U.S. patent application number 16/692676 was filed with the patent office on 2020-03-19 for antigen-binding molecule having regulated conjugation between heavy-chain and light-chain.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Naoka Hironiwa, Tomoyuki Igawa, Meiri Kawazoe, Taichi Kuramochi.
Application Number | 20200087380 16/692676 |
Document ID | / |
Family ID | 48192053 |
Filed Date | 2020-03-19 |
View All Diagrams
United States Patent
Application |
20200087380 |
Kind Code |
A1 |
Kuramochi; Taichi ; et
al. |
March 19, 2020 |
ANTIGEN-BINDING MOLECULE HAVING REGULATED CONJUGATION BETWEEN
HEAVY-CHAIN AND LIGHT-CHAIN
Abstract
It was found that association between CH1 and CL can be
suppressed by substituting amino acids that exist on the interface
between CH1 and CL with electrically-charged amino acids, and that
formation of heterogeneous molecules is enabled more efficiently
than by introducing knobs into holes mutations into CH3 domain.
Inventors: |
Kuramochi; Taichi;
(Shizuoka, JP) ; Kawazoe; Meiri; (Shizuoka,
JP) ; Hironiwa; Naoka; (Shizuoka, JP) ; Igawa;
Tomoyuki; (Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
Chugai Seiyaku Kabushiki
Kaisha
Tokyo
JP
|
Family ID: |
48192053 |
Appl. No.: |
16/692676 |
Filed: |
November 22, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14351654 |
Apr 14, 2014 |
|
|
|
PCT/JP2012/078103 |
Oct 31, 2012 |
|
|
|
16692676 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2866 20130101;
A61P 43/00 20180101; C07K 2317/522 20130101; C07K 16/00 20130101;
C07K 16/468 20130101; C07K 2317/31 20130101; C07K 16/303
20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 16/28 20060101 C07K016/28; C07K 16/30 20060101
C07K016/30; C07K 16/46 20060101 C07K016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 2011 |
JP |
2011-238873 |
Claims
1. An antigen-binding molecule in which association of the heavy
chain and light chain is regulated, wherein one set or two or more
sets of amino acid residues selected from the group consisting of
the sets of amino acid residues shown in (a) to (c) below in the
heavy chain and light chain in the antigen-binding molecule are
amino acid residues that mutually repel electrically: (a) the amino
acid residue comprised in the heavy chain constant region (CH1) at
position 147 as indicated by EU numbering, and the amino acid
residue comprised in the light chain constant region (CL) at
position 180 as indicated by EU numbering; (b) the amino acid
residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and, (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering.
2. The antigen-binding molecule of claim 1, further wherein amino
acid residues in the set of amino acid residues shown in (d) below
are amino acid residues that mutually repel electrically: (d) the
amino acid residue comprised in CH1 at position 213 as indicated by
EU numbering, and the amino acid residue comprised in CL at
position 123 as indicated by EU numbering.
3. The antigen-binding molecule of claim 1 or 2, wherein the amino
acid residues that mutually repel electrically are selected from
amino acid residues comprised in either set of (X) and (Y) below:
(X) glutamic acid (E) or aspartic acid (D); and (Y) lysine (K),
arginine (R) or histidine (H).
4. The antigen-binding molecule of any one of claims 1 to 3,
further wherein two or more amino acid residues forming an
interface between the heavy chain variable region and light chain
variable region are amino acid residues that mutually repel
electrically.
5. The antigen-binding molecule of claim 4, wherein the amino acid
residues that mutually repel electrically are one set or two sets
of amino acid residues selected from the group consisting of the
sets of amino acid residues shown in (a) or (b): (a) the amino acid
residue comprised in the heavy chain variable region at position 39
as indicated by Kabat numbering and the amino acid residue
comprised in the light chain variable region at position 38 as
indicated by Kabat numbering; or (b) the amino acid residue
comprised in the heavy chain variable region at position 45 as
indicated by Kabat numbering, and the amino acid residue comprised
in the light chain variable region at position 44 as indicated by
Kabat numbering.
6. The antigen-binding molecule of claim 4 or 5, wherein the amino
acid residues that mutually repel electrically are selected from
the amino acid residues comprised in either set of (X) and (Y)
below: (X) glutamic acid (E) or aspartic acid (D); and (Y) lysine
(K), arginine (R), or histidine (H).
7. An antigen-binding molecule in which association of the heavy
chain and light chain is regulated, wherein one set or two or more
sets of amino acid residues selected from the group consisting of
the sets of amino acid residues shown in (a) to (c) below in the
associating heavy chain and light chain in the antigen-binding
molecule are amino acid residues that do not mutually repel
electrically: (a) the amino acid residue comprised in the heavy
chain constant region (CH1) at position 147 as indicated by EU
numbering, and the amino acid residue comprised in the light chain
constant region (CL) at position 180 as indicated by EU numbering;
(b) the amino acid residue comprised in CH1 at position 147 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 131 as indicated by EU numbering; and, (c) the amino
acid residue comprised in CH1 at position 175 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
160 as indicated by EU numbering.
8. The antigen-binding molecule of claim 7, further wherein amino
acid residues of the set of amino acid residues shown in (d) below
are amino acid residues that do not mutually repel electrically:
(d) the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 123 as indicated by EU numbering.
9. The antigen-binding molecule of claim 7 or 8, wherein the amino
acid residues that do not mutually repel electrically are amino
acid residues selected from each of two sets selected from the
group consisting of (X) to (Z) below, and wherein the two sets are
selected from among the combinations of (X) and (Y), (X) and (Z),
(Y) and (Z), and (Z) and (Z): (X) glutamic acid (E) or aspartic
acid (D); (Y) lysine (K), arginine (R) or histidine (H); and (Z)
alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine
(G), isoleucine (I), leucine (L), methionine (M), phenylalanine
(F), proline (P), serine (S), threonine (T), tryptophan (W),
tyrosine (Y), or valine (V).
10. The antigen-binding molecule of any one of claims 7 to 9,
wherein the amino acid residues that do not mutually repel
electrically are the amino acid residue comprised in CH1 at
position 175 as indicated by EU numbering which is lysine (K); and
the amino acid residues comprised in CL at position 180, position
131, and position 160 as indicated by EU numbering which are all
glutamic acid (E).
11. The antigen-binding molecule of any one of claims 7 to 9,
wherein the amino acid residues that do not mutually repel
electrically are the amino acid residues comprised in CH1 at
position 147 and position 175 as indicated by EU numbering which
are glutamic acid (E); and the amino acid residues comprised in CL
at position 180, position 131, and position 160 as indicated by EU
numbering which are all lysine (K).
12. The antigen-binding molecule of claim 11, further wherein the
amino acid residue comprised in CH1 at position 213 as indicated by
EU numbering is glutamic acid (E), and the amino acid residue
comprised in CL at position 123 as indicated by EU numbering is
lysine (K).
13. The antigen-binding molecule of any one of claims 7 to 12,
wherein two or more amino acid residues forming the interface
between the heavy chain variable region and light chain variable
region are amino acid residues that do not mutually repel
electrically.
14. The antigen-binding molecule of claim 13, wherein the amino
acid residues that do not mutually repel electrically are one set
or two sets of amino acid residues selected from the group
consisting of the sets of amino acid residues shown in (a) or (b)
below: (a) the amino acid residue comprised in the heavy chain
variable region at position 39 as indicated by Kabat numbering, and
the amino acid residue comprised in the light chain variable region
at position 38 as indicated by Kabat numbering; or (b) the amino
acid residue comprised in the heavy chain variable region at
position 45 as indicated by Kabat numbering, and the amino acid
residue comprised in the light chain variable region at position 44
as indicated by Kabat numbering.
15. The antigen-binding molecule of claim 13 or 14, wherein the
amino acid residues that do not mutually repel electrically are
amino acid residues selected from each of two sets selected from
the group consisting of (X) to (Z) below, and wherein the two sets
are selected from among the combinations of (X) and (Y), (X) and
(Z), (Y) and (Z), and (Z) and (Z): (X) glutamic acid (E) or
aspartic acid (D); (Y) lysine (K), arginine (R) or histidine (H);
and (Z) alanine (A), asparagine (N), cysteine (C), glutamine (Q),
glycine (G), isoleucine (I), leucine (L), methionine (M),
phenylalanine (F), proline (P), serine (S), threonine (T),
tryptophan (W), tyrosine (Y), or valine (V).
16. The antigen-binding molecule of any one of claims 1 to 15,
wherein the antigen-binding molecule is a bispecific antibody.
17. A method for producing an antigen-binding molecule in which
association of the heavy chain and light chain is regulated,
comprising steps of (1) to (3) below: (1) modifying nucleic acids
encoding the heavy chain constant region (CH1) and the light chain
constant region (CL) so that one set or two or more sets of amino
acid residues selected from the group consisting of the sets of
amino acid residues shown in (a) to (c) below mutually repel
electrically: (a) the amino acid residue comprised in CH1 at
position 147 as indicated by EU numbering, and the amino acid
residue comprised in CL at position 180 as indicated by EU
numbering; (b) the amino acid residue comprised in CH1 at position
147 as indicated by EU numbering, and the amino acid residue
comprised in CL at position 131 as indicated by EU numbering; and,
(c) the amino acid residue comprised in CH1 at position 175 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 160 as indicated by EU numbering, (2) introducing
the modified nucleic acids into a host cell and culturing the host
cell so that it expresses the nucleic acids, and (3) collecting the
antigen-binding molecule from a culture of the host cell.
18. The method for producing an antigen-binding molecule of claim
17, further comprising in step (1), modifying nucleic acids so that
the amino acid residues in the set of amino acid residues shown in
(d) below mutually repel electrically: (d) the amino acid residue
comprised in CH1 at position 213 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 123 as indicated
by EU numbering.
19. The method for producing an antigen-binding molecule of claim
17 or 18, comprising in step (1), modifying nucleic acids so that
the amino acid residues that mutually repel electrically are
selected from among amino acid residues comprised in either group
of (X) and (Y) below: (X) glutamic acid (E) or aspartic acid (D);
and (Y) lysine (K), arginine (R), or histidine (H).
20. The method for producing an antigen-binding molecule of any one
of claims 17 to 19, further comprising in step (1), modifying
nucleic acids so that two or more amino acid residues forming the
interface between the heavy chain variable region and light chain
variable region are amino acid residues that mutually repel
electrically.
21. The method for producing an antigen-binding molecule of claim
20, wherein the amino acid residues that mutually repel
electrically are amino acid residues of any one set selected from
the group consisting of the sets of amino acid residues shown in
(a) or (b) below: (a) the amino acid residue comprised in the heavy
chain variable region at position 39 as indicated by Kabat
numbering, and the amino acid residue comprised in the light chain
variable region at position 38 as indicated by Kabat numbering; or
(b) the amino acid residue comprised in the heavy chain variable
region at position 45 as indicated by Kabat numbering, and the
amino acid residue comprised in the light chain variable region at
position 44 as indicated by Kabat numbering.
22. The method for producing an antigen-binding molecule of claim
20 or 21, wherein the amino acid residues that mutually repel
electrically are selected from amino acid residues comprised in
either set of (X) and (Y) below: (X) glutamic acid (E) or aspartic
acid (D); and (Y) lysine (K), arginine (R), or histidine (H).
23. A method for producing an antigen-binding molecule in which
association of the heavy chain and light chain is regulated,
comprising the following steps of (1) to (3): (1) modifying nucleic
acids encoding a heavy chain constant region (CH1) and a light
chain constant region (CL) which associate so that one set or two
or more sets of amino acid residues selected from the group
consisting of the sets of amino acid residues shown in (a) to (c)
below do not mutually repel electrically: (a) the amino acid
residue comprised in the heavy chain constant region (CH1) at
position 147 as indicated by EU numbering, and the amino acid
residue comprised in the light chain constant region (CL) at
position 180 as indicated by EU numbering; (b) the amino acid
residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and, (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering, (2) introducing the modified nucleic acids into a
host cell and culturing the host cell so that it expresses the
nucleic acids, and (3) collecting the antigen-binding molecule from
a culture of the host cell.
24. The method for producing an antigen-binding molecule of claim
23, further comprising in step (1), modifying nucleic acids so that
the amino acid residues in the set of amino acid residues shown in
(d) below do not mutually repel electrically: (d) the amino acid
residue comprised in CH1 at position 213 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
123 as indicated by EU numbering.
25. The method for producing an antigen-binding molecule of claim
23 or 24, comprising in step (1), modifying the nucleic acids so
that the amino acid residues that do not mutually repel
electrically are amino acids residues selected from each of two
sets selected from the group consisting of (X) to (Z) below, and
wherein the two sets are selected from among the combinations of
(X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z): (X)
glutamic acid (E) or aspartic acid (D); (Y) lysine (K), arginine
(R), or histidine (H); and (Z) alanine (A), asparagine (N),
cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine
(L), methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y), or valine (V).
26. The method for producing an antigen-binding molecule of any one
of claims 23 to 25, comprising in step (1), modifying nucleic acids
so that the amino acid residues that do not mutually repel
electrically are the amino acid residue comprised in CH1 at
position 175 as indicated by EU numbering which is lysine (K), and
the amino acid residues comprised in CL at position 180, position
131, and position 160 as indicated by EU numbering which are all
glutamic acid (E).
27. The method for producing an antigen-binding molecule of any one
of claims 23 to 25, comprising in step (1), modifying nucleic acids
so that the amino acid residues that do not mutually repel
electrically are the amino acid residues comprised in CH1 at
position 147 and position 175 as indicated by EU numbering which
are glutamic acid (E), and the amino acid residues comprised in CL
at position 180, position 131, and position 160 as indicated by EU
numbering which are all lysine (K).
28. The method for producing an antigen-binding molecule of claim
27, further comprising modifying nucleic acids so that the amino
acid residue comprised in CH1 at position 213 as indicated by EU
numbering is glutamic acid (E), and the amino acid residue
comprised in CL at position 123 as indicated by EU numbering is
lysine (K).
29. The method for producing an antigen-binding molecule of any one
of claims 23 to 28, further comprising in step (1), modifying
nucleic acids so that two or more amino acid residues forming the
interface between the heavy chain variable region and light chain
variable region are amino acid residues that do not mutually repel
electrically.
30. The method for producing an antigen-binding molecule of claim
29, wherein the amino acid residues that do not mutually repel
electrically are amino acid residues of any one set selected from
the group consisting of the sets of amino acid residues shown in
(a) or (b) below: (a) the amino acid residue comprised in the heavy
chain variable region at position 39 as indicated by Kabat
numbering, and the amino acid residue comprised in the light chain
variable region at position 38 as indicated by Kabat numbering; or
(b) the amino acid residue comprised in the heavy chain variable
region at position 45 as indicated by Kabat numbering, and the
amino acid residue comprised in the light chain variable region at
position 44 as indicated by Kabat numbering.
31. The method for producing an antigen-binding molecule of claim
29 or 30, wherein the amino acid residues that do not mutually
repel electrically are amino acid residues selected from each of
two sets selected from the group consisting of (X) to (Z) below,
and wherein the two sets are selected from among the combinations
of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z): (X)
glutamic acid (E) or aspartic acid (D); (Y) lysine (K), arginine
(R) or histidine (H); and (Z) alanine (A), asparagine (N), cysteine
(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L),
methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y), or valine (V).
32. An antigen-binding molecule produced according to the method
for producing an antigen-binding molecule of any one of claims 17
to 31.
33. The antigen-binding molecule of claim 32, wherein the
antigen-binding molecule is a bispecific antibody.
34. A method for regulating association of the heavy chain and
light chain of an antigen-binding molecule, comprising: modifying
nucleic acids so that one set or two or more sets of amino acid
residues selected from the group consisting of the sets of amino
acid residues shown in (a) to (c) below are amino acid residues
that mutually repel electrically: (a) the amino acid residue
comprised in CH1 at position 147 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 180 as indicated
by EU numbering; (b) the amino acid residue comprised in CH1 at
position 147 as indicated by EU numbering, and the amino acid
residue comprised in CL at position 131 as indicated by EU
numbering; and (c) the amino acid residue comprised in CH1 at
position 175 as indicated by EU numbering, and the amino acid
residue comprised in CL at position 160 as indicated by EU
numbering.
35. The method of claim 34, further comprising modifying nucleic
acids so that the amino acid residues in the set of amino acid
residues shown in (d) below are amino acid residues that mutually
repel electrically: (d) the amino acid residue comprised in CH1 at
position 213 as indicated by EU numbering, and the amino acid
residue comprised in CL at position 123 as indicated by EU
numbering.
36. The method of claim 34 or 35, wherein the amino acid residues
that mutually repel electrically are selected from amino acid
residues comprised in either set of (X) and (Y) below: (X) glutamic
acid (E) or aspartic acid (D); and (Y) lysine (K), arginine (R), or
histidine (H).
37. The method of any one of claims 34 to 36, wherein two or more
amino acid residues forming the interface between the heavy chain
variable region and light chain variable region are amino acid
residues that mutually repel electrically.
38. The method of claim 37, wherein the amino acid residues that
mutually repel electrically are amino acid residues of any one set
selected from the group consisting of the sets of amino acid
residues shown in (a) or (b) below: (a) the amino acid residue
comprised in the heavy chain variable region at position 39 as
indicated by Kabat numbering, and the amino acid residue comprised
in the light chain variable region at position 38 as indicated by
Kabat numbering; or (b) the amino acid residue comprised in the
heavy chain variable region at position 45 as indicated by Kabat
numbering, and the amino acid residue comprised in the light chain
variable region at position 44 as indicated by Kabat numbering.
39. The method of claim 37 or 38, wherein the amino acid residues
that mutually repel electrically are selected from amino acid
residues comprised in either set of (X) and (Y) below: (X) glutamic
acid (E) or aspartic acid (D); and (Y) lysine (K), arginine (R), or
histidine (H).
40. A method for regulating association of the heavy chain and
light chain of an antigen-binding molecule, comprising: modifying
nucleic acids so that one set or two or more sets of amino acid
residues selected from the group consisting of the sets of amino
acid residues shown in (a) to (c) below in the associating heavy
chain and light chain in the antigen-binding molecule are amino
acid residues that do not mutually repel electrically: (a) the
amino acid residue comprised in CH1 at position 147 as indicated by
EU numbering, and the amino acid residue comprised in CL at
position 180 as indicated by EU numbering; (b) the amino acid
residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and, (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering.
41. The method of claim 40, further comprising modifying nucleic
acids so that the amino acid residues in the set of amino acid
residues shown in (d) below are amino acid residues that do not
mutually repel electrically: (d) the amino acid residue comprised
in CH1 at position 213 as indicated by EU numbering, and the amino
acid residue comprised in CL at position 123 as indicated by EU
numbering.
42. The method of claim 40 or 41, wherein the amino acid residues
that do not mutually repel electrically are amino acid residues
selected from each of two sets selected from the group consisting
of (X) to (Z) below, and wherein the two sets are selected from
among the combinations of (X) and (Y), (X) and (Z), (Y) and (Z),
and (Z) and (Z): (X) glutamic acid (E) or aspartic acid (D); (Y)
lysine (K), arginine (R), or histidine (H); and (Z) alanine (A),
asparagine (N), cysteine (C), glutamine (Q), glycine (G),
isoleucine (I), leucine (L), methionine (M), phenylalanine (F),
proline (P), serine (S), threonine (T), tryptophan (W), tyrosine
(Y), or valine (V).
43. The method of any one of claims 40 to 42, wherein the amino
acid residues that do not mutually repel electrically are the amino
acid residue comprised in CH1 at position 175 as indicated by EU
numbering which is lysine (K), and the amino acid residues
comprised in CL at position 180, position 131, and position 160 as
indicated by EU numbering which are all glutamic acid (E).
44. The method of any one of claims 40 to 42, wherein the amino
acid residues that do not mutually repel electrically are the amino
acid residues comprised in CH1 at position 147 and position 175 as
indicated by EU numbering which are glutamic acid (E), and the
amino acid residues comprised in CL at position 180, position 131,
and position 160 as indicated by EU numbering which are all lysine
(K).
45. The method of claim 44, wherein the amino acid residue
comprised in CH1 at position 213 as indicated by EU numbering is
glutamic acid (E), and the amino acid residue contained in CL at
position 123 as indicated by EU numbering is lysine (K).
46. The method of any one of claims 34 to 45, wherein the
antigen-binding molecule is a bispecific antibody.
47. A composition containing the antigen-binding molecule of any
one of claims 1 to 16, 32, and 33, and a pharmaceutically
acceptable carrier.
48. A nucleic acid encoding the antigen-binding molecule of any one
of claims 1 to 16, 32, and 33.
49. A host cell having the nucleic acid of claim 48.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/351,654, filed on Apr. 14, 2014, which is the National Stage
of International Application No. PCT/JP2012/078103, filed on Oct.
31, 2012, which claims the benefit of Japanese Application Serial
No. 2011-238873, filed on Oct. 31, 2011.
BACKGROUND ART
[0002] The present invention relates to antibodies with regulated
association of the heavy chain and light chain, methods for
producing an antibody with regulated association of the heavy chain
and light chain, methods for regulating association of the heavy
chain and light chain of an antibody, pharmaceutical compositions
comprising that antibody as an active ingredient, and the like.
TECHNICAL FIELD
[0003] Several methods have previously been reported as methods for
preparing IgG-type bispecific antibodies having human constant
regions (IgG-type antibodies having a human constant region that
has binding specificity for an antigen A on one arm and binding
specificity for an antigen B on the other arm). In general,
IgG-type bispecific antibodies are composed of two types of H
chains (namely, an H chain for antigen A and an H chain for antigen
B) and two types of L chains (namely, an L chain for antigen A and
an L chain for antigen B). When such IgG-type bispecific antibodies
are expressed, 10 types of combinations are possible as
combinations of H2L2 since two types of H chains and two types of L
chains are expressed. Among these, there is one type of combination
that has the desired binding specificity (IgG having binding
specificity for antigen A on one arm and binding specificity for
antigen B on the other arm). Consequently, in order to acquire the
desired bispecific antibody, it is necessary to purify one type of
antibody of interest from among ten types of antibodies, which is
extremely low in efficiency and difficult.
[0004] Methods have been reported for solving this problem, which
involve preferentially secreting IgG having a heterologous
combination of an H chain for antigen A and an H chain for antigen
B, by substituting amino acids in the CH3 region of the IgG H chain
(Patent Documents 1, 2, 3 and 4, and Non-Patent Documents 1 and 2).
Among these, there have been reported methods that use physical
obstacles in the form of a "knob" and "hole", and those that use
electric charge repulsion.
[0005] A method has also been reported for efficiently obtaining a
desired molecule, which uses a common L chain in which an L chain
for antigen A and an L chain for antigen B are present on a same
amino acid sequence (Patent Documents 5 and 6). However, since the
use of a common L chain has the potential of considerably lowering
the antigen affinity, this is not necessarily the optimum method.
Consequently, in order for a bispecific antibody to bind to two
antigens with high affinity, it is preferable that only the L chain
and H chain for antigen A associate, and only the L chain and H
chain for antigen B associate. Moreover, a method has been reported
to allow the H chains and L chains for each antigen to associate
irrespectively of the variable regions, which comprises
substituting amino acids in the CH1 and CL domains which are
constant regions, instead of those in the variable regions (Patent
Documents 2 and 7). However, this method is still insufficient for
efficiently producing a bispecific antibody of interest.
PRIOR ART DOCUMENTS
Patent Documents
[0006] [Patent Document 1] WO 96/27011 [0007] [Patent Document 2]
WO 2006/106905 [0008] [Patent Document 3] WO 2009/089004 [0009]
[Patent Document 4] WO 2010/129304 [0010] [Patent Document 5] WO
98/050431 [0011] [Patent Document 6] WO 2006/109592 [0012] [Patent
Document 7] WO 2007/147901
Non-Patent Documents
[0012] [0013] [Non-Patent Document 1] Ridgway J B et al., Protein
Engineering, 1996, Vol. 9, p. 617-621 [0014] [Non-Patent Document
2] Merchant A M et al., Nature Biotechnology, 1998, Vol. 16, p.
677-681
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0015] The present invention has been achieved under such
circumstances. An objective of the present invention is to provide
antibodies in which association of the heavy chains and light
chains is regulated, a method for producing antibodies in which the
association of the heavy chains and light chains is regulated, and
a method for regulating association of the heavy chains and light
chains of an antibody. In addition, in one embodiment of the
present invention, an objective of the present invention is to
provide bispecific antibodies in which association at the interface
of CH1 and CL is regulated, and a method for efficiently producing
a bispecific antibody by regulating the association at the
interface of CH1 and CL.
Means for Solving the Problems
[0016] The inventors of the present invention selected a constant
region of the heavy chain, CH1, and a light chain constant region
(CL) as heavy-chain and light-chain regions to be used for
regulating association, and conducted dedicated studies on the
regulation of CH1 and CL association. As a result, the present
inventors found that association of CH1 and CL can be suppressed by
substituting amino acid residues present at the interface of CH1
and CL with amino acid residues that mutually repel electrically or
amino acid residues that do not repel, and that heterologous
molecules are formed more efficiently than by using modifications
which only introduce a knob and hole into CH3 as previously
described.
[0017] Thus, according to findings made by the present inventors,
it is possible to regulate the association of CH1 and CL. In
addition, the present invention can be applied not only to
regulation of the association between CH1 and CL, but also to
regulation of the association between arbitrary polypeptides.
[0018] Moreover, the present inventors also confirmed that a
bispecific antibody of the present invention with regulated
association of the heavy chain and light chain actually retains
function.
[0019] As described above, the present inventors successfully
developed antigen-binding molecules in which association of the
heavy chain and light chain is regulated, and thereby completed the
present invention.
[0020] The present invention relates to antigen-binding molecules
in which the association of the heavy chains and light chains is
regulated, methods for producing an antigen-binding molecule in
which the association of the heavy chains and light chains is
regulated, and methods for regulating the association of the heavy
chains and light chains of an antigen-binding molecule.
