et al. (Mulligan, R. C. et al., Nature (1979)
277, 108-114) when SV40 promoter/enhancer is used, or by the method
of Mizushima et al. (Mizushima, S. and Nagata, S., Nucleic Acids
Res. (1990) 18, 5322) when HEF1.alpha. promoter/enhancer is
used.
[0063] In the case of E. coli, expression may be conducted by
operably linking a commonly used useful promoter, a signal sequence
for antibody secretion, and the antibody gene to be expressed,
followed by expression thereof. As the promoter, for example, there
can be mentioned lacZ promoter and araB promoter. The method of
Ward et al. (Ward, E. S. et al., Nature (1989) 341, 544-546; Ward,
E. S. et al., FASEB J. (1992) 6, 2422-2427) may be used when lacz
promoter is used, and the method of Better et al. (Better, M. et
al., Science (1988) 240, 1041-1043) may be used when araB promoter
is used.
[0064] As the signal sequence for antibody secretion, when produced
in the periplasm of E. coli, the pelB signal sequence (Lei, S. P.
et al., J. Bacteriol. (1987) 169, 4379-4383) can be used. After
separating the antibody produced in the periplasm, the structure of
the antibody is appropriately refolded before use (see, for
example, WO 96/30394).
[0065] As the origin of replication, there can be used those
derived from SV40, polyoma virus, adenovirus, bovine papilloma
virus (BPV) and the like. Furthermore, for the amplification of the
gene copy number in the host cell system, expression vectors can
include, as selectable markers, the aminoglycoside
phosphotransferase (APH) gene, the thymidine kinase (TK) gene, E.
coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, the
dihydrofolate reductase (dhfr) gene and the like.
[0066] For the production of antibody for use in the present
invention, any production system can be used. The production system
for antibody preparation comprises an in vitro or an in vivo
production system. As the in vitro production system, there can be
mentioned a production system which employs eukaryotic cells and a
production system which employs prokaryotic cells.
[0067] When the eukaryotic cells are used, there are production
systems which employ animal cells, plant cells, or fungal cells.
Known animal cells include (1) mammalian cells such as CHO cells,
COS cells, myeloma cells, baby hamster kidney (BHK) cells, HeLa
cells, and Vero cells, (2) amphibian cells such as Xenopus oocytes,
or (3) insect cells such as sf9, sf21, and Tn5. Known plant cells
include, for example, those derived from Nicotiana tabacum, which
may be subjected to callus culture. Known fungal cells include
yeasts such as the genus Saccharomyces, more specifically
Saccharomyces cereviceae, or filamentous fungi such as the genus
Aspergillus, more specifically Aspergillus niger.
[0068] When the prokaryotic cells are used, there are production
systems which employ bacterial cells. Known bacterial cells include
Escherichia coli (E. coli), and Bacillus subtilis.
[0069] By introducing, via transformation, the gene of the desired
antibody into these cells and culturing the transformed cells in
vitro, the antibody can be obtained. Culturing is conducted in the
known methods. For example, as the culture liquid, DMEM, MEM,
RPMI1640, and IMDM can be used, and serum supplements such as fetal
calf serum (FCS) may be used in combination. In addition,
antibodies may be produced in vivo by implanting cells, into which
the antibody gene has been introduced, into the abdominal cavity of
an animal and the like.
[0070] As in vivo production systems, there can be mentioned those
which employ animals and those which employ plants. When animals
are used, there are the production systems which employ mammals and
insects.
[0071] As mammals, goats, pigs, sheep, mice, and cattle can be used
(Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Also as
insects, silkworms can be used. When plants are used, tobacco, for
example, can be used.
[0072] Antibody genes are introduced into these animals or plants,
and the antibodies are produced in such animals or plants and
recovered. For example, an antibody gene is inserted into the
middle of the gene encoding protein which is inherently produced in
the milk such as goat .beta. casein to prepare a fusion gene. The
DNA fragment containing the fusion gene into which the antibody
gene has been inserted is injected into a goat embryo, and the
embryo is introduced into a female goat. The desired antibody is
obtained from the milk produced by the transgenic goat borne to the
goat who received the embryo or offsprings thereof. In order to
increase the amount of milk containing the desired antibody
produced by the transgenic goat, hormones may be given to the
transgenic goat as appropriate. (Ebert, K. M. et al.,
Bio/Technology (1994) 12, 699-702).
[0073] When silkworms are used, baculovirus into which the desired
antibody gene has been inserted is infected to the silkworm, and
the desired antibody can be obtained from the body fluid of the
silkworm (Maeda, S. et al., Nature (1985) 315, 592-594). Moreover,
when tobacco is used, the desired antibody gene is inserted into an
expression vector for plants, for example pMON 530, and then the
vector is introduced into a bacterium such as Agrobacterium
tumefaciens. The bacterium is then infected to tobacco such as
Nicotiana tabacum to obtain the desired antibody from the leaves of
the tobacco (Julian, K.-C. Ma et al., Eur, J. Immunol. (1994) 24,
131-138).
