U.S. patent application number 17/356754 was filed with the patent office on 2021-10-21 for il-15 variants and uses thereof.
This patent application is currently assigned to Pfizer Inc.. The applicant listed for this patent is Pfizer Inc.. Invention is credited to Javier Fernando Chaparro Riggers, Ling Hon Matthew Chu, Ivana Djuretic, Reid Martin Renny Feldman, Laura Lin, Lidia Mosyak, Yik Andy Yeung.
Application Number | 20210324028 17/356754 |
Document ID | / |
Family ID | 1000005678879 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210324028 |
Kind Code |
A1 |
Yeung; Yik Andy ; et
al. |
October 21, 2021 |
IL-15 Variants and Uses Thereof
Abstract
The present invention relates to human interleukin 15 (IL-15)
variants that have therapeutic and diagnostic use, and methods for
making thereof. The present invention also provides fusion proteins
comprising a human IL-15 variant. Also provided are methods of
stimulating or suppressing immune responses in a mammal, and
methods of treating a disorder (e.g., cancer) using the IL-15
variants or the fusion protein of such IL-15 variants.
Inventors: |
Yeung; Yik Andy; (South San
Francisco, CA) ; Feldman; Reid Martin Renny; (San
Francisco, CA) ; Chu; Ling Hon Matthew; (San Jose,
CA) ; Chaparro Riggers; Javier Fernando; (San Mateo,
CA) ; Djuretic; Ivana; (San Bruno, CA) ; Lin;
Laura; (Weston, MA) ; Mosyak; Lidia; (Newton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc. |
New York |
NY |
US |
|
|
Assignee: |
Pfizer Inc.
New York
NY
|
Family ID: |
1000005678879 |
Appl. No.: |
17/356754 |
Filed: |
June 24, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16286158 |
Feb 26, 2019 |
11059876 |
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17356754 |
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62784302 |
Dec 21, 2018 |
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62636371 |
Feb 28, 2018 |
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62636362 |
Feb 28, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2896 20130101;
A61K 38/2086 20130101; C07K 16/2815 20130101; C07K 14/55 20130101;
A61K 45/06 20130101; C07K 16/283 20130101; A61P 35/00 20180101;
C07K 2317/565 20130101; C07K 16/2818 20130101; A61K 39/395
20130101; C07K 2319/31 20130101; A61K 38/191 20130101; A61K 35/17
20130101; A61K 38/19 20130101; C07K 2317/56 20130101; C12N 15/62
20130101; A61K 38/1793 20130101; C07K 16/2827 20130101; C07K 16/32
20130101; C07K 19/00 20130101; C07K 14/5443 20130101; C07K 16/2887
20130101 |
International
Class: |
C07K 14/54 20060101
C07K014/54; A61K 35/17 20060101 A61K035/17; C07K 19/00 20060101
C07K019/00; A61P 35/00 20060101 A61P035/00; A61K 38/17 20060101
A61K038/17; C07K 16/32 20060101 C07K016/32; C07K 16/28 20060101
C07K016/28; C07K 14/55 20060101 C07K014/55; C12N 15/62 20060101
C12N015/62; A61K 38/19 20060101 A61K038/19; A61K 39/395 20060101
A61K039/395; A61K 38/20 20060101 A61K038/20 |
Claims
1. An isolated human interleukin 15 (IL-15) variant comprising
amino acid substitution at positions a) V49 and I51 or b) V49, I50,
and S51 of SEQ ID NO: 1 and further comprising one or more amino
acid substitutions at positions N1, N4, S7, K10, K11, Y26, S29,
D30, V31, H32, E53, G55, E64, I68, L69, E89, L91, M109, and/or I111
of SEQ ID NO: 1, wherein the IL-15 variant has decreased or no
binding to the human IL-15 receptor alpha (IL-15R.alpha.) and the
human IL-2 receptor beta/gamma (IL-2R.beta..gamma.) as compared to
the wild-type human IL-15 polypeptide or a wild-type IL-15 receptor
alpha-IL-15 fusion polypeptide, and wherein the amino acid
substitution at position V49 is glycosylated.
2. The isolated IL-15 variant of claim 1, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) V49, I50, S51, N4, D30, and E64; b) V49,
I50, S51, N4, D30, E64, and I68; c) V49, I50, S51, N4, D30, E64,
M109; d) V49, I50, S51, N4, D30, E64, I68, and M109; e) V49, I50,
S51, D30, E64, and I68; f) V49, I50, S51, D30, E64, M109; g) V49,
I50, S51, D30, E64, I68, and M109; h) N1, V49, I50, and S51; i) N4,
V49, I50, and S51; j) S7, V49, I50, and S51; k) K10, V49, I50, and
S51; l) K11, V49, I50, and S51; m) S29, V49, I50, and S51; n) V31,
V49, I50, and S51; o) H32, V49, I50, and S51; p) V49, I50, S51, and
E64; q) V49, I50, S51, and I68; r) V49, I50, S51, and L69; s) V49,
I50, S51, and I111; t) N4, V49, I50, S51, and E64; u) N1, D30, V49,
I50, and S51; v) N4, D30, V49, I50, and S51; w) S7 D30, V49, I50,
and S51; x) K10, D30, V49, I50, and S51; y) K11, D30, V49, I50, and
S51; z) S29, D30, V49, I50, and S51; aa) D30, V49, I50, S51, and
E64; bb) D30, V49, I50, S51, and I68; cc) D30, V49, I50, S51, and
L69; and dd) D30, V49, I50, S51, and I111.
3. The isolated IL-15 variant of claim 1, wherein the amino acid
substitutions comprise one or more specific substitutions at: a)
V49N, V49K, V49E, V49H, V49Q or V49R; b) I50A or I50G; c) S51T; d)
N1K, N1G, N1Q, N1R, N1E, N1A, or N1D; e) N4K, N4G, N4A, N4S, N4D,
N4E, N4I, N4L, N4R, N4T, N4W, or N4Q; f) S7E, S7G, S7D, S7K, S7N,
S7R, S7H, or S7T; g) K10A, K10S, K10E, K10L, K10M, K10D, or K10G;
h) K11D, K11S, or K11W; i) D30N; j) E64Q, E64K, E64A, E64S, E64N,
E64H, E64T, or E64R; k) E53N; l) G55S or G55T; m) E89N; n) L91S or
L91T; o) Y26K, Y26R, or Y26H; p) S29N; q) V31S, V31D, or V31K; r)
H32G; s) I68S, I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y,
I68E, or I68H; t) L69A, L69S, L69D, L69T, L69M, L69G, L69Q, L69I,
L69E, or L69V; u) M109A, M109S, M109D, or M109K; and/or v) I111A,
I111K, I111S, or I111D.
4. The isolated IL-15 variant of claim 1, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N4K, D30N, V49N, I50A, S51T, and E64Q; b)
N4Q, D30N, V49N, I50A, and S51T; c) D30N, V49N, I50A, S51T, and
E64Q; d) N4Q, D30N, V49N, I50A, S51T, and E64Q; e) N4Q, V49N, I50A,
and S51T; f) V49N, I50A, S51T, and E64Q; and g) N4Q, V49N, I50A,
S51T, and E64Q.
5. An isolated human interleukin 15 (IL-15) variant comprising
amino acid substitutions at positions E46 and V49 of SEQ ID NO: 1,
and at least one or more amino acid substitution(s) at positions
N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32, E53, G55, E64,
I68, L69, E89, E93, M109 and/or I111 of SEQ ID NO: 1, wherein the
IL-15 variant has no binding to the human IL-15 receptor alpha
(IL-15R.alpha.) and decreased binding to the human IL-2 receptor
beta/gamma (IL-2R.beta..gamma.) as compared to the wild-type human
IL-15 polypeptide or a wild-type IL-15 receptor alpha-IL-15 fusion
polypeptide.
6. The isolated IL-15 variant of claim 5, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N1, E46, and V49; b) N4, E46, and V49; c)
S7, E46, and V49; d) K10, E46, and V49; e) K11, E46, and V49; f)
S29, E46, and V49; g) V31, E46, and V49; h) H32, E46, and V49; i)
E46, V49, and E64; j) E46, V49, and I68; k) E46, V49, and L69; l)
E46, V49, and I111; m) N4, E46, V49, and E64; n) E46, V49, N4, D30,
and E64; o) E46, V49, N4, D30, E64, and I68; p) E46, V49, N4, D30,
E64, and M109; q) E46, V49, N4, D30, E64, I68, and M109; r) N1,
D30, E46, and V49; s) N4, D30, E46, and V49; t) S7, D30, E46, and
V49; u) K10, D30, E46, and V49; v) K11, D30, E46, and V49; w) S29,
D30, E46, and V49; x) D30, E46, V49, and E64; y) D30, E46, V49R,
and I68; z) D30, E46, V49R, and L69; aa) D30, E46, V49R, and I111;
bb) N1, D30, E46, V49, and M109; cc) N4, D30, E46, V49, and M109;
dd)S7, D30, E46, V49, and M109; ee) K10, D30, E46, V49, and M109;
if) K11, D30, E46, V49, and M109; gg) D30, E46, V49, E64, and M109;
hh) D30, E46, V49, I68, and M109; ii) D30, E46, V49, L69 and M109;
ii) D30, E46, V49, M109, and I111; kk) D30, E46, V49, E64, I68, and
M109; ll) E46, V49, D30, E64, and I68; mm) E46, V49, E64, and M109;
nn) E46, V49, D30, E64, I68, and M109; oo) D22, Y26, V49, E46, E53,
E89, and E93; and pp) N1, D30, E46, V49, and E64.
7. The isolated IL-15 variant of claim 5, wherein the amino acid
substitutions comprise one or more specific substitutions at: a)
N1Q, N1K, N1R, N1E, N1A, N1D, or N1G; b) N4K, N4G, N4A, N4S, N4D,
N4E, N4R, N4T, n4I, N4L, N4W, or N4Q; c) S7E, S7G, S7D, S7K, S7N,
S7R, S7H, or S7T; d) K10D, K10A, K10S, K10E, K10L, K10M, K10D, or
K10G; e) K11D, K11S, or K11W; f) D22N; g) Y26K, Y26R, or Y26H; h)
S29N; i) D30N; j) V31S, V31D, or V31K; k) H32G; l) E46G or E46Q; m)
V49N, V49K, or V49R V49E, V49H, or V49Q; n) E53Q; o) G55S or G55T
p) E64Q, E64K, E64A, E64S, E64N, E64H, E64T, or E64R; q) I68S,
I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or I68H; r)
L69S, L69A, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or L69V; S)
E89Q; t) E93Q; u) M109A, M109S, M109D, or M109K; and/or v) I111A,
I111K, I111S, or I111D.
8. The isolated IL-15 variant of claim 5, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N1K, E46G, and V49R; b) N4K, E46G, and
V49R; c) N4Q, E46G, and V49R; d) S7T, E46G, and V49R; e) V31S,
E46G, and V49R; f) V31K, E46G, and V49R; g) E46G, V49R, and E64Q;
h) E46G, V49R, and E64K; i) N4Q, E46G, V49R, and E64Q; j) N1G,
D30N, E46G, and V49R; k) N1K, D30N, E46G, and V49R; l) N1Q, D30N,
E46G, and V49R; m) N4G, D30N, E46G, and V49R; n) N4K, D30N, E46G,
and V49R; o) N4Q, D30N, E46G, and V49R; p) S7E, D30N, E46G, and
V49R; q) S7G, D30N, E46G, and V49R; r) S7T, D30N, E46G, and V49R;
S) K10D, D30N, E46G, and V49R; t) D30N, E46G, V49R, and E64A; u)
D30N, E46G, V49R, and E64Q; v) D30N, E46G, V49R, and E64K; w) D30N,
E46G, V49R, and I68S; x) D30N, E46G, V49R, and I68K; y) N4K, D30N,
E46G, V49R, and E64K; z) N4Q, D30N, E46G, V49R, and E64K; aa) N4K,
D30N, E46G, V49R, and E64Q; bb) N4Q, D30N, E46G, V49R, and E64Q;
cc) N4K, D30N, E46G, V49R, and I68S; dd) D30N, E46G, V49R, E64Q,
and I68S; ee) N1A, D30N, E46G, and V49R; and if) N1G, D30N, E46G,
V49R, and E64Q.
9. An isolated fusion protein comprising: 1) an antibody comprising
a Fc domain; and b) a human interleukin 15 (IL-15) variant
comprising amino acid substitution at positions a) V49 and I51 or
b) V49, I50, and S51 of SEQ ID NO: 1 wherein the amino acid
substitution at position V49 of SEQ ID NO: 1 is glycosylated, and
further comprising one or more amino acid substitutions at
positions N1, N4, S7, K10, K11, Y26, S29, D30, V31, H32, E53, G55,
E64, I68, L69, E89, L91, M109, and/or I111 of SEQ ID NO: 1, wherein
the IL-15 variant is covalently linked to the Fc domain of the
antibody, and wherein the Fc domain has decreased or no antibody
dependent cellular cytotoxicity (ADCC) activity compared to the
wild-type Fc.
10. The fusion protein of claim 9, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) V49, I50, S51, N4, D30, and E64; b) V49,
I50, S51, N4, D30, E64, and I68; c) V49, I50, S51, N4, D30, E64,
M109; d) V49, I50, S51, N4, D30, E64, I68, and M109; e) V49, I50,
S51, D30, E64, and I68; f) V49, I50, S51, D30, E64, M109; g) V49,
I50, S51, D30, E64, I68, and M109; h) N1, V49, I50, and S51; i) N4,
V49, I50, and S51; j) S7, V49, I50, and S51; k) K10, V49, I50, and
S51; l) K11, V49, I50, and S51; m) S29, V49, I50, and S51; n) V31,
V49, I50, and S51; o) H32, V49, I50, and S51; p) V49, I50, S51, and
E64; q) V49, I50, S51, and I68; r) V49, I50, S51, and L69; s) V49,
I50, S51, and I111; t) N4, V49, I50, S51, and E64; u) N1, D30, V49,
I50, and S51; v) N4, D30, V49, I50, and S51; w) S7 D30, V49, I50,
and S51; x) K10, D30, V49, I50, and S51; y) K11, D30, V49, I50, and
S51; z) S29, D30, V49, I50, and S51; aa) D30, V49, I50, S51, and
E64; bb) D30, V49, I50, S51, and I68; cc) D30, V49, I50, S51, and
L69; and dd) D30, V49, I50, S51, and I111.
11. The fusion protein of claim 9, wherein the amino acid
substitutions comprise one or more specific substitutions at: a)
V49N, V49K, or V49R; b) I50A or I50G; c) S51T; d) N1K, N1G, N1Q,
N1R, N1E, N1A, or N1D; e) N4K, N4G, N4A, N4S, N4D, N4E, N4L, N4R,
N4T, or N4Q; f) S7E, S7G, S7D, S7K, S7N, S7R, S7H, or S7T; g) K10A,
K10S, K10E, K10L, K10M, K10D, or K10G; h) K11D, K11S, or K11W; i)
D30N; j) E64Q, E64K, E64A, E64S, E64N, E64H, E64T, or E64R; k)
E53N; l) G55S or G55T; m) E89N; n) L91S or L91T; o) Y26K, Y26R, or
Y26H; p) S29N; q) V31S, V31D, or V31K; r) H32G; s) I68S, I68A,
I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or I68H; t) L69S,
L69A, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or L69V; u) M109A,
M109S, M109D, or M109K; and/or v) I111A, I111K, I111S, or
I111D.
12. The fusion protein of claim 9, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N4K, D30N, V49N, I50A, S51T, and E64Q; b)
Y26K, V49R, N4K, D30N, and E64Q; c) Y26K, V49K, N4K, D30N, and
E64Q. d) N4Q, D30N, V49N, I50A, and S51T; e) D30N, V49N, I50A,
S51T, and E64Q; f) N4Q, D30N, V49N, I50A, S51T, and E64Q; g) N4Q,
V49N, I50A, and S51T; h) V49N, I50A, S51T, and E64Q; and i) N4Q,
V49N, I50A, S51T, and E64Q.
13. An isolated fusion protein comprising: 1) an antibody
comprising a Fc domain; and b) a human interleukin 15 (IL-15)
variant comprising amino acid substitutions at positions E46 and
V49 of SEQ ID NO: 1, and at least one or more amino acid
substitution(s) at positions N1, N4, S7, K10, K11, D22, Y26, S29,
D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109 and/or I111
of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to
the Fc domain of the antibody, and wherein the Fc domain has
decreased or no antibody dependent cellular cytotoxicity (ADCC)
activity compared to the wild-type Fc.
14. The fusion protein of claim 13, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N1, E46, and V49; b) N4, E46, and V49; c)
S7, E46, and V49; d) K10, E46, and V49; e) K11, E46, and V49; f)
S29, E46, and V49; g) V31, E46, and V49; h) H32, E46, and V49; i)
E46, V49, and E64; j) E46, V49, and I68; k) E46, V49, and L69; l)
E46, V49, and I111; m) N4, E46, V49, and E64; n) E46, V49, N4, D30,
and E64; o) E46, V49, N4, D30, E64, and I68; p) E46, V49, N4, D30,
E64, and M109; q) E46, V49, N4, D30, E64, I68, and M109; r) N1,
D30, E46, and V49; s) N4, D30, E46, and V49; t) S7, D30, E46, and
V49; u) K10, D30, E46, and V49; v) K11, D30, E46, and V49; w) S29,
D30, E46, and V49; x) D30, E46, V49, and E64; y) D30, E46, V49R,
and I68; z) D30, E46, V49R, and L69; aa) D30, E46, V49R, and I111;
bb) N1, D30, E46, V49, and M109; cc) N4, D30, E46, V49, and M109;
dd)S7, D30, E46, V49, and M109; ee) K10, D30, E46, V49, and M109;
if) K11, D30, E46, V49, and M109; gg) D30, E46, V49, E64, and M109;
hh) D30, E46, V49, I68, and M109; ii) D30, E46, V49, L69, and M109;
jj) D30, E46, V49, M109, and I111; kk) D30, E46, V49, E64, I68, and
M109; ll) E46, V49, D30, E64, and I68; mm) E46, V49, E64, and M109;
nn) E46, V49, D30, E64, I68, and M109; oo) D22, Y26, V49, E46, E53,
E89, and E93; and pp) N1, D30, E46, V49, and E64.
15. The fusion protein of claim 13, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N1Q, N1K, N1R, N1E, N1A, N1D, or N1G; b)
N4K N4G, N4A, N4S, N4D, N4E, N4L, N4R, N4T, or N4Q; c) S7E, S7G,
S7D, S7K, S7N, S7R, S7H, or S7T; d) K10D, K10A, K10S, K10E, K10L,
K10M, K10D, K10G; e) K11D, K11S, or K11W; f) D22N; g) Y26K, Y26R,
or Y26H; h) S29N; i) D30N; j) V31S, V31D, or V31K k) H32G; l) E46G
or E46Q; m) V49N, V49K, or V49R V49E, V49H, or V49Q; n) E53Q; o)
G55S or G55T; p) E64Q, E64K, E64A, E64S, E64N, E64H, E64T, or E64R;
q) I68S, I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E or
I68H; r) L69S, L69A, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or
L69V; s) E89Q; t) E93Q; u) M109A, M109S, M109D, or M109K and v)
I111A, I111K, I111S, or I111D.
16. The fusion protein of claim 13, wherein the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N1K, E46G, and V49R; b) N4K, E46G, and
V49R; c) N4Q, E46G, and V49R; d) S7T, E46G, and V49R; e) V31S,
E46G, and V49R; f) V31K, E46G, and V49R; g) E46G, V49R, and E64Q;
h) E46G, V49R, and E64K; i) N4Q, E46G, V49R, and E64Q; j) N1G,
D30N, E46G, and V49R; k) N1K, D30N, E46G, and V49R; l) N1Q, D30N,
E46G, and V49R; m) N4G, D30N, E46G, and V49R; n) N4K, D30N, E46G,
and V49R; o) N4Q, D30N, E46G, and V49R; p) S7E, D30N, E46G, and
V49R; q) S7G, D30N, E46G, and V49R; r) S7T, D30N, E46G, and V49R;
s) K10D, D30N, E46G, and V49R; t) D30N, E46G, V49R, and E64A; u)
D30N, E46G, V49R, and E64Q; v) D30N, E46G, V49R, and E64K; w) D30N,
E46G, V49R, and I68S; x) D30N, E46G, V49R, and I68K; y) N4K, D30N,
E46G, V49R, and E64K; z) N4Q, D30N, E46G, V49R, and E64K; aa) N4K,
D30N, E46G, V49R, and E64Q; bb) N4Q, D30N, E46G, V49R, and E64Q;
cc) N4K, D30N, E46G, V49R, and I68S; dd) D30N, E46G, V49R, E64Q,
and I68S; ee) N1A, D30N, E46G, and V49R; and if) N1G, D30N, E46G,
V49R, and E64Q.
17. The fusion protein of claim 13 comprising an amino acid
sequence of SEQ ID NO: 86, 87, 89, or 90.
18. The fusion protein of claim 13, wherein the antibody has an
isotype that is selected from the group consisting of IgG.sub.1,
IgG.sub.2, IgG.sub.2.DELTA.a, IgG.sub.4, IgG.sub.4.DELTA.b,
IgG.sub.4.DELTA.c, IgG.sub.4 S228P, IgG.sub.4.DELTA.b S228P, and
IgG.sub.4.DELTA.c S228P.
19. The fusion protein of claim 18, wherein the antibody is a) an
IgG2, and the antibody variable domain comprises amino acid
modifications at positions 223, 225, and/or 228 in the hinge region
and at position 409 and/or 368 (EU numbering scheme) in the CH3
region of the human IgG2 (SEQ ID NO: 3); b) an IgG1, and the
antibody variable domain comprises amino acid modifications at
positions 221 and/or 228 in the hinge region and at position 409
and/or 368 (EU numbering scheme) in the CH3 region of the human
IgG1 (SEQ ID NO: 2); or c) an IgG1, and the antibody variable
domain comprise amino acid modifications at positions 349, 354,
366, 368, and/or 407 (EU numbering scheme) in the CH3 region of the
human IgG1 (SEQ ID NO: 2).
20. The fusion protein of claim 19, wherein the antibody further
comprises an amino acid modification at a) one or more positions
265, 330, and/or 331 of the human IgG2 (SEQ ID NO: 3); or b) one or
more positions 234, 235, and/or 237 of the human IgG1 (SEQ ID NO:
2).
21. The fusion protein of claim 13, wherein the antibody is
selected from the group consisting of an anti-CTLA-4 antibody, an
anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an
anti-4-1 BB antibody, an anti-PD-1 antibody, an anti-PD-L1
antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an
anti-TIGIT antibody, an anti-OX40 antibody, an anti-IL-8 antibody,
an anti-IL-7Ralpha (CD127) antibody, an anti-IL15 antibody, an
anti-HVEM antibody, an anti-BTLA antibody, an anti-CD40 antibody,
an anti-CD40L antibody, anti-CD47 antibody, an anti-CSF1R antibody,
an anti-CSF1 antibody, an anti-MARCO antibody, an anti-CXCR4
antibodies, an anti-VEGFR1 antibody, an anti-VEGFR2 antibody, an
anti-TNFR1 antibody, an anti-TNFR2 antibody, an anti-CD3 bispecific
antibody, an anti-CD19 antibody, an anti-CD20, an anti-Her2
antibody, an anti-EGFR antibody, an anti-ICOS antibody, an
anti-CD22 antibody, an anti-CD 52 antibody, an anti-CCR4 antibody,
an anti-CCR8 antibody, an anti-CD200R antibody, an anti-VISG4
antibody, an anti-CCR2 antibody, an anti-LILRb2 antibody, an
anti-CXCR4 antibody, an anti-CD206 antibody, an anti-CD163
antibody, an anti-KLRG1 antibody, an anti-FLT3 antibody, an
anti-B7-H4 antibody, an anti-B7-H3 antibody, an KLRG1 antibody, and
an anti-GITR antibody.
22. The fusion protein of claim 21, wherein the antibody is a PD-1
antibody comprising a heavy chain variable (VH) region comprising a
VH complementarity determining region one (CDR1) comprising the
amino acid sequence of SEQ ID NO: 14, 15, 80, 81, 91, or 92, a VH
CDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, 82,
or 83, and a VH CDR3 comprising the amino acid sequence shown in
SEQ ID NO: 18 or 62; and/or a VL CDR1 comprising the amino acid
sequence shown in SEQ ID NO: 19 or 31, a VL CDR2 comprising the
amino acid sequence shown in SEQ ID NO: 20 or 32, and a VL CDR3
comprising the amino acid sequence shown in SEQ ID NO: 21 or
33.
23. The fusion protein of claim 21, wherein the antibody is a PD-1
antibody comprising a VH region comprising a CDR1, CDR2, and CDR3
of the VH having an amino acid sequence of SEQ ID NO: 12, 34, 78,
or 36, and/or a VL region comprising a CDR1, CDR2, and CDR3 of the
VL having an amino acid sequence of SEQ ID NO: 13, 35, 79, or
37.
24. The fusion protein of claim 13, wherein the IL-15 variant is
covalently linked to the antibody by a polypeptide linker and/or a
polypeptide tag.
25. An isolated cell line that produces the IL-15 variant of claim
5 or the IL-15 fusion protein of claim 13.
26. An isolated nucleic acid encoding the IL-15 variant of claim 5
or the fusion protein of claim 13.
27. A recombinant expression vector comprising the nucleic acid of
claim 26.
28. A host cell comprising the isolated nucleic acid of claim
26.
29. A pharmaceutical composition comprising the IL-15 variant of
claim or the fusion protein of claim 13, and a pharmaceutically
acceptable carrier.
30. A kit for the treatment of cancer comprising the pharmaceutical
composition of claim 29.
31. A method for treating cancer in a subject in need thereof, the
method comprising administering to the subject an effective amount
of the pharmaceutical composition of claim 29, such that one or
more symptoms associated with the cancer is ameliorated in the
subject.
32. The method of claim 31, wherein the cancer is a solid cancer or
a liquid cancer.
33. The method of claim 32, wherein the solid cancer is selected
from the group consisting of gastric cancer, small intestine
cancer, sarcoma, head and neck cancer, thymic cancer, epithelial
cancer, salivary cancer, liver cancer, biliary cancer,
neuroendocrine tumors, stomach cancer, thyroid cancer, lung cancer,
mesothelioma, ovarian cancer, breast cancer, prostate cancer,
esophageal cancer, pancreatic cancer, glioma, renal cancer, bladder
cancer, cervical cancer, uterine cancer, vulvar cancer, penile
cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal
cancer, oral cancer, skin cancer, Merkel cell carcinoma,
glioblastoma, brain tumor, bone cancer, eye cancer, and
melanoma.
34. The method of claim 32, wherein the liquid cancer is selected
from the group consisting of multiple myeloma, malignant plasma
cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte predominant
Hodgkin's lymphoma, Kahler's disease and Myelomatosis, plasma cell
leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell
leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid
leukemia (AML), chronic lymphocytic leukemia (CLL), acute
lymphocytic leukemia (ALL), chronic myeloid leukemia (CML),
follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma,
mantle cell lymphoma, large cell lymphoma, precursor
B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's
macroglobulienemia, diffuse large B cell lymphoma, follicular
lymphoma, marginal zone lymphoma, mucosa-associated lymphatic
tissue lymphoma, small cell lymphocytic lymphoma, mantle cell
lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large
B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenstrom
macroglobulinemia, nodal marginal zone B cell lymphoma, splenic
marginal zone lymphoma, intravascular large B-cell lymphoma,
primary effusion lymphoma, lymphomatoid granulomatosis, T
cell/histiocyte-rich large B-cell lymphoma, primary central nervous
system lymphoma, primary cutaneous diffuse large B-cell lymphoma
(leg type), EBV positive diffuse large B-cell lymphoma of the
elderly, diffuse large B-cell lymphoma associated with
inflammation, intravascular large B-cell lymphoma, ALK-positive
large B-cell lymphoma, plasmablastic lymphoma, large B-cell
lymphoma arising in HHV8-associated multicentric Castleman disease,
B-cell lymphoma unclassified with features intermediate between
diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma
unclassified with features intermediate between diffuse large
B-cell lymphoma and classical Hodgkin lymphoma, and other
hematopoietic cells related cancer.
35. The method of claim 31, wherein the cancer is relapsed,
refractory, or metastatic.
36. The method of claim 31, wherein the method further comprises
administering an effective amount of a second therapeutic agent,
optionally wherein the administration is separate, sequential, or
simultaneous.
37. The method of claim 36, wherein the second therapeutic agent is
an antibody selected from the group consisting of an anti-CTLA-4
antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8
antibody, an anti-4-1BB antibody, an anti-PD-1 antibody, an
anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody,
an anti-TIGIT antibody, an anti-OX40 antibody, an anti-IL-7Ralpha
(CD127) antibody, an anti-IL-8 antibody, an anti-IL-15 antibody, an
anti-HVEM antibody, an anti-BTLA antibody, an anti-CD40 antibody,
an anti-CD40L antibody, anti-CD47 antibody, an anti-CSF1R antibody,
an anti-CSF1 antibody, an anti-IL-7R antibody, an anti-MARCO
antibody, an anti-CXCR4 antibodies, an anti-VEGF antibody, an
anti-VEGFR1 antibody, an anti-VEGFR2 antibody, an anti-TNFR1
antibody, an anti-TNFR2 antibody, an anti-CD3 bispecific antibody,
an anti-CD19 antibody, an anti-CD20, an anti-Her2 antibody, an
anti-EGFR antibody, an anti-ICOS antibody, an anti-CD22 antibody,
an anti-CD 52 antibody, an anti-CCR4 antibody, an anti-CCR8
antibody, an anti-CD200R antibody, an anti-VISG4 antibody, an
anti-CCR2 antibody, an anti-LILRb2 antibody, an anti-CXCR4
antibody, an anti-CD206 antibody, an anti-CD163 antibody, an
anti-KLRG1 antibody, an anti-FLT3 antibody, an anti-B7-H4 antibody,
an anti-B7-H3 antibody, an KLRG1 antibody, a BTN1A1 antibody, and
an anti-GITR antibody.
38. The method of claim 36, wherein the second therapeutic agent is
a cytokine, an immunocytokine, TNF.alpha., a PAP inhibitor, an
oncolytic virus, a kinase inhibitor, an ALK inhibitor, a MEK
inhibitor, an IDO inhibitor, a GLS1 inhibitor, a tyrosine kinase
inhibitor, a CART cell or T cell therapy, a TLR agonist, or a tumor
vaccine.
39. The pharmaceutical composition of claim 29 for use in the
treatment of cancer, optionally wherein the cancer is a solid
cancer or a liquid cancer and/or the cancer is relapsed,
refractory, or metastatic.
40. The pharmaceutical composition of claim 39, wherein the use is
in combination with a second therapeutic agent, optionally wherein
the combination is for administration simultaneously, concurrently,
or simultaneously.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/636,362 filed Feb. 28, 2018, U.S. Provisional
Application No. 62/636,371 filed Feb. 28, 2018, and U.S.
Provisional Application No. 62/784,302 filed Dec. 21, 2018, the
contents of which are hereby incorporated by reference in their
entireties.
REFERENCE TO SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"PC72383A_SequenceListing_ST25.txt" created on Feb. 25, 2019 and
having a size of 264 KB. The sequence listing contained in this
.txt file is part of the specification and is herein incorporated
by reference in its entirety.
FIELD
[0003] The present invention relates to interleukin 15 (IL-15)
variants that have therapeutic and diagnostic use, and methods for
making thereof. The present invention also provides fusion proteins
comprising such IL-15 variants. Also provided are methods of
stimulating or suppressing immune responses in a mammal, and
methods of treating a disorder (e.g., cancer) using the IL-15
variants or the fusion proteins of such IL-15 variants.
BACKGROUND
[0004] Cytokines are powerful modulators of the immune response and
hold the potential to dramatically affect outcomes to
immune-oncology therapeutic approaches. However, previous efforts
to utilize cytokines in human subjects have yielded only modest
efficacies and significant toxicities. Recent studies have
suggested that a "targeted cytokine", such as an antibody-cytokine
fusion protein, may deliver cytokines to a desired cell type while
minimizing peripheral exposure and thus toxicities. See, e.g., Guo
et al., Cytokine Growth Factor Rev. 38:10-21 (2017); Jakobisiak M,
et al., Cytokine Growth Factor Rev. 22(2):99-108 (2011); Robinson,
T. & Schluns, K. S., Immunol. Lett. 190:159-168 (2017); Rhode
et al., Cancer Immunol. Res. 4(1): 49-60 (2016); Conlon et al., J
Clin. Oncol. 33(1): 74-82 (2015). Accordingly, development of a
therapeutic agent based on a targeted cytokine would be of great
value in treatments of various diseases such as cancer.