Specifically, the present invention relates to the following:
[0021] [1] an antigen-binding molecule in which association of the
heavy chain and light chain is regulated, wherein
[0022] one set or two or more sets of amino acid residues selected
from the group consisting of the sets of amino acid residues shown
in (a) to (c) below in the heavy chain and light chain in the
antigen-binding molecule are amino acid residues that mutually
repel electrically:
(a) the amino acid residue comprised in the heavy chain constant
region (CH1) at position 147 as indicated by EU numbering, and the
amino acid residue comprised in the light chain constant region
(CL) at position 180 as indicated by EU numbering; (b) the amino
acid residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and, (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering;
[0023] [2] the antigen-binding molecule of [1], further wherein
amino acid residues in the set of amino acid residues shown in (d)
below are amino acid residues that mutually repel electrically:
(d) the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 123 as indicated by EU numbering;
[0024] [3] the antigen-binding molecule of [1] or [2], wherein the
amino acid residues that mutually repel electrically are selected
from amino acid residues comprised in either set of (X) and (Y)
below:
(X) glutamic acid (E) or aspartic acid (D); and (Y) lysine (K),
arginine (R) or histidine (H);
[0025] [4] the antigen-binding molecule of any one of [1] to [3],
further wherein two or more amino acid residues forming an
interface between the heavy chain variable region and light chain
variable region are amino acid residues that mutually repel
electrically;
[0026] [5] the antigen-binding molecule of [4], wherein the amino
acid residues that mutually repel electrically are one set or two
sets of amino acid residues selected from the group consisting of
the sets of amino acid residues shown in (a) or (b):
(a) the amino acid residue comprised in the heavy chain variable
region at position 39 as indicated by Kabat numbering and the amino
acid residue comprised in the light chain variable region at
position 38 as indicated by Kabat numbering; or (b) the amino acid
residue comprised in the heavy chain variable region at position 45
as indicated by Kabat numbering, and the amino acid residue
comprised in the light chain variable region at position 44 as
indicated by Kabat numbering;
[0027] [6] the antigen-binding molecule of [4] or [5], wherein the
amino acid residues that mutually repel electrically are selected
from the amino acid residues comprised in either set of (X) and (Y)
below:
(X) glutamic acid (E) or aspartic acid (D); and (Y) lysine (K),
arginine (R), or histidine (H);
[0028] [7] an antigen-binding molecule in which association of the
heavy chain and light chain is regulated, wherein
[0029] one set or two or more sets of amino acid residues selected
from the group consisting of the sets of amino acid residues shown
in (a) to (c) below in the associating heavy chain and light chain
in the antigen-binding molecule are amino acid residues that do not
mutually repel electrically:
(a) the amino acid residue comprised in the heavy chain constant
region (CH1) at position 147 as indicated by EU numbering, and the
amino acid residue comprised in the light chain constant region
(CL) at position 180 as indicated by EU numbering; (b) the amino
acid residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and, (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering;
[0030] [8] the antigen-binding molecule of [7], further wherein
amino acid residues of the set of amino acid residues shown in (d)
below are amino acid residues that do not mutually repel
electrically:
(d) the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 123 as indicated by EU numbering;
[0031] [9] the antigen-binding molecule of [7] or [8], wherein the
amino acid residues that do not mutually repel electrically are
amino acid residues selected from each of two sets selected from
the group consisting of (X) to (Z) below, and wherein the two sets
are selected from among the combinations of (X) and (Y), (X) and
(Z), (Y) and (Z), and (Z) and (Z):
(X) glutamic acid (E) or aspartic acid (D); (Y) lysine (K),
arginine (R) or histidine (H); and (Z) alanine (A), asparagine (N),
cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine
(L), methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y), or valine (V);
[0032] [10] the antigen-binding molecule of any one of [7] to [9],
wherein the amino acid residues that do not mutually repel
electrically are the amino acid residue comprised in CH1 at
position 175 as indicated by EU numbering which is lysine (K); and
the amino acid residues comprised in CL at position 180, position
131, and position 160 as indicated by EU numbering which are all
glutamic acid (E);
[0033] [11] the antigen-binding molecule of any one of [7] to [9],
wherein the amino acid residues that do not mutually repel
electrically are the amino acid residues comprised in CH1 at
position 147 and position 175 as indicated by EU numbering which
are glutamic acid (E); and the amino acid residues comprised in CL
at position 180, position 131, and position 160 as indicated by EU
numbering which are all lysine (K);
[0034] [12] the antigen-binding molecule of [11], further wherein
the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering is glutamic acid (E), and the amino acid
residue comprised in CL at position 123 as indicated by EU
numbering is lysine (K);
[0035] [13] the antigen-binding molecule of any one of [7] to [12],
further wherein two or more amino acid residues forming the
interface between the heavy chain variable region and light chain
variable region are amino acid residues that do not mutually repel
electrically;
[0036] [14] the antigen-binding molecule of [13], wherein the amino
acid residues that do not mutually repel electrically are one set
or two sets of amino acid residues selected from the group
consisting of the sets of amino acid residues shown in (a) or (b)
below:
(a) the amino acid residue comprised in the heavy chain variable
region at position 39 as indicated by Kabat numbering, and the
amino acid residue comprised in the light chain variable region at
position 38 as indicated by Kabat numbering; or (b) the amino acid
residue comprised in the heavy chain variable region at position 45
as indicated by Kabat numbering, and the amino acid residue
comprised in the light chain variable region at position 44 as
indicated by Kabat numbering;
[0037] [15] the antigen-binding molecule of [13] or [14], wherein
the amino acid residues that do not mutually repel electrically are
amino acid residues selected from each of two sets selected from
the group consisting of (X) to (Z) below, and wherein the two sets
are selected from among the combinations of (X) and (Y), (X) and
(Z), (Y) and (Z), and (Z) and (Z):
(X) glutamic acid (E) or aspartic acid (D); (Y) lysine (K),
arginine (R) or histidine (H); and (Z) alanine (A), asparagine (N),
cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine
(L), methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y), or valine (V);
[0038] [16] the antigen-binding molecule of any one of [1] to [15],
wherein the antigen-binding molecule is a bispecific antibody;
[0039] [17] a method for producing an antigen-binding molecule in
which association of the heavy chain and light chain is regulated,
comprising steps of (1) to (3) below:
(1) modifying nucleic acids encoding the heavy chain constant
region (CH1) and the light chain constant region (CL) so that one
set or two or more sets of amino acid residues selected from the
group consisting of the sets of amino acid residues shown in (a) to
(c) below mutually repel electrically: (a) the amino acid residue
comprised in CH1 at position 147 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 180 as indicated
by EU numbering; (b) the amino acid residue comprised in CH1 at
position 147 as indicated by EU numbering, and the amino acid
residue comprised in CL at position 131 as indicated by EU
numbering; and, (c) the amino acid residue comprised in CH1 at
position 175 as indicated by EU numbering, and the amino acid
residue comprised in CL at position 160 as indicated by EU
numbering, (2) introducing the modified nucleic acids into a host
cell and culturing the host cell so that it expresses the nucleic
acids, and (3) collecting the antigen-binding molecule from a
culture of the host cell;
[0040] [18] the method for producing an antigen-binding molecule of
[17], further comprising in step (1), modifying nucleic acids so
that the amino acid residues in the set of amino acid residues
shown in (d) below mutually repel electrically:
(d) the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 123 as indicated by EU numbering;
[0041] [19] the method for producing an antigen-binding molecule of
[17] or [18], comprising in step (1), modifying nucleic acids so
that the amino acid residues that mutually repel electrically are
selected from among amino acid residues comprised in either group
of (X) and (Y) below:
(X) glutamic acid (E) or aspartic acid (D); and (Y) lysine (K),
arginine (R), or histidine (H);
[0042] [20] the method for producing an antigen-binding molecule of
any one of [17] to [19], further comprising in step (1), modifying
nucleic acids so that two or more amino acid residues forming the
interface between the heavy chain variable region and light chain
variable region are amino acid residues that mutually repel
electrically;
[0043] [21] the method for producing an antigen-binding molecule of
[20], wherein the amino acid residues that mutually repel
electrically are amino acid residues of any one set selected from
the group consisting of the sets of amino acid residues shown in
(a) or (b) below:
(a) the amino acid residue comprised in the heavy chain variable
region at position 39 as indicated by Kabat numbering, and the
amino acid residue comprised in the light chain variable region at
position 38 as indicated by Kabat numbering; or (b) the amino acid
residue comprised in the heavy chain variable region at position 45
as indicated by Kabat numbering, and the amino acid residue
comprised in the light chain variable region at position 44 as
indicated by Kabat numbering;
[0044] [22] the method for producing an antigen-binding molecule of
[20] or [21], wherein the amino acid residues that mutually repel
electrically are selected from amino acid residues comprised in
either set of (X) and (Y) below:
(X) glutamic acid (E) or aspartic acid (D); and (Y) lysine (K),
arginine (R), or histidine (H);
[0045] [23] a method for producing an antigen-binding molecule in
which association of the heavy chain and light chain is regulated,
comprising the following steps of (1) to (3):
(1) modifying nucleic acids encoding a heavy chain constant region
(CH1) and a light chain constant region (CL) so that one set or two
or more sets of amino acid residues selected from the group
consisting of the sets of amino acid residues shown in (a) to (c)
below do not mutually repel electrically: (a) the amino acid
residue comprised in the heavy chain constant region (CH1) at
position 147 as indicated by EU numbering, and the amino acid
residue comprised in the light chain constant region (CL) at
position 180 as indicated by EU numbering; (b) the amino acid
residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and, (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering, (2) introducing the modified nucleic acids into a
host cell and culturing the host cell so that it expresses the
nucleic acids, and (3) collecting the antigen-binding molecule from
a culture of the host cell;
[0046] [24] the method for producing an antigen-binding molecule of
[23], further comprising in step (1), modifying nucleic acids so
that the amino acid residues in the set of amino acid residues
shown in (d) below do not mutually repel electrically:
(d) the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 123 as indicated by EU numbering;
[0047] [25] the method for producing an antigen-binding molecule of
[23] or [24], comprising in step (1), modifying the nucleic acids
so that the amino acid residues that do not mutually repel
electrically are amino acids residues selected from each of two
sets selected from the group consisting of (X) to (Z) below, and
wherein the two sets are selected from among the combinations of
(X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z):
(X) glutamic acid (E) or aspartic acid (D); (Y) lysine (K),
arginine (R), or histidine (H); and (Z) alanine (A), asparagine
(N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I),
leucine (L), methionine (M), phenylalanine (F), proline (P), serine
(S), threonine (T), tryptophan (W), tyrosine (Y), or valine
(V);
[0048] [26] the method for producing an antigen-binding molecule of
any one of [23] to [25], comprising in step (1), modifying nucleic
acids so that the amino acid residues that do not mutually repel
electrically are the amino acid residue comprised in CH1 at
position 175 as indicated by EU numbering which is lysine (K), and
the amino acid residues comprised in CL at position 180, position
131, and position 160 as indicated by EU numbering which are all
glutamic acid (E);
[0049] [27] the method for producing an antigen-binding molecule of
any one of [23] to [25], comprising in step (1), modifying nucleic
acids so that the amino acid residues that do not mutually repel
electrically are the amino acid residues comprised in CH1 at
position 147 and position 175 as indicated by EU numbering which
are glutamic acid (E), and the amino acid residues comprised in CL
at position 180, position 131, and position 160 as indicated by EU
numbering which are all lysine (K);
[0050] [28] the method for producing an antigen-binding molecule of
[27], further comprising modifying nucleic acids so that the amino
acid residue comprised in CH1 at position 213 as indicated by EU
numbering is glutamic acid (E), and the amino acid residue
comprised in CL at position 123 as indicated by EU numbering is
lysine (K);
[0051] [29] the method for producing an antigen-binding molecule of
any one of [23] to [28], further comprising in step (1), modifying
nucleic acids so that two or more amino acid residues forming the
interface between the heavy chain variable region and light chain
variable region are amino acid residues that do not mutually repel
electrically;
[0052] [30] the method for producing an antigen-binding molecule of
[29], wherein the amino acid residues that do not mutually repel
electrically are amino acid residues of any one set selected from
the group consisting of the sets of amino acid residues shown in
(a) or (b) below:
(a) the amino acid residue comprised in the heavy chain variable
region at position 39 as indicated by Kabat numbering, and the
amino acid residue comprised in the light chain variable region at
position 38 as indicated by Kabat numbering; or (b) the amino acid
residue comprised in the heavy chain variable region at position 45
as indicated by Kabat numbering, and the amino acid residue
comprised in the light chain variable region at position 44 as
indicated by Kabat numbering;
[0053] [31] the method for producing an antigen-binding molecule of
[29] or [30], wherein the amino acid residues that do not mutually
repel electrically are amino acid residues selected from each of
two sets selected from the group consisting of (X) to (Z) below,
and wherein the two sets are selected from among the combinations
of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and (Z):
(X) glutamic acid (E) or aspartic acid (D); (Y) lysine (K),
arginine (R) or histidine (H); and (Z) alanine (A), asparagine (N),
cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine
(L), methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y), or valine (V);
[0054] [32] an antigen-binding molecule produced according to the
method for producing an antigen-binding molecule of any one of [17]
to [31];
[0055] [33] the antigen-binding molecule of [32], wherein the
antigen-binding molecule is a bispecific antibody;
[0056] [34] a method for regulating association of the heavy chain
and light chain of an antigen-binding molecule, comprising:
[0057] modifying nucleic acids so that one set or two or more sets
of amino acid residues selected from the group consisting of the
sets of amino acid residues shown in (a) to (c) below are amino
acid residues that mutually repel electrically:
(a) the amino acid residue comprised in CH1 at position 147 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 180 as indicated by EU numbering; (b) the amino acid
residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering;
[0058] [35] the method of [34], further comprising modifying
nucleic acids so that the amino acid residues in the set of amino
acid residues shown in (d) below are amino acid residues that
mutually repel electrically:
(d) the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 123 as indicated by EU numbering;
[0059] [36] the method of [34] or [35], wherein the amino acid
residues that mutually repel electrically are selected from amino
acid residues comprised in either set of (X) and (Y) below:
(X) glutamic acid (E) or aspartic acid (D); and (Y) lysine (K),
arginine (R), or histidine (H);
[0060] [37] the method of any one of [34] to [36], further wherein
two or more amino acid residues forming the interface between the
heavy chain variable region and light chain variable region are
amino acid residues that mutually repel electrically;
[0061] [38] the method of [37], wherein the amino acid residues
that mutually repel electrically are amino acid residues of any one
set selected from the group consisting of the sets of amino acid
residues shown in (a) or (b) below:
(a) the amino acid residue comprised in the heavy chain variable
region at position 39 as indicated by Kabat numbering, and the
amino acid residue comprised in the light chain variable region at
position 38 indicated according to the Kabat numbering; or (b) the
amino acid residue comprised in the heavy chain variable region at
position 45 as indicated by Kabat numbering, and the amino acid
residue comprised in the light chain variable region at position 44
as indicated by Kabat numbering;
[0062] [39] the method of [37] or [38], wherein the amino acid
residues that mutually repel electrically are selected from amino
acid residues comprised in either set of (X) and (Y) below:
(X) glutamic acid (E) or aspartic acid (D); and (Y) lysine (K),
arginine (R), or histidine (H);
[0063] [40] a method for regulating association of the heavy chain
and light chain of an antigen-binding molecule, comprising:
[0064] modifying nucleic acids so that one set or two or more sets
of amino acid residues selected from the group consisting of the
sets of amino acid residues shown in (a) to (c) below are amino
acid residues that do not mutually repel electrically:
(a) the amino acid residue comprised in CH1 at position 147 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 180 as indicated by EU numbering; (b) the amino acid
residue comprised in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue comprised in CL at position
131 as indicated by EU numbering; and, (c) the amino acid residue
comprised in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue comprised in CL at position 160 as indicated
by EU numbering;
[0065] [41] the method of [40], further comprising modifying
nucleic acids so that the amino acid residues in the set of amino
acid residues shown in (d) below are amino acid residues that do
not mutually repel electrically:
(d) the amino acid residue comprised in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue comprised in
CL at position 123 as indicated by EU numbering;
[0066] [42] the method of [40] or [41], wherein the amino acid
residues that do not mutually repel electrically are amino acid
residues selected from each of two sets selected from the group
consisting of (X) to (Z) below, and wherein the two sets are
selected from among the combinations of (X) and (Y), (X) and (Z),
(Y) and (Z), and (Z) and (Z):
(X) glutamic acid (E) or aspartic acid (D); (Y) lysine (K),
arginine (R), or histidine (H); and (Z) alanine (A), asparagine
(N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I),
leucine (L), methionine (M), phenylalanine (F), proline (P), serine
(S), threonine (T), tryptophan (W), tyrosine (Y), or valine
(V);
[0067] [43] the method of any one of [40] to [42], wherein the
amino acid residues that do not mutually repel electrically are the
amino acid residue comprised in CH1 at position 175 as indicated by
EU numbering which is lysine (K), and the amino acid residues
comprised in CL at position 180, position 131, and position 160 as
indicated by EU numbering which are all glutamic acid (E);
[0068] [44] the method of any one of [40] to [42], wherein the
amino acid residues that do not mutually repel electrically are the
amino acid residues comprised in CH1 at position 147 and position
175 as indicated by EU numbering which are glutamic acid (E), and
the amino acid residues comprised in CL at position 180, position
131, and position 160 as indicated by EU numbering which are all
lysine (K);
[0069] [45] the method of [44], further wherein the amino acid
residue contained in CH1 at position 213 as indicated by EU
numbering is glutamic acid (E), and the amino acid residue
contained in CL at position 123 as indicated by EU numbering is
lysine (K);
[0070] [46] the method of any one of [34] to [45], wherein the
antigen-binding molecule is a bispecific antibody;
[0071] [47] a composition containing the antigen-binding molecule
of any one of [1] to [16], [32], and [33], and a pharmaceutically
acceptable carrier;
[0072] [48] a nucleic acid encoding the antigen-binding molecule of
any one of [1] to [16], [32], and [33]; and
[0073] [49] a host cell having the nucleic acid of [48].
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] FIG. 1 is a model diagram of an CH1/CL interface.
[0075] FIG. 2 is a conceptual antibody showing possible
combinations of H chain and L chain when the antibody is prepared
by mixing one type of H chain and two types of L chains. Mutated
sites which gave a large proportion of the antibody with the
combination of E and K as shown in the box are thought to interact
electrically.
[0076] FIG. 3 depicts graphs showing results of the AIEX analysis
of each of the antibodies.
[0077] FIG. 4 depicts graphs showing results of the AIEX analysis
of each of the antibodies.
[0078] FIG. 5 depicts graphs showing results of the AIEX analysis
of each of the antibodies.
[0079] FIG. 6 depicts a graph showing results of the AIEX analysis
of each of the antibodies.
[0080] FIG. 7 depicts graphs showing results of the CIEX analysis
of each of the antibodies.
[0081] FIG. 8 depicts graphs showing results of the CIEX analysis
of each of the antibodies.
[0082] FIG. 9-1 depicts graphs showing the results of CIEX analysis
of each of the antibodies. FIG. 9-2 is a continuation of FIG.
9-1.
[0083] FIG. 10 depicts graphs showing results of the CIEX analysis
of each of the antibodies.
[0084] FIG. 11 depicts graphs showing results of the CIEX analysis
of each of the antibodies.
[0085] FIG. 12 is a diagram comparing the H chain CH1 by aligning
the amino acid sequences of human IgA1 (SEQ ID NO: 63), IgA2 (SEQ
ID NO: 64), IgD (SEQ ID NO: 65), IgE (SEQ ID NO: 66), IgG1 (SEQ ID
NO: 67), IgG2 (SEQ ID NO: 68), IgG3 (SEQ ID NO: 69), IgG4 (SEQ ID
NO: 70), and IgM (SEQ ID NO: 71); and the L chain CL by aligning
the amino acid sequences of human IgK (Kappa) (SEQ ID NO: 72), IgL1
(SEQ ID NO: 73), IgL2 (SEQ ID NO: 74), IgL3 (SEQ ID NO: 75), IgL6
(SEQ ID NO: 76), IgL7 (SEQ ID NO: 77) (Lambda).
MODE FOR CARRYING OUT THE INVENTION
[0086] The present invention relates to antibodies in which the
association of the heavy chains and light chains is regulated,
methods for producing an antibody in which the association of the
heavy chains and light chains is regulated, and methods for
regulating the association of the heavy chains and light chains of
an antibody.
[0087] In the present invention, the term "antibody" is used
synonymously with "antigen-binding molecule". That is, in the
present invention, the terms "antibody" and "antigen-binding
molecule" are used in the broadest sense, and include monoclonal
antibodies, polyclonal antibodies, and antibody variants (such as
chimeric antibodies, humanized antibodies, low molecular weight
antibodies (including antibody fragments to which other molecules
may be added arbitrarily), and polyspecific antibodies) provided
that they demonstrate the desired antigen-binding activity or
biological activity. An example of an "antibody" or
"antigen-binding molecule" in the present invention is a molecule
in which an HAS-binding scaffold has been added to the Fab (an
antibody in which only the Fab portion is normal). In addition, in
the present invention, an "antibody" may also be a polypeptide or a
heteromeric multimer. Preferred antibodies are monoclonal
antibodies, chimeric antibodies, humanized antibodies, human
antibodies, Fc-fusion antibodies and low molecular weight
antibodies such as antibody fragments.
[0088] The antibody of the present invention is an antibody in
which the association of the heavy chain and light chain is
regulated, in which the heavy chain and light chain constituting
the antibody are a combination of heavy chain and light chain of
interest, and in which the amino acid residues at given locations
in the constant region of the heavy chain (CH1) and the constant
region of the light chain are mutually electrically repelling amino
acid residues (having the same charge).
[0089] In the present invention, by making amino acid residues at
given locations in the constant region of the heavy chain (CH1) and
constant region of the light chain of an undesired combination of
heavy chain and light chain into amino acid residues that mutually
repel electrically (i.e., that have the same charge), the formation
of undesired combinations of heavy chain and light chain can be
prevented by utilizing this charge repulsion, and as a result, the
desired combination of heavy chain and light chain can be
formed.
[0090] In another embodiment, the antibody of the present invention
is an antibody in which the association of the heavy chain and
light chain is regulated, in which the heavy chain and light chain
constituting the antibody associate as a combination of heavy chain
and light chain of interest, and in which the amino acid residues
at given locations in the constant region of the heavy chain (CH1)
and in the constant region of the light chain do not mutually repel
electrically.
[0091] By making amino acid residues at given locations in the
heavy chain constant region (CH1) and the light chain constant
region of a desired combination of heavy chain and light chain into
amino acid residues that do not mutually repel electrically, a
desired combination of heavy chain and light chain can be formed,
for example, by using the attractive force of the electric charges.
In the present invention, the term "polypeptide" generally refers
to peptides and proteins whose length is about ten amino acids or
longer. Polypeptides are ordinarily derived from organisms but are
not particularly limited thereto, and for example, they may be
composed of an artificially designed sequence. They may also be any
of naturally derived polypeptides, synthetic polypeptides,
recombinant polypeptides, or such. Additionally, fragments of the
above-mentioned polypeptides are also included in the polypeptides
of the present invention.
[0092] In the present invention, the phrases "to regulate
association" and "association is regulated" refer to regulating to
achieve a desired association condition, and more specifically
refers to regulating so that undesirable associations are not
formed between the heavy chain and light chain.
[0093] In the present invention, the term "interface" generally
refers to the association surface that results from association
(interaction), and amino acid residues that form the interface are
ordinarily one or more amino acid residues included in the
polypeptide regions which participate in the association, and are
more preferably amino acid residues that approach each other during
association and are involved in the interaction. More specifically,
this interaction includes, for example, instances where the amino
acid residues come close during the association to form hydrogen
bonds, electrostatic interactions, or salt bridges with each
other.
[0094] In the present invention, the phrase, "amino acid residues
forming an interface" more specifically refers to amino acid
residues included in the polypeptide region that constitutes the
interface. For example, polypeptide regions constituting the
interface refer to polypeptide regions responsible for selective
binding between molecules such as in antibodies, ligands,
receptors, or substrates. More specifically, in antibodies, such
examples include heavy chain constant regions, heavy chain variable
regions, light chain constant regions, and light chain variable
regions.
[0095] "Modification" of amino acid residues in the present
invention specifically refers to substituting original amino acid
residue(s) for other amino acid residue(s), deleting original amino
acid residue(s), adding new amino acid residue(s), and such, but
preferably refers to substituting one or more original amino acid
residues for other amino acid residues.
[0096] In a preferred embodiment of the antibody of the present
invention, the antibody has amino acid residues at given locations
in the heavy chain constant region (CH1) and light chain constant
region of an undesired combination of heavy chain and light chain
before association regulation which electrically repel (which have
the same charge).
[0097] By modifying amino acid residues in the aforementioned
antibody into amino acid residues that mutually repel electrically
(have the same charge), association of these amino acid residues is
thought to be inhibited by the repulsive force of electrical
charges.
[0098] In another preferred embodiment of the antibody of the
present invention, the antibody has amino acid residues involved in
association at the interface of polypeptides that do not mutually
repel electrically.
[0099] In the aforementioned antibody, by modifying amino acid
residues involved in association at the interface of polypeptides
into amino acid residues that do not mutually repel electrically,
association of these amino acid residues is thought to be promoted
by, for example, the attractive force of their electrical
charges.
[0100] Thus, in the aforementioned antibody, the modified amino
acid residues are preferably amino acid residues that approach each
other at association, in the polypeptide regions forming the
interface.
[0101] The amino acid residues that approach during association can
be determined by, for example, analyzing the three-dimensional
structure of a polypeptide, and investigating the amino acid
sequences of the polypeptide regions that form an interface during
polypeptide association. Amino acid residues at the interface that
mutually approach each other are preferable targets of
"modification" in the antibody of the present invention.