[0074] When antibody is produced in the in vitro or the in vivo
production systems, as described above, DNA encoding the heavy
chain (H chain) or the light chain (L chain) of antibody may be
separately integrated into an expression vector and the hosts are
transformed simultaneously, or DNA encoding the H chain and the L
chain may be integrated into a single expression vector, and the
host is transformed therewith (see International Patent
[0075] Publication WO 94-11523).
[0076] In order to produce the subtypes 1 and 2 of the humanized
PM-1 antibody of the present invention, preferably cultured cells,
most preferably CHO cells, of an animal may be used as the host,
and cultured in a culture medium for animal cells. Also the medium
preferably contains peptone, a hydrolyzate of protein, and thus
there can be used peptone derived from beef, pork, soy beans, rice,
fish meat etc. Generally, animal-derived peptone has a high
expression activity, and the use of fish meat-derived peptone (for
example, bonito) has an effect on the amount expressed. In this
case, when a medium containing a mammalian peptone is used to
produce the humanized PM-1 antibody, little production of the
subtypes 1 and 2 is observed, and when a medium containing a fish
meat-derived peptone or a vegetable peptone is used to produce the
humanized PM-1 antibody, the ratio of the subtypes 1 and 2 becomes
higher. Thus, in order to produce the subtypes I or 2 of the
humanized. PM-i antibody of the present invention, preferably a
cultured animal cell of the present invention, most preferably CHO
cells, is used as the host, and it is cultured in a medium
containing a fish meat-derived peptone or a vegetable peptone.
[0077] Antibodies produced and expressed as described above can be
separated from the inside or outside of the host cell and then may
be purified to homogeneity. Separation and purification of the
antibody for use in the present invention may be accomplished by
affinity chromatography. As the column used for such affinity
chromatography, there can be mentioned Protein A column and Protein
G column. Examples of the carriers used in the Protein A column are
Hyper D, POROS, Sepharose F. F. and the like. Alternatively,
methods for separation and purification. conventionally used for
proteins can be used without any limitation.
[0078] It can also be attained by chromatographies other than the
above-mentioned affinity chromatography, such as commonly used
chromatographies, for example, a combination of general column
chromatographies such as ion exchange chromatography, hydrophobic
chromatography, hydroxyapatite chromatography, gel-filtration and
the like.
[0079] Furthermore, by combining, as appropriate, filtration,
ultrafiltration, salting-out, dialysis and the like, the antibody
for use in the present invention can be separated and purified.
These chromatographies can be applied into fast protein liquid
chromatography (FPLC) or high performance liquid chromatography
(HPLC). Alternatively, reverse-phase HPLC can be used.
[0080] The concentration of antibody obtained in the above can be
determined by the measurement of absorbance or by ELISA and the
like. Thus, when absorbance measurement is employed, a sample is
appropriately diluted with PBS(-) and then the absorbance is
measured at 280 nm, followed by calculation with 1.35 OD as 1
mg/ml. When the ELISA method is used, measurement is conducted as
follows. Thus, 100 .mu.l of goat anti-human IgG (manufactured by
TAG) diluted to 1 .mu.g/ml in 0.1 M bicarbonate buffer, pH 9.6, is
added to a 96-well plate (manufactured by Nunc), and is incubated
overnight at 4.degree. C. to immobilize the antibody. After
blocking, 100 .mu.l each of appropriately diluted antibody of the
present invention or a sample containing the antibody, or 100 .mu.l
of human IgG (manufactured by CAPPEL) as the standard is added, and
incubated at room temperature for 1 hour.
[0081] After washing, 100 .mu.l of 5000-fold diluted alkaline
phosphatase-labeled anti-human IgG antibody (manufactured by BIO
SOURCE) is added, and incubated at room temperature for 1 hour.
After washing, the substrate solution is added and incubated,
followed by the measurement of absorbance at 405 nm using the
MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate
the concentration of the desired antibody.
[0082] Since the subtype of the humanized PM-1 antibody of the
present invention has substantially the same antigen-binding
activity as the native humanized PM-1 antibody, it can be used
similarly to the native humanized PM-1 antibody for the treatment
or prevention of various diseases in which IL-6 is involved.