SUMMARY
[0005] The invention disclosed herein is directed to human
interleukin 15 (IL-15) variants and fusion proteins comprising
thereof. It is demonstrated that the IL-15 variants of the present
invention have decreased or no binding to the IL-15 receptor alpha
(CD215), and/or have reduced interaction between IL-15 and its
signaling receptor, comprised of IL-2 receptor beta (CD122) and the
common gamma chain (CD132) as compared to the wild-type human IL-15
polypeptide or a wild-type IL-15 receptor alpha-IL-15 fusion
polypeptide. In a second aspect of the invention, these reduced
affinity IL-15 variants, when presented as an antibody fusion
chimeric protein, are targeted selectively to desired cell types
(those cells expressing the antibody target). Cell types that
express the IL-15 receptor complex, but not the antibody target,
are activated less, or not activated, compared to those cells which
express both components. Accordingly, the IL-15 variants and the
IL-15 fusion proteins of the present invention selectively modulate
the activation of cell subsets to promote biological activity, such
as an anti-tumor activity, efficaciously and safely. In a third
aspect of this invention, these reduced affinity IL-15 variants and
the IL-15 fusion proteins, when expressed as polynucleotides in CAR
(Chimeric Antigen Receptor) T cells, either as secreted or
membrane-tethered versions, are used to enhance CAR T function,
including activity and proliferation.
[0006] Accordingly, in one aspect, the invention provides an
isolated human interleukin (IL-15) variant comprising amino acid
substitution at positions a) V49 and I51 or b) V49, I50, and S51 of
SEQ ID NO: 1, and further comprising one or more amino acid
substitutions at positions N1, N4, S7, K10, K11, Y26, S29, D30,
V31, H32, E53, G55, E64, I68, L69, E89, L91, M109, and/or I111 of
SEQ ID NO: 1, wherein the IL-15 variant has decreased or no binding
to the human IL-15 receptor alpha (IL-15R.alpha.) and the human
IL-2 receptor beta/gamma (IL-2R.beta..gamma.) as compared to the
wild-type human IL-15 polypeptide or a wild-type IL-15 receptor
alpha-IL-15 fusion polypeptide, and wherein the amino acid
substitution at position V49 is glycosylated.
[0007] In another aspect, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) a human
interleukin 15 (IL-15) variant comprising amino acid substitution
at positions a) V49 and I51 or b) V49, I50, and S51 of SEQ ID NO:
1, wherein the amino acid substitution at position V49 of SEQ ID
NO: 1 is glycosylated, and further comprising one or more amino
acid substitutions at positions N1, N4, S7, K10, K11, Y26, S29,
D30, V31, H32, E53, G55, E64, I68, L69, E89, L91, M109, and/or I111
of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to
the Fc domain of the antibody, and wherein the Fc domain has
decreased or no antibody dependent cellular cytotoxicity (ADCC)
activity compared to the wild-type Fc.
[0008] In some embodiments, the IL-15 variant comprises amino acid
substitution at V49N, wherein V49N is glycosylated. In some
embodiments, the amino acid substitution(s) at E53 and/or E89 of
SEQ ID NO: 1 are also glycosylated.
[0009] In some embodiments, the IL-15 variant comprises amino acid
substitutions of SEQ ID NO: 1 at positions selected from the group
consisting of: a) V49, I50, S51, N4, D30, and E64; b) V49, I50,
S51, N4, D30, E64, and I68; c) V49, I50, S51, N4, D30, E64, M109;
d) V49, I50, S51, N4, D30, E64, I68, and M109; e) V49, I50, S51,
D30, E64, and I68; f) V49, I50, S51, D30, E64, M109; g) V49, I50,
S51, D30, E64, I68, and M109; h) N1, V49, I50, and S51; i) N4, V49,
I50, and S51; j) S7, V49, I50, and S51; k) K10, V49, I50, and S51;
l) K11, V49, I50, and S51; m) S29, V49, I50, and S51; n) V31, V49,
I50, and S51; o) H32, V49, I50, and S51; p) V49, I50, S51, and E64;
q) V49, I50, S51, and I68; r) V49, I50, S51, and L69; s) V49, I50,
S51, and I111; t) N4, V49, I50, S51, and E64; u) N1, D30, V49, I50,
and S51; v) N4, D30, V49, I50, and S51; w) S7 D30, V49, I50, and
S51; x) K10, D30, V49, I50, and S51; y) K11, D30, V49, I50, and
S51; z) S29, D30, V49, I50, and S51; aa) D30, V49, I50, S51, and
E64; bb) D30, V49, I50, S51, and I68; cc) D30, V49, I50, S51, and
L69; and dd) D30, V49, I50, S51, and I111.
[0010] In some embodiments, the IL-15 variant comprises amino acid
substitutions comprising one or more specific substitutions at: a)
V49N, V49K, V49E, V49H, V49Q or V49R; b) I50A or I50G; c) S51T; d)
N1K, N1G, N1Q, N1R, N1E, N1A, or N1D; e) N4K, N4G, N4A, N4S, N4D,
N4E, N4L, N4R, N4T, or N4Q; f) S7E, S7G, S7D, S7K, S7N, S7R, S7H,
or S7T; g) K10A, K10S, K10E, K10L, K10M, K10D, or K10G; h) K11D,
K11S, or K11W; i) D30N; j) E64Q, E64K, E64A, E64S, E64N, E64H,
E64T, or E64R; k) E53N; l) G55S or G55T; m) E89N; n) L91S or L91T;
o) Y26K, Y26R, or Y26H; p) S29N; q) V31S, V31D, or V31K; r) H32G;
s) I68S, I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or
I68H; t) L69A, L69S, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or
L69V; u) M109A, M109S, M109D, or M109K; and/or v) I111A, I111K,
I111S, or I111D. In some embodiments, the IL-15 variant comprises
amino acid substitutions at positions selected from the group
consisting of: a) N4K, D30N, V49N, I50A, S51T, and E64Q; b) N4Q,
D30N, V49N, I50A, and S51T; c) D30N, V49N, I50A, S51T, and E64Q; d)
N4Q, D30N, V49N, I50A, S51T, and E64Q; e) N4Q, V49N, I50A, and
S51T; f) V49N, I50A, S51T, and E64Q; and g) N4Q, V49N, I50A, S51T,
and E64Q.
[0011] In another aspect, provided is an isolated human interleukin
15 (IL-15) variant comprising amino acid substitutions at positions
E46 and V49 of SEQ ID NO: 1, and at least one or more amino acid
substitution(s) at positions N1, N4, S7, K10, K11, D22, Y26, S29,
D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109 and/or I111
of SEQ ID NO: 1, wherein the IL-15 variant has no binding to the
human IL-15 receptor alpha (IL-15R.alpha.) and decreased binding to
the human IL-2 receptor beta/gamma (IL-2R.beta..gamma.) as compared
to the wild-type human IL-15 polypeptide or a wild-type IL-15
receptor alpha-IL-15 fusion polypeptide.
[0012] In another aspect, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) a human
interleukin 15 (IL-15) variant comprising amino acid substitutions
at positions E46 and V49 of SEQ ID NO: 1, and at least one or more
amino acid substitution(s) at positions N1, N4, S7, K10, K11, D22,
Y26, S29, D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109
and/or I111 of SEQ ID NO: 1, wherein the IL-15 variant is
covalently linked to the Fc domain of the antibody, and wherein the
Fc domain has decreased or no antibody dependent cellular
cytotoxicity (ADCC) activity compared to the wild-type Fc.
[0013] In some embodiments, the IL-15 variant comprises amino acid
substitutions in SEQ ID NO: 1 at positions selected from the group
consisting of: a) N1, E46, and V49; b) N4, E46, and V49; c) S7,
E46, and V49; d) K10, E46, and V49; e) K11, E46, and V49; f) S29,
E46, and V49; g) V31, E46, and V49; h) H32, E46, and V49; i) E46,
V49, and E64; j) E46, V49, and I68; k) E46, V49, and L69; l) E46,
V49, and I111; m) N4, E46, V49, and E64; n) E46, V49, N4, D30, and
E64; o) E46, V49, N4, D30, E64, and I68; p) E46, V49, N4, D30, E64,
and M109; q) E46, V49, N4, D30, E64, I68, and M109; r) N1, D30,
E46, and V49; s) N4, D30, E46, and V49; t) S7, D30, E46, and V49;
u) K10, D30, E46, and V49; v) K11, D30, E46, and V49; w) S29, D30,
E46, and V49; x) D30, E46, V49, and E64; y) D30, E46, V49R, and
I68; z) D30, E46, V49R, and L69; aa) D30, E46, V49R, and I111; bb)
N1, D30, E46, V49, and M109; cc) N4, D30, E46, V49, and M109; dd)
S7, D30, E46, V49, and M109; ee) K10, D30, E46, V49, and M109; f)
K11, D30, E46, V49, and M109; gg) D30, E46, V49, E64, and M109; hh)
D30, E46, V49, 168, and M109; ii) D30, E46, V49, L69 and M109; jj)
D30, E46, V49, M109, and I111; kk) D30, E46, V49, E64, I68, and
M109; ll) E46, V49, D30, E64, and I68; mm) E46, V49, E64, and M109;
nn) E46, V49, D30, E64, I68, and M109; oo) D22, Y26, V49, E46, E53,
E89, and E93; and pp) N1, D30, E46, V49, and E64.
[0014] In some embodiments, the IL-15 variant comprises amino acid
substitutions comprising one or more specific substitutions at: a)
N1Q, N1K, N1R, N1E, N1A, N1D, or N1G; b) N4K, N4G, N4A, N4S, N4D,
N4E, N4R, N4T, N4I, N4L, N4W, or N4Q; c) S7E, S7G, S7D, S7K, S7N,
S7R, S7H, or S7T; d) K10D, K10A, K10S, K10E, K10L, K10M, K10D, or
K10G; e) K11D, K11S, or K11W; f) D22N; g) Y26K, Y26R, or Y26H; h)
S29N; i) D30N; j) V31S, V31D, or V31K; k) H32G; l) E46G or E46Q; m)
V49N, V49K, or V49R V49E, V49H, or V49Q; n) E53Q; o) G55S or G55T;
p) E64Q, E64K, E64A, E64S, E64N, E64H, E64T, or E64R; q) I68S,
I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or I68H; r)
L69S, L69A, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or L69V; s)
E89Q; t) E93Q; u) M109A, M109S, M109D, or M109K; and/or v) I111A,
I111K, I111S, or I111D. In some embodiments, the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of a) N1K, E46G, and V49R; b) N4K, E46G, and V49R;
c) N4Q, E46G, and V49R; d) S7T, E46G, and V49R; e) V31S, E46G, and
V49R; f) V31K, E46G, and V49R; g) E46G, V49R, and E64Q; h) E46G,
V49R, and E64K; i) N4Q, E46G, V49R, and E64Q; j) N1G, D30N, E46G,
and V49R; k) N1K, D30N, E46G, and V49R; l) N1Q, D30N, E46G, and
V49R; m) N4G, D30N, E46G, and V49R; n) N4K, D30N, E46G, and V49R;
o) N4Q, D30N, E46G, and V49R; p) S7E, D30N, E46G, and V49R; q) S7G,
D30N, E46G, and V49R; r) S7T, D30N, E46G, and V49R; s) K10D, D30N,
E46G, and V49R; t) D30N, E46G, V49R, and E64A; u) D30N, E46G, V49R,
and E64Q; v) D30N, E46G, V49R, and E64K; w) D30N, E46G, V49R, and
I68S; x) D30N, E46G, V49R, and I68K; y) N4K, D30N, E46G, V49R, and
E64K; z) N4Q, D30N, E46G, V49R, and E64K; aa) N4K, D30N, E46G,
V49R, and E64Q; bb) N4Q, D30N, E46G, V49R, and E64Q; cc) N4K, D30N,
E46G, V49R, and I68S; dd) D30N, E46G, V49R, E64Q, and I68S; ee)
N1A, D30N, E46G, and V49R; and ff) N1G, D30N, E46G, V49R, and
E64Q.
[0015] In another aspect, provided is an isolated human IL-15
variant comprising one or more amino acid substitution(s) at
position(s) N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32,
E46, E53, E64, I68, L69, E89, E93, M109, and/or I111 of SEQ ID NO:
1, wherein the IL-15 variant has decreased or no binding to the
human IL-15 receptor alpha (IL-15R.alpha.) and/or the human IL-2
receptor beta (IL-2R.beta.) and/or IL-2 receptor gamma
(IL-2R.gamma.) as compared to the wild-type human IL-15 polypeptide
or a wild-type IL-15 receptor alpha-IL-15 fusion polypeptide.
[0016] In another aspect, provided is an isolated human IL-15
variant comprising the amino acid sequence shown in SEQ ID NO: 93.
In some embodiments, the IL-15 variant further comprises a
transmembrane domain.
[0017] In another aspect, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) a human
interleukin 15 (IL-15) variant comprising one or more amino acid
substitutions at positions N1, N4, S7, K10, K11, D22, Y26, S29,
D30, V31, H32, E46, E53, E64, I68, L69, E89, E93, M109, and/or I111
of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to
the Fc domain of the antibody, and wherein the Fc domain has
decreased or no antibody dependent cellular cytotoxicity (ADCC)
activity compared to the wild-type Fc.
[0018] In another aspect, provided is an isolated fusion protein
comprising: 1) an IL-15 antibody comprising a Fc domain; and b) a
human interleukin 15 (IL-15) protein of SEQ ID NO: 1, wherein the
IL-15 is covalently linked to the Fc domain of the antibody. In
some embodiments, the antibody in the IL-15 fusion protein of the
present invention can be a human antibody, a humanized antibody, a
chimeric antibody, or a bispecific antibody.
[0019] In some embodiments, the antibody in the IL-15 fusion
protein of the present invention is of the human IgG.sub.1,
IgG.sub.2, IgG.sub.2.DELTA.a, IgG.sub.3, IgG.sub.4,
IgG.sub.4.DELTA.b, IgG.sub.4.DELTA.c, IgG.sub.4 S228P,
IgG.sub.4.DELTA.b S228P, and IgG.sub.4.DELTA.c S228P subclass.
[0020] In some embodiments, the antibody of the IL-15 fusion
protein of the present invention is a) an IgG2, and the antibody
variable domain comprises amino acid modifications at positions
223, 225, and 228 in the hinge region and at position 409 or 368
(EU numbering scheme) in the CH3 region of the human IgG2 (SEQ ID
NO: 3); b) an IgG1, and the antibody variable domain comprises
amino acid modifications at positions 221 and 228 in the hinge
region and at position 409 or 368 (EU numbering scheme) in the CH3
region of the human IgG1 (SEQ ID NO: 2); or c) an IgG1, and the
antibody variable domain comprise amino acid modifications at
positions 349, 354, 366, 368, and/or 407 (EU numbering scheme) in
the CH3 region of the human IgG1 (SEQ ID NO: 2). In some
embodiments, the antibody further comprises an amino acid
modification at a) one or more positions 265, 330, and/or 331 of
the human IgG2 (SEQ ID NO: 3); or b) one or more positions 234,
235, 237, and/or 322 of the human IgG1 (SEQ ID NO: 2). In some
embodiments, the antibody further comprises an amino acid
modification at one or more positions 234, 235, 237, 349, 354, 366,
368 and/or 407 of the human IgG1 (SEQ ID NO: 74 and 75).
[0021] In some embodiments, the antibody for the IL-15 fusion
protein is selected from the group consisting of an anti-CTLA-4
antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8
antibody, an anti-4-1 BB antibody, an anti-PD-1 antibody, an
anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody,
an anti-TIGIT antibody, an anti-OX40 antibody, an anti-IL-7Ralpha
(CD127) antibody, an anti-IL-8 antibody, an anti-IL-15 antibody, an
anti-HVEM antibody, an anti-BTLA antibody, an anti-CD40 antibody,
an anti-CD40L antibody, anti-CD47 antibody, an anti-CSF1R antibody,
an anti-CSF1 antibody, an anti-MARCO antibody, an anti-CXCR4
antibodies, an anti-VEGF antibody, an anti-VEGFR1 antibody, an
anti-VEGFR2 antibody, an anti-TNFR1 antibody, an anti-TNFR2
antibody, an anti-CD3 bispecific antibody, an anti-CD19 antibody,
an anti-CD20, an anti-Her2 antibody, an anti-EGFR antibody, an
anti-ICOS antibody, an anti-CD22 antibody, an anti-CD 52 antibody,
an anti-CCR4 antibody, an anti-CCR8 antibody, an anti-CD200R
antibody, an anti-VISG4 antibody, an anti-CCR2 antibody, an
anti-LILRb2 antibody, an anti-CXCR4 antibody, an anti-CD206
antibody, an anti-CD163 antibody, an anti-KLRG1 antibody, an
anti-FLT3 antibody, an anti-B7-H4 antibody, an anti-B7-H3 antibody,
an KLRG1 antibody, and an anti-GITR antibody.
[0022] In some embodiments, the IL-15 fusion protein of the present
invention is covalently linked to the antibody via a polypeptide
linker and/or a polypeptide tag.
[0023] In another aspect, the invention provides a pharmaceutical
composition comprising a therapeutically effective amount of an
IL-15 variant or an IL-15 fusion protein as described herein and a
pharmaceutically acceptable carrier.
[0024] In another aspect, the invention provides an isolated
polynucleotide comprising a nucleotide sequence encoding an IL-15
variant or an IL-15 fusion protein as described herein. In another
aspect, the invention provides a vector comprising the
polynucleotide.
[0025] In another aspect, the invention provides an isolated host
cell or cell line that recombinantly produces an IL-15 variant or
an IL-15 fusion protein as described herein. In some embodiments,
the host cell or cell line is an engineered immune cell, wherein
the engineered immune cell comprises a chimeric antigen receptor
(CAR). In some embodiments, the CAR expressing cells are T cells,
and the T cells express the IL-15 variant or the IL-15 fusion
proteins as described herein in a secreted form or a
membrane-tethered form.
[0026] In another aspect, the invention provides a method of
producing an IL-15 variant or an IL-15 fusion protein, the method
comprising: culturing a cell line that recombinantly produces the
IL-15 variant or the IL-15 fusion protein as described herein under
conditions wherein the protein variant or the fusion protein is
produced, and recovering the protein variant or the fusion
protein.
[0027] In another aspect, the invention provides a method for
treating a condition in a subject comprising administering to the
subject in need thereof an effective amount of the pharmaceutical
composition as described herein. In some embodiments, the condition
is a cancer. In some embodiments, the cancer is a liquid cancer or
a solid cancer. In some embodiments, the cancer is relapsed,
refractory, or metastatic.
[0028] In another aspect, the invention provides a method of
inhibiting tumor growth or progression in a subject who has a
tumor, comprising administering to the subject an effective amount
of the pharmaceutical composition as described herein.
[0029] In another aspect, the invention provides a method of
inhibiting or preventing metastasis of cancer cells in a subject,
comprising administering to the subject in need thereof an
effective amount of the pharmaceutical composition as described
herein.
[0030] In another aspect, the invention provides a method of
inducing tumor regression in a subject in need thereof, comprising
administering to the subject an effective amount of the
pharmaceutical composition as described herein.
[0031] In some embodiments, the IL-15 variants and the IL-15 fusion
proteins described herein can be administered parenterally in a
subject. In some embodiments, the subject is a human.
[0032] In some embodiments, the method can further comprise
administering an effective amount of a second therapeutic agent. In
some embodiments, the second therapeutic agent is a biotherapeutic
agent.
[0033] In some embodiments, the second therapeutic agent is a
cytokine, an immunocytokine (e.g. anti-EDA-IL10 fusion protein), a
TNF.alpha., a PAP inhibitor, an oncolytic virus, a kinase
inhibitor, an ALK inhibitor (e.g., sunitinib or crizotinib), a MEK
inhibitor, an IDO inhibitor, a GLS1 inhibitor, a tyrosine kinase
inhibitor (e.g., axitinib or palbociclib), a CAR (Chimeric Antigen
Receptor)-T cell or T cell therapy, a PRR (Pattern Recognition
Receptor) agonist such as a TLR (Toll-Like Receptor) Agonist (e.g.,
TLR3, TLR4, TLR5, TLR7, TLR9), or a tumor vaccine.
[0034] Also provided is the use of any of the IL-15 variants or the
IL-15 fusion proteins provided herein in the manufacture of a
medicament for the treatment of cancer or for inhibiting tumor
growth or progression in a subject in need thereof.
[0035] In one aspect, the invention provides for a method for
treating cancer in a subject comprising administering to the
subject a combination therapy which comprises a first therapeutic
agent and a second therapeutic agent, wherein the first therapeutic
agent is an IL-15 variant or IL-15 fusion protein.
[0036] In some embodiments, the first therapeutic agent is an IL-15
variant. In some embodiments, the first therapeutic agent is an
IL-15 fusion protein. In some embodiments, the first therapeutic
agent is any IL-15 variant or IL-15 fusion protein of the present
invention. In some embodiments, the first therapeutic agent
comprises the amino acid sequence shown in SEQ ID NO: 84, 85, 86,
87, 89, or 90.
[0037] In some embodiments, the second therapeutic agent is an
immunocytokine. In some embodiments, the second therapeutic agent
is an immunocytokine comprising an antibody, or fragment thereof,
conjugated or fused to a cytokine (e.g. fusion protein). In some
embodiments, the antibody, or fragment thereof, binds the Extra
Domain-A (EDA) isoform of fibronectin (e.g. anti-EDA antibody). In
some embodiments, the anti-EDA antibody, or fragment thereof,
comprises a CDR1, a CDR2 and CDR3 of the heavy chain variable (VH)
region shown in SEQ ID NO: 94 and/or a CDR1, a CDR2 and CDR3 of the
light chain variable (VL) region shown in SEQ ID NO: 96. In some
embodiments, the anti-EDA antibody, or fragment thereof, comprises
a VH region having the amino acid sequence of SEQ ID NO: 94 and/or
a VL region having the amino acid sequence of SEQ ID NO: 96. In
some embodiments, the cytokine is IL-10. In some embodiments, IL-10
comprises the amino acid sequence of SEQ ID NO: 98. In some
embodiments, the immunocytokine comprises at least one linker. In
some embodiments, the linker(s) comprises SEQ ID NO: 95 and/or 97.
In some embodiments, the immunocytokine is an anti-EDA-IL-10 fusion
protein comprising the amino acid sequence shown in SEQ ID NO:
99.
[0038] In some embodiments, the invention provides for a method for
treating cancer in a subject comprising administering to the
subject a combination therapy which comprises a first therapeutic
agent and a second therapeutic agent, wherein the first therapeutic
agent is an IL-15 fusion protein, and wherein the second agent is
an anti-EDA-IL-10 fusion protein. In some embodiments, the IL-15
fusion protein comprises the amino acid sequence shown in SEQ ID
NO: 84, 85, 86, 87, 89, or 90 and the anti-EDA-IL-10 fusion protein
comprises the amino acid sequence shown in SEQ ID NO: 99.
[0039] In some embodiments, the combination therapy may further
comprise 1, 2, 3, 4 or 5 additional therapeutic agents. In some
embodiments, the combination therapy further comprises an anti-PD-1
or anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody
is BCD-100, camrelizumab (SHR-1210), cemiplimab (REGN2810),
genolimzumab (CBT-501), MED10680, nivolumab (OPDIVO.RTM.),
pembrolizumab (KEYTRUDA.RTM.), PF-06801591 (RN888), sintilimab
(IBI-308), spartalizumab (PDR-001), STI-A1110, tislelizunab
(BGB-A317), or TSR-042. In some embodiments, the anti-PD-L1
antibody is atezolizumab (TECENTRIQ.RTM.), durvalumab
(IMFINZI.RTM.), BMS-936559 (MDX-1105), or LY3300054.
[0040] In some embodiments, each therapeutic agent in a combination
therapy may be administered simultaneously, (e.g., in the same
medicament or at the same time), concurrently (i.e., in separate
medicaments administered one right after the other in any order, or
sequentially in any order.
BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS
[0041] FIG. 1A is a schematic drawing depicting an anti-mouse PD1
antibody-IL-15 fusion protein construct comprising the full human
hIgG2.DELTA.a with bivalent Fab at the N-terminus as the targeting
antibody arms; heterodimeric Fc with mutations that abolish
Fc.gamma.R binding at CH2 and CH3 (D265A, A330S, P331S), bispecific
mutations on the hinge (C223R/E, E225R/E, and P228R/E) and CH3
(K409R or L368E) for the monovalent linking of an IL-15 mutein
molecule at the C-terminus through a flexible glycine-serine
(GS)-linker. The mutations in the IL-15 protein include N4K, D30N,
V49N, I50A, S51T and E64Q (of SEQ ID NO: 1); V49N-I50A-S51T are the
N-linked glycosylation sites (as shown with a schematic
carbohydrate motif sticking out of the V49N position.
[0042] FIG. 1B depicts another anti-mouse PD1 antibody-IL-15 mutant
fusion protein construct with the same mutations on the antibody as
shown in FIG. 1A, and the difference in the mutations in the IL-15
protein including N4K, D30N, E46G (or Y26K), V49R (or V49K) and
E64Q.
[0043] FIG. 1C is a schematic drawing depicting an anti-mouse PD1
antibody-IL-15 fusion protein construct ("M2") comprising the full
human IgG1 with bivalent Fab at the N-terminus as the targeting
antibody arms; bispecific mutations on the hinge (L234A, L235A, and
G237A) and CH3 (Y349C, T366W, S354C, T366S, L368A, and Y407V) for
the monovalent linking of an IL-15 mutein molecule at the
C-terminus through a flexible glycine-serine (GS)-linker. The
mutations in the IL-15 protein include E46G, V49R, E64Q, D30N, and
N1G (of SEQ ID NO: 1).
[0044] FIG. 1D depicts another anti-mouse PD1 antibody-IL-15 mutant
fusion protein construct ("M1") with the same mutations on the
antibody as shown in FIG. 1C, and difference is in the mutations in
the IL-15 protein including E46G, V49R, D30N, and N1A (of SEQ ID
NO: 1).
[0045] FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show the direct
binding of antibody or antibody-cytokine fusion molecules to cells
expressing the IL-15 receptor (CD122 plus CD132), and in some
cases, PD-1 as well. The molecules include: anti-mouse PD-1
antibody (FIG. 2A); isotype control antibody (Ab8.8)-aSu-IL-15
(FIG. 2B); anti-mouse PD-1-IL-15RaSu-IL15 (FIG. 2C); and anti-mouse
PD-1-IL-15 NQ (FIG. 2D).
[0046] FIG. 3A, FIG. 3B, and FIG. 3C show the comparison of
untargeted IL-15, xmPD-1-IL-15RaSu-IL15 (wild-type IL-15), and
xmPD-1-IL15 NQ variant in a reporter assay, by plotting the
percentage of cells that are either PD-1+ or PD-1(low) in a given
assay that also stained as positive for pSTAT5 by flow cytometric
analysis.
[0047] FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H show plots of a
range of muteins (mutants or variants, used interchangeably), all
of which are based on the xmPD-1-IL15RaSu-IL15. The specific
molecules assayed are indicated in each plot.
[0048] FIGS. 5A, 5B, 5C, 5D, 5E, 5F, and 5G show plots of a range
of muteins, all of which lack the IL-15RaSu domain and contain
mutations to reduce or eliminate binding to IL-15Ra. The specific
molecules assayed are indicated in each plot.
[0049] FIGS. 6A, 6B, 6C, 6D, 6E, 6F, and 6G show plots of a range
of muteins which have altered activities (pSTAT5 activation)
relative to IL-15 wildtype. The specific molecules assayed are
indicated in each plot.
[0050] FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H show plots of a
range of muteins which have altered activities (pSTAT5 activation)
relative to IL-15 wild-type. The specific molecules assayed are
indicated in each plot.
[0051] FIGS. 8A, 8B, 8C, and 8D show the effects on survival and
body weight of mice treated with either xmPD-1-IL15RaSu-IL15 or
xmPD1-IL15 NQ. More specifically, FIG. 8A shows the changes in body
weight for animals dosed with 2, 1, or 0.2 mg/kg
xmPD1-IL15RaSu-IL15 on days indicated with arrows (d0, 4, 7). FIG.
8B shows overall survival of these same animals. FIG. 8C shows the
changes in body weight for animals dosed on days 0, 3, and 6 with
1, 0.8, or 0.5 mg/kg xmPD1-IL15RaSu-IL15. FIG. 8D shows the changes
in body weight for animals dosed on days 0, 3, and 6 with 3, 2, or
1 mg/kg xmPD1-IL15 NQ.
[0052] FIGS. 9A and 9B plot the percentage of splenocytes or
tumor-infiltrating lymphocytes that were gated as being CD4+ or
CD8+ cells or NK cells and that scored as PD-1+(FIG. 9A); FIG. 9B
shows the same study plotted as actual mean fluorescence
intensities.
[0053] FIGS. 10A, 10B, and 10C show data from an in vivo B16F10
tumor efficacy study, with mice treated with 5, 3, 1, or 0.3 mg/kg
xmPD1-IL15 NQ, or 0.3 mg/kg xmPD1-IL15RaSu-IL15 and compared to a
control (PBS-treated) group. More specifically, in FIG. 10A,
primary tumor volume is plotted for all groups. In FIG. 10B,
overall survival of animals in the study groups is plotted. In FIG.
10C, body weight changes (relative to study start body weight) are
plotted for each group.
[0054] FIGS. 11A and 11B show data from an in vivo B16F10 tumor
efficacy study, with mice treated with either anti-PD-1 antibody,
xmPD1-IL15 NQ, or isotype control (Ab8.8)-IL15 NQ, each at either 1
or 0.3 mg/kg.
[0055] FIGS. 11C, 11D, 11E and 11F show data from an in vivo B16F10
tumor efficacy study, with mice treated with either anti-PD1-IL15
M1 or M2 (as depicted in FIGS. 1D and 1C, respectively), each at
either 0.1, 0.3, 1 or 5 mg/kg.
[0056] FIGS. 11G and 11H show data from an in vivo B16F10 tumor
efficacy study, with mice treated with either anti-PD-1 antibody or
anti-PD1-IL15 M1, each at 1 mg/kg.
[0057] FIGS. 11I, 11J, 11K and 11L show data from an in vivo MC38
tumor efficacy study, with mice treated with either anti-PD1-IL15
M1 or M2, each at either 0.1, 0.3, 1 or 5 mg/kg. FIGS. 11A, 11C,
11E, 11G, 11I and 11K plot the volume of the primary tumor mass
over time for each group; arrows below the X-axis indicated the
dosing schedule. FIGS. 11B, 11D, 11F, 11H, 11J and 11L plot the
averaged changes in body weight from baseline (at beginning of
dosing, d=0) for each group.
[0058] FIGS. 12A, 12B, 12C, and 12D show results from an experiment
to analyze changes in splenic and TIL lymphocytes due to IL-15 drug
administration. FIG. 12A shows the detection, via anti-human Fc, of
anti-PD-1, Ab8.8-IL15 NQ, or xmPD1-IL15 NQ on the cells from
animals treated with the aforementioned molecules. Upper row:
histograms from TIL CD8+(left) or CD4+(right) cells as indicated.
Bottom row: histograms from splenic CD8+(left) or CD4+(right) cells
as indicated. FIG. 12B shows percentages of splenic T cells, CD4+ T
cells, and CD8+ T cells, as indicated, from animals sampled 1 day
after 3 doses of the indicated compound. FIG. 12C shows percentages
of TIL T cells, CD4+ T cells, and CD8+ T cells, as indicated, from
animals sampled 1 day after 3 doses of the indicated compound. FIG.
12D plots the ratios of CD8 to CD4 T cells from spleen and tumor as
calculated from FIGS. 12B and 12C.
[0059] FIGS. 13A and 13B show the absolute counts of NK, CD8+T and
Treg cells in peripheral blood or tumor-infiltrating lymphocytes at
Day 6, which showed to have the maximum effect by xmPD1-IL15 M1 and
xmPD1-IL15 m2, respectively.
[0060] FIGS. 13C and 13D show the respective effects of depleting
CD8 T and NK1.1+ cells on B16F10 tumor efficacy model. Treatment
regimen and average weights of mice during the studies are also
shown.
[0061] FIG. 13E shows the effect of FTY420 treatment, which
inhibits T cell egress, on B16F10 tumor efficacy model. Treatment
regimen and average weights of mice during the studies are also
shown.
[0062] FIGS. 14A and 14B show the effects on in vivo cytokine
production from mice given an IL-15-containing compound. FIG. 14A
shows averaged IFNg levels (n=3 animals) at 6, 24, 48, 72, 96, and
120 hours following a single dose of the indicated IL-compound.
FIG. 14B shows averaged IL-6 levels (n=3 animals) at 6, 24, 48, 72,
96, and 120 hours following a single dose of the indicated IL-15
compound.