[0102] Some amino acids are known to be electrically charged. In
general, lysine (K), arginine (R) and histidine (H) are known to be
amino acids having a positive charge (positively charged amino
acids). Aspartic acid (D), glutamic acid (E), and such are known to
be amino acids having a negative charge (negatively charged amino
acids). In addition, alanine (A), asparagine (N), cysteine (C),
glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine
(M), phenylalanine (F), proline (P), serine (S), threonine (T),
tryptophan (W), tyrosine (Y), valine (V), and the like are known to
be amino acids that do not have a charge, or nonpolar amino
acids.
[0103] Thus, amino acids that mutually repel electrically (have the
same charge) in the present invention refer to:
(1) amino acids in which one of the amino acids is a positively
charged amine acid and the other amino acid is also a positively
charged amino acid, and (2) amino acids in which one of the amino
acids is a negatively charged amino acid and the other amino acid
is also a negatively charged amino acid.
[0104] Further, amino acids that do not mutually repel electrically
in the present invention refer to:
(1) amino acids in which one of the amino acids is a positively
charged amino acid and the other amino acid is a negatively charged
amino acid, (2) amino acids in which one of the amino acids is a
positively charged amino acid and the other amino acid is an
uncharged amino acid or a nonpolar amino acid, (3) amino acids in
which one of the amino acids is a negatively charged amino acid and
the other amino acid is an uncharged amino acid or a nonpolar amino
acid, and (4) amino acids in which both of the amino acids are
uncharged amino acids or nonpolar amino acids.
[0105] Amino acids can be modified according to various methods
known in the field of the art. Examples of these methods include,
but are not limited to site-directed mutagenesis (Hashimoto-Gotoh,
T., Mizuno, T., Ogasahara, Y. and Nakagawa, M. (1995) An
oligodeoxyribonucleotide-directed dual amber method for
site-directed mutagenesis, Gene 152, 271-275; Zoller, M. J. and
Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA
fragments cloned into M13 vectors, Methods Enzymol. 100, 468-500;
Kramer, W., Drutsa, V., Jansen, H. W., Kramer, B., Pflugfelder, M.
and Fritz, H. J. (1984) The gapped duplex DNA approach to
oligonucleotide-directed mutation construction, Nucleic Acids Res.
12, 9441-9456; Kramer, W. and Fritz, H. J. (1987)
Oligonucleotide-directed construction of mutations via gapped
duplex DNA, Methods Enzymol. 154, 350-367; Kunkel, T. A. (1985)
Rapid and efficient site-specific mutagenesis without phenotypic
selection, Proc. Natl. Acad. Sci. USA 82, 488-492), PCR
mutagenesis, cassette mutagenesis, etc.
[0106] Examples of amino acid modifications include modification of
an uncharged amino acid or a nonpolar amino acid into a positively
charged amino acid, modification of an uncharged amino acid or a
nonpolar amino acid into a negatively charged amino acid,
modification of a positively charged amino acid into a negatively
charged amino acid, and modification of a negatively charged amino
acid into a positively charged amino acid. Furthermore,
modification of an uncharged amino acid or a nonpolar amino acid
into a different uncharged or nonpolar amino acid, modification of
a positively charged amino acid into a different positively charged
amino acid, and modification of a negatively charged amino acid
into a different negatively charged amino acid are also included in
the amino acid modifications of the present invention.
[0107] Modifying amino acids in the present invention includes
making one modification in each of the heavy and light chain, or
making multiple modifications to each of the heavy and light chain.
In addition, the number of modifications added to the heavy chain
and light chain may be the same or different.
[0108] Modifying amino acids sin the present invention includes
making multiple modifications into positively charged amino acids
on either the heavy chain or light chain, and making multiple
modifications into negatively charged amino acids on the other
chain. Moreover, multiple modifications into positively charged
amino acids as well as multiple modifications into negatively
charged amino acids may be made on the same heavy chain or light
chain. In these modifications, modifications into uncharged amino
acids or nonpolar amino acids as well as modifications of uncharged
amino acids or nonpolar amino acids may also be suitably
combined.
[0109] In the modifications of the present invention, for example,
the amino acids on one of the chains can be used as they are
without being modified s, and in such cases, the heavy chain and
light chain do not need to be both modified, and only one of the
chains may be modified.
[0110] Although there are no particular limitations to the number
of amino acid residues subjected to modification in the antibody of
the present invention, for example, when modifying the constant
region of the antibody, in order not to reduce the binding activity
toward the antigen and not to increase immunogenicity, it is
preferable to modify as few amino acid residues as possible. The
aforementioned "few" refers to, for example, a number of about 1 to
30, preferably a number of about 1 to 20, even more preferably a
number of about 1 to 15, and most preferably a number of 1 to
5.
[0111] In the present invention, the term "antibody" is used in the
broadest sense, and includes monoclonal antibodies, polyclonal
antibodies, antibody variants (such as chimeric antibodies,
humanized antibodies, low molecular weight antibodies (including
antibody fragments), and polyspecific antibodies) as long as they
demonstrate the desired biological activity. In addition, the
"antibody" in the present invention may be either a polypeptide or
a heteromeric multimer. Preferred antibodies are monoclonal
antibodies, chimeric antibodies, humanized antibodies, human
antibodies, Fc-fusion antibodies, and low molecular weight
antibodies such as antibody fragments.
[0112] In the context of the present invention, the term
"multispecific antibody" (used in the present description to have
the same meaning as "polyspecific antibody") refers to an antibody
that may bind specifically to different types of epitopes. More
specifically, multispecific antibodies are antibodies having
specificity to at least two different types of epitopes, and, in
addition to antibodies recognizing different antigens, antibodies
recognizing different epitopes on the same antigen are also
included. (For example, when the antigens are heterologous
receptors, multispecific antibodies can recognize different domains
constituting the heterologous receptors; alternatively, when the
antigens are monomers, multispecific antibodies recognize multiple
sites on the monomer antigens.) Ordinarily, such molecules bind to
two antigens (bispecific antibodies; used in the present
description to have the same meaning as "dual-specific
antibodies"), but they may even have specificity toward more
antigens (for example three types).
[0113] In addition to the antibodies described above, the
antibodies of the present invention include antibodies whose amino
acid sequences have been modified by amino acid substitutions,
deletions, additions, and/or insertions, or chimerization,
humanization, and such. Such amino acid sequence modifications,
such as amino acid substitutions, deletions, additions, and/or
insertions, and humanization and chimerization, can be achieved by
methods known to those skilled in the art. When the antibodies of
the present invention are prepared as recombinant antibodies,
likewise, the amino acid sequences of the antibody variable and
constant regions may also be modified by amino acid substitutions,
deletions, additions, and/or insertions, or chimerization,
humanization and the like.
[0114] The antibodies of the present invention may be derived from
any animal, such as a mouse, human, rat, rabbit, goat, or camel.
Furthermore, the antibodies may be modified, for example, chimeric
antibodies, and in particular, modified antibodies that include
amino acid substitutions in their sequence, such as humanized
antibodies. The antibodies may be any type of antibody, such as
antibody modification products linked with various molecules,
antibody fragments, and low molecular weight antibodies.
[0115] "Chimeric antibodies" are antibodies prepared by combining
sequences derived from different animals. An example is an antibody
having heavy and light chain variable (V) regions from a mouse
antibody and heavy and light chain constant (C) regions from a
human antibody. Chimeric antibodies can be prepared by known
methods. To obtain such chimeric antibodies, for example, a DNA
encoding an antibody V region may be ligated with a DNA encoding a
human antibody constant region; the resulting ligation product can
be inserted into an expression vector; and the construct can be
introduced into a host to produce the chimeric antibody.
[0116] "Humanized antibodies" are also referred to as reshaped
human antibodies, and can be obtained by substituting the
complementarity determining region (CDR) of a human antibody for
the CDR of an antibody derived from a nonhuman mammal, for example,
a mouse. Methods for identifying CDRs are known in the art (Kabat
et al., Sequence of Proteins of Immunological Interest (1987),
National Institute of Health, Bethesda, Md.; Chothia et al., Nature
(1989) 342:877). General genetic recombination techniques suitable
for this purpose are also known (see European Patent Application
Publication No. EP 125023; and WO 96/02576). For example, the CDR
of a mouse antibody can be determined by known methods, and a DNA
can be prepared such that it encodes an antibody in which the CDR
is ligated with the framework region (FR) of a human antibody. A
humanized antibody can then be produced using a system that uses
conventional expression vectors. Such DNAs can be synthesized by
PCR, using as primers several oligonucleotides designed to include
portions that overlap the ends of both the CDR and FR regions (see
the method described in WO 98/13388). Human antibody FRs linked via
CDRs are selected such that the CDRs form a suitable antigen
binding site. If required, amino acids in the FRs of an antibody
variable region may be modified so that the CDRs of the reshaped
human antibody can form a suitable antigen binding site (Sato, K.
et al., Cancer Res. (1993) 53:851-856). Modifiable amino acid
residues in the FRs include portions that directly bind to an
antigen via non-covalent bonds (Amit et al., Science (1986) 233:
747-53), portions that have some impact or effect on the CDR
structure (Chothia et al., J. Mol. Biol. (1987) 196: 901-17), and
portions involved in the interaction between VH and VL (EP
239400).
[0117] The heavy chain constant region of the antibody of the
present invention is preferably a human heavy chain constant
region. In addition, examples of antibody heavy chain constant
regions include IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and
IgM type constant regions. The heavy-chain constant region of the
antibody of the present invention is preferably an IgG1 type
constant region, and particularly preferably a human IgG1 constant
region, but it is not limited thereto. Several allotype sequences
obtained by genetic polymorphism are described in Sequences of
Proteins of Immunological Interest, NIH Publication No. 91-3242 as
human IgG1 constant region, and any of these may be used in the
present invention.
[0118] Moreover, the light chain constant region of the antibody of
the present invention is preferably a human light chain constant
region. Examples of antibody light chain constant region include
IgK (Kappa), IgL1, IgL2, IgL3, IgL6 and IgL7 (Lambda) type constant
regions. The light chain constant region of the antibody of the
present invention is preferably a human IgK (Kappa) constant
region, but is not limited thereto. The amino acid sequence of the
human IgK (Kappa) constant region is known (SEQ ID NO: 72). Several
allotype sequences obtained by genetic polymorphism are described
in Sequences of Proteins of Immunological Interest, NIH Publication
No. 91-3242 as human IgK (Kappa) constant region and human IgL7
(Lambda) constant region, and any of these may be used in the
present invention.
[0119] Antibody constant regions, in particular, heavy chain
constant regions, may be modified as necessary in order to improve
antibody function or antibody stability. Examples of modifications
for improving antibody function include modifications that
strengthen or weaken the binding between an antibody and an
Fc.gamma. receptor (Fc.gamma.R), modifications that strengthen or
weaken the binding between an antibody and FcRn, modifications that
strengthen or weaken antibody cytotoxic activity (such as ADCC
activity and CDC activity), and such. In addition, modifications
that improve antibody heterogeneity and modifications that improve
immunogenicity and/or pharmacokinetics may also be included.
[0120] Moreover, as the heterogeneity of the heavy chain C-terminal
sequence of the IgG antibody, amidation of the C-terminal carboxyl
group by deletion of the C-terminal amino acid, lysine residue, or
by deletion of the two C-terminal amino acids, glycine and lysine,
has been reported the (Anal. Biochem. 2007 Jan. 1:360(1):75-83).
Thus, in the present invention, to lower heterogeneity of the heavy
chain C terminus, it is preferable to use an IgG in which the
C-terminal lysine or the C-terminal lysine and glycine have been
deleted.
[0121] Since their antigenicity in the human body has been
attenuated, chimeric and humanized antibodies using human-derived
sequences are expected to be useful when administered to humans for
therapeutic purposes or such.
[0122] Moreover, low molecular weight antibodies are useful as the
antibodies because of their in vivo kinetic characteristics and
low-cost production using E. coli, plant cells, or such.
[0123] Antibody fragments are one type of low molecular weight
antibody. The term "low molecular weight antibody" includes
antibodies that include an antibody fragment as a partial
structural unit. The low molecular weight antibodies of the present
invention are not particularly limited by their structure nor their
method of production, so long as they have antigen binding
activity. Some low molecular weight antibodies have an activity
greater than that of a whole antibody (Orita et al., Blood (2005)
105:562-566). Herein, the "antibody fragments" are not particularly
limited, so long as they are a portion of a whole antibody (for
example, whole IgG). However, the antibody fragments preferably
include a heavy chain variable region (VH) or a light chain
variable region (VL), and further include CH1 or CL. Examples of
preferred antibody fragments are: Fab, F(ab').sub.2, and Fab'. The
amino acid sequences of a VH, VL, CH1, and CL in an antibody
fragment may be modified by substitution, deletion, addition,
and/or insertion. Furthermore, some portions of a CH1, CL, VH, and
VL may be deleted, so long as the resulting fragments retain their
antigen binding ability, and antibody fragments such as scFv, Fab,
domain antibody (dAb), and VHH, HAS binding scaffold, PEG, albumin,
cytokines, toxins, and the like (the molecules described in
Biodrugs, 2009, 23(2):93-109; Methods Mol. Med., 2005, 109:347-74;
AAPS J., 2006 Aug. 18, 8(3):E532-51; etc.) may also be added to
increase the pharmacokinetics (PK) or drug efficacy.
[0124] An antibody fragment can be prepared by treating an antibody
with an enzyme, for example, a protease such as papain or pepsin
(see Morimoto et al., J. Biochem. Biophys. Methods (1992) 24:
107-17; Brennan et al., Science (1985) 229:81). Alternatively,
antibody fragments can also be produced by genetic recombination
based on their amino acid sequence.
[0125] A low molecular weight antibody having a structure that
results from modification of an antibody fragment can be prepared
using antibody fragments obtained by enzyme treatment or genetic
recombination. Alternatively, after constructing a gene which
encodes a whole low molecular weight antibody, and introducing the
construct into an expression vector, the low molecular weight
antibody may be expressed in appropriate host cells (see, for
example, Co et al., J. Immunol. (1994) 152: 2968-76; Better and
Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra,
Methods Enzymol. (1989) 178: 497-515; Lamoyi, Methods Enzymol.
(1986) 121: 652-63; Rousseaux et al., Methods Enzymol. (1986) 121:
663-9; Bird and Walker, Trends Biotechnol. (1991) 9: 132-7).
[0126] A preferred example of the antibody of the present invention
is a heteromeric multimer having two or more types of CH1 and two
or more types of CL. This heteromeric multimer preferably
recognizes two or more types of epitopes, and an example thereof is
a polyspecific antibody.
[0127] A preferred example of a polyspecific antibody of the
present invention is a bispecific antibody. Thus, an example of a
preferred embodiment of the antibody of the present invention is a
bispecific antibody composed of two types of heavy chains (a first
heavy chain and a second heavy chain) and two types of light chains
(a first light chain and a second light chain).
[0128] Describing the "bispecific antibodies" of the preferred
embodiments of the antibodies of the present invention more
precisely, the above-mentioned "first heavy chain" refers to one of
the two heavy chains (H chains) forming the antibody, and the
"second H chain" refers to the other H chain that is different from
the first H chain. That is, of the two H chains, one of them can be
arbitrarily defined as the first H chain and the other can be
defined as the second H chain. Similarly, the "first light chain"
refers to one of the two light chains (L chains) forming the
bispecific antibody, and the "second L chain" refers to the other L
chain that is different from the first L chain. Of the two L
chains, one of them can be arbitrarily defined as the first L chain
and the other can be defined as the second L chain. Ordinarily, the
first L chain and the first H chain are derived from a same
antibody that recognizes a certain antigen (or epitope), and the
second L chain and the second H chain are also derived from a same
antibody that recognizes a certain antigen (or epitope). Herein,
the L chain-H chain pair formed by the first H chain and L chain is
called the first pair, and the L chain-H chain pair formed by the
second H chain and L chain is called the second pair. The antigen
(or epitope) used to produce the antibody from which the second
pair derives is preferably different from the antigen used to
produce the antibody from which the first pair derives. More
specifically, antigens recognized by the first pair and the second
pair may be the same, but preferably, the pairs recognize different
antigens (or epitopes). In this case, the H chains and L chains of
the first pair and second pair preferably have amino acid sequences
that differ from each other. When the first pair and the second
pair recognize different epitopes, the first pair and the second
pair may recognize a completely different antigen, or they may
recognize different sites (different epitopes) on the same antigen.
Furthermore, one of them may recognize an antigen such as a
protein, peptide, gene, or sugar, and the other may recognize
cytotoxic substances such as radioactive substances,
chemotherapeutic agents, or cell-derived toxins. However, when one
wishes to produce an antibody having pairs formed by specific
combinations of H chains and L chains, those specific H chains and
L chains may be arbitrary determined to be the first pair and
second pair.
[0129] As for the genes encoding the H chain or L chain of
antibodies before introduction of mutations in the present
invention (herein, it may be simply referred to as "an antibody of
the present invention"), known sequences can be used, or they can
be obtained by methods known to those skilled in the art. For
example, they may be obtained from an antibody library, or they may
be obtained by cloning genes encoding the antibody from hybridomas
producing monoclonal antibodies.
[0130] Regarding antibody libraries, many antibody libraries are
already well known, and since methods for producing antibody
libraries are known, those skilled in the art can appropriately
obtain antibody libraries. For example, regarding antibody phage
libraries, one can refer to the literature such as Clackson et al.,
Nature 1991, 352: 624-8; Marks et al., J. Mol. Biol. 1991, 222:
581-97; Waterhouses et al., Nucleic Acids Res. 1993, 21: 2265-6;
Griffiths et al., EMBO J. 1994, 13: 3245-60; Vaughan et al., Nature
Biotechnology 1996, 14: 309-14; and Japanese Patent Kohyo
Publication No. (JP-A) H10-504970 (unexamined Japanese national
phase publication corresponding to a non-Japanese international
publication). In addition, known methods, such as methods that use
eukaryotic cells as libraries (WO95/15393) and ribosome display
methods, may be used. Furthermore, techniques to obtain human
antibodies by panning using human antibody libraries are also
known. For example, variable regions of human antibodies can be
expressed on the surface of phages as single chain antibodies
(scFvs) using phage display methods, and phages that bind to
antigens can be selected. Genetic analysis of the selected phages
can determine the DNA sequences encoding the variable regions of
human antibodies that bind to the antigens. Once the DNA sequences
of scFvs that bind to the antigens is revealed, suitable expression
vectors can be produced based on these sequences to obtain human
antibodies. These methods are already well known, and one can refer
to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172,
WO95/01438, and WO95/15388.
[0131] As for methods for obtaining genes encoding antibodies from
hybridomas, known techniques may be basically used, which involve
using of desired antigens or cells expressing the desired antigens
as sensitizing antigens, using these to perform immunizations
according to conventional immunization methods, fusing the immune
cells thus obtained with known parent cells by ordinary cell fusion
methods, screening monoclonal antibody producing cells (hybridomas)
by ordinary screening methods, synthesizing cDNAs of antibody
variable regions (V regions) from mRNAs of the obtained hybridomas
using reverse transcriptase, and linking them with DNAs encoding
the desired antibody constant regions.
[0132] The sensitizing antigens for obtaining the aforementioned
antibody genes encoding the H chain and L chain are not
particularly limited to the examples described below, but include
both complete antigens having immunogenicity and incomplete
antigens including haptens and the like that do not demonstrate
immunogenicity. There are no particular limitations on the antigen
for the antibodies of the present invention, and for example, a
full-length protein or a partial peptide of a target protein, as
well as substances composed of polysaccharides, nucleic acids,
lipids, and the like that are known to be able to serve as an
antigen can be used. Antigens can be prepared in accordance with
methods that are known to those skilled in the art, such as methods
using baculoviruses (such as that described in WO 98/46777).
Hybridomas can be produced, for example, following methods of
Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981,
73: 3-46), and such. When the immunogenicity of an antigen is low,
it can be linked to a macromolecule that has immunogenicity, such
as albumin, and then used for immunization. Furthermore, by linking
antigens with other molecules as necessary, they can be converted
into soluble antigens. When transmembrane molecules such as
receptors are used as antigens, portions of the extracellular
regions of the receptors can be used as a fragment, or cells
expressing transmembrane molecules on their cell surface may be
used as immunogens.
[0133] Antibody-producing cells can be obtained by immunizing
animals using suitable sensitizing antigens described above.
Alternatively, antibody-producing cells can be prepared by in vitro
immunization of lymphocytes that can produce antibodies. Various
mammals can be used as the animals for immunization, and rodents,
lagomorphas and primates are generally used. Specific examples of
such animals include mice, rats, and hamsters for rodents, rabbits
for lagomorphas, and monkeys including the cynomolgus monkey,
rhesus monkey, hamadryas, and chimpanzees for primates.
[0134] Transgenic animals carrying human antibody gene repertoires
are also known, and human antibodies can be obtained by using these
animals (see WO96/34096; Mendez et al., Nat. Genet. 1997, 15:
146-56). Instead of using such transgenic animals, for example,
desired human antibodies having binding activity against antigens
can be obtained by in vitro sensitization of human lymphocytes with
desired antigens or cells expressing the desired antigens, and then
fusing the sensitized lymphocytes with human myeloma cells such as
U266 (see Japanese Patent Application Kokoku Publication No. (JP-B)
H1-59878 (examined, approved Japanese patent application published
for opposition)). Furthermore, desired human antibodies can be
obtained by immunizing transgenic animals carrying a complete
repertoire of human antibody genes with desired antigens (see
WO93/12227, WO92/03918, WO94/02602, WO96/34096, and
WO96/33735).
[0135] Animal immunization is carried out by appropriately diluting
and suspending a sensitizing antigen in Phosphate-Buffered Saline
(PBS), physiological saline, or such, forming an emulsion by mixing
an adjuvant if necessary, and intraperitoneally or subcutaneously
injecting this into animals. After that, the sensitizing antigen
mixed with Freund's incomplete adjuvant is preferably administered
several times every four to 21 days. Antibody production can be
confirmed by measuring the target antibody titer in animal sera
using conventional methods.
[0136] Antibody-producing cells obtained from lymphocytes or
animals immunized with a desired antigen can be fused with myeloma
cells to generate hybridomas using conventional fusing agents (for
example, polyethylene glycol) (Goding, Monoclonal Antibodies:
Principles and Practice, Academic Press, 1986, 59-103). Then, as
required, hybridoma cells can be cultured and grown, and the
binding specificity, affinity, or activity of the antibody produced
from these hybridomas can be measured using known analysis methods,
such as immunoprecipitation, radioimmunoassay (RIA), and
enzyme-linked immunosorbent assay (ELISA). Thereafter, hybridomas
that produce antibodies of interest whose binding specificity,
affinity, or activity has been determined can be subcloned by
methods such as limiting dilution.
[0137] Next, genes encoding the antibodies of interest can be
cloned from hybridomas or antibody-producing cells (sensitized
lymphocytes, and such) using probes that may specifically bind to
the antibodies (for example, oligonucleotides complementary to
sequences encoding the antibody constant regions). Cloning from
mRNAs using RT-PCR is also possible. Immunoglobulins are classified
into five different classes, IgA, IgD, IgE, IgG, and IgM. These
classes are further divided into several subclasses (isotypes) (for
example, IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2; and
such). H chains and L chains used in the present invention to
produce antibodies are not particularly limited and may derive from
antibodies belonging to any of these classes or subclasses;
however, IgG is particularly preferred.
[0138] Herein, it is possible to modify H-chain-encoding genes and
L-chain-encoding genes using genetic engineering techniques.
Genetically modified antibodies, such as chimeric antibodies and
humanized antibodies, that have been artificially modified for the
purpose of decreasing heterologous antigenicity and such against
humans, can be appropriately produced for antibodies such as mouse
antibodies, rat antibodies, rabbit antibodies, hamster antibodies,
sheep antibodies, and camel antibodies.
[0139] Chimeric antibodies are antibodies composed of a nonhuman
mammal antibody H chain and L chain variable regions, such as those
of a mouse antibody, and the H chain and L chain constant regions
of a human antibody. They can be obtained by ligating the DNA
encoding a variable region of a mouse antibody to the DNA encoding
a constant region of a human antibody, incorporating them into an
expression vector, and introducing the vector into a host for
production of the antibody. A humanized antibody is also called a
reshaped human antibody. This humanized antibody can be synthesized
by PCR from a number of oligonucleotides produced so that they have
overlapping portions at the ends of DNA sequences designed to link
the complementarity determining regions (CDRs) of an antibody of a
nonhuman mammal (such as a mouse). The obtained DNA can be ligated
to a DNA encoding a human antibody constant region. The ligated DNA
can be incorporated into an expression vector, and the vector can
be introduced into a host to produce the antibody (see EP239400 and
WO96/02576). Human antibody FRs that are ligated via the CDRs are
selected when the CDRs form a favorable antigen-binding site. If
necessary, amino acids in the framework region of an antibody
variable region may be substituted such that the CDRs of the
reshaped human antibody form an appropriate antigen-binding site
(K. Sato et al., Cancer Res. 1993, 53: 851-856).
[0140] In addition to the humanization techniques described above,
antibodies may be modified to improve their biological properties,
for example, antigenic affinity. Such modifications can be carried
out using methods such as site-directed mutagenesis (see for
example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488), PCR
mutagenesis, and cassette mutagenesis. In general, mutant
antibodies whose biological properties have been improved show
amino acid sequence homology and/or similarity of 70% or higher,
more preferably 80% or higher, and even more preferably 90% or
higher (for example, 95% or higher, 97%, 98%, 99%, etc.), when
compared to the amino acid sequence of the original antibody
variable region. Herein, a sequence homology and/or similarity is
defined as the ratio of amino acid residues that are homologous
(same residue) or similar (amino acid residues classified into the
same group based on the general properties of amino acid side
chains) to the original antibody residues, after the sequence
homology value has been maximized by sequence alignment and gap
introduction as necessary. Generally, naturally-occurring amino
acid residues are classified into groups based on the
characteristics of their side chains:
(1) hydrophobic: alanine, isoleucine, norleucine, valine,
methionine, and leucine; (2) neutral hydrophilic: asparagine,
glutamine, cysteine, threonine, and serine; (3) acidic: aspartic
acid, and glutamic acid; (4) basic: arginine, histidine, and
lysine; (5) residues that affect the orientation of the chain:
glycine, and proline; and (6) aromatic: tyrosine, tryptophan, and
phenylalanine.