Examples of IL-6-related diseases include acute and chronic
inflammatory diseases, autoimmune diseases such as nephritis,
mesangial proliferative nephritis, Crohn's disease, ulcerative
colitis, pancreatitis, infantile chronic arthritis or systemic
juvenile idipathic arthritis, vasculitis, Kawasaki disease,
rheumatoid arthritis, systemic lupus erythematosus, psoriasis,
Sjogren's syndrome and adult Still's disease; neoplastic diseases
such as multiple myeloma, Castleman's disease, malignant lymphoma
and renal cancer; infectious diseases such as HIV infection and EBV
infection; cachexia; plasmacytosis, hyperimmunoglobulin disease,
anemia and the like, and preferably rheumatoid arthritis,
plasmacytosis, hyperimmunoglobulin disease, anemia, nephritis,
cachexia, multiple myeloma, Castleman's disease, mesangial
proliferative nephritis, systemic lupus erythematosus, Crohn's
disease, pancreatitis, psoriasis, and infantile chronic arthritis
or systemic juvenile idipathic arthritis.
[0083] The pharmaceutical composition of the present invention may
he administered, either orally or parenterally, systemically or
locally. For example, intravenous injection such as drip infusion,
intramuscular injection, intrapleural injection, intraperitoneal
injection, subcutaneous injection, suppositories, intestinal
lavage, oral enteric coated tablets, and the like can be selected,
and the method of administration may be chosen, as appropriate,
depending on the age and the conditions of the patient. The
effective dosage is Chosen from the range of 0.01 mg to 100 mg per
kg of body weight per administration. Alternatively, the dosage in
the range of 1 to 1000 mg, preferably 5 to 50 mg per patient may be
chosen.
[0084] Preferred dosages and preferred methods of administration
are such that, in the case of anti-IL-6 receptor antibody, the
amounts wherein free antibody is present in the blood are effective
dosages. In specific examples, 0.5 mg to 40 mg per kg of body
weight, preferably 1 mg to 20 mg, per month (4 weeks) are
administered in one to several divided doses, for example in the
administration schedule of twice per week, once per week, once
every two weeks, once every four weeks and the like by intravenous
injection such as drip infusion and subcutaneous injection. The
administration schedule can be adjusted by observing the disease
conditions and blood levels of laboratory tests by, for example,
extending the administration interval from twice per week or once
per week to once per two weeks, once per three weeks, once per four
weeks, and the like.
[0085] The pharmaceutical composition of the present invention may
contain pharmaceutically acceptable carriers or additives depending
on the route of administration. Examples of such carriers or
additives include water, a pharmaceutical acceptable organic
solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a
carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic
sodium, sodium alginate, water-soluble dextran, carboxymethyl
starch sodium, pectin, methyl cellulose, ethyl cellulose, xarithan
gum, gum Arabic, casein, gelatin, agar, diglycerin, propylene
glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol,
stearic acid, human serum albumin (HSA), mannitol, sorbitol,
lactose, a pharmaceutically acceptable surfactant and the like.
Additives used are chosen from, but not limited to, the above or
combinations thereof depending on the dosage form.
EXAMPLES
[0086] The present invention will now be explained in more detail
with reference to the working examples and reference examples. It
should be noted, however, that the present invention is not limited
to them in any way.
Example 1
The Expression of an Antibody Composition Containing the Native
Humanized PM-1 Antibody/Subtype 1/Subtype 2
Construction of Expressing Cells
(1) Preparation of Human IL-6 Receptor Antibody PM-1
[0087] Anti-IL-6R antibody MT18 prepared by the method of Hirata et
al. (J. Immunol., (1989) 143:2900-2906) was bound to CNBr-activated
Sepharose 4B (manufactured by Pharmacia Fine Chemicals, Piscataway,
N.J.) according to the attached regimen, and IL-6R (Yamasaki, K. et
al., Science (1988) 241825-828) was purified.
[0088] Thus, a human myeloma cell line U266 was solubilized with 1
mM p-paraaminophenyl methane sulfonyl fluoride hydrochloride
(manufactured by Wako Chemicals) (digitonin buffer) containing 1%
digitonin (manufactured by Wako Chemicals), 10 mM triethanolamine
(pH 7.8) and 0.15 M NaCl, and mixed with the MT18 antibody bound to
Sepharose 4B beads. Then, the beads were washed six times with the
digitonin buffer to prepare the partially purified IL-6R to be used
for immunization.
[0089] BALB/c mice were immunized four times every ten days with
the above partially purified. IL-6R. obtained from 3.times.10.sup.9
U266 cells, and then a hybridoma was prepared using a standard
method. The culture supernatant of the hybridoma from the
growth-positive well was tested for its activity of binding to
IL-6R according to the method described below. 5.times.10.sup.7
U266 cells were labeled with .sup.35S-methionine (2.5 mCi) and were
solubilized with the above digitonin buffer.