[0063] FIGS. 15A, 15B, 15C, 15D, 15E and 15F show the effects of
adding targeted anti-human PD-1 (xhPD1)-IL15 M1 or M2 or untargeted
isotype control-antibody-IL15 chimeric molecules to the human
peripheral blood mononuclear cells, where they cause different
pSTAT5 activation on human NK cells, CD8.sup.+ effector memory T
cells and CD8.sup.+ central memory T cells, respectively.
[0064] FIGS. 16A, 16B and 16C show an in vivo efficacy study of a
PD-1-targeted IL-15 molecule (xPD1-IL15 M1) in combination with an
anti-EDA-IL-10 fusion protein. FIG. 16A shows the anti-tumor
efficacy of 0.3 mg/kg xPD1-IL15 M1 and 5 mg/kg anti-EDA-IL-10
fusion protein (xEDA-IL 10). FIG. 16B shows the anti-tumor efficacy
of 1 mg/kg xPD1-IL15 M1 and 5 mg/kg anti-EDA-IL-10 fusion protein
(xEDA-IL 10). FIG. 16C plots the averaged body weight changes (from
baseline, at day 0) of animals in each treatment group throughout
the study.
DETAILED DESCRIPTION
[0065] The invention disclosed herein is directed to human
interleukin 15 (IL-15) variants and fusion proteins comprising
thereof. It is demonstrated that the IL-15 variants of the present
invention have decreased or no binding to the IL-15 receptor alpha
(CD215), and have reduced interaction between IL-15 and its
signaling receptor, comprised of IL-2 receptor beta (CD122) and the
common gamma chain (CD132), as compared to the wild-type human
IL-15 polypeptide or a wild-type IL-15 receptor alpha-IL-15 fusion
polypeptide. In a second aspect of the invention, these reduced
affinity IL-variants, when presented as an antibody fusion chimeric
protein, are targeted selectively to desired cell types (those
cells expressing the antibody target). Cell types that express the
IL-15 receptor complex, but not the antibody target, are activated
less, or not activated, compared to those cells which express both
components. Further, it is also demonstrated that the IL-15 fusion
proteins of the present invention preferentially activate
downstream biomarker pSTAT5 in human peripheral CD8 T cells over
natural killer (NK) cells, and have potent and preferential
activation of human CD8 tumor infiltrating T lymphocytes (TILs).
Accordingly, the IL-15 variants and the IL-15 fusion proteins of
the present invention selectively modulate the activation of cell
subsets to promote biological activity, such as an anti-tumor
activity, efficaciously and safely. In a third aspect of this
invention, these reduced affinity IL-15 variants and the IL-15
fusion proteins, when expressed as polynucleotides in CAR T cells,
either as secreted or membrane-tethered versions, are used to
enhance CAR T function, including activity and proliferation.
General Techniques
[0066] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.
J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press;
Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998)
Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987);
Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic
Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and
C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells
(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short
Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.
Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL
Press, 1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
Definitions
[0067] The following terms, unless otherwise indicated, shall be
understood to have the following meanings: the term "isolated
molecule" as referring to a molecule (where the molecule is, for
example, a polypeptide, a polynucleotide, or an antibody) that by
virtue of its origin or source of derivation (1) is not associated
with naturally associated components that accompany it in its
native state, (2) is substantially free of other molecules from the
same source, e.g., species, cell from which it is expressed,
library, etc., (3) is expressed by a cell from a different species,
or (4) does not occur in nature. Thus, a molecule that is
chemically synthesized, or expressed in a cellular system different
from the system from which it naturally originates, will be
"isolated" from its naturally associated components. A molecule
also may be rendered substantially free of naturally associated
components by isolation, using purification techniques well known
in the art. Molecule purity or homogeneity may be assayed by a
number of means well known in the art. For example, the purity of a
polypeptide sample may be assayed using polyacrylamide gel
electrophoresis and staining of the gel to visualize the
polypeptide using techniques well known in the art. For certain
purposes, higher resolution may be provided by using HPLC or other
means well known in the art for purification.
[0068] As used herein, the term "IL-15" refers to any form of IL-15
and variants thereof that retain at least part of the activity of
IL-15. Unless indicated differently, such as by specific reference
to human IL-15, IL-15 includes all mammalian species of native
sequence IL-15, e.g., human, canine, feline, equine, and bovine.
One exemplary wild-type human IL-15 is found as Uniprot Accession
Number P40933 (SEQ ID NO: 5).
[0069] The terms "polypeptide", "oligopeptide", "peptide" and
"protein" are used interchangeably herein to refer to chains of
amino acids of any length. The chain may be linear or branched, it
may comprise modified amino acids, and/or may be interrupted by
non-amino acids. The terms also encompass an amino acid chain that
has been modified naturally or by intervention, for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that the polypeptides can occur as single chains or
associated chains.
[0070] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term encompasses not
only intact polyclonal or monoclonal antibodies, but also, unless
otherwise specified, any antigen binding portion thereof that
competes with the intact antibody for specific binding, fusion
proteins comprising an antigen binding portion, and any other
modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site. Antigen binding portions
include, for example, Fab, Fab', F(ab').sub.2, Fd, Fv, domain
antibodies (dAbs, e.g., shark and camelid antibodies), fragments
including complementarity determining regions (CDRs), single chain
variable fragment antibodies (scFv), maxibodies, minibodies,
intrabodies, diabodies, triabodies, tetrabodies, v-NAR and
bis-scFv, and polypeptides that contain at least a portion of an
immunoglobulin that is sufficient to confer specific antigen
binding to the polypeptide. An antibody includes an antibody of any
class, such as IgG, IgA, or IgM (or sub-class thereof), and the
antibody need not be of any particular class. Depending on the
antibody amino acid sequence of the constant region of its heavy
chains, immunoglobulins can be assigned to different classes. There
are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, and several of these may be further divided into subclasses
(isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1 and IgA.sub.2. The heavy-chain constant regions that
correspond to the different classes of immunoglobulins are called
alpha, delta, epsilon, gamma, and mu, respectively. The subunit
structures and three-dimensional configurations of different
classes of immunoglobulins are well known.
[0071] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. As known in
the art, the variable regions of the heavy and light chains each
consist of four framework regions (FRs) connected by three
complementarity determining regions (CDRs) also known as
hypervariable regions, and contribute to the formation of the
antigen binding site of antibodies. If variants of a subject
variable region are desired, particularly with substitution in
amino acid residues outside of a CDR region (i.e., in the framework
region), appropriate amino acid substitution, preferably,
conservative amino acid substitution, can be identified by
comparing the subject variable region to the variable regions of
other antibodies which contain CDR1 and CDR2 sequences in the same
canonical class as the subject variable region (Chothia and Lesk, J
Mol Biol 196(4): 901-917, 1987).
[0072] In certain embodiments, definitive delineation of a CDR and
identification of residues comprising the binding site of an
antibody is accomplished by solving the structure of the antibody
and/or solving the structure of the antibody-ligand complex. In
certain embodiments, that can be accomplished by any of a variety
of techniques known to those skilled in the art, such as X-ray
crystallography. In certain embodiments, various methods of
analysis can be employed to identify or approximate the CDR
regions. In certain embodiments, various methods of analysis can be
employed to identify or approximate the CDR regions. Examples of
such methods include, but are not limited to, the Kabat definition,
the Chothia definition, the AbM definition, the contact definition,
and the conformational definition.
[0073] The Kabat definition is a standard for numbering the
residues in an antibody and is typically used to identify CDR
regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:
214-8. The Chothia definition is similar to the Kabat definition,
but the Chothia definition takes into account positions of certain
structural loop regions. See, e.g., Chothia et al., 1986, J. Mol.
Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The
AbM definition uses an integrated suite of computer programs
produced by Oxford Molecular Group that model antibody structure.
See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA),
86:9268-9272; "AbM.TM., A Computer Program for Modeling Variable
Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM
definition models the tertiary structure of an antibody from
primary sequence using a combination of knowledge databases and ab
initio methods, such as those described by Samudrala et al., 1999,
"Ab Initio Protein Structure Prediction Using a Combined
Hierarchical Approach," in PROTEINS, Structure, Function and
Genetics Suppl., 3:194-198. The contact definition is based on an
analysis of the available complex crystal structures. See, e.g.,
MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another
approach, referred to herein as the "conformational definition" of
CDRs, the positions of the CDRs may be identified as the residues
that make enthalpic contributions to antigen binding. See, e.g.,
Makabe et al., 2008, Journal of Biological Chemistry,
283:1156-1166. Still other CDR boundary definitions may not
strictly follow one of the above approaches, but will nonetheless
overlap with at least a portion of the Kabat CDRs, although they
may be shortened or lengthened in light of prediction or
experimental findings that particular residues or groups of
residues do not significantly impact antigen binding. As used
herein, a CDR may refer to CDRs defined by any approach known in
the art, including combinations of approaches. The methods used
herein may utilize CDRs defined according to any of these
approaches. For any given embodiment containing more than one CDR,
the CDRs may be defined in accordance with any of Kabat, Chothia,
extended, AbM, contact, and/or conformational definitions.
[0074] As known in the art, a "constant region" of an antibody
refers to the constant region of the antibody light chain or the
constant region of the antibody heavy chain, either alone or in
combination.
[0075] As used herein, "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler and Milstein, 1975,
Nature 256:495, or may be made by recombinant DNA methods such as
described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may
also be isolated from phage libraries generated using the
techniques described in McCafferty et al., 1990, Nature
348:552-554, for example. As used herein, "humanized" antibody
refers to forms of non-human (e.g. murine) antibodies that are
chimeric immunoglobulins, immunoglobulin chains, or fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) that contain minimal sequence derived
from non-human immunoglobulin. Preferably, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
CDR of the recipient are replaced by residues from a CDR of a
non-human species (donor antibody) such as mouse, rat, or rabbit
having the desired specificity, affinity, and capacity. The
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance.
[0076] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen binding residues.
[0077] The term "chimeric antibody" is intended to refer to
antibodies in which the variable region sequences are derived from
one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region
sequences are derived from a mouse antibody and the constant region
sequences are derived from a human antibody.
[0078] As known in the art, "polynucleotide," or "nucleic acid," as
used interchangeably herein, refer to chains of nucleotides of any
length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a chain by DNA or RNA polymerase. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the chain. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications include, for
example, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping group moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0079] An antibody that "preferentially binds" or "specifically
binds" (used interchangeably herein) to an epitope is a term well
understood in the art, and methods to determine such specific or
preferential binding are also well known in the art. A molecule is
said to exhibit "specific binding" or "preferential binding" if it
reacts or associates more frequently, more rapidly, with greater
duration and/or with greater affinity with a particular cell or
substance than it does with alternative cells or substances. An
antibody "specifically binds" or "preferentially binds" to a target
if it binds with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other substances. For
example, an antibody that specifically or preferentially binds to a
target (e.g., PD-1) epitope is an antibody that binds this epitope
with greater affinity, avidity, more readily, and/or with greater
duration than it binds to other target epitopes or non-target
epitopes. It is also understood by reading this definition that,
for example, an antibody (or moiety or epitope) that specifically
or preferentially binds to a first target may or may not
specifically or preferentially bind to a second target. As such,
"specific binding" or "preferential binding" does not necessarily
require (although it can include) exclusive binding. Generally, but
not necessarily, reference to binding means preferential
binding.
[0080] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from contaminants), more
preferably, at least 90% pure, more preferably, at least 95% pure,
yet more preferably, at least 98% pure, and most preferably, at
least 99% pure.
[0081] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
polynucleotide(s) of this invention.
[0082] As known in the art, the term "Fc region" is used to define
a C-terminal region of an immunoglobulin heavy chain. The "Fc
region" may be a native sequence Fc region or a variant Fc region.
Although the boundaries of the Fc region of an immunoglobulin heavy
chain might vary, the human IgG heavy chain Fc region is usually
defined to stretch from an amino acid residue at position Cys226,
or from Pro230, to the carboxyl-terminus thereof. The numbering of
the residues in the Fc region is that of the EU index as in Kabat.
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991. The Fc region of an immunoglobulin generally comprises
two constant domains, CH2 and CH3. As is known in the art, an Fc
region can be present in dimer or monomeric form.
[0083] As used in the art, "Fc receptor" and "FcR" describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof. FcRs are
reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92;
Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al.,
1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the
neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,
117:587; and Kim et al., 1994, J. Immunol., 24:249).
[0084] The term "compete", as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
portion thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
portion thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0085] A "functional Fc region" possesses at least one effector
function of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; complement dependent cytotoxicity;
Fc receptor binding; antibody-dependent cell-mediated cytotoxicity;
phagocytosis; down-regulation of cell surface receptors (e.g. B
cell receptor), etc. Such effector functions generally require the
Fc region to be combined with a binding domain (e.g. an antibody
variable domain) and can be assessed using various assays known in
the art for evaluating such antibody effector functions.
[0086] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" comprises an amino acid sequence
which differs from that of a native sequence Fc region by virtue of
at least one amino acid modification, yet retains at least one
effector function of the native sequence Fc region. Preferably, the
variant Fc region has at least one amino acid substitution compared
to a native sequence Fc region or to the Fc region of a parent
polypeptide, e.g. from about one to about ten amino acid
substitutions, and preferably, from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% sequence identity with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably, at least about 90% sequence
identity therewith, more preferably, at least about 95%, at least
about 96%, at least about 97%, at least about 98%, at least about
99% sequence identity therewith.
[0087] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, one or more of the following: reducing the
proliferation of (or destroying) neoplastic or cancerous cells,
inhibiting metastasis of neoplastic cells, shrinking or decreasing
the size of a tumor, remission of cancer, decreasing symptoms
resulting from cancer, increasing the quality of life of those
suffering from cancer, decreasing the dose of other medications
required to treat cancer, delaying the progression of cancer,
curing a cancer, and/or prolong survival of patients having
cancer.
[0088] "Ameliorating" means a lessening or improvement of one or
more symptoms as compared to not administering an IL-15 variant or
the IL-15 fusion protein as described herein. "Ameliorating" also
includes shortening or reduction in duration of a symptom.
[0089] As used herein, an "effective dosage" or "effective amount"
of drug, compound, or pharmaceutical composition is an amount
sufficient to effect any one or more beneficial or desired results.
In more specific aspects, an effective amount prevents, alleviates
or ameliorates symptoms of disease, and/or prolongs the survival of
the subject being treated. For prophylactic use, beneficial or
desired results include eliminating or reducing the risk, lessening
the severity, or delaying the outset of the disease, including
biochemical, histological and/or behavioral symptoms of the
disease, its complications and intermediate pathological phenotypes
presenting during development of the disease. For therapeutic use,
beneficial or desired results include clinical results such as
reducing one or more symptoms of a disease such as, for example,
solid cancer and liquid cancer including, for example without
limitation, gastric cancer, small intestine cancer, sarcoma, head
and neck cancer, thymic cancer, epithelial cancer, salivary cancer,
liver cancer, biliary cancer, neuroendocrine tumors, stomach
cancer, thyroid cancer, lung cancer, mesothelioma, ovarian cancer,
breast cancer, prostate cancer, esophageal cancer, pancreatic
cancer, glioma, renal cancer (e.g., renal cell carcinoma), bladder
cancer, cervical cancer, uterine cancer, vulvar cancer, penile
cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal
cancer, oral cancer, skin cancer, Merkel cell carcinoma,
glioblastoma, brain tumor, bone cancer, eye cancer, and melanoma,
multiple myeloma, malignant plasma cell neoplasm, Hodgkin's
lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma,
Kahler's disease and Myelomatosis, plasma cell leukemia,
plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia,
B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML),
chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia
(ALL), chronic myeloid leukemia (CML), follicular lymphoma,
Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma,
large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid
leukemia, Waldenstrom's macroglobulienemia, diffuse large B cell
lymphoma, follicular lymphoma, marginal zone lymphoma,
mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic
lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary
mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic
lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell
lymphoma, splenic marginal zone lymphoma, intravascular large
B-cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis, T cell/histiocyte-rich large B-cell lymphoma,
primary central nervous system lymphoma, primary cutaneous diffuse
large B-cell lymphoma (leg type), EBV positive diffuse large B-cell
lymphoma of the elderly, diffuse large B-cell lymphoma associated
with inflammation, intravascular large B-cell lymphoma,
ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large
B-cell lymphoma arising in HHV8-associated multicentric Castleman
disease, B-cell lymphoma unclassified with features intermediate
between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell
lymphoma unclassified with features intermediate between diffuse
large B-cell lymphoma and classical Hodgkin lymphoma, and other
hematopoietic cells related cancer, decreasing the dose of other
medications required to treat the disease, enhancing the effect of
another medication, and/or delaying the progression of the cancer
in patients. An effective dosage can be administered in one or more
administrations. For purposes of this invention, an effective
dosage of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0090] An "individual" or a "subject" is a mammal, more preferably,
a human. Mammals also include, but are not limited to, farm animals
(e.g., cows, pigs, horses, chickens, etc.), sport animals, pets,
primates, horses, dogs, cats, mice and rats.
[0091] As used herein, "vector" means a construct, which is capable
of delivering, and, preferably, expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0092] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0093] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutical acceptable excipient" includes any material which,
when combined with an active ingredient, allows the ingredient to
retain biological activity and is non-reactive with the subject's
immune system. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline (PBS) or
normal (0.9%) saline. Compositions comprising such carriers are
formulated by well-known conventional methods (see, for example,
Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science
and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
The term "effector function" refers to the biological activities
attributable to the Fc region of an antibody. Examples of antibody
effector functions include, but are not limited to,
antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor
binding, complement dependent cytotoxicity (CDC), phagocytosis, C1q
binding, and down regulation of cell surface receptors (e.g., B
cell receptor; BCR). See, e.g., U.S. Pat. No. 6,737,056. Such
effector functions generally require the Fc region to be combined
with a binding domain (e.g., an antibody variable domain) and can
be assessed using various assays known in the art for evaluating
such antibody effector functions. An exemplary measurement of
effector function is through Fc.gamma.3 and/or C1q binding.
[0094] As used herein "antibody-dependent cell-mediated
cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which
nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g.
natural killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC activity of a molecule of interest can be
assessed using an in vitro ADCC assay, such as that described in
U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
NK cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in an animal
model such as that disclosed in Clynes et al., 1998, PNAS (USA),
95:652-656.
[0095] "Complement dependent cytotoxicity" or "CDC" refers to the
lysing of a target in the presence of complement. The complement
activation pathway is initiated by the binding of the first
component of the complement system (C1q) to a molecule (e.g. an
antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al., J. Immunol. Methods, 202: 163 (1996), may be performed.
[0096] The term "k.sub.on" or "k.sub.a", as used herein, refers to
the rate constant for association of an antibody to an antigen.
Specifically, the rate constants (k.sub.on or k.sub.a and k.sub.off
or k.sub.d) and equilibrium dissociation constants are measured
using whole antibody (i.e. bivalent) and monomeric proteins.
[0097] The term "k.sub.off" or "k.sub.d", as used herein, refers to
the rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0098] The term "K.sub.D", as used herein, refers to the
equilibrium dissociation constant of an antibody-antigen
interaction.
[0099] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X." Numeric ranges are inclusive of the
numbers defining the range. Generally speaking, the term "about"
refers to the indicated value of the variable and to all values of
the variable that are within the experimental error of the
indicated value (e.g. within the 95% confidence interval for the
mean) or within 10 percent of the indicated value, whichever is
greater. Where the term "about" is used within the context of a
time period (years, months, weeks, days etc.), the term "about"
means that period of time plus or minus one amount of the next
subordinate time period (e.g. about 1 year means 11-13 months;
about 6 months means 6 months plus or minus 1 week; about 1 week
means 6-8 days; etc.), or within 10 percent of the indicated value,
whichever is greater.
[0100] The term "immune-effector-cell enhancer" or "IEC enhancer"
refers to a substance capable of increasing or enhancing the
number, quality, or function of one or more types of immune
effector cells of a mammal. Examples of immune effector cells
include cytolytic CD8 T cells, CD4 T cells, NK cells, and B
cells.
[0101] The term "immune modulator" refers to a substance capable of
altering (e.g., inhibiting, decreasing, increasing, enhancing, or
stimulating) the immune response (as defined herein) or the working
of any component of the innate, humoral or cellular immune system
of a host mammal. Thus, the term "immune modulator" encompasses the
"immune-effector-cell enhancer" as defined herein and the
"immune-suppressive-cell inhibitor" as defined herein, as well as
substance that affects other components of the immune system of a
mammal.
[0102] The term "immune response" refers to any detectable response
to a particular substance (such as an antigen or immunogen) by the
immune system of a host mammal, such as innate immune responses
(e.g., activation of Toll receptor signaling cascade),
cell-mediated immune responses (e.g., responses mediated by T
cells, such as antigen-specific T cells, and non-specific cells of
the immune system), and humoral immune responses (e.g., responses
mediated by B cells, such as generation and secretion of antibodies
into the plasma, lymph, and/or tissue fluids).
[0103] The term "immunogenic" refers to the ability of a substance
to cause, elicit, stimulate, or induce an immune response, or to
improve, enhance, increase or prolong a pre-existing immune
response, against a particular antigen, whether alone or when
linked to a carrier, in the presence or absence of an adjuvant.
[0104] The term "immune-suppressive-cell inhibitor" or "ISC
inhibitor" refers to a substance capable of reducing or suppressing
the number or function of immune suppressive cells of a mammal.
Examples of immune suppressive cells include regulatory T cells
("Treg"), myeloid-derived suppressor cells, and tumor-associated
macrophages.
[0105] The term "intradermal administration," or "administered
intradermally," in the context of administering a substance to a
mammal including a human, refers to the delivery of the substance
into the dermis layer of the skin of the mammal. The skin of a
mammal is composed of an epidermis layer, a dermis layer, and a
subcutaneous layer. The epidermis is the outer layer of the skin.
The dermis, which is the middle layer of the skin, contains nerve
endings, sweat glands and oil (sebaceous) glands, hair follicles,
and blood vessels. The subcutaneous layer is made up of fat and
connective tissue that houses larger blood vessels and nerves. In
contrast in intradermal administration, "subcutaneous
administration" refers to the administration of a substance into
the subcutaneous layer and "topical administration" refers to the
administration of a substance onto the surface of the skin.
[0106] The term "neoplastic disorder" refers to a condition in
which cells proliferate at an abnormally high and uncontrolled
rate, the rate exceeding and uncoordinated with that of the
surrounding normal tissues. It usually results in a solid lesion or
lump known as "tumor." This term encompasses benign and malignant
neoplastic disorders. The term "malignant neoplastic disorder",
which is used interchangeably with the term "cancer" in the present
disclosure, refers to a neoplastic disorder characterized by the
ability of the tumor cells to spread to other locations in the body
(known as "metastasis"). The term "benign neoplastic disorder"
refers to a neoplastic disorder in which the tumor cells lack the
ability to metastasize.
[0107] The term "preventing" or "prevent" refers to (a) keeping a
disorder from occurring or (b) delaying the onset of a disorder or
onset of symptoms of a disorder.
[0108] The term "tumor-associated antigen" or "TAA" refers to an
antigen which is specifically expressed by tumor cells or expressed
at a higher frequency or density by tumor cells than by non-tumor
cells of the same tissue type. Tumor-associated antigens may be
antigens not normally expressed by the host; they may be mutated,
truncated, misfolded, or otherwise abnormal manifestations of
molecules normally expressed by the host; they may be identical to
molecules normally expressed but expressed at abnormally high
levels; or they may be expressed in a context or milieu that is
abnormal. Tumor-associated antigens may be, for example, proteins
or protein fragments, complex carbohydrates, gangliosides, haptens,
nucleic acids, or any combination of these or other biological
molecules.
[0109] The term "vaccine" refers to an immunogenic composition for
administration to a mammal for eliciting an immune response against
a particular antigen in the mammal. A vaccine typically contains an
agent (known as "antigen" or "immunogen") that resembles, or is
derived from, the target of the immune response, such as a
disease-causing micro-organism or tumor cells. A vaccine intended
for the treatment of a tumor, such as a cancer, typically contains
an antigen that is derived from a TAA found on the target tumor and
is able to elicit immunogenicity against the TAA on the target
tumor.
[0110] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0111] Where aspects or embodiments of the invention are described
in terms of a Markush group or other grouping of alternatives, the
present invention encompasses not only the entire group listed as a
whole, but each member of the group individually and all possible
subgroups of the main group, but also the main group absent one or
more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the claimed invention.
[0112] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present specification, including definitions, will
control. Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising" will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers. Unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular. Any example(s) following the term "e.g." or "for example"
is not meant to be exhaustive or limiting.
[0113] Exemplary methods and materials are described herein,
although methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present invention. The materials, methods, and examples are
illustrative only and not intended to be limiting.
IL-15 Variants
[0114] Provided herein are IL-15 variants (e.g., human IL-15
variants) that have decreased or no binding to the IL-15 receptor
alpha (CD215), and/or reduced interaction between IL-15 and its
signaling receptor, comprised of IL-2 receptor beta (CD122) and the
common gamma chain (CD132), as compared to the wild-type human
IL-15 polypeptide or a wild-type IL-15 receptor alpha-IL-15 fusion
polypeptide, as illustrated in Tables 1-2 under the Example
section.
[0115] In one aspect, the invention provides an isolated human
interleukin 15 (IL-15) variant comprising amino acid substitution
at positions a) V49 and I51 or b) V49, I50, and S51 of SEQ ID NO:
1, and further comprising one or more amino acid substitutions at
positions N1, N4, S7, K10, K11, Y26, S29, D30, V31, H32, E53, G55,
E64, I68, L69, E89, L91, M109, and/or I111 of SEQ ID NO: 1, wherein
the IL-15 variant has decreased or no binding to the human IL-15
receptor alpha (IL-15R.alpha.) and the human IL-2 receptor
beta/gamma (IL-2R.beta..gamma.) as compared to the wild-type human
IL-15 polypeptide or a wild-type IL-15 receptor alpha-IL-15 fusion
polypeptide, and wherein the amino acid substitution at position
V49 is glycosylated.
[0116] Glycosylation of the IL-15 variant is typically either
N-linked or O-linked. N-linked refers to the attachment of the
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine, asparagine-X-threonine,
and asparagine-X-cysteine, where X is any amino acid except
proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0117] Accordingly, in some embodiments, the IL-15 variant
comprises amino acid substitution at V49N, wherein V49N is
glycosylated. In some embodiments, the amino acid substitution(s)
at E53 and/or E89 of SEQ ID NO: 1 are also glycosylated.
[0118] In some embodiments, the IL-15 variant comprises amino acid
substitutions of SEQ ID NO: 1 at positions selected from the group
consisting of: a) V49, I50, S51, N4, D30, and E64; b) V49, I50,
S51, N4, D30, E64, and I68; c) V49, I50, S51, N4, D30, E64, M109;
d) V49, I50, S51, N4, D30, E64, I68, and M109; e) V49, I50, S51,
D30, E64, and I68; f) V49, I50, S51, D30, E64, M109; g) V49, I50,
S51, D30, E64, I68, and M109; h) N1, V49, I50, and S51; i) N4, V49,
I50, and S51; j) S7, V49, I50, and S51; k) K10, V49, I50, and S51;
l) K11, V49, I50, and S51; m) S29, V49, I50, and S51; n) V31, V49,
I50, and S51; o) H32, V49, I50, and S51; p) V49, I50, S51, and E64;
q) V49, I50, S51, and I68; r) V49, I50, S51, and L69; s) V49, I50,
S51, and I111; t) N4, V49, I50, S51, and E64; u) N1, D30, V49, I50,
and S51; v) N4, D30, V49, I50, and S51; w) S7 D30, V49, I50, and
S51; x) K10, D30, V49, I50, and S51; y) K11, D30, V49, I50, and
S51; z) S29, D30, V49, I50, and S51; aa) D30, V49, I50, S51, and
E64; bb) D30, V49, I50, S51, and I68; cc) D30, V49, I50, S51, and
L69; and dd) D30, V49, I50, S51, and I111.
[0119] In some embodiments, the IL-15 variant comprises amino acid
substitutions comprising one or more specific substitutions at: a)
V49N, V49K, V49E, V49H, V49Q or V49R; b) I50A or I50G; c) S51T; d)
N1K, N1G, N1Q, N1R, N1E, N1A, or N1D; e) N4K, N4G, N4A, N4S, N4D,
N4E, N4I, N4L, N4R, N4T, N4W, or N4Q; f) S7E, S7G, S7D, S7K, S7N,
S7R, S7H, or S7T; g) K10A, K10S, K10E, K10L, K10M, K10D, or K10G;
h) K11D, K11S, or K11W; i) D30N; j) E64Q, E64K, E64A, E64S, E64N,
E64H, E64T, or E64R; k) E53N; l) G55S or G55T; m) E89N; n) L91S or
L91T; o) Y26K, Y26R, or Y26H; p) S29N; q) V31S, V31D, or V31K; r)
H32G; s) I68S, I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y,
I68E, or I68H; t) L69A, L69S, L69D, L69T, L69M, L69G, L69Q, L69I,
L69E, or L69V; u) M109A, M109S, M109D, or M109K; and/or v) I111A,
I111K, I111S, or I111D. In some embodiments, the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N4K, D30N, V49N, I50A, S51T, and E64Q; b)
N4Q, D30N, V49N, I50A, and S51T; c) D30N, V49N, I50A, S51T, and
E64Q; d) N4Q, D30N, V49N, I50A, S51T, and E64Q; e) N4Q, V49N, I50A,
and S51T; f) V49N, I50A, S51T, and E64Q; and g) N4Q, V49N, I50A,
S51T, and E64Q.
[0120] In another aspect, provided is an isolated human interleukin
15 (IL-15) variant comprising amino acid substitutions at positions
E46 and V49 of SEQ ID NO: 1, and at least one or more amino acid
substitution(s) at positions N1, N4, S7, K10, K11, D22, Y26, S29,
D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109 and/or I111
of SEQ ID NO: 1, wherein the IL-15 variant has no binding to the
human IL-15 receptor alpha (IL-15R.alpha.) and decreased binding to
the human IL-2 receptor beta/gamma (IL-2R.beta..gamma.) as compared
to the wild-type human IL-15 polypeptide or a wild-type IL-15
receptor alpha-IL-15 fusion polypeptide.
[0121] In some embodiments, the IL-15 variant comprises amino acid
substitutions in SEQ ID NO: 1 at positions selected from the group
consisting of: a) N1, E46, and V49; b) N4, E46, and V49; c) S7,
E46, and V49; d) K10, E46, and V49; e) K11, E46, and V49; f) S29,
E46, and V49; g) V31, E46, and V49; h) H32, E46, and V49; i) E46,
V49, and E64; j) E46, V49, and I68; k) E46, V49, and L69; l) E46,
V49, and I111; m) N4, E46, V49, and E64; n) E46, V49, N4, D30, and
E64; o) E46, V49, N4, D30, E64, and I68; p) E46, V49, N4, D30, E64,
and M109; q) E46, V49, N4, D30, E64, I68, and M109; r) N1, D30,
E46, and V49; s) N4, D30, E46, and V49; t) S7, D30, E46, and V49;
u) K10, D30, E46, and V49; v) K11, D30, E46, and V49; w) S29, D30,
E46, and V49; x) D30, E46, V49, and E64; y) D30, E46, V49R, and
I68; z) D30, E46, V49R, and L69; aa) D30, E46, V49R, and I111; bb)
N1, D30, E46, V49, and M109; cc) N4, D30, E46, V49, and M109; dd)
S7, D30, E46, V49, and M109; ee) K10, D30, E46, V49, and M109; f)
K11, D30, E46, V49, and M109; gg) D30, E46, V49, E64, and M109; hh)
D30, E46, V49, 168, and M109; ii) D30, E46, V49, L69 and M109; jj)
D30, E46, V49, M109, and I111; kk) D30, E46, V49, E64, I68, and
M109; ll) E46, V49, D30, E64, and I68; mm) E46, V49, E64, and M109;
nn) E46, V49, D30, E64, I68, and M109; oo) D22, Y26, V49, E46, E53,
E89, and E93; and pp) N1, D30, E46, V49, and E64.