[0141] Normally, an antigen binding site of an antibody is formed
by the interactions of a total of six complementarity determining
regions (hypervariable portions; CDRs) present in the variable
regions of the H chain and L chain. It is known that even one of
these variable regions has the ability to recognize and bind the
antigen, although the affinity will be lower than that of the
variable regions containing all the binding sites. Thus, with
regard to the antibody genes of the present invention encoding H
chains and L chains, the polypeptides encoded by these genes are
only required to maintain the ability of binding to a desired
antigen and to encode a fragment portion containing the respective
antigen-binding sites of the H chain and L chain.
[0142] A more detailed explanation is provided below on the case of
an IgG-type bispecific antibody having two types of heavy chain
constant regions CH1 (CH1-A and CH1-B) and two types of light chain
constant regions (CL-A and CL-B); however, the present invention
can be similarly applied to other antibodies as well.
[0143] When one wishes to obtain a bispecific antibody that would
recognize one epitope by the first CH1-A and the first CL-A, and
another epitope by the second CH1-B and the second CL-B,
theoretically there is the possibility that 10 types of antibody
molecules may be produced when each of the four types of chains is
expressed for producing that antibody.
[0144] In this case, desired antibody molecules can be
preferentially acquired if, for example, the association is
regulated so that association of CH1-A and CL-B and/or between
CH1-B and CL-A is inhibited.
[0145] An example is modifying amino acid residues forming an
interface between CH1-A and CL-B into positively charged amino acid
residues and modifying amino acid residues forming an interface
between CH1-B and CL-A into negatively charged amino acid residues.
As a result of these modifications, unintended association between
CH1-A and CL-B is inhibited since the amino acid residues forming
the interface are both positively charged, and association between
CH1-B and CL-A is also inhibited since the amino acid residues
forming the interface are both negatively charged. Thus, the
unintended association between CH1-A and CL-B and association
between CH1-B and CL-A are inhibited because the amino acid
residues forming the interfaces mutually have the same charge. As a
result, antibodies having the intended association between CH1-A
and CL-A, and the intended association between CH1-B and CL-B can
be acquired efficiently. Moreover, the intended association between
CH1-A and CL-A is promoted since the amino acid residues forming
the interface have different types of charges from each other; and
the intended association between CH1-B and CL-B is also promoted
since the amino acid residues forming the interface have different
types of charges from each other. Consequently, antibodies with
intended association can be efficiently obtained.
[0146] Another example is modifying the amino acid residues forming
the interface between CH1-A and CL-B into positively charged amino
acid residues, when the amino acid residues forming the interface
between CL-A and CH1-B are mutually uncharged or nonpolar amino
acids. As a result of this modification, the unintended association
between CH1-A and CL-B is inhibited because the amino acid residues
forming the interface are both positively charged. On the other
hand, since the amino acid residues forming the interfaces are
amino acids that do not mutually repel electrically, the intended
association between CH1-A and CL-A, and the intended association
between CH1-B and CL-B will occur more easily than in the case
where the amino acids repel electrically. Consequently, antibodies
having the intended association between CH1-A and CL-A, and the
intended association between CH1-B and CL-B can be efficiently
obtained. Meanwhile, in this example, in the case that the amino
acid residues forming the interface between CL-A and CH1-B are not
mutually uncharged or nonpolar amino acids, they may be modified so
as to become mutually uncharged or nonpolar amino acids.
[0147] In another example, when amino acid residues forming the
interface between CL-B and CH1-B are mutually uncharged or nonpolar
amino acids, one of the amino acid residues forming the interface
between CH1-A and CL-A is modified into a positively charged amino
acid residue, and the other is modified into a negatively charged
amino acid residue. As a result of this modification, while the
intended association between CH1-A and CL-A is promoted because the
amino acid residues forming the interface are a combination of
positive charge and negative charge, the intended association
between CH1-B and CL-B is not inhibited because the amino acid
residues forming the interface are amino acids that do not mutually
repel electrically. As a result, one can efficiently obtain an
antibody having intended association between CH1-A and CL-A, and
intended association between CH1-B and CL-B. Meanwhile, in this
example, when the amino acid residues forming the interface between
CL-B and CH1-B are not mutually uncharged or nonpolar amino acids,
they may be modified so as to become mutually uncharged or nonpolar
amino acids.
[0148] Moreover, in another example, when the amino acid residues
forming the interface between CL-B and CH1-B are uncharged or
nonpolar amino acids in CH1-B, one of the amino acid residues
forming the interface between CH1-A and CL-A is modified into a
positively charged amino acid residue while the other is modified
into a negatively charged amino acid residue; and amino acid
residues forming the interface between CL-B and CH1-B in CL-B are
modified so as to have the same charge as the modification made to
CH1-A. As a result of this modification, while the intended
association between CH1-A and CL-A is promoted because the amino
acid residues forming the interface are a combination of positive
charge and negative charge, the intended association between CH1-B
and CL-B is not inhibited because the amino acid residues forming
the interface are amino acids that do not mutually repel
electrically. As a result, one can efficiently obtain an antibody
having intended association between CH1-A and CL-A, and intended
association between CH1-B and CL-B. Meanwhile, in this example,
when the amino acid residues forming the interface between CL-B and
CH1-B are not uncharged or nonpolar amino acids in CH1-B, they may
be modified so as to become uncharged or nonpolar amino acids.
[0149] In addition, use of the association regulation of the
present invention makes it possible to suppress association between
CHls (CH1-A and CH1-B), or association between CLs (CL-A and
CL-B).
[0150] Those skilled in the art would be able to suitably determine
the types of amino acid residues that come close during association
at the CH1 and CL interface in a desired polypeptide for which
regulation of association by the present invention is desired.
[0151] Further, those skilled in the art can also suitably acquire
sequences that can be used as CH1 or CL of an antibody in an
organism such as a human, monkey, mouse, rabbit, and the like by
using a public database and such. More specifically, the amino acid
sequence information of CH1 or CL can be acquired by means
described in the Examples described below.
[0152] For example, with respect to the bispecific antibodies
described in the Examples below, specific examples of amino acid
residues that come close (that face or are in contact) at the
interface of CH1 and CL upon association include the combinations
shown below: [0153] lysine (K) at position 147 as indicated by EU
numbering in CH1 (for example, position 147 in the amino acid
sequence of SEQ ID NO: 1) and the facing (contacting) threonine (T)
at position 180 as indicated by EU numbering in CL; [0154] lysine
(K) at position 147 as indicated by EU numbering in CH1 and the
facing (contacting) serine (S) at position 131 as indicated by EU
numbering in CL; [0155] glutamine (Q) at position 175 as indicated
by EU numbering in CH1 and the facing (contacting) glutamine (Q) at
position 160 as indicated by EU numbering in CL; and, [0156] lysine
(K) at position 213 as indicated by EU numbering in CH1 and the
facing (contacting) glutamic acid (E) at position 123 as indicated
by EU numbering in CL.
[0157] The numbers described in EU numbering in the present
invention are indicated in accordance with EU numbering (Sequences
of proteins of immunological interest, NIH Publication No.
91-3242). In the present invention, the phrases "an amino acid
residue at position X as indicated by EU numbering" and "an amino
acid at position X as indicated by EU numbering" (where X is an
arbitrary number) can also be read as "an amino acid residue that
corresponds to position X as indicated by EU numbering" and "an
amino acid that corresponds to position X as indicated by EU
numbering".
[0158] As indicated in the Examples described below, desired
antibodies can be preferentially acquired by modifying these amino
acid residues and carrying out the methods of the present
invention.
[0159] Accordingly, the present invention provides an antibody in
which association of the heavy chain and light chain is regulated,
wherein one or two or more sets of amino acid residues selected
from the group consisting of the sets of amino acid residues shown
in (a) to (c) below in the heavy chain and light chain of the
antibody are amino acid residues that mutually repel
electrically:
(a) the amino acid residue contained in the heavy chain constant
region (CH1) at position 147 as indicated by EU numbering, and the
amino acid residue contained in the light chain constant region
(CL) at position 180 as indicated by EU numbering; (b) the amino
acid residue contained in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue contained in CL at position
131 as indicated by EU numbering; and (c) the amino acid residue
contained in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue contained in CL at position 160 as indicated
by EU numbering.
[0160] As another embodiment, the present invention further
provides an antibody in which the amino acid residues in the set of
the amino acid residues of (d) below are amino acid residues that
mutually repel electrically:
(d) the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 123 as indicated by EU numbering.
[0161] In the aforementioned antibody, the "amino acid residues
that mutually repel electrically" or "amino acid residues having
the same charge" are preferably selected from amino acid residues
contained in, for example, either of the set of (X) or (Y)
below:
(X) glutamic acid (E) or aspartic acid (D); or (Y) lysine (K),
arginine (R), or histidine (H).
[0162] In the aforementioned antibody, specific examples of amino
acid residues that mutually repel electrically include the amino
acid residues below: [0163] the amino acid residue contained in CH1
at position 175 as indicated by EU numbering is lysine (K), and the
amino acid residues contained in CL at position 180, position 131,
and position 160 as indicated by EU numbering are all glutamic acid
(E); and, [0164] the amino acid residues contained in CH1 at
position 147 and position 175 as indicated by EU numbering are
glutamic acid (E), and the amino acid residues contained in CL at
position 180, position 131, and position 160 as indicated by EU
numbering are all lysine (K).
[0165] In the aforementioned antibody, examples of amino acid
residues that do not electrically repel further include one in
which the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering is glutamic acid (E), and the amino acid
residue contained in CL at position 123 as indicated by EU
numbering is lysine (K).
[0166] Moreover, methods for producing an aforementioned antibody
and methods of the present invention for regulating association
through modification of the amino acid residues in the sets of
amino acid residues of (a) to (d) mentioned above into amino acid
residues that mutually repel electrically are also preferred
embodiments of the present invention.
[0167] Further, the present invention provides an antibody in which
association of the heavy chain and light chain is regulated,
wherein one or two or more sets of amino acid residues selected
from the group consisting of the sets of amino acid residues shown
in (a) to (c) below in the associating heavy chain and light chain
of the antibody are amino acid residues that do not mutually repel
electrically:
(a) the amino acid residue contained in CH1 at position 147 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 180 as indicated by EU numbering; (b) the amino acid
residue contained in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue contained in CL at position
131 as indicated by EU numbering; and (c) the amino acid residue
contained in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue contained in CL at position 160 as indicated
by EU numbering.
[0168] As another embodiment, the present invention further
provides an antibody in which the amino acid residues in the set of
amino acid residues shown in (d) below are amino acid residues that
do not mutually repel electrically:
(d) the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 123 as indicated by EU numbering.
[0169] As indicated in the Examples described below and FIG. 1,
each of the amino acid residues of the aforementioned combinations
mutually approaches upon association. Those skilled in the art
would be able to find sites corresponding to the amino acid
residues described in (a) to (d) mentioned above for the desired
CH1 or CL by homology modeling and such using commercially
available software, and to suitably modify the amino acid residues
at those sites.
[0170] In the aforementioned antibody, the "amino acid residues
that do not mutually repel electrically" are preferably selected
from, for example, each of the two sets selected from the group
consisting of (X) to (Z) shown below, and where the two sets are
selected from among the combinations of (X) and (Y), (X) and (Z),
(Y) and (Z), and (Z) and (Z):
(X) glutamic acid (E) or aspartic acid (D); (Y) lysine (K),
arginine (R) or histidine (H); (Z) alanine (A), asparagine (N),
cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine
(L), methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y) or valine (V).
[0171] An example involves selecting (X) and (Y) from the group
consisting of (X) to (Z), selecting glutamic acid from (X) and
selecting lysine (K) from (Y); and modifying the amino acid residue
contained in CH1 at position 147 as indicated by EU numbering to
glutamic acid (E), and modifying the amino acid residue contained
in CL at position 180 as indicated by EU numbering to lysine (K).
In this case, there is no need to modify the amino acid residue
contained in CH1 at position 147 as indicated by EU numbering if
the amino acid residue is glutamic acid (E) from before the
modification.
[0172] In the aforementioned antibody, specific examples of amino
acid residues that do not mutually repel electrically include the
amino acid residues shown below: [0173] the amino acid residue
contained in CH1 at position 175 as indicated by EU numbering is
lysine (K), and the amino acid residues contained in CL at position
180, position 131, and position 160 as indicated by EU numbering
are all glutamic acid (E); and [0174] the amino acid residues
contained in CH1 at position 147 and position 175 as indicated by
EU numbering are glutamic acid (E), and the amino acid residues
contained in CL at position 180, position 131, and position 160 as
indicated by EU numbering are all lysine (K).
[0175] In the aforementioned antibody, examples of amino acid
residues that do not electrically repel further include one in
which the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering is glutamic acid (E), and the amino acid
residue contained in CL at position 123 as indicated by EU
numbering is lysine (K).
[0176] Moreover, methods for producing an aforementioned antibody,
and methods of the present invention for regulating association
through modification of the amino acid residues in the sets of
amino acid residues in (a) to (d) mentioned above into amino acid
residues that do not mutually repel electrically are also preferred
embodiments of the present invention.
[0177] A technique for introducing electrical repulsion into the
interface of the second constant region of the heavy chain (CH2) or
the third constant region of the heavy chain (CH3) to suppress
undesired association between heavy chains, a technique for
introducing electrical repulsion into the interface of the heavy
chain variable region and light chain variable region to suppress
unintended association between the heavy chain and light chain, or
a technique for modifying amino acid residues forming a hydrophobic
core present at the interface of the heavy chain variable region
and light chain variable region into polar amino acids having an
electrical charge to suppress unintended association between the
heavy chain and light chain can be further applied to the antibody
of the present invention (see WO 2006/106905).
[0178] In the technique that suppresses unintended association
between heavy chains by introducing electrical repulsion at the
interface of CH2 or CH3, examples of amino acid residues that are
in contact at the interface of other constant regions of the heavy
chain include regions corresponding to position 377 (position 356)
and position 470 (position 439), position 378 (position 357) and
position 393 (position 370), and position 427 (position 399) and
position 440 (position 409) in the CH3 region. For the numbering of
the antibody constant regions, one may refer to the publication by
Kabat et al. (Kabat, E. A., et al., 1991, Sequences of Proteins of
Immunological Interest, NIH); and for the numbering of the heavy
chain constant regions, the EU numbering are shown inside the
parentheses.
[0179] The technique of modifying the amino acid residue at
position 435 as indicated by EU numbering, which is a site related
to binding between IgG and Protein A, to an amino acid having a
different binding strength toward Protein A, such as Arg, may also
be used on the antibody of the present invention in combination
with the aforementioned techniques. By using this technique, the
interaction between the H chain and Protein A can be changed, and
only heterodimeric antibodies can be efficiently purified using a
Protein A column. This technique can also be used independently
without combining with the aforementioned techniques.
[0180] More specifically, for example, in an antibody containing
two types of heavy chain CH3 regions, one to three sets of amino
acid residues in the first heavy chain CH3 region, which are
selected from the sets of amino acid residues of (1) to (3) below,
may be made to mutually repel electrically:
(1) the amino acid residues contained in the heavy chain CH3 region
at position 356 and position 439 as indicated by EU numbering; (2)
the amino acid residues contained in the heavy chain CH3 region at
position 357 and position 370 as indicated by EU numbering; and (3)
the amino acid residues contained in the heavy chain CH3 region at
position 399 and position 409 as indicated by EU numbering.
[0181] Moreover, the antibody can be an antibody having a set of
amino acid residues in the second heavy chain CH3 region distinct
from the aforementioned first heavy chain CH3 region, wherein the
set of amino acid residues is selected from the sets of amino acid
residues shown in (1) to (3) above, and wherein the one to three
sets of amino acid residues that correspond to the sets of amino
acid residues shown in (1) to (3) above, which mutually repel
electrically in the first heavy chain CH3 region, do not
electrically repel from the corresponding amino acid residues in
the first heavy chain CH3 region.
[0182] The amino acid residues described in (1) to (3) above
approach each other upon association. Those skilled in the art
would be able to find sites corresponding to the amino acid
residues described in (1) to (3) mentioned above for a desired
heavy chain CH3 region or heavy chain constant region by homology
modeling and such using commercially available software, and to
suitably modify the amino acid residues at those sites.
[0183] In the aforementioned antibody, "electrically repelling" or
"having a same charge" means that, for example, any two or more
amino acid residues have amino acid residues that are contained in
either one group of (X) and (Y) mentioned above. On the other hand,
"not electrically repelling" means that, for example, the antibody
has amino acid residues that are selected from each of two sets
selected from the group consisting of (X) and (Y) mentioned above
and (Z) below, where the two sets are selected from among the
combinations of (X) and (Y), (X) and (Z), (Y) and (Z), and (Z) and
(Z):
(Z) alanine (A), asparagine (N), cysteine (C), glutamine (Q),
glycine (G), isoleucine (I), leucine (L), methionine (M),
phenylalanine (F), proline (P), serine (S), threonine (T),
tryptophan (W), tyrosine (Y) or valine (V).
[0184] In a preferred embodiment of the aforementioned antibody,
the first heavy chain CH3 region and the second heavy chain CH3
region may be cross-linked by disulfide bonds.
[0185] In the present invention, an amino acid residue subjected to
"modification" is not limited to an amino acid residue of the
antibody variable region or antibody constant region mentioned
above. Those skilled in the art would be able to find amino acid
residues that form an interface in a polypeptide variant or
heteromeric multimer by homology modeling and the like using
commercially available software, and to modify amino acid residues
at those sites so as to regulate association. Homology modeling is
a technique for predicting the three-dimensional structure of a
protein using commercially available software. When constructing
the structure of a protein with unknown three-dimensional
structure, one first searches for a protein that has been
determined to have a highly homologous three-dimensional structure
to the protein. Next, using this three-dimensional structure as a
template, one constructs the structure of the protein with unknown
structure, and the structure is further optimized by molecular
dynamics methods and the like to predict the three-dimensional
structure of the unknown protein.
[0186] In the technique for introducing electrical repulsion into
the interface of the heavy chain variable region and light chain
variable region to suppress undesired association of the heavy
chain and light chain, examples of amino acid residues that are in
contact at the interface of the heavy chain variable region (VH)
and light chain variable region (VL) include glutamine (Q) at
position 39 as indicated by Kabat numbering in the heavy chain
variable region (FR2 region) and the facing (contacting) glutamine
(Q) at position 38 as indicated by Kabat numbering in the light
chain variable region (FR2 region). Moreover, a preferable example
is leucine (L) at position 45 according to the Kabat numbering in
the heavy chain variable region (FR2) and the facing proline (P) at
position 44 according to the Kabat numbering in the light chain
variable region (FR2). The publication by Kabat, et al. (Kabat, E.
A., et al., 1991, Sequence of Proteins of Immunological Interest,
NIH) was referred to for the numbering of these sites.
[0187] Since these amino acid residues are known to be highly
conserved in humans and mice (J. Mol. Recognit. 2003; 16: 113-120),
association of antibody variable regions can be regulated for VH-VL
association of antibodies other than those indicated in the
Examples by modifying amino acid residues corresponding to the
above-mentioned amino acid residues.
[0188] A specific example is an antibody in which two or more amino
acid residues forming the interface of the heavy chain variable
region and light chain variable region are amino acid residues that
mutually repel electrically.
[0189] More specifically, examples include an antibody with one set
or two sets of amino acid residues selected from the group
consisting of the sets of amino acid residues shown in (a) or (b)
below:
(a) the amino acid residue contained in the heavy chain variable
region (1) at position 39 as indicated by Kabat numbering and the
amino acid residue contained in the light chain (2) at position 38
as indicated by Kabat numbering; or (b) the amino acid residue
contained in the heavy chain variable region (3) at position 45 as
indicated by Kabat numbering and the amino acid residue contained
in the light chain variable region (4) at position 44 as indicated
by Kabat numbering.
[0190] Each of the amino acid residues described in the
aforementioned (a) or (b) approaches each other upon association.
Those skilled in the art would be able to find sites that
correspond to the amino acid residues described in the
aforementioned (a) or (b) in a desired heavy chain variable region
or light chain variable region by homology modeling and the like
using commercially available software, and to suitably modify the
amino acid residues at those sites.
[0191] In the aforementioned antibody, "amino acid residues that
mutually repel electrically" are preferably selected from amino
acid residues contained in, for example, either of the sets (X) and
(Y) below:
(X) glutamic acid (E) or aspartic acid (D); or (Y) lysine (K),
arginine (R), or histidine (H).
[0192] In addition, another embodiment of the antibody of the
present invention is, for example, an antibody in which two or more
amino acid residues that form the interface of the heavy chain
variable region and light chain variable region are amino acid
residues that do not electrically repel each other. Specifically,
an example of such an antibody is one having one set or two sets of
amino acid residues selected from the group consisting of the sets
of amino acid residues shown in the aforementioned (a) and (b).
[0193] The respective amino acid residues described in the
aforementioned (a) or (b) are close to each other upon association.
Those skilled in the art would be able to find sites that
correspond to the amino acid residues described in the
aforementioned (a) or (b) for a desired heavy chain variable region
or light chain variable region by homology modeling and the like
using commercially available software, and to suitably subject
amino acid residues at those sites to modification.
[0194] In the aforementioned antibody, "amino acid residues that do
not mutually repel electrically" refers to, for example, amino acid
residues selected from each of two sets selected from the group
consisting of (X) to (Z) mentioned above, and where the two sets
are selected from among the combinations of (X) and (Y), (X) and
(Z), (Y) and (Z), and (Z) and (Z).
[0195] Generally, the amino acid residues described in the
aforementioned (a) or (b) in humans and mice are:
(1) glutamine (Q), (2) glutamine (Q), (3) leucine (L), and (4)
proline (P). Thus, in a preferred embodiment of the present
invention, these amino acid residues are subjected to modification
(such as substitution with amino acids having an electrical
charge). Furthermore, the types of amino acid residues of the
aforementioned (a) or (b) are not necessarily limited to the
aforementioned amino acid residues, but may also be other amino
acids equivalent to these amino acids. For example, the amino acid
at position 38 as indicated by Kabat numbering in the light chain
variable region may be, for example, histidine (H) in the case of
humans.
[0196] In the technique for modifying amino acid residues forming a
hydrophobic core present at the interface of the heavy chain
variable region and light chain variable region into polar amino
acids having an electrical charge to suppress unintended
association of the heavy chain and light chain, preferable examples
of amino acid residues which are able to form a hydrophobic core at
the interface of the heavy chain variable region (VH) and light
chain variable region (VL) include leucine (L) at position 45 as
indicated by Kabat numbering in the heavy chain variable region
(FR2) and the facing proline (P) at position 44 as indicated by
Kabat numbering in light chain variable region (FR2). For the
numbering of these sites, Kabat, et al. (Kabat, E. A., et al.,
1991, Sequences of Proteins of Immunological Interest, NIH) was
used as a reference.
[0197] In general, the term "hydrophobic core" refers to a part
that is formed by an assembly of hydrophobic amino acid side chains
at the interior of associated polypeptides. Examples of hydrophobic
amino acids include alanine, isoleucine, leucine, methionine,
phenylalanine, proline, tryptophan, and valine. Furthermore, amino
acid residues other than hydrophobic amino acids (for example
tyrosine) may be involved in the formation of a hydrophobic core.
This hydrophobic core together with a hydrophilic surface, in which
hydrophilic amino acid side chains are exposed to the exterior,
becomes a driving force for promoting association of water-soluble
polypeptides. When hydrophobic amino acids of two different domains
are present on a molecular surface and are exposed to water
molecules, the entropy will increase and the free energy will
increase. Accordingly, the two domains will associate with each
other to decrease the free energy and become stable, and
hydrophobic amino acids at the interface will be buried into the
interior of the molecule to form a hydrophobic core.
[0198] It is thought that when polypeptide association occurs,
formation of a hydrophobic core is inhibited by modifying
hydrophobic amino acids forming the hydrophobic core to polar amino
acids having an electrical charge; and consequently, polypeptide
association is thought to be inhibited.
[0199] Those skilled in the art would be able to recognize the
presence or absence of a hydrophobic core, the formation site
(region), and the like by analyzing amino acid sequences for a
desired polypeptide. Namely, the antibody of the present invention
is an antibody characterized in that amino acid residues capable of
forming a hydrophobic core at an interface are modified to amino
acid residues having an electrical charge. More specifically,
examples include an antibody in which the amino acid residues shown
in either (1) or (2) below are amino acid residues having an
electrical charge. Side chains of the amino acid residues shown in
(1) and (2) below are adjacent to each other, and can form a
hydrophobic core:
(1) the amino acid residue contained in the heavy chain variable
region at position 45 as indicated by Kabat numbering; and (2) the
amino acid residue contained in the light chain variable region at
position 44 as indicated by Kabat numbering.
[0200] Preferable examples of amino acid residues having an
electrical charge in the aforementioned antibody include glutamic
acid (E), aspartic acid (D), lysine (K), arginine (R) and histidine
(H). More preferable examples include glutamic acid (E) and lysine
(K).
[0201] Generally, the amino acid residues described in the
aforementioned (1) and (2) in humans and mice are respectively:
(1) leucine (L), and (2) proline (P). Thus, in a preferred
embodiment of the present invention, these amino acid residues are
subjected to modification (such as substitution with amino acids
having an electrical charge). Furthermore, the types of the
aforementioned amino acid residues of (1) and (2) are not
necessarily limited to the aforementioned amino acid residues, but
may also be other amino acids equivalent to these amino acid
residues.
[0202] Other known techniques can be applied to the antibodies of
the present invention. For example, in order to promote association
of the first VH (VH1) and the first VL (VL1) and/or the second VH
(VH2) and the second VL (VL2), an amino acid side chain present in
the variable region of one of the H chains can be substituted with
a larger side chain (knob), and an amino acid side chain present in
the opposing variable region of the other H chain can be
substituted with a smaller side chain (hole), so that the knob may
be arranged in the hole, and association of VH1 and VL1 and/or VH2
and VL2 is promoted; and consequently, association of VH1 and VL2
and/or VH2 and VL1 can be further suppressed (WO 1996/027011;
Ridgway, J. B., et al., Protein Engineering (1996) 9, 617-621;
Merchant, A. M., et al., Nature Biotechnology (1998) 16,
677-681).