[0090] The solubilized U266 cells were mixed with a 0.04 ml volume
of MT18 antibody bound to Sepharose 4B beads, and then were washed
six times with the digitonin buffer. .sup.35S-methionine-labeled
IL-6R was eluted with 0.25 ml of the digitonin buffer (pH 3.4) and
was neutralized in 0.025 ml of 1M Tris (pH 7.4). 0.05 ml of the
hybridoma culture supernatant was mixed with 0.01 ml of Protein G
Sepharose (manufactured by Pharmacia).
[0091] After washing, Sepharose was incubated with 0.005 ml of
.sup.35S-labeled IL-6R solution prepared as described above. The
immunoprecipitate was analyzed by SDS-PAGE to investigate the
hybridoma culture supernatant that reacts with IL-6R. As a result,
a reaction-positive hybridoma clone PM-1 was established. The IL-6R
antibody PM-1 produced from the hybridoma PM-1 has a subtype of
IgG1.kappa..
[0092] The inhibitory activity of the antibody produced by the
hybridoma PM-1 on the binding of IL-6 to human IL-6R was studied
using the human myeloma cell line U266. A human recombinant IL-6
was prepared from E. coil (Hirano et al., Immunol. Lett., (1988)
17:41), and was labeled with .sup.1251 using the Bolton-Hunter.
reagent (New England Nuclear, Boston, Mass.) (Taga, T. et al., J.
Exp. Med. (1987) 166:967). 4.times.10.sup.5 U266 cells were
cultured together with the culture supernatant of 70% (v/v)
hybridoma PM-1 and 14,000 cpm of .sup.125I-labeled IL-6 in the
presence of a 100-fold excessive amount of nonlabelled. IL-6 at
room temperature for 1 hour. Seventy .mu.1 of the sample was
layered on 300 .mu.l FCS in a 400 .mu.l microfuge polyethylene
tube. After centrifugation, the radioactivity of the cell was
determined. The result revealed that the antibody produced by the
hybridoma PM-1 inhibits the binding of IL-6 to IL-6R.
(2) Creation of Humanized Antibody hPM-1
[0093] Using a human elongation factor I.alpha. promoter described
in Example 10 of International Patent Publication WO 92/19759 and
according to the method described in Reference Example 2 of
Japanese Unexamined Patent Publication (Kokai) No. 8-99902, a
single expression vector containing both the L chain and H chain
genes was constructed, and was investigated using CHO cells that
produce the humanized PM-1 antibody (anti-human IL-6 receptor
antibody), said cells being prepared by inserting the expression
vector into CHO cells. The ability of the humanized antibody
obtained to bind to human IL-6R was confirmed by ELISA.
Furthermore, hPM-1 inhibited the binding of human IL-6 to human
IL-6R in a similar manner to a mouse antibody or a chimeric
antibody.
Cell Culture and the Expression of Humanized 1 Antibody
[0094] In order to obtain a large quantity of humanized PM-1
antibody, the expressing cells were cultured in a commercially
available serum-free medium and a modified medium. The culture
condition was an environment suitable for the culturing of CHO
cells. In order to increase the amount expressed of the desired
antibody, various additives may be added to the medium. Among them,
various types of peptones are widely used. Peptones derived from
beef, pork, soy beans, rice, fish meat etc. are widely commercially
available. The effect depends on the compatibility with the cell
line. Generally, the effect of expression is high for
animal-derived peptones. In the course of investigating the effect
of various peptones, the use of a peptone derived from fish meat
(bonito) was found to be effective for the amount expressed.
[0095] The purification of the antibody expressed was accomplished
by combinations of commonly used column chromatographies, for
example, affinity chromatography, ion exchange chromatography,
hydrophobic chromatography, hydroxyapatite chromatography,
gel-filtration and the like. In a molecular species of the
humanized PM-1 antibody that was expressed by culturing CHO cells
using a peptone derived from fish meat, there was observed a
molecular species that was present in scarce amounts when
beef-derived peptone was used. This molecular species was also seen
when a vegetable-derived peptone was used.
Example 2
Analysis of the Humanized PM-1 Antibody, the Subtype 1 and the
Subtype 2
Materials and Methods
[0096] As the materials, the native humanized PM-1 antibody
(sometimes referred to as Main), the subtypes 1 and 2 of said
antibody, and, as the reference peptides, a peptide
Ser-Leu-Ser-Leu-Ser-Pro (SLSLSP) (SEQ ID NO: 3) that is present at
the C-terminal of the humanized PM-1 antibody and in which Gly at
the C-terminal has been removed and a peptide SLSLSP-NH.sub.2 (SEQ
ID NO: 4) in which the C-terminal Pro has been amidated were used.