[0122] In some embodiments, the IL-15 variant comprises amino acid
substitutions comprising one or more specific substitutions at: a)
N1Q, N1K, N1R, N1E, N1A, N1D, or N1G; b) N4K, N4G, N4A, N4S, N4D,
N4E, N4R, N4T, N4I, N4L, N4W, or N4Q; c) S7E, S7G, S7D, S7K, S7N,
S7R, S7H, or S7T; d) K10D, K10A, K10S, K10E, K10L, K10M, K10D, or
K10G; e) K11D, K11S, or K11W; f) D22N; g) Y26K, Y26R, or Y26H; h)
S29N; i) D30N; j) V31S, V31D, or V31K; k) H32G; l) E46G or E46Q; m)
V49N, V49K, or V49R V49E, V49H, or V49Q; n) E53Q; o) G55S or G55T;
p) E64Q, E64K, E64A, E64S, E64N, E64H, E64T, or E64R; q) I68S,
I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or I68H; r)
L69S, L69A, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or L69V; s)
E89Q; t) E93Q; u) M109A, M109S, M109S, or M109K; and/or v) I111A,
I111K, I111S, or I111D. In some embodiments, the IL-15 variant
comprises amino acid substitutions at positions selected from the
group consisting of: a) N1K, E46G, and V49R; b) N4K, E46G, and
V49R; c) N4Q, E46G, and V49R; d) S7T, E46G, and V49R; e) V31S,
E46G, and V49R; f) V31K, E46G, and V49R; g) E46G, V49R, and E64Q;
h) E46G, V49R, and E64K; i) N4Q, E46G, V49R, and E64Q; j) N1G,
D30N, E46G, and V49R; k) N1K, D30N, E46G, and V49R; l) N1Q, D30N,
E46G, and V49R; m) N4G, D30N, E46G, and V49R; n) N4K, D30N, E46G,
and V49R; o) N4Q, D30N, E46G, and V49R; p) S7E, D30N, E46G, and
V49R; q) S7G, D30N, E46G, and V49R; r) S7T, D30N, E46G, and V49R;
s) K10D, D30N, E46G, and V49R; t) D30N, E46G, V49R, and E64A; u)
D30N, E46G, V49R, and E64Q; v) D30N, E46G, V49R, and E64K; w) D30N,
E46G, V49R, and I68S; x) D30N, E46G, V49R, and I68K; y) N4K, D30N,
E46G, V49R, and E64K; z) N4Q, D30N, E46G, V49R, and E64K; aa) N4K,
D30N, E46G, V49R, and E64Q; bb) N4Q, D30N, E46G, V49R, and E64Q;
cc) N4K, D30N, E46G, V49R, and I68S; dd) D30N, E46G, V49R, E64Q,
and I68S; ee) N1A, D30N, E46G, and V49R; and ff) N1G, D30N, E46G,
V49R, and E64Q.
[0123] In some embodiments, the IL-15 variant comprises an amino
acid sequence of SEQ ID NO: 84 or 85.
[0124] In another aspect, provided is an isolated human IL-15
variant comprising one or more amino acid substitution(s) at
position(s) N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32,
E46, E53, E64, I68, L69, E89, E93, M109, and/or I111 of SEQ ID NO:
1, wherein the IL-15 variant has decreased or no binding to the
human IL-15 receptor alpha (IL-15R.alpha.) and/or the human IL-2
receptor beta (IL-2R.beta.) and/or IL-2 receptor gamma
(IL-2R.gamma.) as compared to the wild-type human IL-15 polypeptide
or a wild-type IL-15 receptor alpha-IL-15 fusion polypeptide. For
example, the specific substitutions may include, but not limited
to, N1K, N4K, N4Q, S7T, K10S, K11S, D22N, Y26F, D30N, V31S, H32G,
M109A, and/or I111A.
[0125] In some embodiments, the isolated human IL-15 variant
comprises amino acid substitutions at positions D22N, Y26F, E46Q,
E53Q, E89Q, and E93Q (e.g., see SEQ ID NO: 76), wherein the IL-15
variant has decreased or no binding to the human IL-15 receptor
alpha (IL-15R.alpha.) and/or the human IL-2 receptor beta
(IL-2R.beta.) and/or IL-2 receptor gamma (IL-2R.gamma.) as compared
to the wild-type human IL-15 polypeptide.
[0126] In another aspect, provided is an isolated human IL-15
variant comprising the amino acid sequence shown in SEQ ID NO: 93.
In some embodiments, the IL-15 variant further comprises a
transmembrane domain.
IL-15 Fusion Proteins
[0127] Provided herein are IL-15 fusion proteins (e.g.,
antibody-IL-15 fusion proteins) that have decreased or no binding
to the IL-15 receptor alpha (CD215), and reduced interaction
between IL-15 and its signaling receptor, comprised of IL-2
receptor beta (CD122) and the common gamma chain (CD132). Such
IL-15 fusion proteins can deliver cytokines to a desired cell type
while minimizing peripheral exposure (e.g., NK cells which are a
major site of action) and thus toxicities. Further, the IL-15
fusion proteins of the present invention preferentially activate
downstream biomarker pSTAT5 in human peripheral CD8 T cells over
natural killer (NK) cells, and have potent and preferential
activation of human CD8 tumor infiltrating T lymphocytes (TILs).
Accordingly, in some embodiments, an IL-15 variant can be coupled
to a PD-1 antibody, which acts as a marker to antigen-specific
tumor-resident CD8+ cells, thereby maximizing anti-tumor efficacy
and minimizing exposure to peripheral immune cell subsets.
[0128] In one aspect, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) any one
of the IL-15 variants as described herein, wherein the IL-15
variant is covalently linked to the Fc domain of the antibody.
[0129] In some embodiments, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) a human
interleukin 15 (IL-15) variant comprising amino acid substitution
at positions a) V49 and I51 or b) V49, I50, and S51 of SEQ ID NO:
1, wherein the amino acid substitution at position V49 of SEQ ID
NO: 1 is glycosylated, and further comprising one or more amino
acid substitutions at positions N1, N4, S7, K10, K11, Y26, S29,
D30, V31, H32, E53, G55, E64, I68, L69, E89, L91, M109, and/or I111
of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to
the Fc domain of the antibody, and wherein the Fc domain has
decreased or no antibody dependent cellular cytotoxicity (ADCC)
activity compared to the wild-type Fc. In some embodiments, the
IL-15 variant comprises amino acid substitution at V49N, wherein
V49N is glycosylated. In some embodiments, the amino acid
substitution(s) at E53 and/or E89 of SEQ ID NO: 1 are also
glycosylated.
[0130] In some embodiments, the IL-15 fusion protein comprises an
antibody comprising a Fc domain and an IL-15 variant comprises
amino acid substitutions of SEQ ID NO: 1 at positions selected from
the group consisting of: a) V49, I50, S51, N4, D30, and E64; b)
V49, I50, S51, N4, D30, E64, and I68; c) V49, I50, S51, N4, D30,
E64, M109; d) V49, I50, S51, N4, D30, E64, I68, and M109; e) V49,
I50, S51, D30, E64, and I68; f) V49, I50, S51, D30, E64, M109; g)
V49, I50, S51, D30, E64, I68, and M109; h) N1, V49, I50, and S51;
i) N4, V49, I50, and S51; j) S7, V49, I50, and S51; k) K10, V49,
I50, and S51; l) K11, V49, I50, and S51; m) S29, V49, I50, and S51;
n) V31, V49, I50, and S51; o) H32, V49, I50, and S51; p) V49, I50,
S51, and E64; q) V49, I50, S51, and I68; r) V49, I50, S51, and L69;
s) V49, I50, S51, and I111; t) N4, V49, I50, S51, and E64; u) N1,
D30, V49, I50, and S51; v) N4, D30, V49, I50, and S51; w) S7 D30,
V49, I50, and S51; x) K10, D30, V49, I50, and S51; y) K11, D30,
V49, I50, and S51; z) S29, D30, V49, I50, and S51; aa) D30, V49,
I50, S51, and E64; bb) D30, V49, I50, S51, and I68; cc) D30, V49,
I50, S51, and L69; and dd) D30, V49, I50, S51, and I111. In some
embodiments, the IL-15 variant comprises amino acid substitutions
comprising one or more specific substitutions at: a) V49N, V49K,
V49E, V49H, V49Q or V49R; b) I50A or I50G; c) S51T; d) N1K, N1G,
N1Q, N1R, N1E, N1A, or N1D; e) N4K, N4G, N4A, N4S, N4D, N4E, N4I,
N4L, N4R, N4W, N4T, or N4Q; f) S7E, S7G, S7D, S7K, S7N, S7R, S7H,
or S7T; g) K10A, K10S, K10E, K10L, K10M, K10D, or K10G; h) K11D,
K11S, or K11W; i) D30N; j) E64Q, E64K, E64A, E64S, E64N, E64H,
E64T, or E64R; k) E53N; l) G55S or G55T; m) E89N; n) L91S or L91T;
o) Y26K, Y26R, or Y26H; p) S29N; q) V31S, V31D, or V31K; r) H32G;
s) I68S, I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or
I68H; t) L69A, L69S, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or
L69V; u) M109A, M109S, M109D, or M109K; and/or v) I111A, I111K,
I111S, or I111D.
[0131] In some embodiments, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) an IL-15
variant covalently linked to the Fc domain of the antibody, wherein
the IL-15 variant comprises amino acid substitutions at positions
selected from the group consisting of: a) N4K, D30N, V49N, I50A,
S51T, and E64Q; b) N4Q, D30N, V49N, I50A, and S51T; c) D30N, V49N,
I50A, S51T, and E64Q; d) N4Q, D30N, V49N, I50A, S51T, and E64Q; e)
N4Q, V49N, I50A, and S51T; f) V49N, I50A, S51T, and E64Q; and g)
N4Q, V49N, I50A, S51T, and E64Q.
[0132] In another aspect, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) a human
interleukin 15 (IL-15) variant comprising amino acid substitutions
at positions E46 and V49 of SEQ ID NO: 1, and at least one or more
amino acid substitution(s) at positions N1, N4, S7, K10, K11, D22,
Y26, S29, D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109
and/or I111 of SEQ ID NO: 1, wherein the IL-15 variant is
covalently linked to the Fc domain of the antibody, and wherein the
Fc domain has decreased or no antibody dependent cellular
cytotoxicity (ADCC) activity compared to the wild-type Fc.
[0133] In some embodiments, the IL-15 fusion protein comprises an
antibody comprising a Fc domain and an IL-15 variant comprises
amino acid substitutions of SEQ ID NO: 1 at positions selected from
the group consisting of: a) N1, E46, and V49; b) N4, E46, and V49;
c) S7, E46, and V49; d) K10, E46, and V49; e) K11, E46, and V49; f)
S29, E46, and V49; g) V31, E46, and V49; h) H32, E46, and V49; i)
E46, V49, and E64; j) E46, V49, and I68; k) E46, V49, and L69; l)
E46, V49, and I111; m) N4, E46, V49, and E64; n) E46, V49, N4, D30,
and E64; o) E46, V49, N4, D30, E64, and I68; p) E46, V49, N4, D30,
E64, and M109; q) E46, V49, N4, D30, E64, I68, and M109; r) N1,
D30, E46, and V49; s) N4, D30, E46, and V49; t) S7, D30, E46, and
V49; u) K10, D30, E46, and V49; v) K11, D30, E46, and V49; w) S29,
D30, E46, and V49; x) D30, E46, V49, and E64; y) D30, E46, V49R,
and I68; z) D30, E46, V49R, and L69; aa) D30, E46, V49R, and I111;
bb) N1, D30, E46, V49, and M109; cc) N4, D30, E46, V49, and M109;
dd) S7, D30, E46, V49, and M109; ee) K10, D30, E46, V49, and M109;
f) K11, D30, E46, V49, and M109; gg) D30, E46, V49, E64, and M109;
hh) D30, E46, V49, 168, and M109; ii) D30, E46, V49, L69 and M109;
jj) D30, E46, V49, M109, and I111; kk) D30, E46, V49, E64, I68, and
M109; ll) E46, V49, D30, E64, and I68; mm) E46, V49, E64, and M109;
nn) E46, V49, D30, E64, I68, and M109; oo) D22, Y26, V49, E46, E53,
E89, and E93; and pp) N1, D30, E46, V49, and E64. In some
embodiments, the IL-15 variant comprises amino acid substitutions
comprising one or more specific substitutions at: a) N1Q, N1K, N1R,
N1E, N1A, N1D, or N1G; b) N4K, N4G, N4A, N4S, N4D, N4E, N4L, N4I,
N4R, N4T, N4W, or N4Q; c) S7E, S7G, S7D, S7K, S7N, S7R, S7H, or
S7T; d) MOD, K10A, K10S, K10E, K10L, MOM, MOD, or K10G; e) K11D,
K11S, or K11W; f) D22N; g) Y26K, Y26R, or Y26H; h) S29N; i) D30N;
j) V31S, V31D, or V31K; k) H32G; l) E46G or E46Q; m) V49N, V49K, or
V49R V49E, V49H, or V49Q; n) E53Q; o) G55S or G55T; p) E64Q, E64K,
E64A, E64S, E64N, E64H, E64T, or E64R; q) I68S, I68A, I68R, I68T,
I68K, I68N, I68M, I68F, I68Y, I68E, or I68H; r) L69S, L69A, L69D,
L69T, L69M, L69G, L69Q, L69I, L69E, or L69V; s) E89Q; t) E93Q; u)
M109A, M109S, M109D, or M109K; and/or v) I111A, I111K, I111S, or
I111D.
[0134] In some embodiments, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) an IL-15
variant covalently linked to the Fc domain of the antibody, wherein
the IL-15 variant comprises amino acid substitutions at positions
selected from the group consisting of: a) N1K, E46G, and V49R; b)
N4K, E46G, and V49R; c) N4Q, E46G, and V49R; d) S7T, E46G, and
V49R; e) V31S, E46G, and V49R; f) V31K, E46G, and V49R; g) E46G,
V49R, and E64Q; h) E46G, V49R, and E64K; i) N4Q, E46G, V49R, and
E64Q; j) N1G, D30N, E46G, and V49R; k) N1K, D30N, E46G, and V49R;
l) N1Q, D30N, E46G, and V49R; m) N4G, D30N, E46G, and V49R; n) N4K,
D30N, E46G, and V49R; o) N4Q, D30N, E46G, and V49R; p) S7E, D30N,
E46G, and V49R; q) S7G, D30N, E46G, and V49R; r) S7T, D30N, E46G,
and V49R; s) K10D, D30N, E46G, and V49R; t) D30N, E46G, V49R, and
E64A; u) D30N, E46G, V49R, and E64Q; v) D30N, E46G, V49R, and E64K;
w) D30N, E46G, V49R, and I68S; x) D30N, E46G, V49R, and I68K; y)
N4K, D30N, E46G, V49R, and E64K; z) N4Q, D30N, E46G, V49R, and
E64K; aa) N4K, D30N, E46G, V49R, and E64Q; bb) N4Q, D30N, E46G,
V49R, and E64Q; cc) N4K, D30N, E46G, V49R, and I68S; dd) D30N,
E46G, V49R, E64Q, and I68S; ee) N1A, D30N, E46G, and V49R; and ff)
N1G, D30N, E46G, V49R, and E64Q.
[0135] In some embodiments, the isolated fusion protein comprises
an amino acid sequence of SEQ ID NO: 86, 87, 89, or 90.
[0136] In another aspect, provided is an isolated fusion protein
comprising: 1) an antibody comprising a Fc domain; and b) a human
IL-15 variant comprising one or more amino acid substitution(s) at
position(s) N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32,
E46, E53, E64, I68, L69, E89, E93, M109, and/or I111 of SEQ ID NO:
1, wherein the IL-15 variant is covalently linked to the Fc domain
of the antibody, and wherein the Fc domain has decreased or no
antibody dependent cellular cytotoxicity (ADCC) activity compared
to the wild-type Fc. In some embodiments, the human IL-15 variant
comprises amino acid substitutions at positions D22N, Y26F, E46Q,
E53Q, E89Q, and E93Q (e.g., see SEQ ID NO: 76), and the antibody is
an anti-PD-1 antibody.
[0137] In another aspect, provided is an isolated fusion protein
comprising: 1) an IL-15 antibody comprising a Fc domain; and b) a
human interleukin 15 (IL-15) protein of SEQ ID NO: 1, wherein the
IL-15 is covalently linked to the Fc domain of the antibody.
[0138] In some embodiments, one or more polypeptides (e.g.,
heterologous or homologous sequence) can be inserted between the
antibody and the IL-15 variant of the IL-15 fusion proteins as
described herein. In some embodiments, the polypeptide can be
inserted or conjugated at the amino terminus, at the carboxyl
terminus, or both the amino and carboxyl termini of the antibody.
In some embodiments, the polypeptide comprises a polypeptide linker
conjugating the antibody and the IL-15 variant, as depicted in
FIGS. 1A, 1B, 1C, and 1D. For example, the polypeptide linker is a
glycine-serine (GS)-linker as shown in SEQ ID NO: 6, 23, 24, 25, or
77.
[0139] In some embodiments, the polypeptide comprises one or more
linker(s) and tag(s). Examples of a polypeptide tag include, but
not are not limited to a FLAG tag, a 6His tag (i.e., SEQ ID NO:
27), a 8His tag (i.e., SEQ ID NO: 26), or an AVI tag (e.g., SEQ ID
NO: 7).
[0140] Exemplary human IL-15 (hIL-15) variants with a polypeptide
linker and polypeptide tags are provided below.
TABLE-US-00001 hIL-15 V49R-GS-linker-8xHis-Avi tags (SEQ ID NO: 9)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQRI
SLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL
QSFVHIVQMFINTGGGGSGHHHHHHHHGGGLNDIFEAQKIEWHE hIL-15
V49N/150A/S51T-GS-linker-8xHis-Avi tags (SEQ ID NO: 10)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQNA
TLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL
QSFVHIVQMFINTGGGGSGHHHHHHHHGGGLNDIFEAQKIEWHE hIL-15
V49N/I50A/S51T/N79Q-GS-linker- 8xHis-Avi tags (SEQ ID NO: 11)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQNA
TLESGDASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFL
QSFVHIVQMFINTGGGGSGHHHHHHHHGGGLNDIFEAQKIEWHE
* The underlined sequence represents the linker, 8.times.His, and
AVI tags; The bolded letters represent the amino acid mutations on
the hIL-15 protein sequence.
[0141] The antibodies useful in the IL-15 fusion proteins of the
present invention can encompass monoclonal antibodies, polyclonal
antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc,
etc.), chimeric antibodies, bispecific antibodies, heteroconjugate
antibodies, single chain (ScFv), mutants thereof, fusion proteins
comprising an antibody portion (e.g., a domain antibody), humanized
antibodies, and any other modified configuration of the
immunoglobulin molecule that comprises an antigen recognition site
of the required specificity, including glycosylation variants of
antibodies, amino acid sequence variants of antibodies, and
covalently modified antibodies. The antibodies may be murine, rat,
human, or any other origin (including chimeric or humanized
antibodies.
[0142] In some embodiments, the antibodies described herein have an
isotype that is selected from the group consisting of IgG.sub.1,
IgG.sub.2, IgG.sub.2.DELTA.a, IgG.sub.4, IgG.sub.4.DELTA.b,
IgG.sub.4.DELTA.c, IgG.sub.4 S228P, IgG.sub.4.DELTA.b S228P, and
IgG.sub.4.DELTA.c S228P.
[0143] In some embodiments, the antibodies of the IL-15 fusion
proteins as described comprise a Fc domain. In some embodiments,
the Fc domain can be a human IgG1, IgG2, or IgG4.
[0144] In some embodiments, the antibodies as described herein
comprise amino acid modifications at positions 223, 225, and 228
(e.g., (C223E or C223R), (E225R), and (P228E or P228R)) in the
hinge region and at position 409 or 368 (e.g., K409R or L368E (EU
numbering scheme)) in the CH3 region of human IgG2 (SEQ ID NO: 3).
In some embodiments, the antibodies described herein are bispecific
antibodies.
[0145] In some embodiments, the antibodies as described herein
comprise amino acid modifications at positions 221 and 228 (e.g.,
(D221R or D221E) and (P228R or P228E)) in the hinge region and at
position 409 or 368 (e.g., K409R or L368E (EU numbering scheme)) in
the CH3 region of human IgG1 (SEQ ID NO: 2). In some embodiments,
the antibodies described herein are bispecific antibodies.
[0146] In some embodiments, the antibodies as described herein
comprise amino acid modifications at positions 349, 354, 366, 368,
and/or 407 (EU numbering scheme) in the CH3 region of the human
IgG1 (SEQ ID NO: 2). For example, the amino acid modifications
comprise Y349C, S354C, T366W, T366S, L368A, and/or Y407V. In some
embodiments, the antibodies described herein are bispecific
antibodies.
[0147] In some embodiments, the antibodies as described herein
comprise amino acid modifications at positions 228 (e.g., (S228D,
S228E, S228R, or S228K)) in the hinge region and at position 409 or
368 (e.g., R409K, R409, or L368E (EU numbering scheme)) in the CH3
region of human IgG4 (SEQ ID NO: 4). In some embodiments, the
antibodies described herein are bispecific antibodies.
[0148] In some embodiments, the antibodies as described herein
comprise amino acid modifications at one or more of positions 265
(e.g., D265A), 330 (e.g., A330S), and 331 (e.g., P331S) of the
human IgG2 (SEQ ID NO: 3); or one or more positions 234, 235, 237,
and/or 322 of the human IgG1 (SEQ ID NO: 2). In some embodiments,
the antibodies as described herein comprise amino acid
modifications at each of positions 265 (e.g., D265A), 330 (e.g.,
A330S), and 331 (e.g., P331 S) of the human IgG2.
[0149] In some embodiments, the antibodies as described herein
comprise amino acid modifications at one or more of positions 234
(e.g., L234A), 235 (e.g., L235A), and 237 (e.g., G237A) of the
human IgG1 (SEQ ID NO: 2). In some embodiments, the antibodies as
described herein comprise amino acid modifications at each of
positions 234 (e.g., L234A), 235 (e.g., L235A), and 237 (e.g.,
G237A) of the human IgG1 (SEQ ID NO: 2). For example, the antibody
as described herein comprises an amino acid sequence of SEQ ID NO:
88.
[0150] In some embodiments, the antibodies as described herein
comprise amino acid modifications E233F234L235 to P233V234A235
(IgG.sub.4.DELTA.c) of the human IgG4 (SEQ ID NO: 4). In yet
another embodiment, the amino acid modifications are E233F234L235
to P233V234A235 with deletion G236 (IgG.sub.4.DELTA.b) of human
IgG.sub.4 (SEQ ID NO: 4).
[0151] Exemplary antibodies used for the present invention include,
but are not limited to, the sequences listed below.
TABLE-US-00002 TABLE 1.1 SEQ ID NO:/ Sequence 64/CH1-hinge-
ASTKGPSVFPLAPCSRSTSESTAALGOLVKDYFPEPVTVSWNSGALTSGV CH2-CH3 of
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHIKPSNTKVDKTVE
hIgG2.DELTA.a-D265A
RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHED (underlined:
PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK hinge)
CKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK 65/xhPD1
DIQMTQSPSSLSASVGDRVTITCKSSQSLWDSGNQKNFLTVVYQQKPGKA (VK1-39)-
PKLLIYWTSYRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNDYF Light chain
YPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE (bolded:
AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA V-kappa,
CEVTHQGLSSPVTKSFNRGEC underlined: C-kappa) 66/xhPD1
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGN (VH1-69b)-
IYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLT hIgG1-AAA-
TGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF Bsp-R-arm
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCRKTHTCPRCPAPEAAGAPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 74/xhPD1
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGN (VH1-69b)-
IYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLT hIgG1-AAA-
TGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF `knob`
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCT
LPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0152] In some embodiments, the antibodies described herein
comprise a modified constant region that have increased or
decreased binding affinity to a human Fc gamma receptor, are
immunologically inert or partially inert, e.g., do not trigger
complement mediated lysis, do not stimulate antibody-dependent cell
mediated cytotoxicity (ADCC), or do not activate microglia; or have
reduced activities (compared to the unmodified antibody) in any one
or more of the following: triggering complement mediated lysis,
stimulating ADCC, or activating microglia. Different modifications
of the constant region may be used to achieve optimal level and/or
combination of effector functions. See, for example, Morgan et al.,
Immunology 86-319-324, 1995; Lund et al., J. Immunology 157-4963-9
157-4963-4969, 1996; Idusogie et al., J. Immunology 164-4178-4184,
2000; Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis
et al., Immunological Reviews 163-59-76, 1998. In some embodiments,
the constant region is modified as described in Eur. J. Immunol.,
1999, 29-2613-2624; POT Publication No. WO99/058572.
[0153] In some embodiments, an antibody constant region can be
modified to avoid interaction with Fc gamma receptor and the
complement and immune systems. The techniques for preparation of
such antibodies are described in WO 99/58572. For example, the
constant region may be engineered to more resemble human constant
regions to avoid immune response if the antibody is used in
clinical trials and treatments in humans. See, e.g., U.S. Pat. Nos.
5,997,867 and 5,866,692.
[0154] In still other embodiments, the constant region is
aglycosylated for N-linked glycosylation. In some embodiments, the
constant region is aglycosylated for N-linked glycosylation by
mutating the oligosaccharide attachment residue and/or flanking
residues that are part of the N-glycosylation recognition sequence
in the constant region. For example, N-glycosylation site N297 may
be mutated to, e.g., A, Q, K, or H. See, Tao et al., J. Immunology
143: 2595-2601, 1989; and Jefferis et al., Immunological Reviews
163:59-76, 1998. In some embodiments, the constant region is
aglycosylated for N-linked glycosylation. The constant region may
be aglycosylated for N-linked glycosylation enzymatically (such as
removing carbohydrate by enzyme PNGase), or by expression in a
glycosylation deficient host cell.
[0155] Other antibody modifications include antibodies that have
been modified as described in PCT Publication No. WO 99/58572.
These antibodies comprise, in addition to a binding domain directed
at the target molecule, an effector domain having an amino acid
sequence substantially homologous to all or part of a constant
region of a human immunoglobulin heavy chain. These antibodies are
capable of binding the target molecule without triggering
significant complement dependent lysis, or cell-mediated
destruction of the target. In some embodiments, the effector domain
is capable of specifically binding FcRn and/or Fc.gamma.RIIb. These
are typically based on chimeric domains derived from two or more
human immunoglobulin heavy chain CH2 domains. Antibodies modified
in this manner are particularly suitable for use in chronic
antibody therapy, to avoid inflammatory and other adverse reactions
to conventional antibody therapy.
[0156] In some embodiments, the antibody comprises a modified
constant region that has increased binding affinity for FcRn and/or
an increased serum half-life as compared with the unmodified
antibody.
[0157] The antibodies used in the IL-15 fusion proteins of the
present invention include, but are not limited to, an anti-CTLA-4
antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8
antibody, an anti-4-1 BB antibody, an anti-PD-1 antibody, an
anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody,
an anti-TIGIT antibody, an anti-OX40 antibody, an anti-IL-7Ralpha
(CD127) antibody, an anti-IL-8 antibody, an anti-IL-15 antibody, an
anti-HVEM antibody, an anti-BTLA antibody, an anti-CD40 antibody,
an anti-CD40L antibody, anti-CD47 antibody, an anti-CSF1R antibody,
an anti-CSF1 antibody, an anti-MARCO antibody, an anti-CXCR4
antibodies, an anti-VEGFR1 antibody, an anti-VEGFR2 antibody, an
anti-TNFR1 antibody, an anti-TNFR2 antibody, an anti-CD3 bispecific
antibody, an anti-CD19 antibody, an anti-CD20, an anti-Her2
antibody, an anti-EGFR antibody, an anti-ICOS antibody, an
anti-CD22 antibody, an anti-CD 52 antibody, an anti-CCR4 antibody,
an anti-CCR8 antibody, an anti-CD200R antibody, an anti-VISG4
antibody, an anti-CCR2 antibody, an anti-LILRb2 antibody, an
anti-CXCR4 antibody, an anti-CD206 antibody, an anti-CD163
antibody, an anti-KLRG1 antibody, an anti-FLT3 antibody, an
anti-B7-H4 antibody, an anti-B7-H3 antibody, an KLRG1 antibody, an
anti-BTN1A1 antibody, and an anti-GITR antibody. In some
embodiments, the antibody in the IL-15 fusion protein is a PD-1
antibody. For example, the PD-1 antibody comprises a heavy chain
variable (VH) region comprising (i) a VH complementarity
determining region one (CDR1) comprising the amino acid sequence of
SEQ ID NO: 14, 15, 80, 81, or 91, a VH CDR2 comprising the amino
acid sequence of SEQ ID NO: 16, 17, 82, or 83, and a VH CDR3
comprising the amino acid sequence shown in SEQ ID NO: 18 or 62;
and/or a VL CDR1 comprising the amino acid sequence shown in SEQ ID
NO: 19 or 31, a VL CDR2 comprising the amino acid sequence shown in
SEQ ID NO: 20 or 32, and a VL CDR3 comprising the amino acid
sequence shown in SEQ ID NO: 21 or 33. In some embodiments, the
PD-1 antibody comprises a VH region comprising a CDR1, CDR2, and
CDR3 of the VH having an amino acid sequence of SEQ ID NO: 12, 34,
78, or 36 and/or a VL region comprising a CDR1, CDR2, and CDR3 of
the VL having an amino acid sequence of SEQ ID NO: 13, 35, 79, or
37.
[0158] Exemplary IL-15 fusion proteins include, but are not limited
to, the sequences listed below. The IL-15 variants are in bold; and
the linkers are underlined.