[0203] For example, in the case of human IgG1, in order to make an
amino acid side chain in the CH3 region of one H chain a larger
side chain (knob), the modifications of Y349C and T366W are made,
and in order to make an amino acid side chain in the CH3 region of
the other H chain a smaller side chain, the modifications of D356C,
T336S, L368A and Y407V are made.
[0204] Still other known techniques can be applied to the
antibodies of the present invention. A target antibody can be
efficiently prepared by complementary association of CH3 using
strand-exchange engineered domain CH3, in which a portion of CH3 of
one H chain of an antibody is changed to a sequence derived from
IgA corresponding to that portion, and a complementary portion of
CH3 of the other H chain is introduced with a sequence derived from
IgA corresponding to that portion (Protein Engineering Design &
Selection, 23: 195-202, 2010).
[0205] Still other known techniques can be applied to the
antibodies of the present invention. When producing bispecific
antibodies, a target bispecific antibody can be prepared by, for
example, imparting a difference in isoelectric point by making
different amino acid modifications to each of the variable regions
of the two types of H chains, and utilizing that difference in
isoelectric point for purification by ion exchange chromatography
(WO 2007/114325).
[0206] The modifications of the present invention can be used on
antibodies such as the one below, for example, an antibody having a
structure in which, to promote association of a first VH
[0207] (VH1) and a first VL (VL1) and/or a second VH (VH2) and a
second VL (VL2), VH1 is linked to an Fc region through a first CH1
and VL1 is linked to a first CL, and VH2 is linked to another Fc
region through a second CL and VL2 is linked to a second CH1 (WO
09/80254).
[0208] A plurality, for example, two or more of the aforementioned
known techniques can be used in combination for the antibody of the
present invention. Furthermore, the antibody of the present
invention may be prepared based on an antibody to which
modifications of the aforementioned known techniques have been
made.
[0209] The below-mentioned methods of the present invention for
regulating association allow, for example, for the efficient
production of antibodies or polypeptides that are active. Examples
of such activities include binding activity, neutralizing activity,
cytotoxic activity, agonist activity, antagonist activity, and
enzyme activity and such. Agonist activity is an activity that
induces some kind of changes in physiological activity through
binding of an antibody to an antigen, such as a receptor, which
causes signal transduction or such in cells. Examples of the
physiological activity include growth activity, survival activity,
differentiation activity, transcriptional activity, membrane
transport activity, binding activity, proteolytic activity,
phosphorylation/dephosphorylation activity, redox activity,
transfer activity, nucleolytic activity, dehydration activity, cell
death-inducing activity, and apoptosis-inducing activity and such,
but are not limited thereto.
[0210] Antibodies or polypeptides that recognize the desired
antigens or bind to the desired receptors can be produced
efficiently by the methods of the present invention.
[0211] The antigens of the present invention are not particularly
limited, and any type of antigen can be used. Examples of antigens
include receptors or their fragments, cancer antigens, MHC
antigens, and differentiation antigens and the like, but are not
particularly limited thereto.
[0212] Examples of the receptors of the present invention include
receptors belonging to the hematopoietic factor receptor family,
cytokine receptor family, tyrosine kinase-type receptor family,
serine/threonine kinase-type receptor family, TNF receptor family,
G protein-coupled receptor family, GPI-anchored receptor family,
tyrosine phosphatase-type receptor family, adhesion factor family,
hormone receptor family, and such. Reports on the receptors
belonging to these receptor families and their characteristics can
be found in various sources of documents, for example, in Cooke B
A., King R J B., van der Molen H J. ed. New Comprehensive
Biochemistry Vol. 18B "Hormones and their Actions Part II" pp. 1-46
(1988) Elsevier Science Publishers BV., New York, USA; Patthy L.
(1990) Cell, 61: 13-14; Ullrich A., et al. (1990) Cell, 61:
203-212; Massagul J. (1992) Cell, 69: 1067-1070; Miyajima A., et
al. (1992) Annu. Rev. Immunol., 10: 295-331; Taga T. and Kishimoto
T. (1992) FASEB J., 7: 3387-3396; Fantl W I., et al. (1993) Annu.
Rev. Biochem., 62: 453-481; Smith C A., et al. (1994) Cell, 76:
959-962; Flower D R. (1999) Biochim. Biophys. Acta, 1422: 207-234;
Miyasaka M. ed. Cell Technology, Handbook Series "Handbook for
adhesion factors" (1994) Shujunsha, Tokyo, Japan; and such.
Examples of specific receptors belonging to the above-mentioned
receptor families include human or mouse erythropoietin (EPO)
receptor, human or mouse granulocyte-colony stimulating factor
(G-CSF) receptor, human or mouse thrombopoietin (TPO) receptor,
human or mouse insulin receptor, human or mouse Flt-3 ligand
receptor, human or mouse platelet-derived growth factor (PDGF)
receptor, human or mouse interferon (IFN)-.alpha. or
-.beta.receptor, human or mouse leptin receptor, human or mouse
growth hormone (GH) receptor, human or mouse interleukin (IL)-10
receptor, human or mouse insulin-like growth factor (IGF)-I
receptor, human or mouse leukemia inhibitory factor (LIF) receptor,
and human or mouse ciliary neurotrophic factor (CNTF) receptor
(hEPOR: Simon, S. et al. (1990) Blood 76, 31-35; mEPOR: D'Andrea, A
D. et al. (1989) Cell 57, 277-285; hG-CSFR: Fukunaga, R. et al.
(1990) Proc. Natl. Acad. Sci. USA.
[0213] 87, 8702-8706; mG-CSFR: Fukunaga, R. et al. (1990) Cell 61,
341-350; hTPOR: Vigon, I. et al. (1992) 89, 5640-5644.; mTPOR:
Skoda, R C. et al. (1993) 12, 2645-2653; hInsR: Ullrich, A. et al.
(1985) Nature 313, 756-761; hFlt-3: Small, D. et al. (1994) Proc.
Natl. Acad. Sci. USA. 91, 459-463; hPDGFR: Gronwald, R G K. et al.
(1988) Proc. Natl. Acad. Sci. USA. 85, 3435-3439; hIFN
.alpha./.beta. R: Uze, G. et al. (1990) Cell 60, 225-234; and
Novick, D. et al. (1994) Cell 77, 391-400).
[0214] Cancer antigens are antigens that are expressed following
malignant transformation of a cell, and are also referred to as
tumor specific antigens. In addition, abnormal sugar chains which
appear on a cell surface or on a protein molecule when the cell has
become cancerous are also cancer antigens, and they are also
referred to as cancer sugar chain antigens. Examples of cancer
antigens include EpCAM, which is expressed in multiple cancers
including lung cancer (Proc. Natl. Acad. Sci. USA (1989) 86 (1),
27-31) (the polynucleotide sequence thereof is indicated as RefSeq
Accession No. NM_002354.2 (SEQ ID NO: 78) and the polypeptide
sequence thereof is indicated as RefSeq Accession No. NP_002345.2
(SEQ ID NO: 79)), CA19-9, CA15-3, sialyl SSEA-1 (SLX), etc.
[0215] MHC antigens can be classified broadly into MHC class I
antigens and MHC class II antigens: MHC class I antigens include
HLA-A, -B, -C, -E, -F, -G, and -H; and MHC class II antigens
include HLA-DR, -DQ, and -DP.
[0216] Differentiation antigens include CD1, CD2, CD3, CD4, CD5,
CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16,
CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33,
CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44,
CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51,
CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64,
CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, and CDw130.
[0217] The antibodies of the present invention may be a bispecific
antibody; and in that case, two antigens (or epitopes) recognized
by that antibody can be arbitrarily selected from the
aforementioned receptors or fragments thereof, cancer antigens, MHC
antigens, differentiation antigens and the like. For example, two
antigens may be selected from receptors or fragments thereof, two
may be selected from cancer antigens, two may be selected from MHC
antigens, or two may be selected from differentiation antigens. In
addition, one antigen each may be selected from two antigens
arbitrarily selected from, for example, receptors or fragments
thereof, cancer antigens, MHC antigens, and differentiation
antigens.
[0218] In addition, the present invention provides a method for
producing an antibody in which association of the heavy chain and
light chain is regulated.
[0219] A preferred embodiment of the production method of the
present invention is a method for producing an antibody in which
association of the heavy chain and light chain is regulated,
comprising:
(1) modifying nucleic acids encoding CH1 and CL so that one set or
two or more sets of amino acid residues selected from the group
consisting of the sets of amino acid residues shown in (a) to (c)
below become amino acid residues that electrically repel each
other: (a) the amino acid residue contained in CH1 at position 147
as indicated by EU numbering, and the amino acid residue contained
in CL at position 180 as indicated by EU numbering; (b) the amino
acid residue contained in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue contained in CL at position
131 as indicated by EU numbering; and (c) the amino acid residue
contained in CH1 at position 175 as indicated by EU numbering, and
amino acid residue contained in CL at position 160 as indicated by
EU numbering, (2) introducing the modified nucleic acids into host
cells and culturing so that the host cells express the nucleic
acids, and (3) collecting an antibody from a cell culture of the
host cells.
[0220] Another embodiment of the production method of the present
invention includes a method for producing an antibody, wherein step
(1) of the aforementioned production method further comprises
modifying the nucleic acids so that the amino acid residues of the
set of amino acid residues shown in (d) below become amino acid
residues that electrically repel each other:
(d) the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 123 as indicated by EU numbering.
[0221] In addition, the present invention relates to a production
method comprising, in the aforementioned step (1), modifying the
nucleic acids so that the amino acid residues that electrically
repel each other are selected from among the amino acid residues
contained in either of the groups of the aforementioned (X) and
(Y).
[0222] Moreover, the present invention relates to a production
method comprising in the aforementioned step (1), modifying the
nucleic acids so that two or more amino acid residues that form the
interface of the heavy chain variable region and light chain
variable region are amino acid residues that electrically repel
each other. Preferably, the amino acid residues that electrically
repel each other are any set of amino acid residues selected from
the group consisting of, for example, the sets of amino acid
residues shown in (a) and (b) below:
(a) the amino acid residue contained in the heavy chain variable
region at position 39 as indicated by Kabat numbering, and the
amino acid residue contained in the light chain variable region at
position 38 as indicated by Kabat numbering; or (b) the amino acid
residue contained in the heavy chain variable region at position 45
as indicated by Kabat numbering, and the amino acid residue
contained in the light chain variable region at position 44 as
indicated by Kabat numbering.
[0223] The aforementioned amino acid residues which electrically
repel each other are preferably selected from the amino acid
residues contained in either set of the aforementioned (X) and
(Y).
[0224] Another preferred embodiment of the production method of the
present invention includes a method for producing an antibody in
which association of the heavy chain and light chain is regulated,
comprising:
(1) modifying nucleic acids encoding CH1 and CL so that one set or
two or more sets of amino acid residues selected from the group
consisting of the sets of amino acid residues shown in (a) to (c)
below become amino acid residues that do not electrically repel
each other: (a) the amino acid residue contained in CH1 at position
147 as indicated by EU numbering, and the amino acid residue
contained in CL at position 180 as indicated by EU numbering; (b)
the amino acid residue contained in CH1 at position 147 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 131 as indicated by EU numbering; and (c) the amino
acid residue contained in CH1 at position 175 as indicated by EU
numbering, and the amino acid residue contained in CL at position
160 as indicated by EU numbering, (2) introducing the modified
nucleic acids into host cells and culturing so that the host cells
express the nucleic acids, and (3) collecting an antibody from a
culture of the host cells.
[0225] Another embodiment of the production method of the present
invention includes a production method further comprising in step
(1) of the aforementioned production method, modifying the nucleic
acids so that the amino acid residues of the set of amino acid
residues shown in (d) below become amino acid residues that do not
electrically repel each other:
(d) the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 123 as indicated by EU numbering.
[0226] In addition, the present invention relates to a production
method comprising, in the aforementioned step (1), modifying
nucleic acids so that the amino acid residues that do not
electrically repel each other are amino acid residues selected from
each of two sets selected from the group consisting of the
aforementioned (X) to (Z), and where the two sets are selected from
among the combinations of (X) and (Y), (X) and (Z), (Y) and (Z),
and (Z) and (Z).
[0227] In addition, specific examples of the amino acid residues
that do not electrically repel each other in the aforementioned
step (1) of the present invention include the following amino acid
residues: [0228] the amino acid residue contained in CH1 at
position 175 as indicated by EU numbering which is lysine (K); and
the amino acid residues contained in CL at position 180, position
131, and position 160 as indicated by EU numbering which are all
glutamic acid (E); and [0229] the amino acid residues contained in
CH1 at position 147 and position 175 as indicated by EU numbering
which are glutamic acid (E), and the amino acid residues contained
in CL at position 180, position 131, and position 160 as indicated
by EU numbering which are all lysine (K).
[0230] Moreover, in another example, the amino acid residue
contained in CH1 at position 213 as indicated by EU numbering is
glutamic acid (E), and the amino acid residue contained in CL at
position 123 as indicated by EU numbering is lysine (K).
[0231] Moreover, the present invention relates to a production
method, comprising in the aforementioned step (1), modifying the
nucleic acids so that two or more amino acid residues that form the
interface of the heavy chain variable region and light chain
variable region are amino acid residues that do not electrically
repel each other. Preferably, the amino acid residues that do not
electrically repel each other are, for example, amino acid residues
of any set selected from the group consisting of the sets of amino
acid residues indicated in (a) and (b) below:
(a) the amino acid residue contained in the heavy chain variable
region at position 39 as indicated by Kabat numbering, and the
amino acid residue contained in the light chain variable region at
position 38 as indicated by Kabat numbering; and (b) the amino acid
residue contained in the heavy chain variable region at position 45
as indicated by Kabat numbering, and the amino acid residue
contained in the light chain variable region at position 44 as
indicated by Kabat numbering.
[0232] The aforementioned amino acid residues that do not
electrically repel each other are preferably amino acid residues
selected from each of two sets selected from the group consisting
of the aforementioned (X) to (Z), and where the two sets are
selected from among the combinations of (X) and (Y), (X) and (Z),
(Y) and (Z), and (Z) and (Z).
[0233] In addition, the present invention provides a method for
regulating association of the heavy chains and light chains of an
antibody.
[0234] A preferred embodiment of the method for regulating
association of the present invention is a method for regulating
association of the heavy chains and light chains of an antibody,
comprising modifying nucleic acids so that one set or two or more
sets of amino acid residues selected from the group consisting of
the sets of amino acid residues shown in (a) to (c) below become
amino acid residues that electrically repel each other:
(a) the amino acid residue contained in CH1 at position 147 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 180 as indicated by EU numbering; (b) the amino acid
residue contained in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue contained in CL at position
131 as indicated by EU numbering; and (c) the amino acid residue
contained in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue contained in CL at position 160 as indicated
by EU numbering.
[0235] Another embodiment of the present invention provides a
method for regulating association in an antibody, further
comprising modifying nucleic acids so that amino acid residues of
the set of amino acid residues shown in (d) below are amino acid
residues that electrically repel each other:
(d) the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 123 as indicated by EU numbering.
[0236] Another preferred embodiment of the method for regulating
association of the present invention is a method for regulating
association of the heavy chains and light chains of an antibody,
comprising modifying nucleic acids so that one set or two or more
sets of amino acid residues selected from the group consisting of
the sets of amino acid residues shown in (a) to (c) below become
amino acid residues that do not electrically repel each other:
(a) the amino acid residue contained in CH1 at position 147 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 180 as indicated by EU numbering; (b) the amino acid
residue contained in CH1 at position 147 as indicated by EU
numbering, and the amino acid residue contained in CL at position
131 as indicated by EU numbering; and (c) the amino acid residue
contained in CH1 at position 175 as indicated by EU numbering, and
the amino acid residue contained in CL at position 160 as indicated
by EU numbering.
[0237] Another embodiment of the present invention provides a
method for regulating association in an antibody, further
comprising modifying nucleic acids so that amino acid residues of
the set of amino acid residues indicated in (d) below are amino
acid residues that do not electrically repel each other:
(d) the amino acid residue contained in CH1 at position 213 as
indicated by EU numbering, and the amino acid residue contained in
CL at position 123 as indicated by EU numbering.
[0238] According to the method for regulating association of the
present invention, a desired bispecific antibody can be obtained
preferentially and efficiently as previously described. Namely, a
desired heteromeric multimer in the form of a bispecific antibody
can be efficiently formed from a monomer mixture.
[0239] The phrase "modify nucleic acids" in the above-mentioned
methods of the present invention refers to modifying nucleic acids
so that they correspond to amino acid residues introduced by the
"modifications" of the present invention. More specifically, it
refers to modifying the nucleic acids encoding the original
(pre-modified) amino acid residues to the nucleic acids encoding
the amino acid residues that are to be introduced by the
modification. Ordinarily, it means performing gene manipulations or
mutation treatment that would result in at least one nucleotide
insertion, deletion, or substitution to the original nucleic acid
so that codons encoding amino acid residues of interest is formed.
More specifically, codons encoding the original amino acid residues
are substituted with codons encoding the amino acid residues that
are to be introduced by the modification. Such nucleic acid
modification can be performed suitably by those skilled in the art
using known techniques such as site-specific mutagenesis and PCR
mutagenesis.
[0240] In addition, the present invention provides nucleic acids
that encode an antibody of the present invention. Moreover, vectors
carrying the nucleic acids are also included in the present
invention.
[0241] The nucleic acids of the present invention are ordinarily
carried by (inserted into) suitable vectors and then introduced
into host cells. These vectors are not particularly limited so long
as the inserted nucleic acid is stably maintained. For example,
when using E. coli as the host, the cloning vector is preferably a
pBluescript vector (Stratagene) and such, but various commercially
available vectors may be used. Expression vectors are particularly
useful as vectors for producing the polypeptides of the present
invention. Expression vectors are not particularly limited so long
as they can express polypeptides in test tubes, E. coli, cultured
cells, or individual organisms. For example, preferred vectors
include pBEST vector (Promega) for expression in test tubes, pET
vector (Invitrogen) for E. coli, pME18S-FL3 vector (GenBank
Accession No. AB009864) for cultured cells, and pME18S vector (Mol.
Cell Biol. 8:466-472 (1998)) for individual organisms. Insertion of
a DNA of the present invention into vectors can be performed using,
for example, In-Fusion Advantage PCR Cloning Kit (Clontech).
[0242] Further, the present invention provides host cells carrying
the above described nucleic acids. The host cells are not
particularly limited, and various host cells such as E. coli and
various animal cells can be used according to the purpose. The host
cells may be used, for example, as a production system to produce
and express the antibodies or the polypeptides of the present
invention. In vitro and in vivo production systems are available
for polypeptide production systems. Production systems that use
eukaryotic cells or prokaryotic cells are examples of in vitro
production systems.
[0243] Eukaryotic cells that can be used as a host cell include,
for example, animal cells, plant cells, and fungal cells. Animal
cells include: mammalian cells, for example, CHO (J. Exp. Med.
(1995) 108:945), COS, 3T3, myeloma, BHK (baby hamster kidney),
HeLa, C127, HEK293, Bowes melanoma cells, and Vero; amphibian cells
such as Xenopus laevis oocytes (Valle, et al. (1981) Nature
291:338-340); and insect cells (e.g., Drosophila S2, Sf9, Sf21, and
Tn5). In the expression of the antibodies of the present invention,
CHO-DG44, CHO-DX11B, COST cells, and BHK cells can be suitably
used. Among animal cells, CHO cells are particularly preferable for
large-scale expression.
[0244] Vectors can be introduced into a host cell by known methods,
for example, by calcium phosphate methods, the DEAE-dextran
methods, methods using cationic liposome DOTAP
(Boehringer-Mannheim), electroporation methods (Current protocols
in Molecular Biology edit. Ausubel et al. (1987) Publish. John
Wiley & Sons, Section 9.1-9.9), lipofection, lipofectamine
methods (GIBCO-BRL), or microinjection methods. Moreover, gene
introduction to polypeptide expression can also be carried out
using the Free Style 293 Expression System (Invitrogen).
[0245] Plant cells include, for example, Nicotiana tabacum-derived
cells known as a protein production system. Calluses can be
cultured from these cells to produce the antibodies of the present
invention.
[0246] Known protein expression systems are those using fungal
cells including yeast cells, for example, cells of genus
Saccharomyces such as Saccharomyces cerevisiae and Saccharomyces
pombe; and cells of filamentous fungi, for example, cells of genus
Aspergillus such as Aspergillus niger. These cells can be used as a
host to produce the antibodies of the present invention.
[0247] Bacterial cells can be used in the production systems using
prokaryotic cells. Examples of bacterial cells include
Streptococcus, Staphylococcus, E. coli, Steptomyces, Bacillus
subtilis as well as the E. coli described above. Such cells can be
used to produce the antibodies of the present invention.
[0248] When producing an antibody using a host cell of the present
invention, the polynucleotide encoding an antibody of the present
invention may be expressed by culturing the host cells transformed
with the expression vector containing the polynucleotide. The
culture can be performed using known methods. For example, when
using animal cells as a host, DMEM, MEM, RPMI 1640, or IMDM may be
used as the culture medium, and may be used with serum supplements
such as FBS or fetal calf serum (FCS). Serum-free cultures are also
acceptable. The preferred pH is about 6 to 8 during the course of
culturing. Culture is carried out typically at about 30.degree. C.
to 40.degree. C. for about 15 to 200 hours. Medium is exchanged,
aerated, or agitated, as necessary.
[0249] On the other hand, production systems using animals or
plants may be used as systems for producing polypeptides in vivo. A
polynucleotide of interest is introduced into an animal or plant
and the polypeptide is produced in the body of the animal or plant
and then collected. The "host" of the present invention includes
such animals and plants.
[0250] For secreting host cell-expressed polypeptides into the
lumen of the endoplasmic reticulum, periplasmic space, or
extracellular environment, suitable secretion signals can be
incorporated into the polypeptides of interest. These signals may
be intrinsic or foreign to the polypeptides of interest.
[0251] When the polypeptides of the present invention are secreted
into the culture media, the polypeptides produced by the
above-mentioned method can be harvested by collecting the media.
When the polypeptides of the present invention are produced inside
cells, first, the cells are lysed, and then these polypeptides are
collected.
[0252] Animals to be used for the production system include mammals
and insects. Mammals such as goats, pigs, sheep, mice, and cattle
may be used (Vicki Glaser, SPECTRUM Biotechnology Applications
(1993)). Alternatively, the mammals may be transgenic animals.
[0253] For example, a polynucleotide encoding an antibody of the
present invention may be prepared as a fusion gene with a gene
encoding a polypeptide specifically produced in milk, such as the
goat .beta.-casein. Polynucleotide fragments containing the fusion
gene are injected into goat embryos, which are then introduced back
to female goats. The desired antibody can be obtained from milk
produced by the transgenic goats, which are born from the goats
that received the embryos, or from their offspring. Appropriate
hormones may be administered to the transgenic goats to increase
the volume of milk containing the antibody produced by the
transgenic goats (Ebert et al., Bio/Technology 12: 699-702
(1994)).
[0254] Insects such as silkworms may also be used for producing the
antibodies of the present invention. Baculoviruses carrying a
polynucleotide encoding an antibody of interest can be used to
infect silkworms, and the antibody of interest can be obtained from
their body fluids of (Susumu et al., Nature 315: 592-594
(1985)).
[0255] Plants used for producing the antibodies of the present
invention include, for example, tobacco. When tobacco is used, a
polynucleotide encoding an antibody of interest is inserted into a
plant expression vector, for example, pMON 530, and then the vector
is introduced into a bacterium, such as Agrobacterium tumefaciens.
The bacteria are then used to infect tobacco such as Nicotiana
tabacum, and the desired antibodies can be recovered from the
tobacco leaves (Ma et al., Eur. J. Immunol. 24: 131-138
(1994)).
[0256] The resulting antibody may be isolated from the inside or
outside (such as the medium and milk) of host cells, and purified
as a substantially pure and homogenous antibody. Methods are not
limited to any specific method and any standard method for
isolating and purifying antibodies may be used. Antibodies may be
isolated and purified, by appropriately selecting and combining,
for example, ammonium sulfate or ethanol precipitation, acid
extraction, chromatographic columns, filtration, ultrafiltration,
salting out, solvent precipitation, solvent extraction,
distillation, immunoprecipitation, SDS-polyacrylamide gel
electrophoresis, isoelectric focusing, dialysis, recrystallization,
and others.
[0257] Chromatographies include, for example, affinity
chromatographies, ion exchange chromatographies such as anion
exchange chromatographies and cation exchange chromatographies,
phosphocellulose chromatographies, hydrophobic (interaction)
chromatographies, gel filtrations, reverse-phase chromatographies,
adsorption chromatographies, hydroxylapatite chromatographies, and
lectin chromatographies (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). These
chromatographies can be carried out using liquid phase
chromatographies such as HPLC and FPLC. Examples of the affinity
chromatography columns include protein A columns and protein G
columns. Examples of the proteins A columns include Hyper D, POROS,
and Sepharose F. F. (Pharmacia).
[0258] An antibody can be modified freely and peptide portions can
be deleted from it by treating the antibody with an appropriate
protein modifying enzyme before or after antibody purification, as
necessary. Such protein modifying enzymes include, for example,
trypsins, chymotrypsins, lysyl endopeptidases, protein kinases, and
glucosidases.
[0259] In another preferred embodiment, the present invention also
includes methods for producing the antibodies of the present
invention, such methods including the steps of culturing the host
cells of the present invention as described above and collecting
the antibodies from such cell culture.
[0260] Moreover, the present invention relates to pharmaceutical
compositions (pharmaceutical agents) comprising an antibody of the
present invention and a pharmaceutically acceptable carrier. In the
present invention, pharmaceutical compositions ordinarily refer to
pharmaceutical agents for treating or preventing, or testing and
diagnosing diseases.