The peptide SLSLSP (SEQ ID NO: 3) and the amidated peptide
SLSLSP-NH.sub.2 (SEQ ID NO: 4) were chemically synthesized. The
humanized PM-1 antibody Main and the subtypes 1 and 2 of said
antibody were obtained by subjecting the humanized PM-1 antibody
obtained in Example 1 to a column chromatography and
collecting and purifying it by the following method.
[0097] The column used was the Poly CAT A (100.times.4.6 mm)
manufactured by Poly LC, and the guard column used was the Poly CAT
A Javelin guard (10.times.4.0 mm) manufactured by Poly LC. The
mobile phases used were the mobile phase A (25 mM
2-[N-morpholino]ethanesulfonic acid buffer, pH 6.1, containing
0.05% NaN.sub.3) and the mobile phase B (25 mM
2-[N-morpholino]ethanesulfonic acid buffer, pH 6.1, containing 250
mM sodium acetate and 0.05% NaN.sub.3). As the gradient condition,
the ratio of the mobile phase B was 0 min/35%, 5 min/35%, 59
min/60%, and 60 min/100%. The flow rate was 1 ml/min and detected
by UV/VIS absorbance at 280 nm.
The Enzymatic Digestion of the Humanized PM-1 Antibody Main, the
Subtype 1 and the Subtype 2
[0098] 200 .mu.g equivalents of the humanized. PM-1 antibody Main,
the subtype 1 and the subtype 2 were placed in a simple
ultrafiltration cartridge (Minicent, manufactured by Toso), into
which a denaturant solution (100 mM
2-amino-2-hydroxymethyl-1,3-propanediol-hydrochloric acid buffer,
pH 8.3, containing 7 M guanidine and 1 mM
ethylenediaminetetraacetic acid) was added to a liquid volume of
500 .mu.l. The cartridge was centrifuged at 5.degree. C. to a
liquid volume of about 50 .mu.l. The sample was collected into a
microtube, to Which a denaturant solution (the same composition as
above) was added to make a total volume of 300 .mu.l.
[0099] To each solution, 50 .mu.l of a DTT solution (a denaturant
solution containing 162 mM dithiothreitol) was added and the head
space was replaced with N.sub.2, and allowed to stand in an
incubating block at 37.degree. C. for 1 hour. Furthermore, 45 .mu.l
of the IAA solution (0.2 N sodium hydroxide solution containing 417
mM iodoacetic acid) was added and allowed to stand in the dark at
37.degree. C. for 30 minutes. The reaction mixture was recovered,
and each sample was dialyzed, using dialysis tubing, against 500 ml
of the Tris-HCl buffer (100 mM
2-amino-2-hydroxymethyl-1,3-propanediol-hydrochloric acid buffer,
pH 8.0, containing 2 M urea) at 5.degree. C. for 20 hours (dialysis
membrane: M.W.=8000, manufactured by Spectrum). The dialyzed
samples were recovered and to each of them, 20 .mu.l of the trypsin
solution (trypsin is dissolved in the Tris-HCl buffer (the same
composition as above) to make 250 ng/pl) was added and allowed to
stand at 37.degree. C. for 16 hours.
Analysis of the Trypsin Digests and the Reference Peptides
[0100] Forty .mu.l of each sample treated as above was subjected to
the liquid chromatography-mass spectrometry (LC-MS/MS). For the
reference peptide solutions, i.e. the SLSLSP (SEQ ID NO: 3)solution
(SLSLSP (SEQ ID NO: 3) is dissolved in water to make 4 .mu.M) and
the SLSLSP-NH.sub.2 (SEQ ID NO: 4) solution (SLSLSP-NH.sub.2 (SEQ
ID NO: 4) is dissolved in water to make 4 .mu.M), 50 .mu.l is
subjected to the liquid chromatography-mass spectrometry.
[0101] The condition for the liquid chromatography-mass
spectrometry was as follows. Thus, the column used was the YMC-Pack
ODS (250.times.2.0 mm, 5 .mu.m, 300 Angstrom) manufactured by YMC.
The mobile phase used was the mobile phase A (5% acetonitrile
solution containing 0.1% trifluoroacetic acid) and the mobile phase
B (95% acetonitrile solution containing 0.1% trifluoroacetic acid).
As the gradient condition, the ratio of the mobile phase B was 0
min/0%, 10 min/0%, 120 min/35%, and 140 min/35%. The flow rate was
0.2 ml/min and detection was by UV/VIS absorbance at 215 nm.
Result of Analysis of the Trypsin Digests and the Reference
Peptides
(1) Measurement of the Reference Peptide Fragments (SEQ ID NO:
3)
(a) Measurement of the Peptide Fragment SLSLSP
[0102] FIG. 1 to FIG. 3 show the result of liquid chromatography
(LC)-mass spectrometry (MS) of the peptide fragment SLSLSP (SEQ ID
NO: 3). The top of FIG. 1 shows a chromatogram detected with a UV
at 215 nm, and the bottom shows a chromatogram of a base peak
chromatogram. FIG. 2 shows a mass spectrum, and FIG. 3 shows a zoom
scan spectrum. The molecular weight (602.2) obtained was in close
agreement with the theoretical value (602.3; monoisotopic molecular
weight) (FIG. 2 and FIG. 3).