TABLE-US-00003 TABLE 1.2 SEQ ID NO:/ name SEQUENCE 29/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-hIL-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK 15NQ mutant
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV (E arm)
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTATATPGANWVNVISDLKKIEDLIQSMHI ATL TESDVHP SCKVTAMKCFLL
LQVISL SGDASIHDTVENLIILANNSLSSNGNVTE SGCKEC ELE
KNIKEFLQSFVHIVQMFINT (Mutations of NQ are in italics) 38/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLGLQRISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 39/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G-E64Q-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT D30N
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESNVH
PSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 40/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G-E64Q-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT I68S-D30N
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESNVH
PSCKVTAMKCFLLGLGRISLESGDASIHDTVQNLISLANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 41/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G-N4K-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT E64Q-D30N
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVKVISDLKKIEDLIQSMHIDATLYTESNVH
PSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 42/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G-E64Q-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT I68S-D30N-
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK Ml09A
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESNVH
PSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLISLANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQAFINT 43/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G-E64Q-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT N4K-D30N-
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK Ml09A
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVKVISDLKKIEDLIQSMHIDATLYTESNVH
PSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQAFINT 44/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G-E64Q-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT D30N-Ml09A
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESNVH
PSCKVTAMKCFLLGLCIRISLESGDASIHDTVQNLIILANNSLSSNGNV
TESGCKECEELEEKNIKEFLQSFVHIVQAFINT 45/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV Y26K
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLKTESDVH
PSCKVTAMKCFLLELQRISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 46/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV Y26K-E64Q-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT D30N
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLKTESNVH
PSCKVTAMKCFLLELQRISLESGDASIHDTVQNLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 47
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49K-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV Y26K
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLKTESDVH
PSCKVTAMKCFLLELQKISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 48/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49K-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46G
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLGLQKISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 49/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15Rasu-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV hIL15
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT (E64Q/D30N)
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGAITCPPPMSVEHADIWVKSYSLYSRERVICNSG
FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS
GGGGSGGGSGGGGSGGNWVNVISDLKKIEDLIQSMHIDATLYTESNVHP
SCKVTAMKCFLLELQVISLESGDASIHDTVQNLIILANNSLSSNGNVTE
SGCKECEELEEKNIKEFLQSFVHIVQMFINT 50/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15Rasu-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV hIL15
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT (E64Q/I68S/
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK D30N)
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGAITCPPPMSVEHADIWVKSYSLYSRERYICNSG
FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS
GGGGSGGGSGGGGSGGNWVNVISDLKKIEDLIQSMHIDATLYTESNVHP
SCKVTAMKCFLLELQVISLESGDASIHDTVQNLISLANNSLSSNGNVTE
SGCKECEELEEKNIKEFLQSFVHIVQMFINT 51/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15Rasu-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV hIL15
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT (E64Q/N4K/
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK D30N)
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGAITCPPPMSVEHADIWVKSYSLYSRERYICNSG
FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS
GGGGSGGGSGGGGSGGNWVKVISDLKKIEDLICISNIHMATLYTESNVH
PSCKVTAMKCFLLELQVISLESGDASIHDTVQNLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLCISFVHWQMFINT 52/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15Rasu-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV hIL15
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT (E64Q/D30N/
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK M109A)
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGAITCPPPMSVEHADIWVKSYSLYSRERVICNSG
FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS
GGGGSGGGSGGGGSGGNWVNVISDLKKIEDLIQSMHIDATLYTESNVHP
SCKVTAMKCFLLELQVISLESGDASIFIDTVQNLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVCAFINT 53/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15Rasu-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV hIL15
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT (E64Q/I68S/
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK D30N/M109A)
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGAITCPPPMSVEHADIWVKSYSLYSRERVICNSG
FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS
GGGGSGGGSGGGGSGGNWVNVISDLKKIEDLIQSMHIDATLYTESNVHP
SCKVTAMKCFLLELQVISLESGDASIHDTVQNLISLANNSLSSNGNVTE
SGCKECEELEEKNIKEFLQSFVHIVQAFINT 54/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15Rasu-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV hIL15
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT (E64Q/N4K/
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK D30N/M109A)
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGAITCPPPMSVEHADIWVKSYSLYSRERYICNSG
FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS
GGGGSGGGSGGGGSGGNWVKVISDLKKIEDUQSMHIDATLYTESNVHPS
CKVTAMKCFLLELQVISLESGDASIHDWQNLIILANNSLSSNGNVTESG
CKECEELEEKNIKEFLQSFVHIVQAFINT 55/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E46Q
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLQLQRISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 56/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E53Q
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLELQRISLQSGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 57/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15_V49R-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV E93Q
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLELQRISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEQKNIKEFLQSFVHIVQMFINT 58/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15NQ-2a
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV (E46Q/E93Q)
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLQLQVISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEQKNIKEFLQSFVHIVQMFINT 59/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15NQ-2b
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV (E46Q/E53Q)
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLQLQVISLQSGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 60/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15NQ-2c
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV (E53Q/E93Q)
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLELQVISLQSGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEQKNIKEFLQSFVHIVQMFINT 61/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL15NQ-3d
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV (E46Q/E53Q/
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT E93Q)
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGSGGGGGTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLQLQVISLQSGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEQKNIKEFLQSFVHIVQMFINT 63/
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD-1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIL-15V49R
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV fusion
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPK
DTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGKGGGGRTSATATPGANWVNVISDLKKIEDLIQSMHIDATLYTESDVH
PSCKVTAMKCFLLELQRISLESGDASIHDTVENLIILANNSLSSNGNVT
ESGCKECEELEEKNIKEFLQSFVHIVQMFINT 67/
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ Bsp-E-arm-
TYICNVNHKPSNTKVDKKVEPKSCEKTHTCPECPAPEAAGAPSVFLFPP 3GS-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE hIL15Rasu-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP hIL15
REPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGITCPPPMSVEHADIWVKSYSLYSRERYICB
SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSG
GSGGGGSGGGSGGGGSGGNWVNVISDLKKIEDLIQSMHIDATLYTESDV
HPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNV
TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 68/
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ Bsp-E-arm-
TYICNVNHKPSNTKVDKKVEPKSCEKTHTCPECPAPEAAGAPSVFLFPP 3GS-hIL15NQ
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGNWVNVISDLKKIEDLIQSMHINATLFTESD
VHPSCKVTAMKCFLLQLQVISLQSGDASIHDTVENLIILANNSLSSNGN
VTESGCKECQELEQKNIKEFLQSFVHIVQMFINTS 69/
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ Bsp-E-arm-
TYICNVNHKPSNTKVDKKVEPKSCEKTHTCPECPAPEAAGAPSVFLFPP 3GS-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE hIL15V49R-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP E46G-E64Q
REPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGNWVNVISDLKKIEDLIQSMHIDATLYTESD
VHPSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSNGN
VTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 70/
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ Bsp-E-arm-
TYICNVNHKPSNTKVDKKVEPKSCEKTHTCPECPAPEAAGAPSVFLFPP 3GS-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE hIL15V49R-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP E46G-E64Q-
REPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYK D30N
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGNWVNVISDLKKIEDLIQSMHIDATLYTESN
VHPSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSNGN
VTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 71/
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ Bsp-E-arm-
TYICNVNHKPSNTKVDKKVEPKSCEKTHTCPECPAPEAAGAPSVFLFPP 3GS-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE hIL15V49R-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP E46G-N4Q-
REPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYK D30N
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGNWVCAMSDLKKIEDLIQSMHIDATLYTESN
VHPSCKVTAMKCFLLGLQRISLESGDASINDTVENLIILANNSLSSNGN
VTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 72
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ Bsp-E-arm-
TYICNVNHKPSNTKVDKKVEPKSCEKTHTCPECPAPEAAGAPSVFLFPP 3GS-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE hIL15V49R-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP E46G-N1K-
REPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYK D30N
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGKWVNVISDLKKIEDLIQSMHIDATLYTESN
VHPSCKVTAMKCFLLGLQRISLESGDASIHDTVENLIILANNSLSSNGN
VTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 73
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ Bsp-E-arm-
TYICNVNHKPSNTKVDKKVEPKSCEKTHTCPECPAPEAAGAPSVFLFPP 3GS-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE hIL15V49R-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP E46G-S7T-
REPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYK D30N
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGNWVNVITDLKKIEDLIQSMHDATLYTESNV
HPSCKVTAMKCFLLGLQRISLESGDASIHDTVENLIILANNSLSSNGNV
TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 75/
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ `hole`-3GS-
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPP hIL15V49R-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE E46G-E64Q-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP D30N
REPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGGGGGSGGGGSGGGGNWVNVISDLKKIEDLIQSMHIDATLYTESN
VHPSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSNGN
VTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 86
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIgG1-AAA-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV `hole`-3GS-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT hIL15V49R-
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFP E46G-N1A-
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE D30N(m1)
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGSGGGGSGGGGSGGGGAWVNVISDLKKIEDLIQSMHIDATLYTE
SNVHPSCKVTAMKCFLLGLQRISLESGDASIHDTVENLIILANNSLSSN
GNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 87
EVQLVESGGGLVKPGGSLELSCAASGFTFSSYWMSWVRQAPEKGLEWVA xmPD1-
AISPSGGSTYYADSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAK hIgG1-AAA-
ESWGAYYDLWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV `hole`-3GS-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT hIL15V49R-
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFP E46G-N1G-
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE E64Q-D30N
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ (m2)
PREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGSGGGGSGGGGSGGGGGWVNVISDLKKIEDLIQSMHIDATLYTE
SNVHPSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSN
GNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 89
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG xhPD1
NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ `hole`-3GS-
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPP hIL15V49R-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE E46G-N1A-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP D30N(m1)
REPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGSGGGGSGGGGSGGGGAWVNVISDLKKIEDLIQSMHIDATLYTES
NVHPSCKVTAMKCFLLGLQRISLESGDASIHDTVENLIILANNSLSSNG
NVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 90
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG
xhPD1 NIYPGSSITNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (VH1-69b)-
LTTGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK hIgG1-AAA-
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ `hole`-3GS-
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPP hIL15V49R-
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE E46G-N1G-
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP E64Q-D30N
REPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK (m2)
TTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGSGGGGSGGGGSGGGGGWVNVISDLKKIEDLIQSMHIDATLYTES
NVHPSCKVTAMKCFLLGLQRISLESGDASIHDTVQNLIILANNSLSSNG
NVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
[0159] The IL-15 fusion protein as described herein can be created
by methods known in the art, for example, synthetically or
recombinantly. Typically, the fusion proteins of this invention are
made by preparing and expressing a polynucleotide encoding them
using recombinant methods described herein, although they may also
be prepared by other means known in the art, including, for
example, chemical synthesis.
[0160] The antibodies as described herein can also be made by any
method known in the art. For the production of hybridoma cell
lines, the route and schedule of immunization of the host animal
are generally in keeping with established and conventional
techniques for antibody stimulation and production, as further
described herein. General techniques for production of human and
mouse antibodies are known in the art and/or are described
herein.
[0161] It is contemplated that any mammalian subject including
humans or antibody producing cells therefrom can be manipulated to
serve as the basis for production of mammalian, including human and
hybridoma cell lines. Typically, the host animal is inoculated
intraperitoneally, intramuscularly, orally, subcutaneously,
intraplantar, and/or intradermally with an amount of immunogen,
including as described herein.
[0162] Hybridomas can be prepared from the lymphocytes and
immortalized myeloma cells using the general somatic cell
hybridization technique of Kohler, B. and Milstein, C., Nature
256:495-497, 1975 or as modified by Buck, D. W., et al., In Vitro,
18:377-381, 1982. Available myeloma lines, including but not
limited to X63-Ag8.653 and those from the Salk Institute, Cell
Distribution Center, San Diego, Calif., USA, may be used in the
hybridization. Generally, the technique involves fusing myeloma
cells and lymphoid cells using a fusogen such as polyethylene
glycol, or by electrical means well known to those skilled in the
art. After the fusion, the cells are separated from the fusion
medium and grown in a selective growth medium, such as
hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate
unhybridized parent cells. Any of the media described herein,
supplemented with or without serum, can be used for culturing
hybridomas that secrete monoclonal antibodies. As another
alternative to the cell fusion technique, EBV immortalized B cells
may be used to produce the monoclonal antibodies of the subject
invention. The hybridomas are expanded and subcloned, if desired,
and supernatants are assayed for anti-immunogen activity by
conventional immunoassay procedures (e.g., radioimmunoassay, enzyme
immunoassay, or fluorescence immunoassay).
[0163] Hybridomas that may be used as source of antibodies
encompass all derivatives, progeny cells of the parent hybridomas
that produce monoclonal antibodies.
[0164] Hybridomas that produce antibodies used for the present
invention may be grown in vitro or in vivo using known procedures.
The monoclonal antibodies may be isolated from the culture media or
body fluids, by conventional immunoglobulin purification procedures
such as ammonium sulfate precipitation, gel electrophoresis,
dialysis, chromatography, and ultrafiltration, if desired.
Undesired activity, if present, can be removed, for example, by
running the preparation over adsorbents made of the immunogen
attached to a solid phase and eluting or releasing the desired
antibodies off the immunogen. Immunization of a host animal with
cells expressing the antibody target (e.g., PD-1), a human target
protein (e.g., PD-1), or a fragment containing the target amino
acid sequence conjugated to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups, can yield a population of
antibodies (e.g., monoclonal antibodies).
[0165] If desired, the antibody (monoclonal or polyclonal) of
interest may be sequenced and the polynucleotide sequence may then
be cloned into a vector for expression or propagation. The sequence
encoding the antibody of interest may be maintained in vector in a
host cell and the host cell can then be expanded and frozen for
future use. Production of recombinant monoclonal antibodies in cell
culture can be carried out through cloning of antibody genes from B
cells by means known in the art. See, e.g. Tiller et al., J.
Immunol. Methods 329, 112, 2008; U.S. Pat. No. 7,314,622.
[0166] In some embodiments, antibodies may be made using hybridoma
technology. It is contemplated that any mammalian subject including
humans or antibody producing cells therefrom can be manipulated to
serve as the basis for production of mammalian, including human,
hybridoma cell lines. The route and schedule of immunization of the
host animal are generally in keeping with established and
conventional techniques for antibody stimulation and production, as
further described herein. Typically, the host animal is inoculated
intraperitoneally, intramuscularly, orally, subcutaneously,
intraplantar, and/or intradermally with an amount of immunogen,
including as described herein.
[0167] In some embodiments, antibodies as described herein are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and
Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains
of the immunoglobulins affect the protein's function (Boyd et al.,
1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
29:4175-4180) and the intramolecular interaction between portions
of the glycoprotein, which can affect the conformation and
presented three-dimensional surface of the glycoprotein (Jefferis
and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.
7:409-416). Oligosaccharides may also serve to target a given
glycoprotein to certain molecules based upon specific recognition
structures. Glycosylation of antibodies has also been reported to
affect antibody-dependent cellular cytotoxicity (ADCC). In
particular, antibodies produced by CHO cells with
tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GIcNAc, was
reported to have improved ADCC activity (Umana et al., 1999, Nature
Biotech. 17:176-180).
[0168] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0169] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0170] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, variations in the glycosylation pattern of the
antibodies can be expected (see, e.g. Hse et al., 1997, J. Biol.
Chem. 272:9062-9070).
[0171] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example, using endoglycosidase H (Endo H),
N-glycosidase F, endoglycosidase F1, endoglycosidase F2,
endoglycosidase F3. In addition, the recombinant host cell can be
genetically engineered to be defective in processing certain types
of polysaccharides. These and similar techniques are well known in
the art.
[0172] Other methods of modification include using coupling
techniques known in the art, including, but not limited to,
enzymatic means, oxidative substitution and chelation.
Modifications can be used, for example, for attachment of labels
for immunoassay. Modified polypeptides are made using established
procedures in the art and can be screened using standard assays
known in the art, some of which are described below and in the
Examples.
[0173] The IL-15 fusion proteins or the Il-15 variants of this
invention may be linked to a labeling agent such as a fluorescent
molecule, a radioactive molecule or any others labels known in the
art. Labels are known in the art which generally provide (either
directly or indirectly) a signal.
Polynucleotides, Vectors, and Host Cells
[0174] The invention also provides polynucleotides encoding any of
the IL-15 variant and IL-15 fusion proteins as described herein. In
one aspect, the invention provides a method of making any of the
polynucleotides described herein. Polynucleotides can be made and
expressed by procedures known in the art.
[0175] In another aspect, the invention provides compositions (such
as a pharmaceutical compositions) comprising any of the
polynucleotides of the invention. In some embodiments, the
composition comprises an expression vector comprising a
polynucleotide encoding any of the IL-15 variant and IL-15 fusion
proteins described herein.
[0176] In another aspect, provided is an isolated cell line that
produces the IL-15 variants and the IL-15 fusion proteins as
described herein. In some embodiments, the cell line is an
engineered immune cell, wherein the engineered immune cell
comprises a chimeric antigen receptor (CAR). In some embodiments,
the IL-15 variants and the IL-15 fusion proteins, when expressed as
polynucleotides in CAR T cells, either as secreted or
membrane-tethered versions, are used to enhance CAR T function,
including activity and proliferation. In some embodiments, the
Il-15 variants or the IL-15 fusion proteins comprising thereof
comprise amino acid substitutions at positions D22N, Y26F, E46Q,
E53Q, E89Q, and E93Q (e.g., see SEQ ID NO: 76).
[0177] Immune cells producing the IL-15 variants and the IL-15
fusion proteins as described herein may be made by introducing a
CAR into immune cells, and expanding the cells. For example, the
immune cells can be engineered by: providing a cell and expressing
at the surface of the cell at least one CAR and at least one IL-15
variant or IL-15 fusion protein as described herein. Methods for
engineering immune cells are described in, for example, PCT Patent
Application Publication Nos. WO/2014/039523, WO/2014/184741,
WO/2014/191128, WO/2014/184744, and WO/2014/184143, each of which
is incorporated herein by reference in its entirety. In some
embodiments, the cell can be transformed with at least one
polynucleotide encoding a CAR, one polynucleotide encoding the
IL-15 variant or IL-15 fusion protein as described herein, followed
by expressing the polynucleotides in the cell.
[0178] Polynucleotides complementary to any such sequences are also
encompassed by the present invention. Polynucleotides may be
single-stranded (coding or antisense) or double-stranded, and may
be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules
include HnRNA molecules, which contain introns and correspond to a
DNA molecule in a one-to-one manner, and mRNA molecules, which do
not contain introns. Additional coding or non-coding sequences may,
but need not, be present within a polynucleotide of the present
invention, and a polynucleotide may, but need not, be linked to
other molecules and/or support materials.
[0179] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an antibody or a fragment thereof)
or may comprise a variant of such a sequence. Polynucleotide
variants contain one or more substitutions, additions, deletions
and/or insertions such that the immunoreactivity of the encoded
polypeptide is not diminished, relative to a native immunoreactive
molecule. The effect on the immunoreactivity of the encoded
polypeptide may generally be assessed as described herein. Variants
preferably exhibit at least about 70% identity, more preferably, at
least about 80% identity, yet more preferably, at least about 90%
identity, and most preferably, at least about 95% identity to a
polynucleotide sequence that encodes a native antibody or a
fragment thereof.
[0180] Two polynucleotide or polypeptide sequences are said to be
"identical" if the sequence of nucleotides or amino acids in the
two sequences is the same when aligned for maximum correspondence
as described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, or 40 to
about 50, in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned.
[0181] Optimal alignment of sequences for comparison may be
conducted using the MegAlign.RTM. program in the Lasergene.RTM.
suite of bioinformatics software (DNASTAR.RTM., Inc., Madison,
Wis.), using default parameters. This program embodies several
alignment schemes described in the following references: Dayhoff,
M. O., 1978, A model of evolutionary change in proteins--Matrices
for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas
of Protein Sequence and Structure, National Biomedical Research
Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J.,
1990, Unified Approach to Alignment and Phylogenes pp. 626-645
Methods in Enzymology vol. 183, Academic Press, Inc., San Diego,
Calif.; Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153;
Myers, E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D.,
1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol.
Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical
Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman
Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J.,
1983, Proc. Natl. Acad. Sci. USA 80:726-730.
[0182] Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e. the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[0183] Variants may also, or alternatively, be substantially
homologous to a native gene, or a portion or complement thereof.
Such polynucleotide variants are capable of hybridizing under
moderately stringent conditions to a naturally occurring DNA
sequence encoding a native antibody (or a complementary
sequence).
[0184] Suitable "moderately stringent conditions" include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC
containing 0.1% SDS.
[0185] As used herein, "highly stringent conditions" or "high
stringency conditions" are those that: (1) employ low ionic
strength and high temperature for washing, for example 0.015 M
sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate
at 50.degree. C.; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash consisting of
0.1.times.SSC containing EDTA at 55.degree. C. The skilled artisan
will recognize how to adjust the temperature, ionic strength, etc.
as necessary to accommodate factors such as probe length and the
like.
[0186] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless,
polynucleotides that vary due to differences in codon usage are
specifically contemplated by the present invention. Further,
alleles of the genes comprising the polynucleotide sequences
provided herein are within the scope of the present invention.
Alleles are endogenous genes that are altered as a result of one or
more mutations, such as deletions, additions and/or substitutions
of nucleotides. The resulting mRNA and protein may, but need not,
have an altered structure or function. Alleles may be identified
using standard techniques (such as hybridization, amplification
and/or database sequence comparison).
[0187] The polynucleotides of this invention can be obtained using
chemical synthesis, recombinant methods, or PCR. Methods of
chemical polynucleotide synthesis are well known in the art and
need not be described in detail herein. One of skill in the art can
use the sequences provided herein and a commercial DNA synthesizer
to produce a desired DNA sequence.
[0188] For preparing polynucleotides using recombinant methods, a
polynucleotide comprising a desired sequence can be inserted into a
suitable vector, and the vector in turn can be introduced into a
suitable host cell for replication and amplification, as further
discussed herein. Polynucleotides may be inserted into host cells
by any means known in the art. Cells are transformed by introducing
an exogenous polynucleotide by direct uptake, endocytosis,
transfection, F-mating or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. The polynucleotide so amplified can be isolated
from the host cell by methods well known within the art. See, e.g.,
Sambrook et al., 1989.
[0189] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art and is described in U.S. Pat.
Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR:
The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer
Press, Boston, 1994.
[0190] RNA can be obtained by using the isolated DNA in an
appropriate vector and inserting it into a suitable host cell. When
the cell replicates and the DNA is transcribed into RNA, the RNA
can then be isolated using methods well known to those of skill in
the art, as set forth in Sambrook et al., 1989, supra, for
example.
[0191] Suitable cloning vectors may be constructed according to
standard techniques, or may be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected may vary according to the host cell intended to be used,
useful cloning vectors will generally have the ability to
self-replicate, may possess a single target for a particular
restriction endonuclease, and/or may carry genes for a marker that
can be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,
pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors
such as pSA3 and pAT28. These and many other cloning vectors are
available from commercial vendors such as BioRad, Strategene, and
Invitrogen.
[0192] Expression vectors are further provided. Expression vectors
generally are replicable polynucleotide constructs that contain a
polynucleotide according to the invention. It is implied that an
expression vector must be replicable in the host cells either as
episomes or as an integral part of the chromosomal DNA. Suitable
expression vectors include but are not limited to plasmids, viral
vectors, including adenoviruses, adeno-associated viruses,
retroviruses, cosmids, and expression vector(s) disclosed in PCT
Publication No. WO 87/04462. Vector components may generally
include, but are not limited to, one or more of the following: a
signal sequence; an origin of replication; one or more marker
genes; suitable transcriptional controlling elements (such as
promoters, enhancers and terminator). For expression (i.e.,
translation), one or more translational controlling elements are
also usually required, such as ribosome binding sites, translation
initiation sites, and stop codons.
[0193] The vectors containing the polynucleotides of interest can
be introduced into the host cell by any of a number of appropriate
means, including electroporation, transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (e.g., where the vector is an infectious agent such as
vaccinia virus). The choice of introducing vectors or
polynucleotides will often depend on features of the host cell.
[0194] The invention also provides host cells comprising any of the
polynucleotides described herein. Any host cells capable of
over-expressing heterologous DNAs can be used for the purpose of
isolating the genes encoding the antibody, polypeptide or protein
of interest. Non-limiting examples of mammalian host cells include
but not limited to COS, HeLa, and CHO cells. See also PCT
Publication No. WO 87/04462. Suitable non-mammalian host cells
include prokaryotes (such as E. coli or B. subtilis) and yeast
(such as S. cerevisae, S. pombe; or K. lactis). Preferably, the
host cells express the cDNAs at a level of about 5 fold higher,
more preferably, 10 fold higher, even more preferably, 20 fold
higher than that of the corresponding endogenous antibody or
protein of interest, if present, in the host cells. Screening the
host cells for a specific binding to IL-15 or a IL-15 domain is
effected by an immunoassay or FACS. A cell overexpressing the
antibody or protein of interest can be identified.
[0195] An expression vector can be used to direct expression of an
IL-15 variant or an IL-15 fusion protein. One skilled in the art is
familiar with administration of expression vectors to obtain
expression of an exogenous protein in vivo. See, e.g., U.S. Pat.
Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of
expression vectors includes local or systemic administration,
including injection, oral administration, particle gun or
catheterized administration, and topical administration. In another
embodiment, the expression vector is administered directly to the
sympathetic trunk or ganglion, or into a coronary artery, atrium,
ventrical, or pericardium.
[0196] Targeted delivery of therapeutic compositions containing an
expression vector, or subgenomic polynucleotides can also be used.
Receptor-mediated DNA delivery techniques are described in, for
example, Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et
al., Gene Therapeutics: Methods And Applications Of Direct Gene
Transfer, J. A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988,
263:621; Wu et al., J. Biol. Chem., 1994, 269:542; Zenke et al.,
Proc. Natl. Acad. Sci. USA, 1990, 87:3655; Wu et al., J. Biol.
Chem., 1991, 266:338. Therapeutic compositions containing a
polynucleotide are administered in a range of about 100 ng to about
200 mg of DNA for local administration in a gene therapy protocol.
Concentration ranges of about 500 ng to about 50 mg, about 1 pg to
about 2 mg, about 5 pg to about 500 pg, and about 20 pg to about
100 pg of DNA can also be used during a gene therapy protocol. The
therapeutic polynucleotides and polypeptides can be delivered using
gene delivery vehicles. The gene delivery vehicle can be of viral
or non-viral origin (see generally, Jolly, Cancer Gene Therapy,
1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845; Connelly,
Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature Genetics,
1994, 6:148). Expression of such coding sequences can be induced
using endogenous mammalian or heterologous promoters. Expression of
the coding sequence can be either constitutive or regulated.
[0197] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publication Nos. WO
90/07936: WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO
93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB
Patent No. 2,200,651; and EP Patent No. 0 345 242),
alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki
forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC
VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus
(ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and
adeno-associated virus (AAV) vectors (see, e.g., PCT Publication
Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO
95/11984 and WO 95/00655). Administration of DNA linked to killed
adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 can
also be employed.
[0198] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther., 1992, 3:147); ligand-linked DNA (see, e.g., Wu, J.
Biol. Chem., 1989, 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional
approaches are described in Philip, Mol. Cell Biol., 1994, 14:2411,
and in Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.
Compositions
[0199] The invention also provides pharmaceutical compositions
comprising an effective amount of an IL-15 variant or an IL-15
fusion protein as described herein. Examples of such compositions,
as well as how to formulate, are also described herein. In some
embodiments, the composition comprises one or more IL-15 variant or
IL-15 fusion protein. In some embodiments, the composition
comprises an IL-15 fusion protein comprising an PD-1 antibody and a
human IL-15 variant comprising amino acid substitutions at
positions V49, I50, S51, N4, D30, and E64 (e.g., V49N, I50A, S51T,
N4K, D30N, and E64Q), wherein the human IL-15 variant is covalently
linked to the Fc domain of the antibody. In some embodiments, the
composition comprises a human IL-fusion protein comprising an PD-1
antibody and a human IL-15 variant comprising amino acid
substitutions at positions E46, V49, E64, D30, and N4 (e.g., E46G,
V49R, E64Q, D30N, and N4K), wherein the IL-15 variant is covalently
linked to the Fc domain of the antibody. In some embodiments, the
composition comprises a human IL-15 fusion protein comprising an
PD-1 antibody and a human IL-15 variant comprising amino acid
substitutions at positions N1, D30, E46, and V49 (e.g., N1A, D30N,
E46G, and V49R), wherein the IL-15 variant is covalently linked to
the Fc domain of the antibody. In some embodiments, the composition
comprises a human IL-15 fusion protein comprising an PD-1 antibody
and a human IL-15 variant comprising amino acid substitutions at
positions N1, D30, E46, V49, and E64 (e.g., N1G, D30N, E46G, V49R,
and E64Q), wherein the IL-15 variant is covalently linked to the Fc
domain of the antibody.
[0200] It is understood that the compositions can comprise more
than one IL-15 variant or IL-15 fusion protein (e.g., a mixture of
IL-15 variants or IL-15 fusions comprising different IL-15 variants
and/or different antibodies). For example, the composition
comprises 1) a human IL-15 fusion protein comprising a PD-1
antibody and a human IL-variant comprising amino acid substitutions
at positions E46G, V49R, E64Q, D30N, and N4K; and 2) an IL-15
fusion protein comprising a PD-1 antibody and a human IL-15 variant
comprising amino acid substitutions at positions V49N, I50A, S51T,
N4K, D30N, and E64Q. In another example, the composition comprises
1) a human IL-15 fusion protein comprising a PD-1 antibody and a
human IL-15 variant comprising amino acid substitutions at
positions N1A, D30N, E46G, and V49R; and 2) an IL-15 fusion protein
comprising a PD-1 antibody and a human IL-15 variant comprising
amino acid substitutions at positions N1G, D30N, E46G, V49R, and
E64Q.
[0201] The composition used in the present invention can further
comprise pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and practice of Pharmacy 20th
Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, and may comprise
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrans; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
Pharmaceutically acceptable excipients are further described
herein.
[0202] The IL-15 variants, IL-15 fusion proteins, and compositions
thereof can also be used in conjunction with, or administered
separately, simultaneously, or sequentially with other agents that
serve to enhance and/or complement the effectiveness of the
agents.
[0203] The invention also provides compositions, including
pharmaceutical compositions, comprising any of the polynucleotides
of the invention. In some embodiments, the composition comprises an
expression vector comprising a polynucleotide encoding the IL-15
variants and IL-15 fusion proteins as described herein. In other
embodiment, the composition comprises an expression vector
comprising a polynucleotide encoding any of the IL-15 variants and
IL-15 fusion proteins described herein.
Methods for Preventing or Treating Conditions Using IL-15 Variants
and IL-15 Fusion Proteins
[0204] The IL-15 variants and the IL-15 fusion proteins of the
present invention are useful in various applications including, but
are not limited to, therapeutic treatment methods and diagnostic
treatment methods.
[0205] In one aspect, the invention provides a method for treating
a cancer. In some embodiments, the method of treating a cancer in a
subject comprises administering to the subject in need thereof an
effective amount of a composition (e.g., pharmaceutical
composition) comprising any of the IL-15 variants and the IL-15
fusion proteins as described herein. As used herein, a cancer can
be a solid cancer or a liquid cancer. Solid cancers include, but
are not limited to, gastric cancer, small intestine cancer,
sarcoma, head and neck cancer (e.g., squamous cell head and neck
cancer), thymic cancer, epithelial cancer, salivary cancer, liver
cancer, biliary cancer, neuroendocrine tumors, stomach cancer,
thyroid cancer, lung cancer, mesothelioma, ovarian cancer, breast
cancer, prostate cancer, esophageal cancer, pancreatic cancer,
glioma, renal cancer (e.g., renal cell carcinoma), bladder cancer,
cervical cancer, uterine cancer, vulvar cancer, penile cancer,
testicular cancer, anal cancer, choriocarcinoma, colorectal cancer,
oral cancer, skin cancer, Merkel cell carcinoma, glioblastoma,
brain tumor, bone cancer, eye cancer, and melanoma.
[0206] Liquid cancers include, but not limited to, multiple
myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma,
nodular lymphocyte predominant Hodgkin's lymphoma, Kahler's disease
and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell
prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's
lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic
leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid
leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal
zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor
B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's
macroglobulienemia, diffuse large B cell lymphoma, follicular
lymphoma, marginal zone lymphoma, mucosa-associated lymphatic
tissue lymphoma, small cell lymphocytic lymphoma, mantle cell
lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large
B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenstrom
macroglobulinemia, nodal marginal zone B cell lymphoma, splenic
marginal zone lymphoma, intravascular large B-cell lymphoma,
primary effusion lymphoma, lymphomatoid granulomatosis, T
cell/histiocyte-rich large B-cell lymphoma, primary central nervous
system lymphoma, primary cutaneous diffuse large B-cell lymphoma
(leg type), EBV positive diffuse large B-cell lymphoma of the
elderly, diffuse large B-cell lymphoma associated with
inflammation, intravascular large B-cell lymphoma, ALK-positive
large B-cell lymphoma, plasmablastic lymphoma, large B-cell
lymphoma arising in HHV8-associated multicentric Castleman disease,
B-cell lymphoma unclassified with features intermediate between
diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma
unclassified with features intermediate between diffuse large
B-cell lymphoma and classical Hodgkin lymphoma, and other
hematopoietic cells related cancer.
[0207] In some embodiments, the cancer is relapsed, refractory, or
metastatic.
[0208] In some embodiments, provided is a method of inhibiting
tumor growth or progression in a subject, comprising administering
to the subject in need thereof an effective amount of a composition
comprising the IL-15 variants or IL-15 fusion proteins as described
herein. In some embodiments, provided is a method of inhibiting
metastasis of cancer cells in a subject, comprising administering
to the subject in need thereof an effective amount of a composition
comprising any of the IL-15 variants or IL-fusion proteins as
described herein. In other embodiments, provided is a method of
inducing regression of a tumor in a subject, comprising
administering to the subject in need thereof an effective amount of
a composition comprising any of the IL-15 variants or IL-15 fusion
proteins as described herein.
[0209] In another aspect, provided is a method of detecting,
diagnosing, and/or monitoring a cancer. For example, the IL-15
variants or IL-15 fusion proteins as described herein can be
labeled with a detectable moiety such as an imaging agent and an
enzyme-substrate label. The IL-15 variants or IL-15 fusion proteins
as described herein can also be used for in vivo diagnostic assays,
such as in vivo imaging (e.g., PET or SPECT), or a staining
reagent.
[0210] In some embodiments, the methods described herein further
comprise a step of treating a subject with an additional form of
therapy. In some embodiments, the additional form of therapy is an
additional anti-cancer therapy including, but not limited to,
chemotherapy, radiation, surgery, hormone therapy, and/or
additional immunotherapy.
[0211] With respect to all methods described herein, reference to
IL-15 variants or IL-15 fusion proteins also includes compositions
comprising one or more additional agents. These compositions may
further comprise suitable excipients, such as pharmaceutically
acceptable excipients including buffers, which are well known in
the art. The present invention can be used alone or in combination
with other methods of treatment.
[0212] The IL-15 variants or IL-15 fusion proteins as described
herein can be administered to a subject via any suitable route. It
should be apparent to a person skilled in the art that the examples
described herein are not intended to be limiting but to be
illustrative of the techniques available. Accordingly, in some
embodiments, the IL-variant or IL-15 fusion protein is administered
to a subject in accord with known methods, such as intravenous
administration, e.g., as a bolus or by continuous infusion over a
period of time, by intramuscular, intraperitoneal,
intracerebrospinal, transdermal, subcutaneous, intra-articular,
sublingually, intrasynovial, via insufflation, intrathecal, oral,
inhalation or topical routes. Administration can be systemic, e.g.,
intravenous administration, or localized. Commercially available
nebulizers for liquid formulations, including jet nebulizers and
ultrasonic nebulizers are useful for administration. Liquid
formulations can be directly nebulized and lyophilized powder can
be nebulized after reconstitution. Alternatively, the IL-15
variants or IL-15 fusion proteins can be aerosolized using a
fluorocarbon formulation and a metered dose inhaler, or inhaled as
a lyophilized and milled powder.