[0261] The pharmaceutical compositions of the present invention can
be formulated by methods known to those skilled in the art.
Moreover, the antibodies of the present invention can be formulated
in combination with other pharmaceutical substances, as required.
For example, they can be used parenterally in the form of an
injection of a sterile solution or suspension with water or another
pharmaceutically acceptable liquid. For example, they may be
formulated as unit doses that meet the requirements for the
preparation of pharmaceuticals by appropriately combining with
pharmaceutically acceptable carriers or media, specifically with
sterile water, physiological saline, a vegetable oil, emulsifier,
suspension, detergent, stabilizer, flavoring agent, excipient,
vehicle, preservative, binder, or such. In such preparations, the
amount of active ingredient is adjusted such that the dose falls
within an appropriately pre-determined range.
[0262] Sterile compositions for injection can be formulated using
vehicles such as distilled water for injection, according to
standard protocols for formulation.
[0263] Aqueous solutions for injection include, for example,
physiological saline and isotonic solutions containing dextrose or
other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol,
and sodium chloride). Appropriate solubilizers, for example,
alcohols (ethanol and such), polyalcohols (propylene glycol,
polyethylene glycol, and such), non-ionic detergents (polysorbate
80.TM., HCO-50, and such), may be used in combination.
[0264] Oils include sesame and soybean oils. Benzyl benzoate and/or
benzyl alcohol can be used in combination as solubilizers. Buffers
(for example, phosphate buffer and sodium acetate buffer), soothing
agents (for example, procaine hydrochloride), stabilizers (for
example, benzyl alcohol and phenol), and/or antioxidants can also
be combined. Prepared injectables are generally filled into
appropriate ampules.
[0265] The pharmaceutical compositions of the present invention are
preferably administered parenterally. For example, the compositions
may be injections, transnasal compositions, transpulmonary
compositions or transdermal compositions. For example, such
compositions can be administered systemically or locally by
intravenous injection, intramuscular injection, intraperitoneal
injection, subcutaneous injection, or such.
[0266] The administration methods can be appropriately selected in
consideration of a patient's age and symptoms. The dose of a
pharmaceutical composition containing an antibody or a
polynucleotide encoding an antibody may be, for example, from
0.0001 to 1000 mg/kg for each administration. Alternatively, the
dose may be, for example, from 0.001 to 100,000 mg per patient.
However, the doses are not necessarily limited to the ranges
described above. The doses and administration methods vary
depending on a patient's weight, age, symptoms, and such. Those
skilled in the art can select appropriate doses and administration
methods in consideration of the factors described above.
[0267] Amino acids contained in the amino acid sequences of the
present invention may be post-translationally modified (for
example, the modification of an N-terminal glutamine into a
pyroglutamic acid by pyroglutamylation is well-known to those
skilled in the art). Naturally, such post-translationally modified
amino acids are included in the amino acid sequences in the present
invention.
[0268] All prior art documents cited in the present specification
are incorporated herein by reference.
EXAMPLES
[0269] Hereinbelow, the present invention will be specifically
described with reference to the Examples, but the present invention
is not limited thereto.
[Example 1] Search for Sites that Regulate the CH1/CL Interface
[0270] It was thought that by introducing mutations into each of
the CH1 and CL domains of a bispecific antibody, and utilizing the
electrical charge at the CH1/CL interface to regulate the CH1/CL
interface, only the H chain and L chain for an antigen A
specifically associate, and only the H chain and L chain for an
antigen B specifically associate. Hereinafter, it is referred to as
regulation of the CH1/CL interface. A search to found out the
positions where the CH1/CL interface can be controlled was carried
out using a crystal structure model. Amino acids maintain
interactions between side chains through hydrophobic interaction,
electrical interaction, hydrogen bonding and the like. These
interactions are known to occur between side chains present within
a range of about 4 .ANG.. Therefore, amino acids were found in a
PDB model 1HZH, wherein the distance between amino acids present in
CH1 and amino acids present in CL at the interface between CH1 and
CL is about 4 .ANG.. The sites at which the amino acids are found
are each summarized in FIG. 1 and Table 1 (Summary of Modified
Sites). The amino acid numbers shown in Table 1 are indicated in
accordance with EU numbering (Sequences of proteins of
immunological interest, NIH Publication No. 91-3242). In addition,
subsequent amino acid numbers are also indicated in accordance with
EU numbering. In the present example, IgG1 was used for the H
chain, and IgK (Kappa) was used for the L chain.
TABLE-US-00001 TABLE 1 CH CL 1 K147 T180 2 Q175 Q160 3 K213 E123 4
K133 N138 5 K147 S131 6 H168 T164 7 F170 L135
[0271] In order to regulate the CH1/CL interface using the
electrical charge of amino acids, the amino acids found in CH1 of
the H chain or CL of the L chain (Table 1) were substituted with
positively charged Lys or His and negatively charged Glu or Asp.
More specifically, constant regions: TH2 and TH11, in which amino
acids of CH1 of human Gld (SEQ ID NO: 1) were substituted with
positively charged Lys; and TH1, TH3, TH4, TH9, TH10, and TH12, in
which amino acids of CH1 of human G1d (SEQ ID NO: 1) were
substituted with negatively charged Glu or Asp were prepared.
Similarly, constant regions: TL2, TL4, TL5, TL6, TL8, and TL12, in
which amino acids of human CL (SEQ ID NO: 13) were substituted with
positively charged Lys; TL11, in which amino acids of human CL (SEQ
ID NO: 13) were substituted with His; and TL1, TL3, TL7, TL9, TL10,
and TL13, in which amino acids of human CL (SEQ ID NO: 13) were
substituted with negatively charged Glu or Asp were prepared. The
names (name), sites of mutation (mutation) and sequence numbers of
the prepared constant regions are summarized in Table 2 (Summary of
Modified Sites).
TABLE-US-00002 TABLE 2 CH1 muta- CL name tion SEQ ID NO name
mutation SEQ ID NO TH1 K147E SEQ ID NO: 002 TL1 T180E SEQ ID NO:
014 TH2 Q175K SEQ ID NO: 003 TL2 T180K SEQ ID NO: 015 TH3 Q175E SEQ
ID NO: 004 TL3 Q160E SEQ ID NO: 016 TH4 K213E SEQ ID NO: 005 TL4
Q160K SEQ ID NO: 017 TH9 K133E SEQ ID NO: 006 TL5 E123K SEQ ID NO:
018 TH10 H168D SEQ ID NO: 007 TL6 N138K SEQ ID NO: 019 TH11 F170K
SEQ ID NO: 008 TL7 N138E SEQ ID NO: 020 TH12 F170E SEQ ID NO: 009
TL8 S131K SEQ ID NO: 021 TL9 S131E SEQ ID NO: 022 TL10 T164D SEQ ID
NO: 023 TL11 T164H SEQ ID NO: 024 TL12 L135K SEQ ID NO: 025 TL13
L135E SEQ ID NO: 026
[Example 2] Method for Screening Sites that Regulate the CH1/CL
Interface, and Preparation and Analysis of Each Antibody
[0272] Effects on the regulation of the CH1/CL interface of the
found amino acids were confirmed using the method described below.
Screening was carried out using an anti-GPC3 antibody. First,
expression vectors of the H chain and L chain were constructed. An
H chain expression vector having the H chain variable region GpH7
(SEQ ID NO: 34) and the H chain constant region prepared in Example
1, and an L chain expression vector having the L chain variable
region GpL16 (SEQ ID NO: 35) and the L chain constant region
prepared in Example 1 were each constructed in accordance with
Reference Example 1. Next, combinations of the prepared H chain and
L chain expression vectors were selected in the manner described
below. A single H chain in which the amino acid at the found site
has a positive charge or negative charge was selected from among
the constant regions prepared in Example 1. In this case, a
mutation was not always introduced. For example, although position
147 of TH1 is substituted with Glu, a mutation is not introduced
because the amino acid at position 147 of G1d is Lys and it
initially has a positive charge. Next, L chains, which have
mutations at the positions corresponding to the mutated positions
in CH1 of the selected H chains according Table 1, were selected
from Table 2. For example, when TH1 is selected for the H chain,
TL1 and TL2 were selected for the L chain, since the amino acid at
position 147 of CH1 and the amino acid at position 180 of CL are
expected to interact as the CH1/CL interface. Subsequently, the
selected two L chains were mixed with the selected one H chain, and
antibodies were expressed in accordance with Reference Example 1.
Finally, the expressed antibodies were analyzed in accordance with
Reference Example 3 or Reference Example 4, and modifications
effective for regulating the CH1/CL interface were screened
according to the expression ratio of each antibody. Since IgG is
composed of a complex of two H chains and two L chains, when one
type of H chain and two types of L chains are mixed and expressed,
three combinations are expected to be expressed. For example, when
combinations of TH1 as the H chain, and TL1 and TL2 as the L chains
are expressed, three combinations below are expressed (FIG. 2): H
chain_1:H chain_2: L chain_1:L chain_2=TH1:TH1:TL1:TL1 (indicated
as TH1/TL1), H chain_1:H chain_2: L chain_1:L
chain_2=TH1:TH1:TL1:TL2 (indicated as TH1/TL1_TL2) H chain_1:H
chain_2: L chain_1:L chain_2=TH1:TH1:TL2:TL2 (indicated as
TH1/TL2). In the case that association of the H chain and L chain
is not selective, the H chain and L chains are expected to be
expressed in the ratio TH1/TL1:TH1/TL1_TL2:TH1/TL2=1:2:1, since the
two L chains are present in equal amounts. However, in the case
that the H chain preferentially binds to only either one of the L
chains at the CH1/CL interface, it is thought that only that
combination is expressed preferentially. For example, in the case
that the amino acid at position 147 of CH1 and the amino acid at
position 180 of CL are involved at the CH1/CL interface, when TH1
is expressed as the H chain, and TL1 and TL2 are expressed as the L
chains, the combination of TH1, in which the amino acid at position
147 of CH1 is Glu (negatively charged), and TL2, in which the amino
acid at position 180 of the L chain CL is Lys (positively charged),
is expected to be expressed preferentially. However, in the case
that the amino acid at position 147 of CH1 and the amino acid at
position 180 of CL are not interacting on the CH1/CL interface,
since the association of the H chain and L chain is not selective,
they are expected to be expressed in the ratio of
TH1/TL1:TH1/TL1_TL2:TH1/TL2=1:2:1. In this manner, modifications
effective for regulation of the CH1/CL interface (modifications
involved in the CH1/CL interface) were screened by mixing and
expressing one type of H chain and two types of L chains, and using
the expression balance thereof as an indicator.
[0273] Combinations of H chain and L chains are summarized in Table
3 (Combinations of H Chain and L Chains Used in Expression; Sites
of Mutation in H Chain and L Chain also Shown).
TABLE-US-00003 TABLE 3 Hch Lch name mutation SEQ ID NO name
mutation SEQ ID NO G1d K147K SEQ ID NO: 001 TL1_TL2 T180E_T180K SEQ
ID NO: 014, SEQ ID NO: 015 TH1 K147E SEQ ID NO: 002 TL1_TL2
T180E_T180K SEQ ID NO: 014, SEQ ID NO: 015 TH2 Q175K SEQ ID NO: 003
TL3_TL4 Q160E_Q160K SEQ ID NO: 016, SEQ ID NO: 017 TH3 Q175E SEQ ID
NO: 004 TL3_TL4 Q160E_Q160K SEQ ID NO: 016, SEQ ID NO: 017 G1d
K213K SEQ ID NO: 001 k0_TL5 E123E_E123K SEQ ID NO: 013, SEQ ID NO:
018 TH4 K213E SEQ ID NO: 005 k0_TL5 E123E_E123K SEQ ID NO: 013, SEQ
ID NO: 018 G1d K133K SEQ ID NO: 001 TL6_TL7 N138K_N138E SEQ ID NO:
019, SEQ ID NO: 020 TH9 K133E SEQ ID NO: 006 TL6_TL7 N138K_N138E
SEQ ID NO: 019, SEQ ID NO: 020 G1d K147K SEQ ID NO: 001 TL8_TL9
S131K_S138E SEQ ID NO: 021, SEQ ID NO: 022 TH1 K147E SEQ ID NO: 002
TL8_TL9 S131K_S138E SEQ ID NO: 021, SEQ ID NO: 022 G1d H168H SEQ ID
NO: 001 TL10_TL11 T164D_T164H SEQ ID NO: 023, SEQ ID NO: 024 TH10
H168D SEQ ID NO: 007 TL10_TL11 T164D_T164H SEQ ID NO: 023, SEQ ID
NO: 024 TH11 F170K SEQ ID NO: 008 TL12_TL13 L135K_L135E SEQ ID NO:
025, SEQ ID NO: 026 TH12 F170E SEQ ID NO: 009 TL12_TL13 L135K_L135E
SEQ ID NO: 025, SEQ ID NO: 026
[0274] Antibodies were expressed in accordance with the
combinations shown in Table 3, and the effects on regulation of the
selected CH1/CL interface were confirmed. At that time, antibodies
of one type of H chain and one type of L chain were simultaneously
expressed, and used as a control in analyses. Antibodies were
expressed in accordance with the method of Reference Example 1.
[0275] The prepared antibodies were analyzed by AIEX in accordance
with the method of Reference Example 3. Data of the AIEX analysis
are summarized in FIG. 3. Since an anion exchange column is used in
the AIEX analysis, positively charged antibodies are eluted more
rapidly. The analyzed data are summarized and shown in Table 4. The
peaks are indicated as peaks 1, 2, and 3 in the order of increasing
elution time. The ratio of each peak was calculated with the total
of the peak areas being 100%.
[0276] As shown in FIG. 2, in the case that the positions
introduced with mutations form electrical interaction, the ratio of
antibody at the position of the gray-colored peak increases.
Namely, mutation sites at which the ratio of the gray-colored
antibody is greater than 25% are thought to be interacting
electrically.
TABLE-US-00004 TABLE 4 Hch Lch peak1 peak2 peak3 name mutation name
mutation % % % G1d K147K TL1 T180E 100 G1d K147K TL2 T180K 100 G1d
K147K TL1_TL2 T180E_T180K 20.2 44.2 35.6 TH1 K147E TL1 T180E 100
TH1 K147E TL2 T180K 100 TH1 K147E TL1_TL2 T180E_T180K 28.5 46.7
24.8 TH2 Q175K TL3 Q160E 100 TH2 Q175K TL4 Q160K 100 TH2 Q175K
TL3_TL4 Q160E_Q160K 13 44 43 TH3 Q175E TL3 Q160E 100 TH3 Q175E TL4
Q160K 100 TH3 Q175E TL3_TL4 Q160E_Q160K 25 49.9 24.8 G1d K213K k0
E123E 100 G1d K213K TL5 E123K 100 G1d K213K k0_TL5 E123E_E123K 15.1
39.3 45.6 TH4 K213E k0 E123E 100 TH4 K213E TL5 E123K 100 TH4 K213E
k0_TL5 E123E_E123K 17.5 41.3 41.2 G1d K133K TL6 N138K 100 G1d K133K
TL7 N138E 100 G1d K133K TL6_TL7 N138K_N138E 27.3 44 28.7 TH9 K133E
TL6 N138K 100 TH9 K133E TL7 N138E 100 TH9 K133E TL6_TL7 N138K_N138E
29.1 44 26.9 G1d K213K TL8 S131K 100 G1d K213K TL9 S131E 100 G1d
K213K TL8_TL9 S131K_S131E 17.8 45.7 36.7 TH1 K147E TL8 S131K 100
TH1 K147E TL9 S131E 100 TH1 K147E TL8_TL9 S131K_S131E 36.9 44.1 19
G1d H168H TL10 T164D 100 G1d H168H TL11 T164H 100 G1d H168H
TL10_TL11 T164D_T164H 27 43.3 29.7 TH10 H168D TL10 T164D 100 TH10
H168D TL11 T164H 100 TH10 H168D TL10_TL11 T164D_T164H 27 44.4 28.6
TH11 F170K TL12 L135K 100 TH11 F170K TL13 L135E 100 TH11 F170K
TL12_TL13 L135K_L135E not expressed TH12 F170E TL12 L135K 100 TH12
F170E TL13 L135E 100 TH12 F170E TL12_TL13 L135K_L135E 38.1 41.6
20.3
[0277] As a result of examining various sites of modification in
this manner, positions 147, 175, and 213 of the H chain, and
positions 123, 131, 160, and 180 of the L chain were thought to be
effective for regulating the CH1/CL interface. In addition, it was
found that modifications of only position 147 of the H chain and
position 123 of the L chain reported in WO 2006/106905 and WO
2007/147901 were inadequate for causing specific association of the
H chain and L chain, and the specific association is possible only
by combining modifications found in the present example.
[Example 3] Preparation and Analysis of Antibodies with Combined
Sites of Modification
[0278] It was thought that the CH1/CL interface is regulated more
effectively by combining the sites of K147, Q175, and K213 in CH1
and the sites of E123, 5131, Q160, and T180 in CL as found in
Example 2, which were thought to have considerable effects in
regulating the CH1/CL interface. The combinations of modifications
in the prepared antibodies, and the expressed antibodies are
summarized in Table 5 (Summary of Modification Sites).
TABLE-US-00005 TABLE 5 Hch Lch name mutation SEQ ID NO name
mutation SEQ ID NO G1d K147K SEQ ID NO: 001 TL14_TL15 T180K,
S131K_T180E, S131E SEQ ID NO: 027, SEQ ID NO: 028 TH1 K147E SEQ ID
NO: 002 TL14_TL15 T180K, S131K_T180E, S131E SEQ ID NO: 027, SEQ ID
NO: 028 TH2 Q175K SEQ ID NO: 003 TL16_TL17 T180K, S131K,
Q160K_T180E, S131E, Q160E SEQ ID NO: 029, SEQ ID NO: 030 TH13
K147E, Q175E SEQ ID NO: 010 TL16_TL17 T180K, S131K, Q160K_T180E,
S131E, Q160E SEQ ID NO: 029, SEQ ID NO: 030 G1d K147K SEQ ID NO:
001 TL18_TL15 T180K, S131K, E123K_T180E, S131E, E123E SEQ ID NO:
031, SEQ ID NO: 028 TH14 K147E, K213E SEQ ID NO: 011 TL18_TL15
T180K, S131K, E123K_T180E, S131E, E123E SEQ ID NO: 031, SEQ ID NO:
028 TH2 Q175K SEQ ID NO: 003 TL19_TL17 T180K, S131K, Q160K,
E123K_T180E, S131E, SEQ ID NO: 032, SEQ ID NO: 030 Q160E TH15
K147E, Q175E, SEQ ID NO: 012 TL19_TL17 T180K, S131K, Q160K,
E123K_T180E, S131E, SEQ ID NO: 032, SEQ ID NO: 030 K213E Q160E,
E123E
[0279] Preparation of expression vectors of the H chain or L chain
introduced with mutations, and antibody expression were carried out
in accordance with Reference Example 1, and analyses of the
prepared antibodies were carried out in accordance with Reference
Example 3 or Reference Example 4. The results are summarized in
Table 6.
TABLE-US-00006 TABLE 6 Hch Lch peak 1 peak 2 peak 3 name mutation
name mutation % % % G1d K147K TL14 T180K, S131K 100 G1d K147K TL15
T180E, S131E 100.0 G1d K147K TL14_TL15 T180K, S131K, T180E, S131E
11.5 43.6 44.9 TH1 K147E TL14 T180K, S131K 100.0 TH1 K147E TL15
T180E, S131E 100.0 TH1 K147E TL14_TL15 T180K, S131K_T180E, S131E
46.1 43.4 10.4 TH2 Q175K TL16 T180K, S131K, Q160K not expressed TH2
Q175K TL17 T180E, S131E, Q160E 100.0 TH2 Q175K TL16_TL17 T180K,
S131K, Q160K_T180E, 1.4 16.5 82.0 S131E, Q160E TH13 K147E, Q175E
TL16 T180K, S131K, Q160K 100.0 TH13 K147E, Q175E TL17 T180E, S131E,
Q160E not expressed TH13 K147E, Q175E TL16_TL17 T180K, S131K,
Q160K_T180E, 70.2 26.4 3.4 S131E, Q160E G1d K147K TL18 T180K,
S131K, E123K not expressed G1d K147K TL15 T180E, S131E 100.0 G1d
K147K TL18_TL15 T180K, S131K, E123K_T180E, 5.1 35.3 59.6 S131E,
E123E TH14 K147E, K213E TL18 T180K, S13SK, E123K 100.0 TH14 K147E,
K213E TL15 T180E, S131E 100.0 TH14 K147E, K213E TL18_TL15 T180K,
S131K, E123K_T180E, 44.5 44.4 11.1 S131E, E123E TH2 Q175K TL19
T180K, S131K, Q160K, E123K not expressed TH2 Q175K TL17 T180E,
S131E, Q160E 100.0 TH2 Q175K TL19_TL17 T180K, S131K, Q160K, 93.1
E123K_T180E, S131E, Q160E TH15 K147E, Q175E, TL19 T180K, S131K,
Q160K, E123K 100.0 K213E TH15 K147E, Q175E, TL17 T180E, S131E,
Q160E not K213E expressed TH15 K147E, Q175E, TL19_TL17 T180K,
S131K, Q160K, 78.1 19.9 2.0 K213E E123K_T180E, S131E, Q160E
[0280] When Table 4 and Table 6 are compared, it is understood that
the ratio of targeted combinations of the H chain and L chain is
increased by combining modifications, as compared with the
introduction of a single modification. Consequently, it is thought
that an antibody in which only the H chain and L chain of interest
have associated can be efficiently prepared by combining
modifications.
[Example 4] Expression and Analysis of Bispecific Antibodies
[0281] In Example 3, preparation of bispecific antibodies
(bispecific Abs) for TH2, TH13, and TH15 of H chains, and TL16,
TL17, TL19, and TL20 of L chains that showed considerable effects
for regulating the CH1/CL interface was taken thought. In this
example, bispecific antibodies were prepared using an anti-IL6R
antibody and an anti-GPC3 antibody.
[0282] The constant regions of H chain (SEQ ID NO: 59) and L chain
(SEQ ID NO: 60) recognizing anti-IL6R, and the constant regions of
H chain (SEQ ID NO: 61) and L chain (SEQ ID NO: 62) recognizing
anti-GPC3 were substituted with TH2, TH13, and TH15 for the
constant regions of the H chain, and with TL16, TL17, TL19, and
TL20 for CL of the L chain. Moreover, an H chain introduced with a
Knob into Hole (KiH) modification (WO 96/27011) was prepared to
avoid association between homogeneous H chains. The mutation sites
in these prepared antibodies and the expressed antibodies are
summarized in Table 7 (Combinations of H Chain and L Chain of Each
Bispecific Antibody).
TABLE-US-00007 TABLE 7 Ach Bch NAME VH CH VL CL VH CH VL CL comment
4ch_001 MH0 G1d ML0 k0 GpH7 G1d GpL16 k0 4ch_002 MH0 G1dk ML0 k0
GpH7 G1dh GpL16 k0 KiH 4ch_003 MH0 TH2 ML0 TL17 GpH7 TH13 GpL16
TL16 CH1/CL_1 4ch_004 MH0 TH2k ML0 TL17 GpH7 TH13h GpL16 TL16 KiH +
CH1/CL_1 4ch_011 MH0 TH2k ML0 TL17 GpH7 TH13h ML0 TL17 4ch_012 MH0
TH2k GpL 16 TL16 GpH7 TH13h GpL16 TL16 4ch_005 MH0 TH2 ML0 TL17
GpH7 TH15 GpL16 TL19 CH1/CL_2 4ch_006 MH0 TH2k ML0 TL17 GpH7 TH15h
GpL16 TL19 KiH + CH1/CL_2 4ch_015 MH0 TH2k ML0 TL17 GpH7 TH15h ML0
TL17 4ch_016 MH0 TH2k GpL 16 TL19 GpH7 TH15h GpL16 TL19 4ch_001 MH0
G1d ML0 k0 GdH7 G1d GpL16 k0 4ch_002 MH0 G1dk ML0 k0 GpH7 G1dh
GpL16 k0 KiH 4ch_007 MH0 TH13 ML0 TL16 GpH7 TH2 GpL16 TL17 CH1/CL_1
4ch_008 MH0 TH13k ML0 TL16 GpH7 TH2h GpL16 TL17 KiH + CH1/CL_1
4ch_013 MH0 TH13k ML0 TL16 GpH7 TH2h ML0 TL16 4ch_014 MH0 TH13k GpL
16 TL17 GpH7 TH2h GpL16 TL17 4ch_009 MH0 TH15 ML0 TL19 GpH7 TH2
GpL16 TL17 CH1/CL_2 4ch_010 MH0 TH15k ML0 TL19 GpH7 TH2h GpL16 TL17
KiH + CH1/CL_2 4ch_017 MH0 TH15k ML0 TL19 GpH7 TH2h ML0 TL19
4ch_018 MH0 TH15k GpL 16 TL17 GpH7 TH2h GpL16 TL17
[0283] In Table 7 above, "k" is added after the variant name for
those constant regions in which a "knob" modification was
introduced into the H chain, and "h" is added after the variant
name for those constant regions in which a "hole" modification was
introduced. For example, "THlk" indicates that a "knob"
modification was introduced in addition to the TH1 mutation, and
"TH1h" indicates that a "hole" modification was introduced in
addition to the TH1 mutation. Preparation of expression vectors of
the H chain or L chain into which a mutation has been introduced as
well as expression of antibodies were carried out in accordance
with Reference Example 1, and analyses of the prepared antibodies
were carried out in accordance with the CIEX analysis method shown
in Reference Example 4.
[0284] The combinations of H chains and L chains of the anti-IL6R
antibody and anti-GPC3 antibody used in the bispecific antibodies
are summarized in Table 8.