(b) Measurement of the Peptide Fragment SLSLSP-NH.sub.2 (SEQ ID NO:
4)
[0103] FIG. 4 to FIG. 6 show the result of liquid chromatography
(LC)-mass spectrometry (MS) of the peptide fragment SLSLSP (SEQ ID
NO: 3). The top of FIG. 4 shows a chromatogram detected with a UV
at 215 nm, and the bottom shows a chromatogram of a base peak
chromatogram. FIG. 5 shows a mass spectrum, and FIG. 6 shows a zoom
scan spectrum. The molecular weight (601.2) obtained was in close
agreement with the theoretical value (601.3; monoisotopic molecular
weight) (FIG. 5 and FIG. 6).
(c) Measurement of the Mixture of the Peptide Fragments SLSLSP (SEQ
ID NO: 3) and SLSLSP-NH2 (SEQ ID NO: 4)
[0104] FIG. 7 to FIG. 9 show the result of liquid chromatography
(LC)-mass spectrometry (MS) of the mixture of the peptide fragment
SLSLSP (SEQ ID NO: 3) and SLSLSP-NH.sub.2(SEQ ID NO: 4). The top of
FIG. 7 shows a chromatogram detected with a UV at 215 nm, and the
bottom shows a chromatogram of a base peak chromatogram. FIG. 8
shows the mass spectrum of a peak at a retention time of 44 minutes
in FIG. 7, and FIG. 9 shows the mass spectrum of a peak at a
retention time of 51 minutes in FIG. 7. The both peptide fragments
were completely separated under the condition of the above liquid
chromatography.
(2) Analysis of the Structure of the H Chain C-Terminal of the
Humanized PM-1 Antibody
[0105] (a)Analysis of the Structure of the H Chain C-Terminal of
the Humanized PM-1 Antibody (Main)
[0106] FIG. 10 A shows a peptide map of peptides obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody (Main)
followed by trypsin digestion. In order to investigate the
structure of the C-terminal fragment of the H chain, the MS
chromatogram of SLSLSPG (SEQ ID NO: 5) (selective monitoring at m/z
660.3.+-.0.5) is shown in FIG. 10 B, that of SLSLSP-NH.sub.2 (SEQ
ID NO: 4) (selective monitoring at m/z 602.3.+-.0.5) in FIG. 10 C,
and that of SLSLSP (SEQ ID NO: 4) (selective monitoring at m/z
603.3.+-.0.5) in FIG. 10 D. A peak corresponding to SLSLSPG (SEQ ID
NO: 5) was detected at 49.7 minutes, but no peptide fragments
having the molecular weight of SLSLSP-NH.sub.2 (SEQ ID NO: 4) and
SLSLSP (SEQ ID NO: 3) were found.
[0107] FIG. 11 to FIG. 13 show the result of LC-MS/MS analysis of a
peptide obtained by the reduction/carboxymethylation of the
humanized PM-1 antibody (Main) followed by trypsin digestion. The
top in FIG. 11 shows a chromatogram detected by a UV at 215 nm and
the bottom shows a base peak chromatogram. FIG. 12 shows a mass
spectrum of the peak at a retention time of 50 minutes in FIG. 11,
and FIG. 13 shows a zoom scan spectrum of the same peak as in FIG.
11. From these results, the detected peak was shown to have the
amino acid sequence SLSLSPG (SEQ ID NO: 5). Thus, it was
demonstrated that both C-terminals of the H chain of the humanized
PM-1 antibody (Main) have the -SLSLSPG (SEQ ID NO: 5) sequence.
(d) Analysis of the Structure of the H Chain C-Terminal of the
Humanized PM-1 Antibody Subtype 1
[0108] FIG. 14 A shows a peptide map of peptides obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody subtype
1 followed by trypsin digestion. In order to investigate the
structure of the C-terminal fragment of the H chain, FIG. 14 B
shows the MS chromatogram of molecular weight of SLSLSPG (SEQ ID
NO: 5) (selective monitoring at m/z 660.3.+-.0.5). FIG. 14 C shows
that of SLSLSP-NH.sub.2 (SEQ ID NO: 4) (selective monitoring at m/z
602.3.+-.0.5), and FIG. 14 D shows that of SLSLSP (SEQ ID NO: 3)
(selective monitoring at m/z 603.3.+-.0.5). In addition to a peak
corresponding to SLSLSPG (SEQ ID NO: 5) at 47.7 minutes, a peak
corresponding to SLSLSP-NH.sub.2 (SEQ ID NO: 4) at 46.2 minutes was
noted (though a peak with a
molecular weight of 603.3 was noted at about 46 minutes in FIG. 14
D, it is not SLSLSP (SEQ ID NO: 3), based on the retention
time).