[0213] In some embodiments, an IL-15 variant or IL-15 fusion
protein is administered via site-specific or targeted local
delivery techniques. Examples of site-specific or targeted local
delivery techniques include various implantable depot sources of
the IL-variants or IL-15 fusion proteins or local delivery
catheters, such as infusion catheters, indwelling catheters, or
needle catheters, synthetic grafts, adventitial wraps, shunts and
stents or other implantable devices, site specific carriers, direct
injection, or direct application. See, e.g., PCT Publication No. WO
00/53211 and U.S. Pat. No. 5,981,568.
[0214] Various formulations of an IL-15 variant or IL-15 fusion
protein may be used for administration. In some embodiments, the
IL-15 variant or IL-15 fusion protein may be administered neat. In
some embodiments, the IL-15 variant or IL-15 fusion protein and a
pharmaceutically acceptable excipient may be in various
formulations. Pharmaceutically acceptable excipients are known in
the art, and are relatively inert substances that facilitate
administration of a pharmacologically effective substance. For
example, an excipient can give form or consistency, or act as a
diluent. Suitable excipients include but are not limited to
stabilizing agents, wetting and emulsifying agents, salts for
varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers. Excipients as well as formulations for
parenteral and nonparenteral drug delivery are set forth in
Remington, The Science and Practice of Pharmacy 20th Ed. Mack
Publishing, 2000.
[0215] In some embodiments, these agents are formulated for
administration by injection (e.g., intraperitoneally,
intravenously, subcutaneously, intramuscularly, etc.). Accordingly,
these agents can be combined with pharmaceutically acceptable
vehicles such as saline, Ringer's solution, dextrose solution, and
the like. The particular dosage regimen, i.e., dose, timing and
repetition, will depend on the particular individual and that
individual's medical history.
[0216] The IL-15 variants or IL-15 fusion proteins described herein
can be administered using any suitable method, including by
injection (e.g., intraperitoneally, intravenously, subcutaneously,
intramuscularly, etc.). The IL-15 variants or IL-15 fusion proteins
can also be administered topically or via inhalation, as described
herein. Generally, for administration of IL-15 variants or IL-15
fusion proteins, the candidate dosage can be administered daily,
every week, every other week, every three weeks, every four weeks,
every five weeks, every six weeks, every seven weeks, every eight
weeks, every ten weeks, every twelve weeks, or more than every
twelve weeks. For repeated administrations over several days or
longer, depending on the condition, the treatment is sustained
until a desired suppression of symptoms occurs or until sufficient
therapeutic levels are achieved, for example, to reduce symptoms
associated with cancer. The progress of this therapy is easily
monitored by conventional techniques and assays. The dosing regimen
(including the specific IL-15 variants or IL-15 fusion proteins
used) can vary over time.
[0217] In some embodiments, the candidate dosage is administered
daily with the dosage ranging from about any of 1 pg/kg to 30 pg/kg
to 300 pg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more,
depending on the factors mentioned above. For example, daily dosage
of about 0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3
mg/kg, about 1 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 5
mg/kg, about 10 mg/kg, about 15 mg/kg, and about 25 mg/kg may be
used.
[0218] In some embodiments, the candidate dosage is administered
every week with the dosage ranging from about any of 1 pg/kg to 30
pg/kg to 300 pg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more,
depending on the factors mentioned above. For example, a weekly
dosage of about 0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg,
about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2.5 mg/kg,
about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about
25 mg/kg, and about 30 mg/kg may be used.
[0219] In some embodiments, the candidate dosage is administered
every two weeks with the dosage ranging from about any of 1 pg/kg
to 30 pg/kg to 300 pg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or
more, depending on the factors mentioned above. For example, a
bi-weekly dosage of about 0.1 mg/kg, about 0.3 mg/kg, about 1
mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10
mg/kg, about 15 mg/kg, about 25 mg/kg, and about 30 mg/kg may be
used.
[0220] In some embodiments, the candidate dosage is administered
every three weeks with the dosage ranging from about any of 1 pg/kg
to 30 pg/kg to 300 pg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or
more, depending on the factors mentioned above. For example, a
tri-weekly dosage of about 0.1 mg/kg, about 0.3 mg/kg, about 1
mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10
mg/kg, about 15 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35
mg/kg, about 40 mg/kg, about 45 mg/kg, and about 50 mg/kg may be
used.
[0221] In some embodiments, the candidate dosage is administered
every month or every four weeks with the dosage ranging from about
any of 1 pg/kg to 30 pg/kg to 300 pg/kg to 3 mg/kg, to 30 mg/kg, to
100 mg/kg or more, depending on the factors mentioned above. For
example, a monthly dosage of about 0.1 mg/kg, about 0.3 mg/kg,
about 1 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 5 mg/kg, about
10 mg/kg, about 15 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35
mg/kg, about 40 mg/kg, about 45 mg/kg, and about 50 mg/kg may be
used.
[0222] In other embodiments, the candidate dosage is administered
daily with the dosage ranging from about 0.01 mg to about 1200 mg
or more, depending on the factors mentioned above. For example,
daily dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10
mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about
400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg,
about 900 mg, about 1000 mg, about 1100 mg, or about 1200 mg may be
used.
[0223] In other embodiments, the candidate dosage is administered
every week with the dosage ranging from about 0.01 mg to about 2000
mg or more, depending on the factors mentioned above. For example,
weekly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10
mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about
400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg,
about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about
1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700
mg, about 1800 mg, about 1900 mg, or about 2000 mg may be used.
[0224] In other embodiments, the candidate dosage is administered
every two weeks with the dosage ranging from about 0.01 mg to about
2000 mg or more, depending on the factors mentioned above. For
example, bi-weekly dosage of about 0.01 mg, about 0.1 mg, about 1
mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300
mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about
800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg,
about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about
1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg may be
used.
[0225] In other embodiments, the candidate dosage is administered
every three weeks with the dosage ranging from about 0.01 mg to
about 2500 mg or more, depending on the factors mentioned above.
For example, tri-weekly dosage of about 0.01 mg, about 0.1 mg,
about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg,
about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700
mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about
1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600
mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg,
about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, or
about 2500 mg may be used.
[0226] In other embodiments, the candidate dosage is administered
every four weeks or month with the dosage ranging from about 0.01
mg to about 3000 mg or more, depending on the factors mentioned
above. For example, monthly dosage of about 0.01 mg, about 0.1 mg,
about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg,
about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700
mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about
1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600
mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg,
about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about
2500, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg,
or about 3000 mg may be used.
[0227] In some embodiments, a therapeutic of the present invention
is administered at a dose ranging from about 1 .mu.g/kg to about
600 .mu.g/kg or more, about 6 .mu.g/kg to about 600 .mu.g/kg, about
6 .mu.g/kg to about 300 .mu.g/kg, about 30 .mu.g/kg to about 600
.mu.g/kg or about 30 .mu.g/kg to about 300 .mu.g/kg. For example,
the dose is administered at about 1 .mu.g/kg, about 2 .mu.g/kg,
about 3 .mu.g/kg, about 4 .mu.g/kg, about 5 .mu.g/kg, about 6
.mu.g/kg, about 7 .mu.g/kg, about 8 .mu.g/kg, about 9 .mu.g/kg,
about 10 .mu.g/kg, about 15 .mu.g/kg, about 20 .mu.g/kg, about 25
.mu.g/kg, about 30 .mu.g/kg, about 35 .mu.g/kg, about 40 .mu.g/kg,
about 45 .mu.g/kg, about 50 .mu.g/kg, about 55 .mu.g/kg, about 60
.mu.g/kg, about 65 .mu.g/kg, about 70 .mu.g/kg, about 75 .mu.g/kg,
about 80 .mu.g/kg, about 85 .mu.g/kg, about 90 .mu.g/kg, about 95
.mu.g/kg, about 100 .mu.g, about 110 .mu.g/kg, about 120 .mu.g/kg,
about 130 .mu.g/kg, about 140 .mu.g/kg, about 150 .mu.g/kg, about
160 .mu.g/kg, about 170 .mu.g/kg, about 180 .mu.g/kg, about 190
.mu.g/kg, about 200 .mu.g/kg, about 210 .mu.g/kg, about 220
.mu.g/kg, about 230 .mu.g/kg, about 240 .mu.g/kg, about 250
.mu.g/kg, about 260 .mu.g/kg, about 270 .mu.g/kg, about 280
.mu.g/kg, about 290 .mu.g/kg, about 300 .mu.g/kg, about 350
.mu.g/kg, about 400 .mu.g/kg, about 450 .mu.g/kg, about 500
.mu.g/kg, about 550 .mu.g/kg or about 600 .mu.g/kg may be used.
[0228] For the purpose of the present invention, the appropriate
dosage of an IL-15 variant or an IL-15 fusion protein will depend
on the IL-15 variant or an IL-15 fusion protein (or compositions
thereof) employed, the type and severity of symptoms to be treated,
whether the agent is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the agent, the patient's clearance rate for the
administered agent, and the discretion of the attending physician.
Typically the clinician will administer an IL-15 variant or an
IL-15 fusion protein until a dosage is reached that achieves the
desired result. Dose and/or frequency can vary over course of
treatment. Empirical considerations, such as the half-life,
generally will contribute to the determination of the dosage.
Frequency of administration may be determined and adjusted over the
course of therapy, and is generally, but not necessarily, based on
treatment and/or suppression and/or amelioration and/or delay of
symptoms. Alternatively, sustained continuous release formulations
of IL-15 variants or IL-15 fusion proteins may be appropriate.
Various formulations and devices for achieving sustained release
are known in the art.
[0229] In one embodiment, dosages for an IL-15 variant or an IL-15
fusion protein may be determined empirically in individuals who
have been given one or more administration(s) of an IL-15 variant
or an IL-15 fusion protein. For example, individuals are given
incremental dosages of an IL-15 variant or an IL-15 fusion protein.
To assess efficacy, an indicator of the disease can be
followed.
[0230] Administration of an IL-15 variant or an IL-15 fusion
protein as described herein in accordance with the method in the
present invention can be continuous or intermittent, depending, for
example, upon the recipient's physiological condition, whether the
purpose of the administration is therapeutic or prophylactic, and
other factors known to skilled practitioners. The administration of
an IL-15 variant or an IL-15 fusion protein may be essentially
continuous over a preselected period of time or may be in a series
of spaced doses.
[0231] In some embodiments, more than one IL-15 variant or IL-15
fusion protein may be present. At least one, at least two, at least
three, at least four, at least five different, or more IL-15
variants or an IL-15 fusion proteins can be present. Generally,
those IL-variants or IL-15 fusion proteins may have complementary
activities that do not adversely affect each other.
[0232] In some embodiments, the IL-15 variant or the IL-15 fusion
protein may be administered in combination with the administration
of one or more additional therapeutic agents. These include, but
are not limited to, the administration of a biotherapeutic agent, a
chemotherapeutic agent, a vaccine, a CAR-T cell-based therapy,
radiotherapy, another cytokine therapy (e.g., immunostimulatory
cytokines including various signaling proteins that stimulate
immune response, such as interferons, interleukins, and
hematopoietic growth factors), a vaccine, an inhibitor of other
immunosuppressive pathways, an inhibitors of angiogenesis, a T cell
activator, an inhibitor of a metabolic pathway, an mTOR
(mechanistic target of rapamycin) inhibitor (e.g., rapamycin,
rapamycin derivatives, sirolimus, temsirolimus, everolimus, and
deforolimus), an inhibitor of an adenosine pathway, a tyrosine
kinase inhibitor including but not limited to inlyta, ALK
(anaplastic lymphoma kinase) inhibitors (e.g., crizotinib,
ceritinib, alectinib, and sunitinib), a BRAF inhibitor (e.g.,
vemurafenib and dabrafenib), an epigenetic modifier, an inhibitors
or depletor of Treg cells and/or of myeloid-derived suppressor
cells, a JAK (Janus Kinase) inhibitor (e.g., ruxolitinib and
tofacitinb, varicitinib, filgotinib, gandotinib, lestaurtinib,
momelotinib, pacritinib, and upadacitinib), a STAT (Signal
Transducers and Activators of Transcription) inhibitor (e.g.,
STAT1, STAT3, and STAT5 inhibitors such as fludarabine), a
cyclin-dependent kinase inhibitor, an immunogenic agent (for
example, attenuated cancerous cells, tumor antigens, antigen
presenting cells such as dendritic cells pulsed with tumor derived
antigen or nucleic acids, a MEK inhibitor (e.g., trametinib,
cobimetinib, binimetinib, and selumetinib), a GLS1 inhibitor, a PAP
inhibitor, an oncolytic virus, an IDO (Indoleamine-pyrrole
2,3-dioxygenase) inhibitor, a PRR (Pattern Recognition Receptors)
agonist, and cells transfected with genes encoding immune
stimulating cytokines such as but not limited to GM-CSF).
[0233] In some embodiments, exemplary immunostimulatory cytokines
include, but are not limited to, GM-CSF, G-CSF, IFN.gamma.,
IFN.alpha.; IL-2 (e.g. denileukin difitox), IL-6, IL-7, IL-10,
IL-11, IL-12, IL-15, IL-18, IL-21, and TNF.alpha.. In some
embodiments, the cytokines are pegylated (e.g., pegylated IL-2,
IL-10, IFN.gamma., and IFN.alpha.).
[0234] Pattern recognition receptors (PRRs) are receptors that are
expressed by cells of the immune system and that recognize a
variety of molecules associated with pathogens and/or cell damage
or death. PRRs are involved in both the innate immune response and
the adaptive immune response. PRR agonists may be used to stimulate
the immune response in a subject. There are multiple classes of PRR
molecules, including toll-like receptors (TLRs), RIG-1-like
receptors (RLRs), nucleotide-binding oligomerization domain
(NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), and
Stimulator of Interferon Genes (STING) protein.
[0235] The terms "TLR" and "toll-like receptor" refer to any
toll-like receptor. Toll-like receptors are receptors involved in
activating immune responses. TLRs recognize, for example,
pathogen-associated molecular patterns (PAMPs) expressed in
microbes, as well as endogenous damage-associated molecular
patterns (DAMPs), which are released from dead or dying cells.
[0236] Molecules which activate TLRs (and thereby activate immune
responses) are referred to herein as "TLR agonists". TLR agonists
can include, for example, small molecules (e.g. organic molecule
having a molecular weight under about 1000 Daltons), as well as
large molecules (e.g. oligonucleotides and proteins). Some TLR
agonists are specific for a single type of TLR (e.g. TLR3 or TLR9),
while some TLR agonists activate two or more types of TLR (e.g.
both TLR7 and TLR8).
[0237] Exemplary TLR agonists provided herein include agonists of
TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.
[0238] Exemplary small molecule TLR agonists include those
disclosed in, for example, U.S. Pat. Nos. 4,689,338; 4,929,624;
5,266,575; 5,268,376; 5,346,905; 5,352,784; 5,389,640; 5,446,153;
5,482,936; 5,756,747; 6,110,929; 6,194,425; 6,331,539; 6,376,669;
6,451,810; 6,525,064; 6,541,485; 6,545,016; 6,545,017; 6,573,273;
6,656,938; 6,660,735; 6,660,747; 6,664,260; 6,664,264; 6,664,265;
6,667,312; 6,670,372; 6,677,347; 6,677,348; 6,677,349; 6,683,088;
6,756,382; 6,797,718; 6,818,650; and 7,7091,214; U.S. Patent
Publication Nos. 2004/0091491, 2004/0176367, and 2006/0100229; and
International Publication Nos. WO 2005/18551, WO 2005/18556, WO
2005/20999, WO 2005/032484, WO 2005/048933, WO 2005/048945, WO
2005/051317, WO 2005/051324, WO 2005/066169, WO 2005/066170, WO
2005/066172, WO 2005/076783, WO 2005/079195, WO 2005/094531, WO
2005/123079, WO 2005/123080, WO 2006/009826, WO 2006/009832, WO
2006/026760, WO 2006/028451, WO 2006/028545, WO 2006/028962, WO
2006/029115, WO 2006/038923, WO 2006/065280, WO 2006/074003, WO
2006/083440, WO 2006/086449, WO 2006/091394, WO 2006/086633, WO
2006/086634, WO 2006/091567, WO 2006/091568, WO 2006/091647, WO
2006/093514, and WO 2006/098852.
[0239] Additional examples of small molecule TLR agonists include
certain purine derivatives (such as those described in U.S. Pat.
Nos. 6,376,501, and 6,028,076), certain imidazoquinoline amide
derivatives (such as those described in U.S. Pat. No. 6,069,149),
certain imidazopyridine derivatives (such as those described in
U.S. Pat. No. 6,518,265), certain benzimidazole derivatives (such
as those described in U.S. Pat. No. 6,387,938), certain derivatives
of a 4-aminopyrimidine fused to a five membered nitrogen containing
heterocyclic ring (such as adenine derivatives described in U.S.
Pat. Nos. 6,376,501; 6,028,076 and 6,329,381; and in WO 02/08905),
and certain 3-.beta.-D-ribofuranosylthiazolo [4,5-d]pyrimidine
derivatives (such as those described in U.S. Publication No.
2003/0199461), and certain small molecule immuno-potentiator
compounds such as those described, for example, in U.S. Patent
Publication No. 2005/0136065.
[0240] Exemplary large molecule TLR agonists include as
oligonucleotide sequences. Some TLR agonist oligonucleotide
sequences contain cytosine-guanine dinucleotides (CpG) and are
described, for example, in U.S. Pat. Nos. 6,194,388; 6,207,646;
6,239,116; 6,339,068; and 6,406,705. Some CpG-containing
oligonucleotides can include synthetic immunomodulatory structural
motifs such as those described, for example, in U.S. Pat. Nos.
6,426,334 and 6,476,000. Other TLR agonist nucleotide sequences
lack CpG sequences and are described, for example, in International
Patent Publication No. WO 00/75304. Still other TLR agonist
nucleotide sequences include guanosine- and uridine-rich
single-stranded RNA (ssRNA) such as those described, for example,
in Heil et ah, Science, vol. 303, pp. 1526-1529, Mar. 5, 2004.
[0241] Other TLR agonists include biological molecules such as
aminoalkyl glucosaminide phosphates (AGPs) and are described, for
example, in U.S. Pat. Nos. 6,113,918; 6,303,347; 6,525,028; and
6,649,172.
[0242] TLR agonists also include inactivated pathogens or fractions
thereof, which may activate multiple different types of TLR
receptor. Exemplary pathogen-derived TLR agonists include BCG,
Mycobacterium obuense extract, Talimogene laherparepvec (T-Vec)
(derived from HSV-1), and Pexa-Vec (derived from vaccina
virus).
[0243] In some embodiments, a TLR agonist may be an agonist
antibody that binds specifically to the TLR.
[0244] In some embodiments, the biotherapeutic agent is an
antibody, including but not limited to, an anti-CTLA-4 antibody, an
anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an
anti-4-1 BB antibody, an anti-PD-1 antibody, an anti-PD-L1
antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an
anti-TIGIT antibody, an anti-OX40 antibody, an anti-IL-7Ralpha
(CD127) antibody, an anti-IL-8 antibody, an anti-IL-15 antibody, an
anti-HVEM antibody, an anti-BTLA antibody, an anti-CD40 antibody,
an anti-CD40L antibody, anti-CD47 antibody, an anti-CSF1R antibody,
an anti-CSF1 antibody, an anti-IL-7R antibody, an anti-MARCO
antibody, an anti-CXCR4 antibodies, an anti-VEGF antibody, an
anti-VEGFR1 antibody, an anti-VEGFR2 antibody, an anti-TNFR1
antibody, an anti-TNFR2 antibody, an anti-CD3 bispecific antibody,
an anti-CD19 antibody, an anti-CD20, an anti-Her2 antibody, an
anti-EGFR antibody, an anti-ICOS antibody, an anti-CD22 antibody,
an anti-CD 52 antibody, an anti-CCR4 antibody, an anti-CCR8
antibody, an anti-CD200R antibody, an anti-VISG4 antibody, an
anti-CCR2 antibody, an anti-LILRb2 antibody, an anti-CXCR4
antibody, an anti-CD206 antibody, an anti-CD163 antibody, an
anti-KLRG1 antibody, an anti-FLT3 antibody, an anti-B7-H4 antibody,
an anti-B7-H3 antibody, an KLRG1 antibody, a BTN1A1 antibody, a
BCMA antibody, or an anti-GITR antibody.
[0245] In some embodiments, an IL-15 variant or an IL-15 fusion
protein is used in combination with an immunocytokine. In some
embodiments, the immunocytokine comprises an antibody, or fragment
thereof, conjugated or fused to a cytokine (e.g. fusion protein).
In some embodiments, the antibody, or fragment thereof, binds the
Extra Domain-A (EDA) isoform of fibronectin (e.g. anti-EDA
antibody). For example, the anti-EDA antibody, or fragment thereof,
comprises a CDR1, a CDR2 and CDR3 of the heavy chain variable (VH)
region shown in SEQ ID NO: 94 and/or a CDR1, a CDR2 and CDR3 of the
light chain variable (VL) region shown in SEQ ID NO: 96. In some
embodiments, the anti-EDA antibody, or fragment thereof, comprises
a VH region having the amino acid sequence of SEQ ID NO: 94 and/or
a VL region having the amino acid sequence of SEQ ID NO: 96. In
some embodiments the cytokine is IL-10. For example, IL-10 may
comprise the amino acid sequence of SEQ ID NO: 98. In some
embodiments, the immunocytokine comprises at least one linker. For
example, the linker(s) may comprise SEQ ID NO: 95 and/or 97. In
some embodiments, the immunocytokine is an anti-EDA-IL-10 fusion
protein comprising the amino acid sequence of SEQ ID NO: 99 (Table
1.3, CDRs underlined).
TABLE-US-00004 TABLE 1.3 SEQ ID NO: SEQUENCE 94
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLWVSAI Anti-EDA
SGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTH Ab VH
LYLFDYWGQGTLVTVSS 95 ggsgg Linker 96
EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIY Anti-EDA
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFG Ab VL QGTKVEIK
97 ssssgssssgssssg Linker 98
SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKE IL-10
SLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKT
LRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYI EAYMTMKIRN 99
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSA Anti-EDA-
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKST IL-10
HLYLFDYWGQGTLVTVSSqqsqqEIVLTQSPGTLSLSPGERATLSCRASQ Fusion
SVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS Protein
RLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKssssgssssgssssgSPGQ
GTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLE
DFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLR
LRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYM TMKIRN
[0246] Accordingly, in some embodiments, an IL-15 variant or an
IL-15 fusion protein is used in conjunction with, for example, an
anti-PD-L1 antagonist antibody; an anti-PD-1 antagonist antibody
such as for example, nivolumab (OPDIVO.RTM.), pembrolizumab
(KEYTRUDA.RTM.), mAb7 (e.g., as described in US Pub. No.
US20i60i59905, hereby incorporated by reference), and pidilizumab;
an anti-CTLA-4 antagonist antibody such as for example ipilimumab
(YERVOY.RTM.); an anti-LAG-3 antagonist antibody such as BMS-9860i6
and IMP70i; an anti-TIM-3 antagonist antibody; an anti-17-1H3
antagonist antibody such as for example MGA27i; an-anti-VISTA
antagonist antibody; an anti-TIGIT antagonist antibody; an
anti-CD28 antagonist antibody; an anti-CD80 antibody; an anti-CD86
antibody; an anti-B7-H4 antagonist antibody; an anti-ICOS agonist
antibody; an anti-CD28 agonist antibody; an innate immune response
modulator (e.g., TLRs, KIR, NKG2A), and an IDO inhibitor. In some
embodiments, an IL-15 variant or an IL-15 fusion protein is used in
conjunction with a 4-1BB (CD137) agonist such as, for example,
PF-05082566 or urelumab (BMS-663513). In some embodiments, an IL-15
variant or an IL-15 fusion protein is used in conjunction with an
OX40 agonist such as, for example, an anti-OX-40 agonist antibody.
In some embodiments, an IL-15 variant or an IL-15 fusion protein is
used in conjunction with a GITR agonist such as, for example,
TRX518. In some embodiments, an IL-15 variant or an IL-15 fusion
protein is used in conjunction with an IDO inhibitor. In some
embodiments, an IL-15 variant or an IL-15 fusion protein is used in
conjunction with a cytokine therapy such as, for example without
limitation, (pegylated or non-pegylated) IL-2, IL-10, IL-12, IL-7,
IL-15, IL-21, IL-33, CSF-1, MCSF-1, etc.
[0247] In some embodiments, other examples of the antibody for the
combination use with the IL-15 variant or the IL-15 fusion protein
of the present invention can be directed to, 5T4; A33; alpha-folate
receptor 1 (e.g. mirvetuximab soravtansine); Alk-1; CA-125 (e.g.
abagovomab); Carboanhydrase IX; CCR2; CCR4 (e.g. mogamulizumab);
CCR5 (e.g. Ieronlimab); CCR8; CD3 [e.g. blinatumomab (CD3/CD19
bispecific), PF-06671008 (CD3/P-cadherin bispecific), PF-06863135
(CD3/BCMA bispecific), CD25; CD28; CD30 (e.g. brentuximab vedotin);
CD33 (e.g. gemtuzumab ozogamicin); CD38 (e.g. daratumumab,
isatuximab), CD44v6; CD63; CD79 (e.g. polatuzumab vedotin); CD80;
CD123; CD276/B7-H3 (e.g. omburtamab); CDH17; CEA; ClhCG; desmoglein
4; DLL3 (e.g. rovalpituzumab tesirine); DLL4; E-cadherin; EDA; EDB;
EFNA4; EGFR (e.g. cetuximab, depatuxizumab mafodotin, necitumumab,
panitumumab); EGFRvIII; Endosialin; EpCAM (e.g. oportuzumab
monatox); FAP; Fetal Acetylcholine Receptor; FLT3 (e.g. see
WO2018/220584); GD2 (e.g. dinutuximab, 3F8); GD3; GloboH; GM1; GM2;
HER2/neu [e.g. margetuximab, pertuzumab, trastuzumab;
ado-trastuzumab emtansine, trastuzumab duocarmazine, PF-06804103
(see U.S. Pat. No. 8,828,401)]; HER3; HER4; ICOS; IL-10; ITG-AvB6;
LAG-3 (e.g. relatlimab); Lewis-Y; LG; Ly-6; M-CSF [e.g. PD-0360324
(see U.S. Pat. No. 7,326,414)]; MCSP; mesothelin; MUC1; MUC2; MUC3;
MUC4; MUCSAC; MUCSB; MUC7; MUC16; Notch1; Notch3; Nectin-4 (e.g.
enfortumab vedotin); P-Cadherein [e.g. PF-06671008 (see
WO2016/001810)]; PCDHB2; PDGFRA (e.g. olaratumab); Plasma Cell
Antigen; PolySA; PSCA; PSMA; PTK7 [e.g. PF-06647020 (see U.S. Pat.
No. 9,409,995)]; Ror1; SAS; SCRx6; SLAMF7 (e.g. elotuzumab); SHH;
SIRPa (e.g. ED9, Effi-DEM); STEAP; TGF-beta; TIGIT; TMPRSS3;
TNF-alpha precursor; TROP-2 (e.g., sacituzumab govitecan); TSPAN8;
and Wue-1.
[0248] Examples of chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
the enediyne antibiotics (e.g. calicheamicin, especially
calicheamicin gamma1I and calicheamicin phil1, see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromomophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidamine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,
2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoids such as retinoic acid;
capecitabine; and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Also included are anti-hormonal
agents that act to regulate or inhibit hormone action on tumors
such as anti-estrogens and selective estrogen receptor modulators
(SERMs), including, for example, tamoxifen, raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and toremifene (Fareston); aromatase inhibitors that
inhibit the enzyme aromatase, which regulates estrogen production
in the adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, megestrol acetate, exemestane, formestane,
fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens
such as flutamide, nilutamide, bicalutamide, leuprolide, fluridil,
apalutamide, enzalutamide, cimetidine and goserelin; KRAS
inhibitors; MCT4 inhibitors; MAT2a inhibitors; tyrosine kinase
inhibitors such as sunitinib, axitinib; alk/c-Met/ROS inhibitors
such as crizotinib, lorlatinib; mTOR inhibitors such as
temsirolimus, gedatolisib; src/abl inhibitors such as bosutinib;
cyclin-dependent kinase (CDK) inhibitors such as palbociclib,
PF-06873600; erb inhibitors such as dacomitinib; PARP inhibitors
such as talazoparib; SMO inhibitors such as glasdegib, PF-5274857;
EGFR T790M inhibitors such as PF-06747775; EZH2 inhibitors such as
PF-06821497; PRMT5 inhibitors such as PF-06939999; TGFR.beta.r1
inhibitors such as PF-06952229; and pharmaceutically acceptable
salts, acids or derivatives of any of the above.
[0249] In some embodiments, an IL-15 variant or an IL-15 fusion
protein is used in conjunction with one or more other therapeutic
agents targeting an immune checkpoint modulator, such as, for
example without limitation, an agent targeting PD-1, PD-1, CTLA-4,
LAG-3, B7-H3, B7-H4, B7-DC (PD-L2), B7-H5, B7-H6, B7-H8, B7-H2,
B7-1, B7-2, ICOS, ICOS-L, TIGIT, CD2, CD47, CD80, CD86, CD48, CD58,
CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4,
VISTA (PD-H1), OX40, OX40L, GITRL, CD70, CD27, 4-1BB, 4-BBL, DR3,
TL1A, CD40, CD40L, CD30, CD30L, LIGHT, HVEM, SLAM (SLAMF1, CD150),
SLAMF2 (CD48), SLAMF3 (CD229), SLAMF4 (2B4, CD244), SLAMF5 (CD84),
SLAMF6 (NTB-A), SLAMCF7 (CS1), SLAMF8 (BLAME), SLAMF9 (CD2F), CD28,
CEACAM1(CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7,
CEACAM8, CEACAM1-3AS CEACAM3C2, CEACAM1-15, PSG1-11, CEACAM1-4C1,
CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, CD39-CD73-adenosine pathway
(A2AR), BTKs, TIKs, CXCR2, CCR4, CCR8, CCR5, VEGF pathway, CSF-1,
or an innate immune response modulator.
[0250] In some embodiments, an IL-15 variant or an IL-15 fusion
protein is used in conjunction with a biotherapeutic agent and a
chemotherapeutic agent. For example, provided is a method for
treating cancer in a subject in need thereof comprising
administering to the subject an effective amount of the IL-15
variant or IL-15 fusion protein as described wherein, an anti-PD-L1
antagonist antibody, and a chemotherapeutic agent (e.g.,
gemcitabine, methotrexate, or a platinum analog). In some
embodiments, provided is a method for treating cancer in a subject
in need thereof comprising administering to the subject an
effective amount of the IL-15 variant or IL-15 fusion protein as
described wherein, an anti-PD-1 antagonist antibody (e.g.,
nivolumab (OPDIVO.RTM.), mAb7 (e.g., as described in US Pub. No.
US20160159905, hereby incorporated by reference), or pembrolizumab
(KEYTRUDA.RTM.), and a chemotherapeutic agent (e.g., gemcitabine,
methotrexate, or a platinum analog). In some embodiments, provided
is a method for treating cancer in a subject in need thereof
comprising administering to the subject an effective amount of the
IL-15 variant or IL-15 fusion protein as described wherein, an
anti-CTLA-4 antagonist antibody (e.g., ipilimumab (YERVOY.RTM.)),
and a chemotherapeutic agent (e.g., gemcitabine, methotrexate, or a
platinum analog).
[0251] In some embodiments, the IL-15 variant or IL-15 fusion
protein therapy may precede or follow the other agent treatment by
intervals ranging from minutes to weeks. In embodiments where the
other agents and/or a proteins or polynucleotides are administered
separately, one would generally ensure that a significant period of
time did not expire between each delivery, such that the agent and
the composition of the present invention would still be able to
exert an advantageously combined effect on the subject. In such
instances, it is contemplated that one may administer both
modalities within about 12-24 h of each other and, more preferably,
within about 6-12 h of each other. In some situations, it may be
desirable to extend the time period for administration
significantly, however, where several days (2, 3, 4, 5, 6 or 7) to
several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0252] In some embodiments, an IL-15 variant or an IL-15 fusion
protein composition comprises a second agent selected from
crizotinib, palbociclib, gemcitabine, cyclophosphamide,
fluorouracil, FOLFOX, folinic acid, oxaliplatin, axitinib,
sunitinib malate, tofacitinib, bevacizumab, rituximab, and
trastuzumab.
[0253] In some embodiments, an IL-15 variant or IL-15 fusion
protein composition is combined with a treatment regimen further
comprising a traditional therapy selected from the group consisting
of: surgery, radiation therapy, chemotherapy, targeted therapy,
immunotherapy, hormonal therapy, angiogenesis inhibition and
palliative care.