TABLE-US-00008 TABLE 8 VH CH VL CL NAME chain name mutation SEQ ID
NO name mutation SEQ ID NO name mutation SEQ ID NO name mutation
SEQ ID NO 4ch_001 Ach MH0 -- SEQ ID NO: 036 G1d K147K SEQ ID NO:
001 ML0 -- SEQ ID NO: 037 k0 -- SEQ ID NO: 013 4ch_002 Ach MH0 --
SEQ ID NO: 036 G1dk -- SEQ ID NO: 038 ML0 -- SEQ ID NO: 037 k0 --
SEQ ID NO: 013 4ch_003 Ach MH0 -- SEQ ID NO: 036 TH2 Q175K SEQ ID
NO: 003 ML0 -- SEQ ID NO: 037 TL17 T180E, S131E, Q160E SEQ ID NO:
030 4ch_004 Ach MH0 -- SEQ ID NO: 036 TH2k Q175K SEQ ID NO: 039 ML0
-- SEQ ID NO: 037 TL17 T180E, S131E, Q160E SEQ ID NO: 030 4ch_011
Ach MH0 -- SEQ ID NO: 036 TH2k Q175K SEQ ID NO: 039 ML0 -- SEQ ID
NO: 037 TL17 T180E, S131E, Q160E SEQ ID NO: 030 4ch_012 Ach MH0 --
SEQ ID NO: 036 TH2k Q175K SEQ ID NO: 039 GpL16 -- SEQ ID NO: 035
TL16 T180K, S131K, Q160K SEQ ID NO: 029 4ch_005 Ach MH0 -- SEQ ID
NO: 036 TH2 Q175K SEQ ID NO: 003 ML0 -- SEQ ID NO: 037 TL17 T180E,
S131E, Q160E SEQ ID NO: 030 4ch_006 Ach MH0 -- SEQ ID NO: 036 TH2k
Q175K SEQ ID NO: 039 ML0 -- SEQ ID NO: 037 TL17 T180E, S131E, Q160E
SEQ ID NO: 030 4ch_015 Ach MH0 -- SEQ ID NO: 036 TH2k Q175K SEQ ID
NO: 039 ML0 -- SEQ ID NO: 037 TL17 T180E, S131E, Q160E SEQ ID NO:
030 4ch_016 Ach MH0 -- SEQ ID NO: 036 TH2k Q175K SEQ ID NO: 039
GpL16 -- SEQ ID NO: 035 TL19 T180K, S131K, Q160K, SEQ ID NO: 032
E123K 4ch_001 Ach MH0 -- SEQ ID NO: 036 G1d K147K SEQ ID NO: 001
ML0 -- SEQ ID NO: 037 k0 -- SEQ ID NO: 013 4ch_002 Ach MH0 -- SEQ
ID NO: 036 G1dk -- SEQ ID NO: 038 ML0 -- SEQ ID NO: 037 k0 -- SEQ
ID NO: 013 4ch_007 Ach MH0 -- SEQ ID NO: 036 TH13 K147E, Q175E SEQ
ID NO: 010 ML0 -- SEQ ID NO: 037 TL16 T180K, S131K, Q160K SEQ ID
NO: 029 4ch_008 Ach MH0 -- SEQ ID NO: 036 TH13k K147E, Q175E SEQ ID
NO: 040 ML0 -- SEQ ID NO: 037 TL16 T180K, S131K, Q160K SEQ ID NO:
029 4ch_013 Ach MH0 -- SEQ ID NO: 036 TH13k K147E, Q175E SEQ ID NO:
040 ML0 -- SEQ ID NO: 037 TL16 T180K, S131K, Q160K SEQ ID NO: 029
4ch_014 Ach MH0 -- SEQ ID NO: 036 TH13k K147E, Q175E SEQ ID NO: 040
GpL16 -- SEQ ID NO: 035 TL17 T180E, S131E, Q160E SEQ ID NO: 030
4ch_009 Ach MH0 -- SEQ ID NO: 036 TH15 K147E, Q175E, K213E SEQ ID
NO: 012 ML0 -- SEQ ID NO: 037 TL19 T180K, S131K, Q160K, SEQ ID NO:
032 E123K 4ch_010 Ach MH0 -- SEQ ID NO: 036 TH15k K147E, Q175E,
K213E SEQ ID NO: 041 ML0 -- SEQ ID NO: 037 TL19 T180K, S131K,
Q160K, SEQ ID NO: 032 E123K 4ch_017 Ach MH0 -- SEQ ID NO: 036 TH15k
K147E, Q175E, K213E SEQ ID NO: 041 ML0 -- SEQ ID NO: 037 TL19
T180K, S131K, Q160K, SEQ ID NO: 032 E123K 4ch_018 Ach MH0 -- SEQ ID
NO: 036 TH15k K147E, Q175E, K213E SEQ ID NO: 041 GpL16 -- SEQ ID
NO: 035 TL17 T180E, S131E, Q160E SEQ ID NO: 030 4ch_001 Bch GpH7 --
SEQ ID NO: 034 G1d K147K SEQ ID NO: 001 GpL16 -- SEQ ID NO: 035 k0
-- SEQ ID NO: 013 4ch_002 Bch GpH7 -- SEQ ID NO: 034 G1dh -- SEQ ID
NO: 042 GpL16 -- SEQ ID NO: 035 k0 -- SEQ ID NO: 013 4ch_003 Bch
GpH7 -- SEQ ID NO: 034 TH13 K147E, Q175E SEQ ID NO: 010 GpL16 --
SEQ ID NO: 035 TL16 T180K, S131K, Q160K SEQ ID NO: 029 4ch_004 Bch
GpH7 -- SEQ ID NO: 034 TH13h K147E, Q175E SEQ ID NO: 044 GpL16 --
SEQ ID NO: 035 TL16 T180K, S131K, Q160K SEQ ID NO: 029 4ch_011 Bch
GpH7 -- SEQ ID NO: 034 TH13h K147E, Q175E SEQ ID NO: 044 ML0 -- SEQ
ID NO: 037 TL17 T180E, S131E, Q160E SEQ ID NO: 030 4ch_012 Bch GpH7
-- SEQ ID NO: 034 TH13h K147E, Q175E SEQ ID NO: 044 GpL16 -- SEQ ID
NO: 035 TL16 T180K, S131K, Q160K SEQ ID NO: 029 4ch_005 Bch GpH7 --
SEQ ID NO: 034 TH15 K147E, Q175E, K213E SEQ ID NO: 012 GpL16 -- SEQ
ID NO: 035 TL19 T180K, S131K, Q160K, SEQ ID NO: 032 E123K 4ch_006
Bch GpH7 -- SEQ ID NO: 034 TH15h K147E, Q175E, K213E SEQ ID NO: 045
GpL16 -- SEQ ID NO: 035 TL19 T180K, S131K, Q160K, SEQ ID NO: 032
E123K 4ch_015 Bch GpH7 -- SEQ ID NO: 034 TH15h K147E, Q175E, K213E
SEQ ID NO: 045 ML0 -- SEQ ID NO: 037 TL17 T180E, S131E, Q160E SEQ
ID NO: 030 4ch_016 Bch GpH7 -- SEQ ID NO: 034 TH15h K147E, Q175E,
K213E SEQ ID NO: 045 GpL16 -- SEQ ID NO: 035 TL19 T180K, S131K,
Q160K, SEQ ID NO: 032 E123K 4ch_001 Bch GpH7 -- SEQ ID NO: 034 G1d
K147K SEQ ID NO: 001 GpL16 -- SEQ ID NO: 035 k0 -- SEQ ID NO: 013
4ch_002 Bch GpH7 -- SEQ ID NO: 034 G1dh -- SEQ ID NO: 042 GpL16 --
SEQ ID NO: 035 k0 -- SEQ ID NO: 013 4ch_007 Bch GpH7 -- SEQ ID NO:
034 TH2 Q175K SEQ ID NO: 003 GpL16 -- SEQ ID NO: 035 TL17 T180E,
S131E, Q160E SEQ ID NO: 030 4ch_008 Bch GpH7 -- SEQ ID NO: 034 TH2h
Q175K SEQ ID NO: 043 GpL16 -- SEQ ID NO: 035 TL17 T180E, S131E,
Q160E SEQ ID NO: 030 4ch_013 Bch GpH7 -- SEQ ID NO: 034 TH2h Q175K
SEQ ID NO: 043 ML0 -- SEQ ID NO: 037 TL16 T180K, S131K, Q160K SEQ
ID NO: 029 4ch_014 Bch GpH7 -- SEQ ID NO: 034 TH2h Q175K SEQ ID NO:
043 GpL16 -- SEQ ID NO: 035 TL17 T180E, S131E, Q160E SEQ ID NO: 030
4ch_009 Bch GpH7 -- SEQ ID NO: 034 TH2 Q175K SEQ ID NO: 003 GpL16
-- SEQ ID NO: 035 TL17 T180E, S131E, Q160E SEQ ID NO: 030 4ch_010
Bch GpH7 -- SEQ ID NO: 034 TH2h Q175K SEQ ID NO: 043 GpL16 -- SEQ
ID NO: 035 TL17 T180E, S131E, Q160E SEQ ID NO: 030 4ch_017 Bch GpH7
-- SEQ ID NO: 034 TH2h Q175K SEQ ID NO: 043 ML0 -- SEQ ID NO: 037
TL19 T180K, S131K, Q160K, SEQ ID NO: 032 E123K 4ch_018 Bch GpH7 --
SEQ ID NO: 034 TH2h Q175K SEQ ID NO: 043 GpL16 -- SEQ ID NO: 035
TL17 T180E, S131E, Q160E SEQ ID NO: 030
[0285] Each combination is explained herein using 4ch_001, 4
ch_002, 4 ch_003, 4ch_004, 4 ch_011, and 4ch_012 as examples.
Modifications that regulate the CH1/CL interface are summarized in
Table 8.
[0286] 4ch_001 was expressed using H chains and L chains that do
not have the introduction of modifications for regulating the
CH1/CL interface and the KiH modification. 4ch_002 was expressed
using H chains and L chain into which the KiH modification was
introduced. 4ch_003 was expressed using H chains and L chains into
which modifications for regulating the CH1/CL interface were
introduced. 4ch_004 was expressed using H chains and an L chains
into which the KiH modification and modifications for regulating
the CH1/CL interface were introduced. In addition, 4ch_011 was
expressed using the H chains of an anti-IL6R antibody and the H
chains of an anti-GPC3 antibody into which the KiH modification and
modifications for regulating the CH1/CL interface were introduced,
and the L chains of an anti-IL6R antibody into which modifications
for regulating the CH1/CL interface were introduced. 4ch_012 was
expressed using the H chains of an anti-IL6R antibody and the H
chains of an anti-GPC3 antibody into which the KiH modification and
modifications for regulating the CH1/CL interface were introduced,
and the L chains of an anti-GPC3 antibody into which modifications
for regulating the CH1/CL interface were introduced. Each antibody
was expressed in accordance with Reference Example 1, and analyzed
by CIEX in accordance with Reference Example 4; and the results are
summarized in FIGS. 9-1 and 9-2. The case of using the H-chain
variable region of an anti-IL6R antibody is indicated with MHO, and
the case of using the H chain variable region of an anti-GPC3
antibody is indicated with GpH7. The case of using the L chain
variable region of an anti-IL6R antibody is indicated with ML0, and
the case of using the L chain variable region of an anti-GPC3
antibody is indicated with GpL16. Multiple heterogeneous components
which are thought to be various combinations of H chain and L chain
were detected by chromatography for 4ch_001 which is not introduced
with the mutations for CH1/CL interface control and KiH. In
contrast, since the association of homogeneous H chains was
suppressed in 4ch_002 with KiH mutation, the number of
chromatographic peaks which are thought to be impurities are
reduced. In addition, since the association of H chains and L
chains was suppressed in 4ch_003 which uses TH2 and TH13 in H
chains and TL16 and TL17 in L chains, into which modifications for
regulating the CH1/CL interface were introduced, the number of
chromatography peaks which are thought to represent impurities
decreased. Moreover, it was revealed that 4ch_004 combining the
mutations of KiH and modifications for regulating the CH1/CL
interface is mostly the main peak. The reason that the
chromatography peaks of 4ch_004 nearly coincide with the
chromatography peaks of 4ch_011 is thought to be that their peaks
are unable to be separated by chromatography due to their similar
isoelectric points (pI). Studies were also conducted on 4ch_005,
4ch_006, 4ch_015, and 4ch_016, to which a different regulation of
the CH1/CL interface from that for 4ch_004 had been applied;
4ch_007, 4ch_008, 4ch_013, and 4ch_014, in which modifications for
regulating the CH1/CL interface introduced into 4ch_004 were
interchanged between the anti-GPC3 antibody and the anti-IL6R
antibody; and 4ch_009, 4ch_010, 4ch_017, and 4ch_018, in which
modifications for regulating the CH1/CL interface introduced into
4ch_006 were interchanged between the anti-GPC3 antibody and the
anti-IL6R antibody, using the same methods as 4ch_003, 4ch_004,
4ch_011, and 4ch_012. As a result, heterogeneous components
presented in chromatogram were reduced significantly in comparison
with 4ch_001.
[0287] From the above, it became apparent that bispecific
antibodies can be efficiently prepared by combining modifications
for regulating the CH1/CL interface and the KiH modification.
[Example 5] Effects of Modifying Regulation of the CH1/CL Interface
Using Different Antibodies
[0288] It became apparent from Example 4 that the interface
regulation using CH1/CL is useful for preparing bispecific
antibodies. Therefore, the effect of regulating the CH1/CL
interface was confirmed using an anti-CD3 antibody, M12 (H chain:
SEQ ID NO: 54, L chain: SEQ ID NO: 57) and an anti-GPC3 antibody,
GC33(2) (H chain: SEQ ID NO: 55, L chain: SEQ ID NO: 58). As with
Example 4, bispecific antibodies were prepared using TH2, TH13, and
TH15 in H chains and TL16, TL17, and TL19 in L chains which
demonstrated considerable effects for regulating the CH1/CL
interface.
[0289] The constant regions of the H chain (SEQ ID NO: 54) and the
L chain (SEQ ID NO: 57) of anti-CD3 recognizing antibody M12, and
the constant regions of the H chain (SEQ ID NO: 55) and the L chain
(SEQ ID NO: 58) of anti-GPC3 recognizing antibody GC33(2), were
substituted with TH2, TH13, and TH15 for CH1 of the H chain, and
with TL16, TL17, and TL19 for CL of the L chain. Moreover, an H
chain with Knob into Hole (KiH) modifications (Patent Document 1)
was prepared to avoid association between homogeneous H chains. The
mutation sites of these prepared antibodies and the expressed
antibodies are summarized in Table 9 (Summary of Modification
Sites).
TABLE-US-00009 Ach Bch NAME VH CH VL CL VH CH VL CL 4ch_001 M12VH
G1d M12VL k0 G1d k0 4ch_004 M12VH TH2k M12VL TL17 TH h TL16 4ch_
M12VH TH2k M12VL TL17 TH h TL19 4ch_001 MH0 G1d ML0 k0 GpH7 G1d
GpL16 k0 4ch_ MH0 TH13k ML0 TL18 GpH7 TH2h GpL16 TL17 4ch_ MH0
TH15k ML0 TL19 GdH7 TH2h GpL16 TL17 VH CH NAME chain name mutation
SEQ ID NO name mutation SEQ ID NO 4ch_001 Ach M12VH -- SEQ ID NO:
SEQ ID NO: 4ch_004 Ach M12VH -- SEQ ID NO: Q175K SEQ ID NO: 4ch_
Ach M12VH -- SEQ ID NO: Q175K SEQ ID NO: 4ch_001 Ach MH0 -- SEQ ID
NO: SEQ ID NO: 4ch_ Ach MH0 -- SEQ ID NO: SEQ ID NO: 4ch_010 Ach
MH0 -- SEQ ID NO: SEQ ID NO: 4ch_001 Bch -- SEQ ID NO: SEQ ID NO:
4ch_004 Bch -- SEQ ID NO: SEQ ID NO: 4ch_ Bch -- SEQ ID NO: SEQ ID
NO: 4ch_001 Bch GpH7 -- SEQ ID NO: SEQ ID NO: 4ch_ Bch GpH7 -- SEQ
ID NO: Q175K SEQ ID NO: 4ch_010 Bch GpH7 -- SEQ ID NO: Q175K SEQ ID
NO: VL CL NAME chain name mutation SEQ ID NO name mutation SEQ ID
NO 4ch_001 Ach M12VL -- SEQ ID NO: k0 -- SEQ ID NO: 4ch_004 Ach
M12VL -- SEQ ID NO: TL17 SEQ ID NO: 4ch_ Ach M12VL -- SEQ ID NO:
TL17 SEQ ID NO: 4ch_001 Ach MH0 -- SEQ ID NO: k0 -- SEQ ID NO: 4ch_
Ach ML0 -- SEQ ID NO: TL18 SEQ ID NO: 4ch_010 Ach ML0 -- SEQ ID NO:
TL19 SEQ ID NO: 4ch_001 Bch -- SEQ ID NO: k0 -- SEQ ID NO: 4ch_004
Bch -- SEQ ID NO: TL1 SEQ ID NO: 4ch_ Bch -- SEQ ID NO: TL19 SEQ ID
NO: 4ch_001 Bch GpL16 -- SEQ ID NO: k0 -- SEQ ID NO: 4ch_ Bch GpL16
-- SEQ ID NO: TL17 SEQ ID NO: 4ch_010 Bch GpL16 -- SEQ ID NO: TL17
SEQ ID NO: indicates data missing or illegible when filed
[0290] Preparation of expression vectors of H chains and L chains
with mutations and expression of antibodies were carried out in
accordance with Reference Example 1, and analyses of the prepared
antibodies were carried out in accordance with the CIEX analysis
method shown in Reference Example 4.
[0291] It is apparent that regulation of the CH1/CL interface is
also useful for preparing bispecific antibodies with an anti-CD3
antibody and an anti-GPC3 antibody.
[Example 5] Combination of Regulation of the CH1/CL Interface and
Regulation of the Variable Region Interface
[0292] When preparing bispecific antibodies, introducing electrical
repulsion into the variable regions VH and VL is known as a
technique for allowing specific association of target H chains and
L chains (Patent Document WO 2006/106905). Therefore, in order to
efficiently express only target components, one is to cause
repulsion between variable regions of the H chain and L chain, in
addition to regulation of the CH1/CL interface. This is referred to
as VH/VL interface regulation. MH01 (SEQ ID NO: 46), in which Gln
at position 39 as indicated by Kabat numbering of the H chain of
anti-IL6R was substituted with Lys; MH02 (SEQ ID NO: 47), in which
the Gln was substituted with Glu; ML01 (SEQ ID NO: 50), in which
Gln at position 38 as indicated by Kabat numbering of the L chain
was substituted with Glu; and ML02 (SEQ ID NO: 51), in which the
Glu was substituted with Lys, were prepared. Moreover, GpH71 (SEQ
ID NO: 48), in which Gln at position 39 as indicated by Kabat
numbering of the H chain of anti-GPC3 was substituted with Lys;
GpH72 (SEQ ID NO: 49), in which the Gln was substituted with Glu;
GpL161 (SEQ ID NO: 52), in which Gln at position 38 as indicated by
Kabat numbering of the L chain was substituted with Glu; and GpL162
(SEQ ID NO: 53), in which the Gln was substituted with Lys, were
respectively prepared. Preparation of antibody expression vectors
was carried out in accordance with the method of Reference Example
1. Bispecific antibodies were expressed using the prepared
antibodies. The combinations of modifications in the prepared
antibodies and the expressed antibodies are summarized in Table
10.
TABLE-US-00010 TABLE 10 VH CH VL CL SEQ SEQ SEQ SEQ ID ID ID ID
NAME chain name mutation NO name mutation NO name mutation NO name
mutation NO 4ch_1,2_004 Ach MH01 Q39K 46 TH2k Q175K 39 MH01 Q38E 50
TL17 T180E, S131E, 30 Q160E 4ch_1,2_006 Ach MH01 O39K 46 TH2k Q175K
39 MH01 Q38E 50 TL17 T180E, S131E, 30 Q160E 4ch_2,1_008 Ach MH02
Q39E 47 TH13k K147E, Q175E 49 MH02 Q38K 51 TL16 T180K, S131K, 29
Q160K 4ch_2,1_010 Ach MH02 Q39E 47 TH15k K147E, Q175E, 41 MH02 Q38K
51 TL19 T180K, S131K, 32 K213E Q160K, E123K 4ch_1,2_004 Bch GpH72
Q39E 49 TH13h K147E, Q175E 44 GpL162 Q38K 53 TL16 T180K, S131K, 29
Q160K 4ch_1,2_006 Bch GpH72 Q39E 49 TH15h K147E, Q175E, 45 GpL162
Q38K 53 TL19 T180K, S131K, 32 K213E Q160K, E123K 4ch_2,1_008 Bch
GpH71 Q39K 48 TH2h Q175K 43 GpL161 Q38E 52 TL17 T180E, S131E, 30
Q160E 4ch_2,1_010 Bch GpH71 Q39K 48 TH2h Q175K 43 GpL161 Q38E 52
TL17 T180E, S131E, 30 Q160E
[0293] Antibody expression was carried out in accordance with
Reference Example 1, and analyses of the prepared antibodies were
carried out in accordance with Reference Example 4.
[0294] Since the peaks observed in the chromatograms of 4ch_006 and
4ch_008, into which mutations for regulating the CH1/CL interface
had been introduced, which are thought to represent heterogeneous
components, had disappeared in 4ch_1,2 006 and 4ch_2,1 008 into
which mutations for regulating the VH/VL interface had been
introduced, it became apparent that only target components can be
efficiently prepared by applying regulation of the VH/VL interface
in addition to regulation of the CH1/CL interface (FIGS. 10 and
11). In addition, the peaks which are thought to represent new
heterogeneous components were not detected even if one further made
mutations for regulating the VH/VL interface in 4ch_004 and
4ch_010, which had mutations introduced into CH1/CL only, and in
which only components that were considered to be target components
were thought to be purified (FIGS. 10 and 11).
[0295] From the above, it became apparent that the addition of
regulation of the VH/VL interface to regulation of the CH1/CL
interface further facilitates purification of target components,
while it does not have a detrimental effect on the purification
when only the target components are thought to be purified
already.
[Example 6] Measurement of the Tm of Antibodies with Combined
Modification Sites
[0296] Modifications for regulating the CH1/CL interface may have
an effect on Fab stability. Therefore, Fab stability or Tm was
measured in accordance with the method of Reference Example 2, for
the combinations of TH2/TL17, TH13/TL16, and TH15/TL19. Antibodies
in which the H chain/L chain consisted of TH2/TL17, TH13/TL16, and
TH15/TL19 were prepared using an anti-IL6R antibody. The
combinations of antibody modifications and the expressed antibodies
are summarized in Table 11.
TABLE-US-00011 TABLE 11 Hch Lch SEQ SEQ name mutation ID NO name
mutation ID NO Tm G1d -- 1 k0 -- 13 95.0 TH2 Q175K 3 TL17 T180E,
S131E, Q160E 30 93.1 TH13 K147E, Q175E 10 TL16 T180K, S131K, Q160K
29 95.1 TH15 K147E, Q175E, K213E 12 TL19 T180K, S131K, Q160K, E123K
32 94.8
[0297] Expression of antibodies was measured in accordance with
Reference Example 1, and Tm (.degree. C.) of each of the prepared
antibodies was measured in accordance with Reference Example 2. The
result shows that the values of Tm for the Fab of G1d/k0 which had
no introduction of mutations, and for the Fab of TH2/TL17,
TH13/TL16, and TH15/TL19, into which mutations were introduced into
CH1/CL, were 95.0.degree. C., 93.1.degree. C., 95.1.degree. C., and
94.8.degree. C., respectively. It revealed that mutations for
regulating the CH1/CL interface do not have an effect on Fab
stability.
[Example 7] Effect of Introducing Mutations for Regulating the
CH1/CL Interface on Binding Activity
[0298] The possibility of modifications for regulating the CH1/CL
interface having an effect on antigen binding cannot be completely
ruled out. Therefore, in order to measure the affinity for IL-6R
and GPC3, binding activities of TH2/TL17, TH13/TL16, and TH15/TL19
were measured in accordance with the method of Reference Example 5
(Table 12).
[0299] Since the IL-6R-binding activity and GPC3-binding activity
of TH2/TL17, TH13/TL16, and TH15/TL19, into which mutations for
regulating the CH1/CL interface have been introduced, were not
different from the binding activities of native Gld/k0 to IL-6R and
GPC3, it became apparent that modifications for regulating the
CH1/CL interface do not affect the affinities. Moreover, when the
affinities for the two antigens, IL-6R and GPC3, were measured in
accordance with Reference Example 5 using 4ch_004, 4ch_006,
4ch_008, and 4ch_010 prepared in Example 4, it was found that their
affinities are equal to that of native Gld/k0 shown in Table 12
(Tables 13 and 14).
[0300] According to the studies conducted in Examples 1 to 7, it
became apparent that only target components could be efficiently
purified by introducing a mutation into CH1/CL, without lowering
Fab stability and without lowering binding activity.