[0109] FIG. 15 to FIG. 17 show the result (on the peak in FIG. 14
B) of LC-MS/MS analysis of a peptide obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody subtype
1 followed by trypsin digestion. In FIG. 15, the top is a
chromatogram detected by a UV at 215 nm and the bottom is a base
peak chromatogram. FIG. 16 shows a mass spectrum of the peak at a
retention time of 48 minutes in FIG. 15, and FIG. 17 shows a zoom
scan spectrum of the same peak as in FIG. 16.
[0110] FIG. 18 to FIG. 20 show the result (on the peak in FIG. 14
C) of LC-MS/MS analysis of a peptide obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody subtype
1 followed by trypsin digestion. In FIG. 18, the top is a
chromatogram detected by a UV at 215 nm and the bottom is a base
peak chromatogram. FIG. 19 shows a mass spectrum of the peak at a
retention time of 46 minutes in FIG. 18, and FIG. 20 shows a zoom
scan spectrum of the same peak as in FIG. 19.
[0111] From these results, the detected peak was shown to have the
amino acid sequences SLSLSPG (SEQ ID NO: 5) and SLSLSP-NH.sub.2
(SEQ ID NO: 4). Thus, it was demonstrated that one of the H chain
C-terminals of the humanized PM-1 antibody subtype 1 has the
-SLSLSPG sequence (SEQ ID NO: 5), and the other has the
-SLSLSPG-NH.sub.2 sequence (SEQ ID NO: 6).
(e) Analysis of the Structure of the H Chain C-Terminal of the
Humanized PM-1 Antibody Subtype 2
[0112] FIG. 21 A shows a peptide map of peptides obtained by the
reduction/carboxymethylation of the humanized PM-1 antibody subtype
2 followed by trypsin digestion. In order to investigate the
structure of the C-terminal fragment of the H chain, FIG. 21 B
shows the MS chromatogram of molecular weight of SLSLSPG (SEQ ID
NO: 5) (selective monitoring at m/z 660.3.+-.0.5), FIG. 21 C shows
that of SLSLSP-NH.sub.2 (SEQ ID NO: 4) (selective monitoring at m/z
602.3.+-.0.5), and
FIG. 21 D shows that of SLSLSP (SEQ ID NO: 3) (selective monitoring
at m/z 603.3.+-.0.5). Though a peak corresponding to SLSLSPG (SEQ
ID NO: 5) was slightly detected, a peak corresponding to
SLSLSP-NH.sub.2 (SEQ ID NO: 4) was more strongly noted (though a
peak with a molecular weight of 603.3 was noted at about 45 minutes
in FIG. 21 D, it is not SLSLSP (SEQ ID NO: 3), based on the
retention time).
[0113] FIG. 22 to FIG. 24 show the result of LC-MS/MS analysis of a
peptide obtained by the reduction/carboxymethylation of the
humanized PM-1 antibody subtype 2 followed by trypsin digestion. In
FIG. 22, the top is a chromatogram detected by a UV at 215 nm and
the bottom is a base peak chromatogram. FIG. 23 shows a mass
spectrum of the peak at a retention time of 45 minutes in FIG. 22,
and FIG. 24 shows a zoom scan spectrum of the same peak as in FIG.
23. From these results, the detected peak was shown to have the
amino acid sequence SLSLSP-NH.sub.2 (SEQ ID NO: 4). Thus, it was
demonstrated that both of the H chain C-terminals of the humanized
PM-1 antibody subtype 2 have the -SLSLSPG-NH.sub.2 (SEQ ID NO: 6)
sequence.
Example 3
Measurement of Biological Activity of the Humanized PM-1 Antibody
Subtype 1 and Subtype 2
(1) Determination of IL-6 Receptor-Binding Activity
(a) Method of Determination
[0114] The method of determination is as described in the following
steps.
[0115] 1) One hundred .mu.l of anti-IL-6 receptor antibody diluted
to 5 .mu.g/ml with a sodium carbonate buffer, pH 9.6, is added to
each well of an immunoplate, and allowed to stand in a cold place
overnight or longer.
[0116] 2) Each well is washed three times with 300 .mu.l of a
phosphate buffered saline (hereinafter referred to as RB)
containing 0.05% polysorbate 20.
[0117] 3) To each well, 200 '.mu.l of a Tris-HCl buffer, pH 8.1
(hereinafter referred to as DB), containing 1% bovine serum albumin
is added, and allowed to stand at room temperature for 2 hours or
longer.