Formulations
[0254] Therapeutic formulations of the IL-15 variant or IL-15
fusion protein used in accordance with the present invention are
prepared for storage by mixing the protein having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing, 2000), in the form
of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and may comprise buffers
such as phosphate, citrate, and other organic acids; salts such as
sodium chloride; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens, such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0255] Liposomes containing the IL-15 variant or IL-15 fusion
protein are prepared by methods known in the art, such as described
in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985);
Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S.
Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced
circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising
phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0256] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington, The Science and Practice of
Pharmacy 20th Ed. Mack Publishing (2000).
[0257] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0258] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes. Therapeutic IL-15
variant or IL-15 fusion protein compositions are generally placed
into a container having a sterile access port, for example, an
intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
[0259] The compositions according to the present invention may be
in unit dosage forms such as tablets, pills, capsules, powders,
granules, solutions or suspensions, or suppositories, for oral,
parenteral or rectal administration, or administration by
inhalation or insufflation.
[0260] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g. conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g. water, to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
about 0.1 to about 500 mg of the active ingredient of the present
invention. The tablets or pills of the novel composition can be
coated or otherwise compounded to provide a dosage form affording
the advantage of prolonged action. For example, the tablet or pill
can comprise an inner dosage and an outer dosage component, the
latter being in the form of an envelope over the former. The two
components can be separated by an enteric layer that serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0261] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g. Span.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently comprise between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0262] Suitable emulsions may be prepared using commercially
available fat emulsions, such as Intralipid.TM., Liposyn.TM.,
Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can comprise
fat droplets between 0.1 and 1.0 .mu.m, particularly 0.1 and 0.5
.mu.m, and have a pH in the range of 5.5 to 8.0.
[0263] The emulsion compositions can be those prepared by mixing an
IL-15 variant or IL-15 fusion protein with Intralipid.TM. or the
components thereof (soybean oil, egg phospholipids, glycerol and
water).
[0264] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in preferably
sterile pharmaceutically acceptable solvents may be nebulised by
use of gases. Nebulised solutions may be breathed directly from the
nebulising device or the nebulising device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner. Kits
[0265] The invention also provides kits comprising any or all of
the IL-15 variants or IL-fusion proteins described herein. Kits of
the invention include one or more containers comprising an IL-15
variant or IL-15 fusion protein described herein and instructions
for use in accordance with any of the methods of the invention
described herein. Generally, these instructions comprise a
description of administration of the IL-variant or IL-15 fusion
protein for the above described therapeutic treatments. In some
embodiments, kits are provided for producing a single-dose
administration unit. In certain embodiments, the kit can contain
both a first container having a dried protein and a second
container having an aqueous formulation. In certain embodiments,
kits containing single and multi-chambered pre-filled syringes
(e.g., liquid syringes and lyosyringes) are included.
[0266] The instructions relating to the use of an IL-15 variant or
an IL-15 fusion protein generally include information as to dosage,
dosing schedule, and route of administration for the intended
treatment. The containers may be unit doses, bulk packages (e.g.,
multi-dose packages) or sub-unit doses. Instructions supplied in
the kits of the invention are typically written instructions on a
label or package insert (e.g., a paper sheet included in the kit),
but machine-readable instructions (e.g., instructions carried on a
magnetic or optical storage disk) are also acceptable.
[0267] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). A t least one active
agent in the composition is an IL-15 variant or an IL-15 fusion
protein. The container may further comprise a second
pharmaceutically active agent.
[0268] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container.
Biological Deposit
[0269] Representative materials of the present invention were
deposited in the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110-2209, USA, on Jan. 23, 2019. Vector
VK1-39 having ATCC Accession No. PTA-125686 is a polynucleotide
encoding the antibody VK1-39 light chain variable region, and
vector VH1-69b-hole-m2 having ATCC Accession No. PTA-125685 is a
polynucleotide encoding the VH1-69 heavy chain variable region and
the IL-15 variant M2. The deposits were made under the provisions
of the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purpose of Patent Procedure and
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposit will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between Pfizer,
Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 U.S.C. .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 C.F.R. .sctn.
1.14 with particular reference to 886 OG 638).
[0270] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0271] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
EXAMPLES
Example 1: Preparation of Human IL-15 Variants in Condition Media
and the Purification of Antibody-IL-15 Fusion Proteins
[0272] This example illustrates the production of human IL-15
variants in condition media and the purification of various IL-15
fusion proteins as described herein. Four micrograms of DNA of each
carboxyl-terminal 8-histidine-AVI-tagged IL-15 mutants having the
mutations of as showing in Tables 1-2 were transiently transfected
into Expi-293 cells at a density of about 3.times.10.sup.6 cells
per mL in each well of 24-well blocks. After five days of
post-transfection, condition media supernatant of the cells that
contain the secreted IL-15 mutants was harvested by centrifugation,
transferred to a 96-well block and subsequently filtered and ready
for kinetics and affinity determination.
[0273] Antibody-IL-15 fusion proteins (e.g., PD-1 antibody with
IL-15 mutations as shown in Tables 1-2) were produced by transient
transfection with the DNA plasmids of antibody heavy chain-IL-15
chimera, antibody heavy chain and antibody light chain in Expi-293
cells at a density of about 3.times.10.sup.6 cells per mL. After
five days of post-transfection, supernatant of the cells that
contained the secreted antibody-IL-15 fusion proteins was harvested
by centrifugation and subsequently filtered. The antibody-IL-15
fusion proteins were then purified from the cell supernatant using
Protein A affinity chromatography on a MabSelectSuRe column (GE
Lifesciences, Marlborough, Mass.), nickel affinity chromatography,
ion-exchange chromatography on a Mono S 5/50 GL or Mono S 10/100 GL
column (GE Lifesciences, Marlborough, Mass.) and size exclusion
chromatography on a HiLoad 16/600 Superdex 200 prep grade or HiLoad
26/600 Superdex 200 prep grade column (GE Lifesciences,
Marlborough, Mass.).
Example 2: Determination of Kinetics and Affinity of Human IL-15
Variants or Anti-PD-1-hIL-15 Fusion Proteins/IL-15R.alpha. and
IL-2R.beta. Interactions at 37.degree. C.
[0274] This example illustrates the kinetics and affinities of
various human IL-15 variants binding to human IL-15R.alpha. and
human IL-2R.beta. at 37.degree. C.
[0275] All experiments were performed on a Biacore 8K or Biacore
4000 Surface Plasmon Resonance based biosensor (GE Lifesciences,
Marlborough, Mass.). The anti-AVI tag sensor chips were prepared at
25.degree. C. with a running buffer of 10 mM HEPES, 150 mM NaCl,
0.05% (v/v) Tween-20, pH 7.4. All surfaces of a Biacore CM4 sensor
chip were activated with a 1:1 (v/v) mixture of 400 mM EDC and 100
mM NHS for 7 minutes, at a flow rate of 10 .mu.L/min. An anti-Avi
reagent (Rabbit Anti-Avi-tag, Genscript Catalog #A00674-200) was
diluted to 30 .mu.g/mL in 10 mM sodium acetate (pH 4.5) and
injected on all flow cells for 7 minutes at 20 .mu.L/min. All flow
cells were blocked with 100 mM ethylenediamine in 200 mM Borate
buffer pH 8.5 for 7 minutes at 10 .mu.L/min.
[0276] All protein interaction experiments were performed at
37.degree. C. using a running buffer of 10 mM HEPES, 150 mM NaCl,
0.05% (v/v) Tween-20, pH 7.4, 1 mg/mL BSA. For experiments
performed on a Biacore 4000, Avi-tagged IL-15 variants were
captured from undiluted supernatants onto spots 1 and 5 on flow
cells 1, 2, 3 and 4 at a flow rate of 10 .mu.L/min for 2 minutes.
Different mutants were captured on each spot. Spots 2 and 4 on flow
cells 1, 2, 3 and 4 were used as reference surfaces. Following
capture of IL15 variants, analyte (buffer, 12.3 nM, 37 nM, 111 nM,
333 nM and 1 pM concentrations of human IL-15R.alpha. or human
IL-2R.beta.) was injected at a flow rate of 30 .mu.L/min in all
flow cells for two minutes. After each analyte injection,
dissociation was monitored for 5 minutes, followed by regeneration
of all flow cells with three 30-second injections of 75 mM
phosphoric acid. Buffer cycles were collected for each captured
IL-15V49R mutant for double-referencing purposes
(double-referencing as described in Myszka, D. G., Improving
biosensor analysis. J. Mol. Recognit. 12, 279-284 (1999)). For
kinetic analysis, the double-referenced sensorgrams were fit
globally to a simple 1:1 Langmuir with mass transport binding model
using Biacore 4000 Evaluation Software version 1.1 For steady-state
affinity analysis, the double-referenced equilibrium binding
responses were fit with a 1:1 Langmuir steady-state model using
Biacore 4000 Evaluation Software version 1.1.
[0277] For experiments performed on a Biacore 8K, Avi-tagged IL-15
variants were captured from undiluted supernatants for 2 minutes at
10 .mu.L/min on flow cell 2 in each channel, while flow cell 1 was
used as a reference surface. A different IL-15 variants was
captured in each channel. Following the capture of IL-15V49R
mutants, analyte (buffer, 3.2 nM, 16 nM, 80 nM, 400 nM, 1840 nM
human IL-15R.alpha. and 0.8 nM, 4 nM, 20 nM, 100 nM, 500 nM human
IL-2R.beta.) was injected over both flow cells for 2 minutes at a
flow rate of 30 .mu.L/min. After each analyte injection,
dissociation was monitored for 10 minutes followed by regeneration
of all flow cells with three 30-second injections of 75 mM
phosphoric acid. Buffer cycles were collected for each captured
IL-15 variants for double-referencing purposes (double-referencing
as described in Myszka, D. G. Improving biosensor analysis. J. Mol.
Recognit. 12, 279-284 (1999)). For kinetic analysis, the
double-referenced sensorgrams were fit globally to a simple 1:1
Langmuir with mass transport binding model using Biacore 8K
Evaluation Software version 1.1.1.7442. For steady-state affinity
analysis, the double-referenced equilibrium binding responses were
fit with a 1:1 Langmuir steady-state model using Biacore 8K
Evaluation Software version 1.1.1.7442.
[0278] The kinetics and affinity parameters for tested IL-15
variants are shown in Tables 1-2. The term "IL-15Rasu" refers to
IL-15R alpha sushi domain.
[0279] The below example determines the kinetics and affinity of
various anti-PD-1-hIL-fusion proteins binding to human
IL-15R.alpha. and human IL-2R.beta. at 37.degree. C. Experiments
were performed on a Biacore T200 Surface Plasmon Resonance based
biosensor (GE Lifesciences, Marlborough, Mass.).
[0280] Anti-human Fc sensor chips were prepared at 25.degree. C.
with a running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v/v)
Tween-20, pH 7.4. All surfaces of a Biacore CM4 sensor chip were
activated with a 1:1 (v/v) mixture of 400 mM EDC and 100 mM NHS for
7 minutes, at a flow rate of 10 .mu.L/min. An anti-human Fc reagent
(Goat anti-human IgG Fc-.gamma. specific, SouthernBiotech,
Birmingham, Ala., Catalog #2014-01) was diluted to 50 .mu.g/mL in
10 mM sodium acetate (pH 4.5) and injected on all flow cells for 7
minutes at 20 .mu.L/min. All flow cells were blocked with 100 mM
ethylenediamine in 200 mM Borate buffer pH 8.5 for 7 minutes at 10
.mu.L/min.
[0281] All protein interaction experiments were performed at
37.degree. C. using a running buffer of 10 mM HEPES, 150 mM NaCl,
0.05% (v/v) Tween-20, pH 7.4, 1 mg/mL BSA. Anti-PD-1-hIL-15 fusion
proteins were captured at 10 .mu.g/mL at a flow rate of 10
.mu.L/min for 2 minutes. Different fusion proteins were captured in
flow cells 2, 3 and 4. No protein was captured onto flow cell 1
which was used as a reference surface. Following capture of fusion
proteins, analyte (buffer, 12.3 nM, 37 nM, 111 nM, 333 nM, 1000 nM
and 3000 nM concentrations of human IL-15R.alpha. or human
IL-2R.beta.) was injected at a flow rate of 30 .mu.L/min in all
flow cells for two minutes. After each analyte injection,
dissociation was monitored for 5 minutes followed by regeneration
of all flow cells with three 60-second injections of 75 mM
phosphoric acid. Buffer cycles were collected for each captured
fusion protein for double-referencing purposes (double-referencing
as described in Myszka, D. G. Improving biosensor analysis. J. Mol.
Recognit. 12, 279-284 (1999)). For kinetic analysis, the
double-referenced sensorgrams were fit globally to a simple 1:1
Langmuir with mass transport binding model using Biacore T200
Evaluation Software version 2.0. For steady-state affinity
analysis, the double-referenced equilibrium binding responses were
fit with a 1:1 Langmuir steady-state model using Biacore T200
Evaluation Software version 2.0.
[0282] The kinetics and affinity parameters for anti-PD-1-hIL-15
variant fusion proteins are shown in Tables 3-5.
TABLE-US-00005 TABLE 1 Kinetics and affinity parameters for
non-fusion human IL-15 variants that reduced human IL-15.alpha.
binding: hIL-15.alpha. kinetics hIL-15R.alpha. hIL-2R.beta. Lower
hIL-15R.alpha. Mutants k.sub.a (1/Ms) k.sub.d (1/s) t.sub.1/2 (min)
K.sub.D (nM) K.sub.D (nM) affinity than V49R? IL-15Rasu-IL-15 N/A
N/A N/A No 69.6* -- binding IL-15 (wild-type) 9.0E+06 2.1E-04 55.85
0.023 31 -- V49R N/A N/A N/A 91.9* 109* -- V49A 1.27E+06 1.11E-03
10.42 0.874 N/A No V49E N/A N/A N/A 43.4* N/A No V49G 1.69E+06
7.33E-03 1.58 4.32 N/A No V49H N/A N/A N/A 28.5* N/A No V49K N/A
N/A N/A 241* 86.6* Yes V49N 2.07E+06 3.85E-03 3.00 1.86 N/A No V49Q
1.56E+06 1.28E-02 0.91 8.17 N/A No V49S 2.06E+06 2.77E-03 4.17 1.34
N/A No Y26H N/A N/A N/A 10.9* 87* No Y26K N/A N/A N/A 146* 75.7*
Yes Y26R N/A N/A N/A 96.8* 57.9* Yes E46G N/A N/A N/A 267* 141* Yes
*A steady state affinity analysis was used due to fast kinetics
TABLE-US-00006 TABLE 2 Affinity parameters for non-fusion human
IL-15 variants in the background of V49R mutation that reduced
human IL-2R.beta. binding: Reduced hIL-2R.beta. hIL-15R.alpha.
hIL-2R.beta. Position Mutations K.sub.D (nM) K.sub.D (nM) K.sub.D
to >300 nM IL-15 {circumflex over ( )} WT 21.6 0.023 N/A
IL-15Rasu- WT 54.4 N/A N/A IL15 {circumflex over ( )} V49 R 97.5
94.2 No N1 A 398 175 Yes N1 D 311 105 Yes N1 K >500 264 Yes N1 S
194 140 No N1 H 235 151 No N1 R .sup. 479 * 243 Yes N1 E 340 171
Yes N1 T 249 104 No N1 Q >500 115 Yes N1 G >500 160 Yes N1 P
Ambiguous Binds .sup.# (LC) N/A (LC) N1 I Ambiguous Ambiguous N/A
(LC) (LC) N1 L Binds .sup.# (LC) Binds .sup.# (LC) N/A N1 M Binds
.sup.# (LC) Binds .sup.# (LC) N/A N1 F Binds .sup.# (LC) Binds
.sup.# (LC) N/A N1 Y Binds .sup.# Binds .sup.# N/A N1 W Ambiguous
Ambiguous N/A (LC) (LC) N1 V Binds .sup.# (LC) Binds .sup.# (LC)
N/A N4 A >500 98 Yes N4 D 365 103 Yes N4 K No binding 73.1 Yes
N4 S No binding 94.7 Yes N4 H 96.4 81.5 No N4 R Binds .sup.# 56.1
N/A N4 E 311 79.8 Yes N4 T Ambiguous 88.6 No (approx.) N4 Q >500
59.5 Yes N4 G >500 105 Yes N4 P Binds .sup.# 356 N/A N4 I No
binding Binds .sup.# Yes N4 L 429 98.6 Yes N4 M 233 88.1 No N4 F
247 Binds .sup.# No N4 Y .sup. 117 * Binds .sup.# Yes N4 W 438 143
No N4 V Binds .sup.# Binds .sup.# N/A S7 A 220 84 No S7 D >500
121 Yes S7 K >500 103 Yes S7 N >500 93.2 Yes S7 H 354 75.6
Yes S7 R >500 77 Yes S7 E >500 87.6 Yes S7 T >500 93.9 Yes
S7 Q 215 88.1 No S7 G >500 147 Yes S7 P Binds .sup.# >500 N/A
S7 I Ambiguous Ambiguous N/A (LC) (LC) S7 L Ambiguous Ambiguous N/A
(LC) (LC) S7 M Ambiguous Ambiguous N/A (LC) (LC) S7 F Ambiguous
Ambiguous N/A (LC) (LC) S7 Y Ambiguous Ambiguous N/A (LC) (LC) S7 W
Ambiguous Ambiguous N/A (LC) (LC) S7 V Ambiguous Ambiguous N/A (LC)
(LC) D8 A No binding 102 Yes D8 K No binding 101 Yes D8 S No
binding 124 Yes D8 N No binding 119 Yes K10 A 321 150 Yes K10 D
>500 270 Yes K10 S 496 195 Yes K10 N 230 128 No K10 H 114 85.6
No K10 R 146 94.5 No K10 E 360 126 Yes K10 T Ambiguous Ambiguous
N/A (LC) (LC) K10 Q 250 100 No K10 G >500 209 Yes K10 P No
binding Binds .sup.# Yes K10 I Ambiguous Binds .sup.# N/A K10 L 348
124 Yes K10 M 391 122 Yes K10 F Binds .sup.# (LC) Binds .sup.# (LC)
N/A K10 Y Ambiguous Ambiguous N/A (LC) (LC) K10 W 185 101 No K10 V
Binds .sup.# Binds .sup.# N/A K11 A 206 191 No K11 D >500 242
Yes K11 S 453 194 Yes K11 N 291 217 No K11 H 104 147 No K11 R 138
140 No K11 E 160 159 No K11 T 225 169 No K11 Q 107 156 No K11 G
Binds .sup.# Binds .sup.# N/A K11 P No binding Binds .sup.# N/A K11
I 76 95.8 No K11 L 92.2 142 No K11 M 180 Binds .sup.# No K11 F 162
Binds .sup.# No K11 Y 143 219 No K11 W .sup. 346 * 361 Yes K11 V
110 142 No S29 A 274 154 No S29 D 238 121 No S29 K 248 261 No S29 N
322 154 Yes D30 A 273 151 No D30 K 351 158 Yes D30 S 249 146 No D30
N 163 94 No D30 H 149 121 No D30 R 290 155 No D30 E 165 124 No D30
T 269 184 No D30 Q 197 111 No D30 G .sup. 513 * Binds .sup.# Yes
D30 P Binds .sup.# No binding N/A D30 I 201 197 No D30 L 277 237 No
D30 M 238 142 No D30 F 213 166 No D30 Y 231 159 No D30 W 321 Binds
.sup.# Yes D30 V 258 153 No V31 A 269 129 No V31 K >500 No
binding Yes V31 S >500 265 Yes V31 D >500 >500 Yes H32 A
218 152 No H32 K 149 92.8 No H32 S 176 110 No H32 D 123 85.5 No H32
N 124 85.9 No H32 R 156 131 No (approx.) H32 E 130 108 No H32 T 186
Binds .sup.# No H32 Q 147 64 No H32 G 459 249 Yes H32 P 146 162 No
H32 I 110 113 No H32 L 159 189 No H32 M 161 134 No H32 F .sup. 315
* Binds .sup.# Yes H32 Y Binds .sup.# Binds .sup.# N/A H32 W 376
Binds .sup.# Yes H32 V 247 99.8 No D61 A >500 78.2 Yes D61 K No
binding 116 Yes D61 S No binding 102 Yes D61 N No binding 121 Yes
E64 A >500 130 Yes E64 K No binding 164 Yes E64 N/S >500 130
Yes E64 N >500 115 Yes E64 D 291 108 No E64 H >1000 112 Yes
E64 R Binds .sup.# Binds .sup.# N/A E64 T >500 141 Yes E64 Q
>500 127 Yes E64 G Binds .sup.# Binds .sup.# N/A E64 P Ambiguous
Ambiguous N/A (LC) (LC) E64 I Ambiguous Ambiguous N/A (LC) (LC) E64
L Ambiguous Ambiguous N/A (LC) (LC) E64 M Ambiguous Ambiguous N/A
(LC) (LC) E64 F Ambiguous Ambiguous N/A (LC) (LC) E64 Y Ambiguous
Ambiguous N/A (LC) (LC) E64 W Ambiguous Ambiguous N/A (LC) (LC) E64
V Ambiguous Binds .sup.# N/A (LC) N65 A No binding 202 Yes N65 K
193 201 No N65 S No binding 158 Yes N65 D >500 121 Yes 168 A
>500 113 Yes 168 K No binding 94.2 Yes 168 S No binding 104 Yes
168 D No binding 106 Yes 168 H >1000 87.6 Yes 168 R >500 102
Yes 168 T >500 76.3 Yes 168 Q Ambiguous 132 N/A 168 G Binds
.sup.# 101 N/A 168 P No binding No binding Yes 168 N >1000 85
Yes 168 L 245 73.6 No 168 M >1000 96.2 Yes 168 F >1000 78.5
Yes 168 Y >1000 92.9 Yes 168 W Binds .sup.# 93.7 N/A 168 V 296
104 No 168 E Binds .sup.# 122 N/A L69 A >500 82.9 Yes L69 K No
binding No binding Yes L69 S >500 78.2 Yes L69 D >500 65.6
Yes L69 H Binds .sup.# 54.5 N/A L69 R Ambiguous 111 N/A L69 T
>1000 63.6 Yes L69 Q Binds .sup.# 43.3 N/A (approx.) L69 G Binds
.sup.# Binds .sup.# N/A L69 P No binding No binding Yes L69 I 306
93.1 Yes L69 N Binds .sup.# 44 N/A L69 M >1000 74.5 Yes L69 F
Ambiguous 106 N/A L69 Y Ambiguous 137 N/A L69 W Ambiguous Ambiguous
N/A (LC) (LC) L69 V >1000 85.5 Yes L69 E >1000 47.8 Yes N72 A
148 117 No N72 D 101 105 No N72 K >500 101 Yes N72 S 182 118 No
Q108 A 462 161 Yes Q108 D 215 125 No Q108 K 249 85.4 No Q108 S 290
144 No M109 A >500 198 Yes M109 K >500 215 Yes M109 S 362 190
Yes M109 D 339 288 Yes I111 A 375 173 Yes I111 K 397 199 Yes
I111 S >500 223 Yes I111 D >500 214 Yes {circumflex over ( )}
Wild-type IL-15 and IL-15Rasu-IL15 do not contain the V49R mutation
* Affinities reported are from kinetic fits (all other K.sub.D
values mentioned are steady state K.sub.D values) .sup.# Fits could
not be obtained because of low responses LC Low Capture of
supernatant
TABLE-US-00007 TABLE 3 Affinity parameters for anti-PD-1-hIL-15
variant fusion proteins to human IL-2R.beta. binding: hIL-2R.beta.
kinetics Mutants k.sub.a (1/Ms) k.sub.d (1/s) t.sub.1/2 (S) K.sub.D
(nM) xmPD-1-hIL15Rasu-hIL15 2.36E+06 9.88E-02 7.00 41.8
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N) N/A N/A N/A 271*
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/l68S) N/A N/A N/A >3000
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N N4K) N/A N/A N/A 767*
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/M109A) N/A N/A N/A 315*
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/I68S/M109A) N/A N/A N/A >1500
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/N4K/M109A) N/A N/A N/A >3000
Ab8.8-hIL15Raus-hIL15 2.62E+06 9.71E-02 7.14 37.1
xmPD-1-hIL15(wild-type) N/A N/A N/A 561 xmPD-1-hIL15 V49R N/A N/A
N/A 494* xmPD-1-hIL15 V49R/E46G N/A N/A N/A 437* xmPD-1-hIL15
V49R/E46G/N1A/D30N N/A N/A N/A 433* xmPD-1-hIL15
V49R/E46G/N1G/E64Q/D30N N/A N/A N/A >1500 xmPD-1-hIL15
V49R/E46G/N1G/N4Q/D30N N/A N/A N/A >1500 xmPD-1-hIL15
V49R/E46G/E64Q/D30N N/A N/A N/A >3000 xmPD-1-hIL15
V49R/Y26K/E64Q/D30N N/A N/A N/A >1500 xmPD-1-hIL15
V49R/E46G/E64Q/D30N/I68S N/A N/A N/A No binding xmPD-1-hIL15
V49R/E46G/E64Q/D30N/N4K N/A N/A N/A No binding xmPD-1-hIL15
V49R/E46G/E64Q/D30N/M109A N/A N/A N/A >3000 xmPD-1-hIL15
V49R/E46G/E64Q/D30N/I68S/M109A N/A N/A N/A No binding xmPD-1-hIL15
V49R/E46G/E64Q/D30N/N4K/M109A N/A N/A N/A No binding xmPD-1-hIL15
V49R/E46Q N/A N/A N/A 560* xmPD-1-hIL15 V49R/E53Q N/A N/A N/A 590*
xmPD-1-hIL15 V49R/E93Q N/A N/A N/A 451* xmPD-1-hIL15 NQ mutant N/A
N/A N/A 518* xmPD-1-hIL15 NQ-3d N/A N/A N/A 446* xmPD-1-hIL15 NQ-2a
N/A N/A N/A 514* xmPD-1-hIL15 NQ-2b N/A N/A N/A 724* xmPD-1-hIL15
NQ-2c N/A N/A N/A 262* Ab8.8-hIL15 NQ mutant N/A N/A N/A 506* *A
steady state affinity analysis was used due to fast kinetics
TABLE-US-00008 TABLE 4 Affinity parameters for anti-PD-1-hIL-15
variant fusion proteins to human IL-15R.alpha. binding:
hIL-15R.alpha. kinetics Mutants k.sub.a (1/Ms) k.sub.d (1/s)
t.sub.1/2 (s) K.sub.D (nM) xmPD-1-hIL15Rasu-hIL15 No binding
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N) No binding
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/l68S) No binding
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N N4K) No binding
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/M109A) No binding
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/I68S/M109A) No binding
xmPD-1-hIL15Rasu-hIL15(E64Q/D30N/N4K/M109A) No binding
Ab8.8-hIL15Raus-hIL15 No binding xmPD-1-hIL15(wild-type) 4.31E+06
3.63E-04 N/A 0.084 xmPD-1-hIL15 V49R N/A N/A N/A 119* xmPD-1-hIL15
V49R/E46G No binding xmPD-1-hIL15 V49R/E46G/N1A/D30N No binding
xmPD-1-hIL15 V49R/E46G/N1G/E64Q/D30N No binding xmPD-1-hIL15
V49R/E46G/N1G/N4Q/D30N No binding xmPD-1-hIL15 V49R/E46G/E64Q/D30N
No binding xmPD-1-hIL15 V49R/Y26K/E64Q/D30N No binding xmPD-1-hIL15
V49R/E46G/E64Q/D30N/I68S No binding xmPD-1-hIL15
V49R/E46G/E64Q/D30N/N4K No binding xmPD-1-hIL15
V49R/E46G/E64Q/D30N/M109A No binding xmPD-1-hIL15
V49R/E46G/E64Q/D30N/I68S/M109A No binding xmPD-1-hIL15 No binding
VS49R/E46G/E64Q/D30N/N4K/M109A xmPD-1-hIL15 V49R/E46Q No binding
xmPD-1-hIL15 V49R/E53Q N/A N/A N/A >1500 xmPD-1-hIL15 V49R/E93Q
N/A N/A N/A 943* xmPD-1-hIL15 NQ mutant No binding xmPD-1-hIL15
NQ-3d No binding xmPD-1-hIL15 NQ-2a N/A N/A N/A >3000
xmPD-1-hIL15 NQ-2b No binding xmPD-1-hIL15 NQ-2c 1.06E+06 2.84E-02
24.4 26.8 Ab8.8-hIL15 NQ mutant No binding *A steady state affinity
analysis was used due to fast kinetics
TABLE-US-00009 TABLE 5 Affinity parameters for anti-human
PD-1-hIL-15 variant fusion proteins to human PD-1 binding: hPD-1
kinetics Mutants k.sub.a (1/Ms) k.sub.d (1/s) t.sub.1/2 (S) K.sub.D
(nM) Ab8.8-hIL15Raus-IL15 N/A N/A N/A No binding
xhPD1-hIL15Rasu-IL15 1.63E+05 5.41E-04 1281.2 3.32 xhPD1-hIL15 NQ
mutant 1.73E+05 5.96E-04 1163.8 3.44
Example 3: Construction of Reporter Cell Lines with Constitutive
Expression of the IL-15 Receptor and Inducible Expression of
PD-1
[0283] This example demonstrates a method for establishing useful
reporter cell lines to evaluate the bioactivity of IL-15-based
molecules.
[0284] To assay the IL-15-based molecules described herein, a
reporter cell line that has a functional response to IL-15 was
generated and was used to assay the effect of PD-1-target-driven
activity. To this end, reporter cell lines were constructed with
the following properties: (1) constitutive expression of functional
IL-15 signaling receptors: IL-2R.beta. (also known as CD122), and
common gamma chain (also known as CD132); and (2) inducible
expression of either human or mouse PD-1.
[0285] A lentiviral system was used as described previously by
Metzger, T., et al., Cancer Res. July 1; 76 (13):3684-9. (2016)).
To express full-length human IL-2R.beta., plasmid pRF791 was also
generated. Briefly, cDNA sequences encoding the TurboGFP
fluorescent protein followed by a short glycine-serine linker
sequence, the viral T2A sequence, and the full length human CD122
(IL-2R.beta.) sequence were cloned into a lentiviral transfer
vector so that they were expressed under the control of the
EF1alpha promoter. pRF791 was used to generate lentiviral particles
as described in Metzger, T., et al. (2016), and the resulting virus
was used to transduce the murine 32D cell line, which is normally
grown in media as described by American Type Culture Collection
(ATCC). This cell line was chosen because it expresses only the
common gamma chain, and not mouse IL2Rb or mouse IL-15Ralpha, and
has also been used as a host to assay IL-15 bioactivity previously.
(See, e.g., Zhu, X., et. al., J Immunol. 2009 Sep. 15;
183(6):3598-607. Transduced cells were identified by resistance to
100 .mu.g/mL blasticidin and by GFP fluorescence. A
blasticidin-resistant, GFP-expressing population of cells were
selected and maintained as line polyclonal line 32D[pRF791].
[0286] To express human or mouse PD-1 under the control of the
doxycycline-inducible TetOn3G promoter, cDNA sequences encoding the
mCherry fluorescent protein followed by a short glycine-serine
linker sequence, the viral T2A sequence, and full-length human or
mouse PD-1 were cloned into the lentiviral transfer vector
pLVX-SFFV-Puro-P2A-TetOn3G (see, e.g., Metzger et al. (2016)). The
resulting vectors, pRF768 (expressing human PD-1) and pRF770
(expressing mouse PD-1) were each used to generate lentiviral
particles as described in Metzger, T., et al. (2016), and the
resulting viruses were used to transduce the 32D[pRF791] cell line.
Transduced cells were identified by resistance to 100 .mu.g/mL
blasticidin plus 10 ug/mL Puromycin, and by GFP and mCherry
fluorescence. Blasticidin-resistant, puromycin-resistant,
GFP-expressing population of cells were selected and maintained as
polyclonal line 32D[pRF768+pRF791] or 32D[pRF770+[pRF791],
depending on whether they were transduced with virus from pRF768 or
pRF770, respectively.
[0287] Inducible expression of mCherry-PD-1 was confirmed in
32D[pRF768+pRF791] and 32D[pRF770+pRF791] by addition of
doxycycline (to 1 ug/mL final concentration) for 12-16 hours,
followed by analysis of mCherry expression by flow cytometry.
Expression of IL-2Rb and PD-1 were also confirmed by staining with
antibodies specific for CD122(IL-2R.beta.) or PD-1,
respectively.
[0288] To establish clonal cell lines, 32D[pRF768+pRF791] or
32D[pRF770+pRF791] cells were induced with 1 ug/mL doxycycline for
12-16 hours, and then analyzed by flow cytometry for GFP and
mCherry fluorescence. Single cells of each cell line that fell
within the top decile of fluorescence intensity were sorted into
unique wells of a 96-well plate and cultured for 2 weeks until
small colonies of cells appeared. These colonies were expanded to
generate single cell cloned lines, which were confirmed by flow
cytometry for transgene expression.