TABLE-US-00012 TABLE 12 Affinity to GPC3 Hch Lch name mutation SEQ
ID NO name mutation SEQ ID NO ka kd KD(nM) G1d -- SEQ ID NO: 001 k0
-- SEQ ID NO: 013 2.7E+05 3.6E-04 1.3E-09 TH2 Q175K SEQ ID NO: 003
TL17 T180E, S131E, SEQ ID NO: 030 2.8E+05 3.9E-04 1.4E-09 Q160E
TH13 K147E, Q175E SEQ ID NO: 010 TL16 T180K, S131K, SEQ ID NO: 029
2.7E+05 4.0E-04 1.5E-09 Q160K TH15 K147E, Q175E, SEQ ID NO: 012
TL19 T180K, S131K, SEQ ID NO: 032 3.9E+05 3.8E-04 9.9E-10 K213E
Q160K, E123K Affinity to IL6R Hch Lch name mutation SEQ ID NO name
mutation SEQ ID NO KLon Koff KD(nM) G1d -- SEQ ID NO: 001 k0 -- SEQ
ID NO: 013 1.5E+05 4.1E-04 2.8E-09 TH2 Q175K SEQ ID NO: 003 TL17
T180E, S131E, SEQ ID NO: 030 1.3E+05 5.0E-04 3.8E-09 Q160E TH13
K147E, Q175E SEQ ID NO: 010 TL16 T180K, S131K, SEQ ID NO: 029
1.6E+05 4.4E-04 2.9E-09 Q160K TH15 K147E, Q175E, SEQ ID NO: 012
TL19 T180K, S131K, SEQ ID NO: 032 2.1E+05 4.8E-04 2.3E-09 K213E
Q160K, E123K
TABLE-US-00013 TABLE 13 Ach Bch Affinity to GPC3 Affinity to IL6R
NAME VH CH VL CL VH CH VL CL ka kd KD(nM) ka kd KD(nM) 4ch_001 MH0
G1d ML0 k0 GpH7 G1d GpL16 k0 2.7E+05 3.6E-04 1.3E-09 1 5E+05
4.1E-04 2.6E-09 4ch_004 MH0 TH2k ML0 TL17 GpH7 TH13h GpL16 TL16
2.7E+05 3.8E-04 1.4E-09 1.7E+05 4.7E-04 2.8E-09 4ch_006 MH0 TH2k
ML0 TL17 GpH7 TH15h GpL16 TL19 3.4E+05 4.2E-04 1.2E-09 2.0E+05
4.3E-04 2.2E-09 4ch_001 MH0 G1d ML0 k0 GpH7 G1d GpL16 k0 2.7E+05
3.6E-04 1.3E-09 1.5E+05 4.1E-04 2.8E-09 4ch_008 MH0 TH13k ML0 TL16
GpH7 TH2h GpL16 TL17 2.6E+05 3.9E-04 1.5E-09 1.6E+05 4.6E-04
2.8E-09 4ch_010 MH0 TH15k ML0 TL19 GpH7 TH2h GpL16 TL17 2.9E+05
4.1E-04 1.4E-09 2.4E+05 5.9E-04 2.4E-09
TABLE-US-00014 TABLE 14 VH CH NAME chain name mutation SEQ ID NO
name mutation SEQ ID NO 4ch_001 Ach MH0 -- SEQ ID NO: 036 G1d K147K
SEQ ID NO: 001 4ch_004 Ach MH0 -- SEQ ID NO: 036 TH2k Q175K SEQ ID
NO: 039 4ch_006 Ach MH0 -- SEQ ID NO: 036 TH2k Q175K SEQ ID NO: 039
4ch_001 Ach MH0 -- SEQ ID NO: 036 G1d K147K SEQ ID NO: 001 4ch_008
Ach MH0 -- SEQ ID NO: 036 TH13k K147E, Q175E SEQ ID NO: 040 4ch_010
Ach MH0 -- SEQ ID NO: 036 TH15k K147E, Q175E, SEQ ID NO: 041 K213E
4ch_001 Bch GpH7 -- SEQ ID NO: 034 G1d K147K SEQ ID NO: 001 4ch_004
Bch GpH7 -- SEQ ID NO: 034 TH13h K147E, Q175E SEQ ID NO: 044
4ch_006 Bch GpH7 -- SEQ ID NO: 034 TH15h K147E, Q175E, SEQ ID NO:
045 K213E 4ch_001 Bch GpH7 -- SEQ ID NO: 034 G1d K147K SEQ ID NO:
001 4ch_008 Bch GpH7 -- SEQ ID NO: 034 TH2h Q175K SEQ ID NO: 043
4ch_010 Bch GpH7 -- SEQ ID NO: 034 TH2h Q175K SEQ ID NO: 043 VL CL
NAME chain name mutation SEQ ID NO name mutation SEQ ID NO 4ch_001
Ach ML0 -- SEQ ID NO: 037 k0 -- SEQ ID NO: 013 4ch_004 Ach ML0 --
SEQ ID NO: 037 TL17 T180E, S131E, SEQ ID NO: 030 Q160E 4ch_006 Ach
ML0 -- SEQ ID NO: 037 TL17 T180E, S131E, SEQ ID NO: 030 Q160E
4ch_001 Ach ML0 -- SEQ ID NO: 037 k0 -- SEQ ID NO: 013 4ch_008 Ach
ML0 -- SEQ ID NO: 037 TL16 T180K, S131K, SEQ ID NO: 029 Q160K
4ch_010 Ach ML0 -- SEQ ID NO: 037 TL19 T180K, S131K, SEQ ID NO: 032
Q160K, E123K 4ch_001 Bch GpL16 -- SEQ ID NO: 035 k0 -- SEQ ID NO:
013 4ch_004 Bch GpL16 -- SEQ ID NO: 035 TL16 T180K, S131K, SEQ ID
NO: 029 Q160K 4ch_006 Bch GpL16 -- SEQ ID NO: 035 TL19 T180K,
S131K, SEQ ID NO: 032 Q160K, E123K 4ch_001 Bch GpL16 -- SEQ ID NO:
035 k0 -- SEQ ID NO: 013 4ch_008 Bch GpL16 -- SEQ ID NO: 035 TL17
T180E, S131E, SEQ ID NO: 030 Q160E 4ch_010 Bch GpL16 -- SEQ ID NO:
035 TL17 T180E, S131E, SEQ ID NO: 030 Q160E
Example 8
[0301] Amino acid sequences of human IgA1 (SEQ ID NO: 63), IgA2
(SEQ ID NO: 64), IgD (SEQ ID NO: 65), IgE (SEQ ID NO: 66), IgG1
(SEQ ID NO: 67), IgG2 (SEQ ID NO: 68), IgG3 (SEQ ID NO: 69), IgG4
(SEQ ID NO: 70), and IgM (SEQ ID NO: 71) were aligned with respect
to CH1 of the H chain; and amino acid sequences of human IgK
(Kappa) (SEQ ID NO: 72), IgL1 (SEQ ID NO: 73), IgL2 (SEQ ID NO:
74), IgL3 (SEQ ID NO: 75), IgL6 (SEQ ID NO: 76), and IgL7 (SEQ ID
NO: 77) (Lambda) were aligned with respect to CL of the L chain,
followed by their respective comparisons. The results are shown in
FIG. 12. Modifications discovered in the present example are
indicated with arrows. As a result of introducing amino acids
having different charges into the H chain and L chain so that the
amino acids indicated with the arrows repel between CH1 of the H
chain and CL of the L chain as indicated in the present example, it
is thought that the target H chain and L chain can be specifically
associated.
[Reference Example 1] Preparation of Antibody Expression Vectors,
and Expression and Purification of Antibodies
[0302] Amino acid substitutions were introduced according to a
method known among those skilled in the art using the QuikChange
Site-Directed Mutagenesis Kit (Stratagene), PCR or the In-fusion
Advantage PCR Cloning Kit (Takara), etc., followed by construction
of expression vectors. The base sequences of the obtained
expression vectors were determined according to a method known
among those skilled in the art. Antibodies were expressed by
transiently transfecting the prepared plasmids into human embryonic
kidney cancer cell-derived HEK293H cell lines (Invitrogen) or
FreeStyle 293 cells (Invitrogen). Antibodies were purified from the
obtained culture supernatant according to a method known among
those skilled in the art using rProtein A Sepharose.TM. Fast Flow
(GE Healthcare). The concentration of purified antibodies was
determined by measuring absorbance at 280 nm using a
spectrophotometer, and the antibody concentration was calculated
from the obtained value using an absorption coefficient calculated
according to the PACE method (Protein Science 1995; 4:
2411-2423).
[Reference Example 2] Evaluation of the Melting Temperature (Tm) of
Modified Antibodies by Differential Scanning Calorimetry
[0303] In this study, thermal stability was evaluated by measuring
the melting temperature (Tm) of antibodies using a differential
scanning calorimeter; MicroCal Capillary DSC (DKSH).
[0304] 500 .mu.L aliquot of each antibody solution was placed in a
measuring plate and the temperature was increased from 20.degree.
C. to 115.degree. C. The rate of temperature increase 120.degree.
C./hour and the change in heat capacity was monitored.
[0305] The data was analyzed using Origin? (Light Stone), and the
temperature at which a change in heat capacity was observed was
calculated and defined as the value of Tm.
[Reference Example 3] Anion Exchange Chromatography (AIEX)
Analysis
[0306] Prepared antibodies were analyzed by the AIEX method using
the Alliance System (Waters). Analyses were carried out according
to the two-liquid gradient method using TSK-gel DEAE-NPR (Tosoh)
for the analytical column, 10 mmol/L Tris-HCl (pH 7.5) for mobile
phase A, and 10 mmol/L Tris-HCl and 500 mmol/L NaCl (pH 7.5) for
mobile phase B. Measurements were carried out at a wavelength of
280 nm.
[0307] Data was analyzed using Empower2 (Waters), followed by
calculation of the ratio of each detected peak.
[Reference Example 4] Cation Exchange Chromatography (CIEX)
Analysis
[0308] Prepared antibodies were analyzed by the CIEX method using
the Alliance System (Waters). Analyses were carried out according
to the two-liquid gradient method using WCX-10 (Dionex) for the
analytical column, 25 mmol/L MES (pH 6.1) for mobile phase A, and
25 mmol/L MES and 500 mmol/L NaCl (pH 6.1) for mobile phase B.
Measurements were carried out at a wavelength of 280 nm.
[0309] Data was analyzed using Empower2 (Waters), followed by
calculation of the ratio of each detected peak.
[Reference Example 5] Measurement of Affinity for IL6R and GPC3
[0310] Interactions between a target antibody and hIL6R or GPC3
were analyzed using Biacore T100 (GE Healthcare). HBS-EP+(GE
Healthcare) was used for the running buffer, and the measurement
temperature was 25.degree. C. Protein A/G (Thermo Scientific) was
immobilized on the Series S Sensor Chip CMS (GE Healthcare) by
amine coupling, and was used as a chip. A target antibody was
captured onto the chip, and interacted with each antigen diluted
with running buffer. Antibodies captured on the chip were washed
off by reacting with 10 mM glycine-HCl (pH 1.5) to regenerate the
chip to be used repeatedly.
[0311] The dissociation constant (KD) of each antibody for antigen
was calculated by carrying out kinetic analysis on the results of
Biacore measurement. More specifically, the association rate
constant ka (L/mol/s) and dissociation rate constant kd (1/s) were
calculated by global fitting sensorgrams obtained by measuring with
the Biacore Evaluation Software in a 1:1 Langmuir binding model,
followed by calculation of dissociation constant KD (mol/L) from
those values.
INDUSTRIAL APPLICABILITY
[0312] The method provided by the present invention enables one to
regulate association without altering the structure, function,
activity and the like of the original polypeptide (antibody), and
is extremely useful since it requires only a small number of amino
acid substitutions. In addition, the method also has little
influence on antigenicity.
[0313] Use of the present invention enables efficient acquisition
of bispecific antibodies that actually retain activity.
Sequence CWU 1
1
791328PRTHomo sapiens 1Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3252328PRTArtificial Sequencean artificially
synthesized sequence 2Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Glu Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3253328PRTArtificial Sequencean artificially
synthesized sequence 3Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Lys Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3254328PRTArtificial Sequencean artificially
synthesized sequence 4Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Glu Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3255328PRTArtificial Sequencean artificially
synthesized sequence 5Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3256328PRTArtificial Sequencean artificially
synthesized sequence 6Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Glu1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3257328PRTArtificial Sequencean artificially
synthesized sequence 7Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val Asp Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3258328PRTArtificial Sequencean artificially
synthesized sequence 8Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Lys Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 3259328PRTArtificial Sequencean artificially
synthesized sequence 9Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Glu Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 32510328PRTArtificial Sequencean artificially
synthesized sequence 10Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Glu Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Glu Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 32511328PRTArtificial Sequencean artificially
synthesized sequence 11Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Glu Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 32512328PRTArtificial Sequencean artificially
synthesized sequence 12Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Glu Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Glu Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro 32513107PRTHomo sapiens 13Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10514107PRTArtificial Sequencean artificially synthesized sequence
14Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Glu Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10515107PRTArtificial Sequencean artificially
synthesized sequence 15Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr
Leu Lys Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10516107PRTArtificial Sequencean
artificially synthesized sequence 16Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Glu
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10517107PRTArtificial
Sequencean artificially synthesized sequence 17Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly
Asn Ser Lys Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10518107PRTArtificial Sequencean artificially synthesized sequence
18Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10519107PRTArtificial Sequencean artificially
synthesized sequence 19Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Lys Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10520107PRTArtificial Sequencean
artificially synthesized sequence 20Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Glu Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10521107PRTArtificial
Sequencean artificially synthesized sequence 21Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Lys Val Val Cys
Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 100 10522107PRTArtificial Sequencean
artificially synthesized sequence 22Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Glu Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10523107PRTArtificial
Sequencean artificially synthesized sequence 23Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly
Asn Ser Gln Glu Ser Val Asp Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10524107PRTArtificial Sequencean artificially synthesized sequence
24Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val His Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10525107PRTArtificial Sequencean artificially
synthesized sequence 25Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Lys Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10526107PRTArtificial Sequencean
artificially synthesized sequence 26Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Glu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10527107PRTArtificial
Sequencean artificially synthesized sequence 27Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser
Gly Thr Ala Lys Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr
Tyr Ser Leu Ser Ser Thr Leu Lys Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10528107PRTArtificial Sequencean artificially synthesized sequence
28Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Glu Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Glu Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10529107PRTArtificial Sequencean artificially
synthesized sequence 29Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Lys Val Val
Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Lys Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr
Leu Lys Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10530107PRTArtificial Sequencean
artificially synthesized sequence 30Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Glu Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Glu
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Glu Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10531107PRTArtificial
Sequencean artificially synthesized sequence 31Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys1 5 10 15Gln Leu Lys Ser
Gly Thr Ala Lys Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr
Tyr Ser Leu Ser Ser Thr Leu Lys Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10532107PRTArtificial Sequencean artificially synthesized sequence
32Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys1
5 10 15Gln Leu Lys Ser Gly Thr Ala Lys Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Lys Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Lys Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10533107PRTArtificial Sequencean artificially
synthesized sequence 33Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Glu Val Val
Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Glu Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr
Leu Glu Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10534115PRTHomo sapiens 34Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Glu Met His Trp Ile Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile
35 40 45Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser
Phe 50 55 60Gln Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11535112PRTHomo sapiens
35Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Gln Ala Ser Glu Ser Leu Val His
Ser 20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Glu 100 105 11036119PRTHomo sapiens 36Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu
Trp 35 40 45Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro
Ser Leu 50 55 60Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
Gln Phe Ser65 70 75 80Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
Asp Tyr Trp Gly Gln Gly 100 105 110Ser Leu Val Thr Val Ser Ser
11537107PRTHomo sapiens 37Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 10538328PRTArtificial
Sequencean artificially synthesized sequence 38Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg
Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro 32539328PRTArtificial Sequencean
artificially synthesized sequence 39Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Lys Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180
185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu
Trp Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32540328PRTArtificial Sequencean artificially synthesized sequence
40Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Cys
Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32541328PRTArtificial Sequencean artificially synthesized sequence
41Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Glu 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Cys
Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32542328PRTArtificial Sequencean artificially synthesized sequence
42Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Cys Glu225 230 235 240Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32543328PRTArtificial Sequencean artificially synthesized sequence
43Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Lys Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Cys Glu225 230 235 240Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32544328PRTArtificial Sequencean artificially synthesized sequence
44Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Cys Glu225 230 235 240Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32545328PRTArtificial Sequencean artificially synthesized sequence
45Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Glu 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Cys Glu225 230 235 240Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32546119PRTArtificial Sequencean artificially synthesized sequence
46Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser
Asp 20 25 30His Ala Trp Ser Trp Val Arg Lys Pro Pro Gly Arg Gly Leu
Glu Trp 35 40 45Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn
Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala
Met Asp Tyr Trp Gly Gln Gly 100 105 110Ser Leu Val Thr Val Ser Ser
11547119PRTArtificial Sequencean artificially synthesized sequence
47Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser
Asp 20 25 30His Ala Trp Ser Trp Val Arg Glu Pro Pro Gly Arg Gly Leu
Glu Trp 35 40 45Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn
Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala
Met Asp Tyr Trp Gly Gln Gly 100 105 110Ser Leu Val Thr Val Ser Ser
11548115PRTArtificial Sequencean artificially synthesized sequence
48Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Glu Met His Trp Ile Arg Lys Pro Pro Gly Glu Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser
Glu Ser Phe 50 55
60Gln Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu
Val Thr 100 105 110Val Ser Ser 11549115PRTArtificial Sequencean
artificially synthesized sequence 49Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Thr Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Ile Arg
Glu Pro Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro
Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser Phe 50 55 60Gln Asp Arg Val
Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 11550107PRTArtificial Sequencean artificially
synthesized sequence 50Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Glu Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 10551107PRTArtificial Sequencean
artificially synthesized sequence 51Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Lys
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10552112PRTArtificial
Sequencean artificially synthesized sequence 52Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser
Ile Ser Cys Gln Ala Ser Glu Ser Leu Val His Ser 20 25 30Asn Arg Asn
Thr Tyr Leu His Trp Tyr Leu Glu Lys Pro Gly Gln Ser 35 40 45Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Glu 100 105 11053112PRTArtificial Sequencean artificially
synthesized sequence 53Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Gln Ala Ser
Glu Ser Leu Val His Ser 20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr
Leu Lys Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 100 105
11054125PRTHomo sapiens 54Glu Val Lys Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Lys Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr
Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile65 70 75 80Leu Tyr Leu Gln
Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val
Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120
12555115PRTHomo sapiens 55Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Ile Arg Gln Pro
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro Lys Thr
Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe
Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser 11556328PRTArtificial Sequencean artificially synthesized
sequence 56Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32557109PRTHomo sapiens 57Gln Ala Val Val Thr Gln Glu Ser Ala Leu
Thr Thr Ser Pro Gly Glu1 5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser
Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu
Lys Pro Asp His Leu Phe Thr Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys
Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly
Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75 80Gln Thr Glu Asp
Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10558112PRTHomo sapiens
58Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 11059447PRTHomo sapiens 59Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu
Trp 35 40 45Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro
Ser Leu 50 55 60Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
Gln Phe Ser65 70 75 80Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
Asp Tyr Trp Gly Gln Gly 100 105 110Ser Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 44560214PRTHomo sapiens 60Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser
Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 21061443PRTHomo sapiens 61Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Thr Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His
Trp Ile Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45Gly Ala
Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser Phe 50 55 60Gln
Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200
205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315
320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser
340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 44062219PRTHomo sapiens 62Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Gln Ala Ser Glu Ser Leu Val His Ser
20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Glu 100 105 110Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21563102PRTHomo sapiens 63Ala Ser Pro Thr Ser Pro Lys Val Phe Pro
Leu Ser Leu Cys Ser Thr1 5 10 15Gln Pro Asp Gly Asn Val Val Ile Ala
Cys Leu Val Gln Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu Ser Val Thr
Trp Ser Glu Ser Gly Gln Gly Val 35 40 45Thr Ala Arg Asn Phe Pro Pro
Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser Ser Gln Leu
Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly65 70 75 80Lys Ser Val Thr
Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95Val Thr Val
Pro Cys Pro 10064102PRTHomo sapiens 64Ala Ser Pro Thr Ser Pro Lys
Val Phe Pro Leu Ser Leu Asp Ser Thr1 5 10 15Pro Gln Asp Gly Asn Val
Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu
Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45Thr Ala Arg Asn
Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser
Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly65 70 75 80Lys
Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90
95Val Thr Val Pro Cys Pro 10065101PRTHomo sapiens 65Ala Pro Thr Lys
Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg1 5 10 15His Pro Lys
Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly 20 25 30Tyr His
Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser 35 40 45Gln
Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr 50 55
60Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly65
70 75 80Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys
Glu 85 90 95Ile Phe Arg Trp Pro 10066103PRTHomo sapiens 66Ala Ser
Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys Cys Lys1 5 10 15Asn
Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr 20 25
30Gly Tyr Phe Pro Glu Pro Val Met Val Thr Cys Asp Thr Gly Ser Leu
35 40 45Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu Ser
Gly 50 55 60His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala Trp
Ala Lys65 70 75 80Gln Met Phe Thr Cys Arg Val Ala His Thr Pro Ser
Ser Thr Asp Trp 85 90 95Val Asp Asn Lys Thr Phe Ser 1006798PRTHomo
sapiens 67Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val6898PRTHomo sapiens
68Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1
5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Thr Val6998PRTHomo sapiens 69Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Arg Val7098PRTHomo sapiens 70Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Arg Val71104PRTHomo sapiens 71Gly Ser Ala
Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn1 5 10 15Ser Pro
Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30Phe
Leu Pro Asp Ser Ile Thr Leu Ser Trp Lys Tyr Lys Asn Asn Ser 35 40
45Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys
50 55 60Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met
Gln65 70 75 80Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro
Asn Gly Asn 85 90 95Lys Glu Lys Asn Val Pro Leu Pro 10072107PRTHomo
sapiens 72Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 10573106PRTHomo sapiens 73Gly Gln Pro Lys Ala
Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45Val Lys
Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75
80Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 10574106PRTHomo
sapiens 74Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala
Asp Ser Ser Pro 35 40 45Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Arg Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 100 10575106PRTHomo sapiens 75Gly Gln Pro Lys Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45Val Lys Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75
80Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 10576106PRTHomo
sapiens 76Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Lys Val Ala Trp Lys Ala
Asp Gly Ser Pro 35 40 45Val Asn Thr Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Arg Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val Ala Pro Ala
Glu Cys Ser 100 10577106PRTHomo sapiens 77Gly Gln Pro Lys Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp 20 25 30Phe Tyr Pro Gly
Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45Val Lys Val
Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75
80Ser His Arg Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val
85 90 95Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 100
105781731DNAHomo sapiens 78aactgcagcg ccggggctgg gggaggggag
cctactcact cccccaactc ccgggcggtg 60actcatcaac gagcaccagc ggccagaggt
gagcagtccc gggaaggggc cgagaggcgg 120ggccgccagg tcgggcaggt
gtgcgctccg ccccgccgcg cgcacagagc gctagtcctt 180cggcgagcga
gcaccttcga cgcggtccgg ggaccccctc gtcgctgtcc tcccgacgcg
240gacccgcgtg ccccaggcct cgcgctgccc ggccggctcc tcgtgtccca
ctcccggcgc 300acgccctccc gcgagtcccg ggcccctccc gcgcccctct
tctcggcgcg cgcgcagcat 360ggcgcccccg caggtcctcg cgttcgggct
tctgcttgcc gcggcgacgg cgacttttgc 420cgcagctcag gaagaatgtg
tctgtgaaaa ctacaagctg gccgtaaact gctttgtgaa 480taataatcgt
caatgccagt gtacttcagt tggtgcacaa aatactgtca tttgctcaaa
540gctggctgcc aaatgtttgg tgatgaaggc agaaatgaat ggctcaaaac
ttgggagaag 600agcaaaacct gaaggggccc tccagaacaa tgatgggctt
tatgatcctg actgcgatga 660gagcgggctc tttaaggcca agcagtgcaa
cggcacctcc atgtgctggt gtgtgaacac 720tgctggggtc agaagaacag
acaaggacac tgaaataacc tgctctgagc gagtgagaac 780ctactggatc
atcattgaac taaaacacaa agcaagagaa aaaccttatg atagtaaaag
840tttgcggact gcacttcaga aggagatcac aacgcgttat caactggatc
caaaatttat 900cacgagtatt ttgtatgaga ataatgttat cactattgat
ctggttcaaa attcttctca 960aaaaactcag aatgatgtgg acatagctga
tgtggcttat tattttgaaa aagatgttaa 1020aggtgaatcc ttgtttcatt
ctaagaaaat ggacctgaca gtaaatgggg aacaactgga 1080tctggatcct
ggtcaaactt taatttatta tgttgatgaa aaagcacctg aattctcaat
1140gcagggtcta aaagctggtg ttattgctgt tattgtggtt gtggtgatag
cagttgttgc 1200tggaattgtt gtgctggtta tttccagaaa gaagagaatg
gcaaagtatg agaaggctga 1260gataaaggag atgggtgaga tgcataggga
actcaatgca taactatata atttgaagat 1320tatagaagaa gggaaatagc
aaatggacac aaattacaaa tgtgtgtgcg tgggacgaag 1380acatctttga
aggtcatgag tttgttagtt taacatcata tatttgtaat agtgaaacct
1440gtactcaaaa tataagcagc ttgaaactgg ctttaccaat cttgaaattt
gaccacaagt 1500gtcttatata tgcagatcta atgtaaaatc cagaacttgg
actccatcgt taaaattatt 1560tatgtgtaac attcaaatgt gtgcattaaa
tatgcttcca cagtaaaatc tgaaaaactg 1620atttgtgatt gaaagctgcc
tttctattta cttgagtctt gtacatacat acttttttat 1680gagctatgaa
ataaaacatt ttaaactgaa tttcttaaaa aaaaaaaaaa a 173179314PRTHomo
sapiens 79Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala
Ala Ala1 5 10 15Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys
Glu Asn Tyr 20 25 30Lys Leu Ala Val Asn Cys Phe Val Asn Asn Asn Arg
Gln Cys Gln Cys 35 40 45Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys
Ser Lys Leu Ala Ala 50 55 60Lys Cys Leu Val Met Lys Ala Glu Met Asn
Gly Ser Lys Leu Gly Arg65 70 75 80Arg Ala Lys Pro Glu Gly Ala Leu
Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95Pro Asp Cys Asp Glu Ser Gly
Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110Thr Ser Met Cys Trp
Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125Lys Asp Thr
Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140Ile
Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys145 150
155 160Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln
Leu 165 170 175Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn
Val Ile Thr 180 185 190Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr
Gln Asn Asp Val Asp 195 200 205Ile Ala Asp Val Ala Tyr Tyr Phe Glu
Lys Asp Val Lys Gly Glu Ser 210 215 220Leu Phe His Ser Lys Lys Met
Asp Leu Thr Val Asn Gly Glu Gln Leu225 230 235 240Asp Leu Asp Pro
Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala 245 250 255Pro Glu
Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile 260 265
270Val Val Val Val Ile Ala Val Val Ala Gly Ile Val Val Leu Val Ile
275 280 285Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile
Lys Glu 290
295 300Met Gly Glu Met His Arg Glu Leu Asn Ala305 310
* * * * *