[0118] 4) After the liquid in the well is discarded, 100 .mu.l of a
soluble IL-6 receptor diluted to 0.1 .mu.g/ml with DB is added to
each well, and allowed to stand at room temperature for 2
hours.
[0119] 5) Each well is washed three times with 300 .mu.l of RB.
[0120] 6) To each well, 100 .mu.l of a sample solution serially
diluted with a DB containing a given amount of biotinylated MRA is
added, and allowed to stand at room temperature for 1 hour.
[0121] 7) Each well is washed three times with 300 .mu.l of RB.
[0122] 8) To each well, 100 .mu.l of a alkaline
phosphatase-labelled streptoavidin diluted to 0.5 .mu.g/ml with DB
is added, and allowed to stand at room temperature for 1 hour.
[0123] 9) Each well of the immunoplate is washed five times with
300 .mu.l of RB.
[0124] 10) A chromogenic reagent (SIGMA FAST p-nitrophenyl
phosphate) is dissolved with water, and 100 .mu.l of it is added to
each well, and allowed to stand at room temperature for 30
minutes.
[0125] 11) The difference in absorbance at 405 nm and that at 655
nm of the reaction in each well is determined.
[0126] 12) From the absorbance obtained, the binding activity of
each sample is calculated using a parallel line test (3+3).
(b) Result
[0127] The result is shown in Table 1.
TABLE-US-00001 TABLE 1 Antigen-binding activity of humanized PM-1
antibody subtypes Subtype Activity Specific activity Native (Main)
1.04 .times. 10.sup.3 100% Subtype 1 1.13 .times. 10.sup.3 109%
Subtype 2 1.12 .times. 10.sup.3 108%
[0128] The result in Table 1 reveals that the humanized PM-1
antibody (Main), Subtype 1, and Subtype 2 have substantially the
same antigen-binding activity.
(2) Inhibition of KT-3 Cell Growth
(a) Method of Determination
[0129] The method of determination is as described in the following
steps.
[0130] 1) To each well of a microplate, 50 .mu.l of an IL-6
solution diluted to 2 ng/ml with a RPMI medium is added, and then
50 .mu.l of a sample solution serially diluted with a RPMI medium
is added. To a blank well, 50 .mu.l of the RPMI medium is
added.
[0131] 2) Furthermore, 100 .mu.l of a KT-3 cell suspension adjusted
to 5.times.10.sup.4 cells/ml with the RPMI medium is added to each
well, and cultured in a CO.sub.2 incubator for 3 days.
[0132] 3) To each well, 50 .mu.l of a .sup.3H-thymidine solution
appropriately diluted with the RPMI medium is added, and cultured
in the CO.sub.2 incubator for 6 days.
[0133] 4) The cells in the microplate are collected on a glass
filter using a cell harvester.
[0134] 5) After drying the glass filter in a microwave oven for 10
minutes, a solid scintillator is impregnated into the glass filter
under heating with a hot plate etc.
[0135] 6) Using a liquid scintillation counter, radioactivity (cpm)
is measured.
[0136] 7) From the radioactivity obtained, the biological activity
of each sample is calculated by a parallel line test (4+4).
[0137] The result is shown in Table 2.
TABLE-US-00002 TABLE 2 Activity of inhibiting cell growth of
humanized PM-1 antibody subtypes Subtype Activity Specific activity
Native (Main) 1.00 .times. 10.sup.3 100% Subtype 1 1.02 .times.
10.sup.3 102% Subtype 2 1.01 .times. 10.sup.3 101%
[0138] The result in. Table 2 reveals that the humanized. PM-1
antibody (Main), Subtype 1, and Subtype 2 have substantially the
same activity of inhibiting cell growth.
Sequence CWU 1
1
61448PRTArtificial SequenceAmino acid sequence of H chain of
humanized antibody PM-1 against interleukin-6 receptor 1Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10 15 Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25
30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro
Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr
Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405
410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 435 440 445 2214PRTArtificial SequenceAmino acid
sequence of L chain of humanized antibody PM-1 against
interleukin-6 receptor 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 36PRTArtificial SequenceSynthesized
Construct 3Ser Leu Ser Leu Ser Pro1 5 46PRTArtificial
SequenceSynthesized ConstructVARIANT(1)...(6)Xaa = Amidated Proline
4Ser Leu Ser Leu Ser Xaa1 5 57PRTArtificial SequenceSynthesized
Construct 5Ser Leu Ser Leu Ser Pro Gly1 5 67PRTArtificial
SequenceSynthesized ConstructVARIANT(1)...(7)Xaa = Amidated Glycine
6Ser Leu Ser Leu Ser Pro Xaa1 5
* * * * *