Example 4: Binding Assay of Antibody-IL15 Chimeric Molecules to a
Reporter Cell Line
[0289] This Example describes a method to assay the direct binding
of antibody-cytokine fusion molecules to cells expressing the IL-15
receptor (CD122 plus CD132), either with or without expression of
the antibody binding target.
[0290] To detect the binding of antibody-IL-15 molecules to cells,
approximately 200 micrograms of each of the purified, recombinant
molecules were labeled with Alexa647 fluorophore using a the
Click-IT Alexa Fluor 647 sDIBO Alkyne kit and following the
manufacturer's protocol. The antibodies and the chimeric molecules
described in this and all subsequent Examples are summarized in
Table 6, below:
TABLE-US-00010 TABLE 6 List of molecules utilized in all following
Examples: Name Relevant SEQ IDs Brief description xmPD-1 12(VH) +
64 (CH1- Anti-mouse PD-1 (clone F12.3) with hinge-IgG2dA-D265A-
human IgG2dA constant regions CH2-CH3) + 30 (LC: VK-CK)
xmPD-1-IL15RaSu- 28 (R-arm) + 22 (E- Anti-mouse PD-1 (clone F12.3)
IgG IL15* arm) + 30 fusion with human IL15Ra Sushi domain - human
IL15 (wildtype mature protein sequence) xmPD-1-IL15 NQ* 28 + 29 +
30 Anti-mouse PD-1 (clone F12.3) IgG fusion with human IL15 NQ
variant (6 mutations D22N/Y26F/E46Q/E53Q/E89Q/E93Q of SEQ ID NO: 1,
eliminate binding to IL-15Ralpha) xmPD-1-IL15 NQ-2a 28 + 30 + 58
Anti-mouse PD-1 (clone F12.3) IgG fusion with human IL15 NQ-2a
variant (reduce binding to IL-15Ralpha) xmPD-1-IL15 NQ-2b 28 + 30 +
59 Anti-mouse PD-1 (clone F12.3) IgG fusion with human IL15 NQ
variant (reduce binding to IL-15Ralpha) xmPD-1-IL15 NQ-3d 28 + 30 +
61 Anti-mouse PD-1 (clone F12.3) IgG fusion with human IL15 NQ
variant (reduce binding to IL-15Ralpha) xmPD-1-IL15 m1 86 + 30 + 88
Anti-mouse PD-1 (clone F12.3) IgG fusion with human IL15 ml variant
(4 mutations V49R/E46G/N1A/D30N of SEQ ID NO: 1, eliminate binding
to IL- 15Ralpha, reduce binding to IL- 2Rbeta-gamma) xmPD-1-IL15 m2
87 + 30 + 88 Anti-mouse PD-1 (clone F12.3) IgG fusion with human
IL15 m2 variant (5 mutations V49R/E46G/N1G/E64Q/D30N of SEQ ID NO:
1, eliminate binding to IL- 15Ralpha, reduce binding to IL-
2Rbeta-gamma) xhPD-1-IL15 m1 74 + 65 + 89 Anti-human PD-1
(VH1-69b/VK1-39) IgG fusion with human IL15 m1 variant (4 mutations
V49R/E46G/N1A/D30N of SEQ ID NO: 1, eliminate binding to IL-
15Ralpha, reduce binding to IL- 2Rbeta-gamma) xhPD-1-IL15 m2 74 +
65 + 90 Anti-human PD-1 (VH1-69b/VK1-39) IgG fusion with human IL15
m2 variant (5 mutations V49R/E46G/N1G/E64Q/D30N of SEQ ID NO: 1,
eliminate binding to IL- 15Ralpha, reduce binding to IL-
2Rbeta-gamma) *Mutant versions of these proteins are indicated in
the text with the format "original amino acid-residue number-new
amino acid", where residue number corresponds to the amino acid
sequence listing of human IL-15 as shown in SEQ ID 1.
[0291] The following cell lines were suspended in growth medium to
0.7*10{circumflex over ( )}6 cells/mL and, cultured for 16 hrs: 1)
untransduced 32D (parental line); 2) 32D[pRF770+pRF791]; and 3)
32D[pRF770+pRF791] grown in the presence of 1 ug/mL doxycycline, to
induce PD-1 expression. Cultures were then resuspended to a final
concentration of 1.0*10{circumflex over ( )}6 cells/mL, and 0.1 mL
of each suspension was plated into separate wells of a 96-well
round-bottom plate, of sufficient number required for the
experiment. Each Alexa647-labeled antibody or antibody-cytokine
chimeric proteins were then added to a unique well to obtain a
final concentration of 500 nM, 100 nM, 10 mM, 1 nM, 0.1 nM, 0.01
nM, or 0.001 nM in a given well. Plates were incubated at 4.degree.
C. for 30', and then the cells were pelleted by centrifugation
(300.times.g, 5') and resuspended in 0.2 mL of PBS. This wash step
was repeated twice. Upon final resuspension, the cells were
analyzed on an LSRII flow cytometer. Data were analyzed using
FlowJo version 10. Cells that exhibited a fluorescence in the
Alexa647 channel above background (as defined by cells to which no
labeled antibody-cytokine chimera had been added) were scored as
positive for binding. The percent of Alexa647-positive cells as a
function of added labeled protein concentration is shown in FIGS.
2A-2D.
[0292] Each labeled molecule that featured an anti-mouse PD-1
moiety was able to bind to PD-1 expressing cells
("[pRF770+pRF791]+Dox") in a concentration-dependent manner, even
at low picomolar final concentrations. The xmPD1-IL15RaSu-IL-15
fusion molecule (FIG. 2C) also exhibited more modest binding to
cells expressing only CD122 plus CD132 ("[pRF770+pRF791]"); in
contrast, the anti-mouse PD1-IL15 NQ variant showed reduced binding
to these same type of cells (FIG. 2D). The isotype control antibody
version (Ab8.8-IL15RaSu-IL15) only exhibited binding to any of the
cells at the highest tested final protein concentrations (FIG. 2B).
All four recombinant proteins tested showed background levels of
binding to the 32D parent line at relatively high concentrations
(>=10 nM of each compound) as well.
[0293] This example demonstrates that the anti-PD-1 antibody
portion of the antibody-IL-15 chimeric proteins is a key
determinant of directing protein binding to cells. The assay also
reveals a difference between wildtype IL-15 and the NQ mutein in
the ability of each to bind to cells expressing CD122 plus CD132
(in an antibody or PD-1-independent manner).
Example 5: In Vitro Functional Assay of Anti-Mouse PD-1-IL15
Chimeric Molecules Using a Reporter Cell Line
[0294] This example describes a functional activity assay of an
antibody-cytokine fusion molecule to cells expressing the IL-15
receptor (CD122 plus CD132), either with or without expression of
the antibody binding target.
[0295] In this example, the ability of anti-mouse PD-1-IL15
chimeric fusion proteins to trigger downstream intracellular
signaling in a reporter cell line 32D[pRF770+pRF791] was measured.
The cell line 32D[pRF770+pRF791] expresses human CD122 along with
endogenous mouse CD132 (common gamma chain), and can express
doxycycline-inducible mouse PD-1. The activation of CD122/CD132
downstream signaling was measured by monitoring relative changes in
phosphorylated STAT5 (pSTAT5), which is known to be a downstream
consequence of IL-15 signaling (see for example: Steel J C,
Waldmann T A, Morris J C. Trends Pharmacol Sci. 2012 January;
33(1):35-41.) The 32D [pRF770+pRF791] cells were resuspended to a
concentration of 0.4-0.8*10e6 cells/mL in growth medium and grown
overnight (12-16 hrs) either in the presence or absence of 1
.mu.g/mL final concentration of doxycycline. Cells were collected
by centrifugation (230.times.g, 5 min), washed once into growth
medium lacking IL-3 and calf serum, and then resuspended at a final
concentration of 1.0*10{circumflex over ( )}6 cells/mL and
incubated at 37.degree. C. for 4 hours. Of the single-cell cloned
lines, approximately 40,000-50,000 of the doxycycline-induced cells
and 150,000-160,000 of the non-doxycycline cells were combined and
added to each well. For non-single-cell-sorted lines, we routinely
utilized cell populations which, upon doxycycline induction,
demonstrated approximately 20-30% detectable mCherry expression by
flow cytometric analysis. Recombinant protein compounds of interest
were added to each well such that the final concentration of each
was between 500 nM and 0.1 pM. Triplicate wells for each condition
were set up identically. Plates were incubated in a cell culture
incubator at 37.degree. C. for 30', and cells were then pelleted by
centrifugation (230.times.g, 5'). Paraformaldehyde was then
immediately added to each well (4% final concentration, in PBS),
the cells were mixed gently by pipetting up and down once to
prevent clumping, and then incubated at 37.degree. C. for 15 min.
Cells were then pelleted by centrifugation (230.times.g, 5') and
washed with Phosphate Buffered Saline (PBS) solution. This step was
repeated two additional times. Residual PBS was carefully aspirated
and 0.1 mL of cold (-20.degree. C.) methanol was added, followed by
gentle pipetting once to prevent clumping. Plates were sealed with
foil and immediately placed in a -20.degree. C. freezer for at
least 1 hr and up to several days (generally, for 12-16 hours). On
the day of analysis, cells were washed cells three times with PBS
or FACS buffer (cells were spun at 300.times.g RCF to ensure
pellet). The final cell pellet was resuspended in 50 ul of a 1:50
dilution of anti-pSTAT5 antibody (anti-Stat5 (pY694)-A647 (BD
Biosciences, Clone: 47/Stat5(pY694)). The cells were incubated for
1 hr at room temperature in the dark. The plate was then
centrifuged and washed three times with FACS buffer as described
above. Cells were resuspended in 125-150 .mu.l FACS buffer per well
and the cells were analyzed on an LSRII flow cytometer. Data were
analyzed using FlowJo version 10.
[0296] Because plasmid pRF770 contains a cassette for the
doxycycline-inducible expression of mCherry followed by mouse PD-1
(separated by a viral 2A cleavage sequence, which results in two
independent polypeptides), cells expressing mCherry (as detected on
an appropriate equipped flow cytometer using the PE-Texas Red
channel) are reliably scored as "PD-1+"; cells that do not
fluoresce in this channel above background were considered "PD-1-"
or "PD-1 (low)". Analysis of pSTAT5 positive cells was therefore
gated on PD-1+ or PD-1- (or PD-1 (low)) cells, as present within
the same experimental reaction.
[0297] FIG. 3 depicts the use of the 32D [pRF770+791] reporter cell
line to assay several IL-15-containing molecules, according to the
method described above. FIG. 3A shows the effects of adding
Ab8.8-IL15RaSu-IL15 to the mixed population of 32D[pRF770+791]
PD-1+ and PD-1(low) reporter cells, where it causes equivalent
pSTAT5 activation of both PD-1+ and PD-1(low) cells. FIG. 3B
depicts the effects of adding xmPD-1-IL15RaSu-IL15 to the mixed
population of 32D[pRF770+791] PD-1+ and PD-1(low) reporter cells.
In this case, PD-1+ cells exhibit a much greater sensitivity, and
lower EC50, to xmPD-1-IL15RaSu-IL15 as compared to PD-1(low) cells,
as measured by pSTAT5 levels. FIG. 3C depicts the effects of adding
xmPD-1-IL15 NQ to the mixed population of 32D[pRF770+791]
PD-1+(i.e., doxycycline-induced) and PD-1(low) (i.e.,
non-doxycycline induced) reporter cells. PD-1+ cells exhibit a much
greater sensitivity, and lower EC50, to xmPD-1-IL15 NQ as compared
to PD-1(low) cells, as measured by pSTAT5 levels. Additionally, the
EC50 values for anti-mouse PD-1-IL15 NQ are approximately 10-fold
greater as compared to those for xmPD-1-IL15RaSu-IL15, on both
PD-1+ and PD-1(low) cells, indicating that xmPD-1-IL15 NQ
represents a mutein variant with reduced activity (as measured by
CD122/CD132-dependent pSTAT5 activation), in addition to having the
property of eliminating detectable binding to CD215 (IL-15 receptor
alpha). Table 7, below, summarizes the calculated EC50 values for
the aforementioned molecules, and also displays the fold change as
the ratio of EC50 between PD-1+ and PD-1(low) cells.
TABLE-US-00011 TABLE 7 EC50 (nM) Molecule PD-1+ PD-1(low) Fold
change* Ab8.8-IL15RaSu-IL15 0.3 0.15 0.51 xmPD1-IL15RaSu-IL15 0.01
2.19 365.32 xmPD1-IL15 NQ 0.09 21.14 223.07 *Fold change = EC50
(PD-1(low))/EC50 (PD-1+)
[0298] To identify additional variants of an antibody-targeted
IL-15, wherein the IL-15 moiety contains mutations that confer a
range of functional activities or binding to xD122 and/or CD132,
the following experiment was conducted:
[0299] As listed in Table 8 (below) and as displayed in FIG. 4,
various IL-15 mutein forms of xmPD-IL15RaSu-L15 were generated and
assayed using the 32D[pRF770+pRF791] reporter cell line as
detailed, above. Note that for each mutein, the mutations present
relative to wildtype mature human IL-15 protein sequence are
denoted (in the format of original amino acid, amino acid reside
number as present in SEQ ID #1, new amino acid at that position).
Percentages of PD-1+ or PD-1(low) cells that were positive for
pSTAT5 (by flow cytometric analysis) are plotted in FIG. 4A-H, and
the resulting plots used to calculate EC50 values, as shown below
in Table 8.
TABLE-US-00012 TABLE 8 EC50 (nM) Molecule PD-1+ PD-1(low) Fold
change* xmPD1-hIL15Rasu-hIL-15 0.05 5.42 108 xmPD1-hIL15Rasu-hIL-15
0.42 18.76 44 K11S/D30N xmPD1-hIL15Rasu-hIL-15 0.16 22.04 139
D61N/D30N xmPD1-hIL15Rasu-hIL-15 0.069 24.89 360 E64Q/D30N
xmPD1-hIL15Rasu-hIL-15 0.16 3.13 19 K11S/M109A
xmPD1-hIL15Rasu-hIL-15 0.07 12.88 193 D61N/M109A
xmPD1-hIL15Rasu-hIL-15 0.06 10.05 163 E64Q/M109A
xmPD1-hIL15Rasu-hIL-15 D61N 0.38 6.91 18 *Fold change = EC50
(PD-1(low))/EC50 (PD-1+)
[0300] Thus, the present in vitro assay method can identify those
mutants with either non-target-driven or antibody target-driven
activities that differ from a corresponding chimeric protein
containing a wildtype IL-15 sequence.
[0301] FIG. 5 depicts the result of an experiment wherein the 32D
[pRF770+791] reporter cell line was used to assay several
additional antibody-IL-15 molecules, according to the method
described above in this Example. In this instance, none of the
molecules included the IL-15 receptor alpha Sushi domain
(IL-15RaSu). Percentages of PD-1+ or PD-1(low) cells that were
positive for pSTAT5 are plotted in FIG. 5A-G, and the resulting
plots used to calculate EC50 values, as shown below in Table 9.
TABLE-US-00013 TABLE 9 EC50 (nM) Fold Molecule (relevant
mutation(s) listed) PD-1+ PD-1(low) change* xmPD-IL15 NQ 0.00574
1.724 300.3484 xmPD-IL15 NQ2a 0.009495 2.939 309.5313 xmPD-IL15
NQ2b 0.01349 8.001 593.106 xmPD-IL15 NQ3d 0.02294 3.731 162.6417
xmPD-IL15 V49R 0.05476 1.511 27.59313 xmPD-IL15 V49R/E46G 0.01491
3.432 230.1811 xmPD-IL15 V49R/E46G/E64Q/ 0.1371 97.98 714.6608 D30N
*Fold change = EC50 (PD-1(low))/EC50 (PD-1+)
[0302] Thus, the in vitro assay method was used to identify those
mutants which lack IL-15RaSu and possess either non-target-driven
or antibody target-driven activities that differ from the
comparator xmPD1-IL15 NQ.
[0303] FIG. 6 depicts the result of an additional experiment
wherein the 32D [pRF770+791] reporter cell line was used to assay
several additional antibody-IL-15 molecules, according to the
method described above in this Example. In this instance, most of
the molecules lacked the IL-15 receptor alpha Sushi domain
(IL-15RaSu); xmPD-1-IL15RaSu-IL15 was included as an
IL15RsSu-containing control comparator. Percentages of PD-1+ or
PD-1(low) cells that were positive for pSTAT5 are plotted in FIG.
6A-G, and the resulting plots used to calculate EC50 values, as
shown below in Table 10.
TABLE-US-00014 TABLE 10 EC50 (nM) Fold Molecule (relevant
mutation(s) listed) PD-1+ PD-1(low) change* xmPD-IL15 NQ 0.03026
7.29 240.91 xmPD1-V49R 0.1634 4.378 26.79 xmPD1-V49R/E64G 0.0188
5.45 289.75 xmPD1 -V49R/Y26K/E64Q/D30N 0.05614 21.43 381.72
xmPD1-IL15 V49R/E46Q 0.002937 1.335 454.55 xmPD1-IL15 V49R/Y26K
0.00412 0.8539 207.21 xmPD1-IL15Rasu-IL15 0.0054 0.602 110.9 *Fold
change = EC50 (PD-1(low))/EC50 (PD-1+)
[0304] FIG. 7 depicts the result of an additional experiment
wherein the 32D [pRF770+791] reporter cell line was used to assay
several additional antibody-IL-15 molecules, according to the
method described above in this Example. In this instance, most of
the molecules contained a mutation at position V49 which modulated
the interaction between IL-15 and IL-15Ralpha. Percentages of PD-1+
or PD-1(low) cells that were positive for pSTAT5 are plotted in
FIG. 7A-7E, and the resulting plots used to calculate EC50 values,
as shown below in Table 11.
TABLE-US-00015 TABLE 11 EC50 (nM) Fold Molecule (relevant
mutation(s) listed) PD-1+ PD-1(low) change* xmPD1-IL15 V49R 0.05573
0.3284 5.89 xmPD1-IL15 V49R E46G 0.01182 4.535 383.67 xmPD1-IL15
V49K E46G 0.007902 3.977 503.29 xmPD1-IL15 V49K Y26K 0.01751 3.975
227.01 xmPD1-IL15 V49NAT 0.04394 3.047 69.34 *Fold change = EC50
(PD-1(low))/EC50 (PD-1+)
[0305] FIG. 7F-7H further depict the result of an additional
experiment wherein the 32D [pRF770+791] reporter cell line was used
to assay several additional antibody-IL-molecules, including the
molecules contained the mutations at positions N1, D30 and E64
which modulated the interaction between IL-15 and IL-2Rbeta-gamma,
in comparison to IL-15 NQ. Percentages of PD-1+ or PD-1(low) cells
that were positive for pSTAT5 are plotted in FIG. 7F-7H, and the
resulting plots used to calculate EC50 values, as shown below in
Table 11A.
TABLE-US-00016 TABLE 11A EC50 (nM) Fold Molecule (relevant
mutation(s) listed) PD-1+ PD-1(low) change* xmPD1-IL15 NQ 0.01088
13.35 1227.02 xmPD1-IL15 M1 (V49R E46G N1A 0.05314 76.91 1447.31
D30N) xmPD1-IL15 M2 (V49K E46G N1G 0.3097 734.3 2371 E64Q D30N)
*Fold change = EC50 (PD-1(low))/EC50 (PD-1+)
Example 6: Determination of Maximum Tolerated Dose (MTD) in
Non-Tumor-Bearing Mice
[0306] This Example describes the determination of the in vivo
maximum tolerated dose of an antibody-IL-15 fusion protein as
described herein.
[0307] Healthy female 6-8 week-old C56/B16 mice were treated with 3
doses each of either xmPD-1-IL-15RaSu-IL15, or xmPD-1-IL-15 NQ, as
indicated. On days 0, 3 and 6 of the experiment, mice were injected
subcutaneously (into the neck scruff) with sufficient recombinant
protein to yield the indicated concentration of each molecule,
based on each animal's body weight. Each compound was administered
to a cohort of animals. Each animal's changes in body weight
(compared to its weight at study start) were measured and the
average values for each group were recorded. FIGS. 8A and 8B plot
the changes in body weight and total survival, respectively, of
animals in the xmPD1-IL15RaSu-IL15 groups dosed at 2 mg/kg, 1
mg/kg, or 0.2 mg/kg. Mice that received 2 mg/kg or 1 mg/kg
experienced abrupt weight loss and ensuing mortality, such that by
day 8 of the study, all animals in these groups had died or needed
to be sacrificed. In an additional study, shown in FIG. 8C, mice
were treated with xmPD1-IL15RaSu-IL15 at a final dosing
concentration 1 mg/kg, 0.8 mg/kg, or 0.5 mg/kg. Mice in the 1 mg/kg
and 0.8 mg/kg groups lost 15-18% of their starting body weight and
appeared hunched and distressed, animals in the 0.5 mg/kg dosing
group experienced more modest, transient weight loss. The MTD for
xmPD1-IL15RaSu-IL15 was established as at or below 0.5 mg/kg. FIG.
8D depicts the changes in body weight following administration of
xmPD1-IL15 NQ at a final concentration of 3 mg/kg, 2 mg/kg, or 1
mg/kg. Animals in all groups experienced transient weight loss
(which rebounded following cessation of dosing) which was more
modest than see with lower doses of xmPD1-IL15RaSu-IL15, and
without any apparent signs of distress. The MTD for xmPD1-IL15 NQ
was thus established as at or below 3 mg/kg, indicating that it was
better tolerated than xmPD1-IL15RaSu-IL15.
Example 7: Characterization of the B16F10 Murine Syngeneic Tumor
Model
[0308] Various mouse tumor cell lines are commonly available (for
example, from the American Type Culture Collection, ATCC) and the
immune cell infiltrate profiles have been evaluated for many of
these (for example, see Mosley, S. I., et al., Cancer Immunol Res.
2017 January; 5(1):29-41). Mosley et al. and others have shown that
the tumor cell line B16F10, when implanted subcutaneously into
C57/B16 mice, develops a tumor that contains a relatively low
abundance of T cells (as a fraction of total immune cell
infiltrate) and is also poorly responsive to anti-PD-1 antibody
therapy. To evaluate the expression of PD-1 on tumor-infiltrating
lymphocytes (TILs) as well as peripheral immune cells, the
following experiment was carried out:
[0309] Female C57/B16 mice were subcutaneously implanted in the
upper thigh with approximately 500,000 B16F10 cells, which had been
freshly thawed from a single, low-passage vial (of 1*10{circumflex
over ( )}7 cells) and cultured for the minimum time required to
establish sufficient cells for implantation. When a cohort of
animals with visible tumor masses of 300-400 mm.sup.3 (as defined
by (length.times.width.sup.2)/2) was obtained, mice were euthanized
and sacrificed, and the spleen and primary tumor mass of each
animal were obtained. Splenocytes were obtained by mashing the
spleens through a 40 uM mesh filter, followed by addition of 10 mL
of PBS and collection by centrifugation (250.times.g, 5'). Cells
were resuspended in 2 mL of ACK buffer for 5 min followed by
addition of 10 mL of PBS and collected by centrifugation as above.
The cell pellet was resuspended in 2 mL of PBS, passed through a 40
uM mesh filter, and the total cells counted. Tumor cell suspensions
were obtained using the Miltenyi mouse tumor dissociation kit and
the Octomax dissociator according to the manufacturer's protocol,
except 1/10.sup.th of the amount of enzyme "R" was utilized.
Following dissociation, cells were washed in PBS and collected by
centrifugation, and then counted as above. Approximately 5 million
splenocytes and the entire collected amount of tumor cells
(typically, 1-5 million) from each mouse were then stained using
the cocktail of antibodies as described in Table 12, below:
TABLE-US-00017 TABLE 12 Antibodies used for staining splenocytes
and tumor cell suspensions for flow cytometry. Target Label Clone
Vendor CD8a APC-Cy7 53-6.7 Biolegend PD1 BV421 29F.1A12 Biolegend
CD4 FITC GK1.5 Biolegend NKp46 PerCP-Cy5.5 29A1.4 BD CD45 BV786
30-F11 Biolegend
[0310] For flow cytometric analysis, after defining cells as live,
singlet, CD45+ lymphocytes, T cells were gated as either CD4+ or
CD8+; NK cells were defined as NKp46+ cells. FIG. 9A shows the mean
fluorescence intensity (MFI) of anti-PD-1 staining on each of the
aforementioned cell populations; FIG. 9B shows the percent of each
population that stained positive (as defined by gating on an FMO
control sample).
[0311] Thus, despite the reported low abundance of T cells amongst
B16F10 TILs, the PD-1 expression pattern on TIL and peripheral T
cells presented an opportunity to evaluate the effects of
PD-1-driven targeting of an IL-15 moiety.
Example 8: Anti-Tumor Efficacy of Targeted IL-15 Molecules in the
Murine Syngeneic B16F10 Melanoma Tumor Model
[0312] This Example demonstrates the effects of various compounds,
including 3 PD-1-targeted IL-15 muteins, on tumor growth, body
weight changes, and overall survival of mice in the murine
syngeneic B16F10 melanoma tumor model. To evaluate the effects of
PD-1-targeted IL-15 muteins in this tumor model, the following
experiment was carried out:
[0313] Female C57/B16 mice were subcutaneously implanted in the
upper thigh with approximately 500,000 B16F10 cells, which had been
freshly thawed from a single, low-passage vial (of 1*10{circumflex
over ( )}7 cells) and cultured for the minimum time required to
establish sufficient cells for implantation. When sufficient
animals with visible tumor masses of 60-100 mm.sup.3 (as defined by
(length.times.width.sup.2)/2) was obtained, mice were randomized
into groups (of n=10 animals) immediately prior to dosing. Each
compound of interest was injected subcutaneously (neck scruff),
every 3-4 days, for a total of 3 doses (day 0, defined as the day
of first dosing), into each animal of a given group. Tumor volumes,
body weight, and animal survival were tracked throughout the course
of the experiment. Animals were sacrificed once their tumor volume
measured at least 2000 mm.sup.3. Overall survival of mice in each
treatment group, as well as the body weight of animals within each
treatment group, were recorded.
[0314] FIG. 10 depicts the results of a study wherein tumor-bearing
mice were dosed with xmPD1-IL15 NQ at concentrations of 5, 3, 1, or
0.3 mg/kg; or, with xmPD1-IL15RaSu-IL15 at 0.3 mg/kg; or, with PBS
as a vehicle control. FIG. 10A plots the size of implanted tumors
starting at day 0 (first day of drug injection). Animals in groups
receiving 5, 3, 1, or 0.3 mg/kg of anti-mouse PD-1-IL15 NQ
experienced a dose-dependent inhibition in average tumor volume and
in tumor volume per individual animal. Furthermore, animals in
various groups experienced durable tumor regression (no tumor mass
detectable more than 45 days from completion of drug
administration), with long-term survival as indicated in Table 13,
below:
TABLE-US-00018 TABLE 13 Long-term survival from animals treated
with anti-PD-1-IL15 compounds. Treatment group* Long-term surviving
animals* PBS 0/10 xmPD-1-IL15 NQ, 5 mg/kg 5/6 xmPD-1-IL15 NQ, 3
mg/kg 8/10 xmPD-1-IL15 NQ, 1 mg/kg 6/10 xmPD-1-IL15 NQ, 0.3 mg/kg
5/9 xmPD-1-IL15RaSu-IL15, 0.3 mg/kg 3/8 *Mice in each treatment
group were scored for long-term survival (more than 45 days
tumor-free beyond the cessation of drug treatment).
[0315] Animals that experienced long-term survival following
xmPD-1-IL15 NQ treatment were re-challenged with B16F10 tumor cells
as described in this Example. No detectable tumors grew in any of
the long-term surviving animals (in contrast to tumor growth in
100% of naive mice implanted with B16F10 cells in parallel). FIG.
10B plots the proportion of viable animals throughout the study. In
the 5 mg/kg xmPD-1-IL15 NQ treatment group, 4 of 10 animals died
in-study with tumor volumes less than 500 mm.sup.3. FIG. 10C plots
the averaged weight changes (from baseline, at day 0) of animals in
each treatment group throughout the study. Animals in the 5 mg/kg
and 3 mg/kg treatment groups experienced transient weight loss
(maximum, 15% drop relative to day 0) which returned to baseline
upon completion of dosing (FIG. 9C). The maximum tolerated dose of
xmPD-1-IL15 NQ was defined as at or below 3 mg/kg (consistent with
observations described in Example 6).
[0316] To compare and contrast the effects on tumor-bearing mice
of: an anti-PD-1 antibody; an untargeted IL-15 NQ compound; and a
PD-1-targeted IL-15 NQ, the following experiment was conducted:
[0317] A B16F10 tumor model study was established as previously
described herein. The study included groups of n=10 animals, as
summarized in Table 14, below:
TABLE-US-00019 TABLE 14 Treatment groups for B16 tumor efficacy
study. Group Treatment 1 PBS 2 xmPD1-IL15 NQ, 1 mg/kg 3 xmPD1-IL15
NQ, 0.3 mg/kg 4 xmPD1, 1 mg/kg 5 xmPD1, 0.3 mg/kg 6 Ab8.8-IL15 NQ,
1 mg/kg 7 Ab8.8-IL15 NQ, 0.3 mg/kg
[0318] FIG. 11 depicts the results of a study wherein tumor-bearing
mice were dosed with the compounds listed in Table 14. FIG. 11A
plots the size of implanted tumors starting at day 0 (first day of
drug injection). Treatment with either anti-PD-1 antibody, or with
untargeted IL-15 NQ (Ab8.8-IL15 NQ) did not significantly alter the
tumor growth characteristics compared to PBS treated animals. In
contrast, mice treated with xmPD1-IL15 NQ exhibited significant
tumor growth inhibition, in a dose-dependent manner. FIG. 11B plots
the averaged body weight changes (from baseline, at day 0) of
animals in each treatment group throughout the study. Mice treated
with xmPD1-IL15 NQ or Ab8.8-IL15 NQ at 1 mg/kg exhibited modest
body weight loss (less than 10% from baseline) at day 6,
immediately prior to receiving the third dose. This weight loss was
transient, with animals returning to baseline weight upon cessation
of treatment.
[0319] Thus, at equivalent doses, xmPD1-IL15 NQ provided superior
tumor growth inhibition versus xPD-1 or Ab8.8-IL15 NQ, and was well
tolerated.
[0320] To determine the effects of a single dose of the xmPD1-IL-15
M1, the following experiment was conducted. A B16F10 tumor model
study was established as previously described herein, with the
exception that instead of previously giving 3 doses of each
compound, only a single dose was administered in the experiment
described below. The study included groups of n=10 animals, as
summarized in Table 15, below. Table 15. Treatment groups for B16
tumor efficacy study
TABLE-US-00020 TABLE 15 Treatment groups for B16 tumor efficacy
study. Group Treatment 1 PBS 2 xmPD1-IL15 M1 5 mg/kg 3 xmPD1-IL15
M1 1 mg/kg 4 xmPD1-IL15 M1 0.3 mg/kg 5 xmPD1-IL15 M1 0.1 mg/kg
[0321] FIGS. 11C and 11D depict the results of a study wherein
tumor-bearing mice were singly-dosed with either concentrations of
xmPD1-IL-15 M1 listed in table 15, or PBS as a vehicle control.
FIG. 11C demonstrates xPD1-IL15 M1 reduces B16F10 tumor growth in a
dose-dependent manner. All xmPD1-IL15 M1 doses tested (0.1-5 mg/kg)
demonstrated significant inhibition of tumor growth relative to
control. Table 15 below, summarizes the number of mice at the end
of the study that were determined to be tumor-free relative to each
treatment. 5 mg/kg of xmPD1-IL15 M1 yielded the highest number of
tumor free mice (6/10) by the end of the study. FIG. 11D plots the
averaged body weight changes (from baseline, at day 0) of animals
in each treatment group throughout the study. Mice treated with a
single dose of xmPD1-IL15 M1 at 5 mg/kg exhibited average body
weight loss of 13.5% from baseline at day 6. This weight loss was
transient, with body weights rising by the next measurement (3 days
later) and back to baseline upon cessation of treatment. No other
treatment groups exhibited any weight loss for the duration of the
study.
TABLE-US-00021 TABLE 16 Number of Tumor-free mice at the end of
study. Treatment group Tumor-free mice* PBS 0/10 xmPD1-IL15 M1 5
mg/kg 2/10 xmPD1-IL15 M1 1 mg/kg 3/10 xmPD1-IL15 M1 0.3 mg/kg 3/10
xmPD1-IL15 M1 0.1 mg/kg 6/10 *Tumor-free mice were determined based
upon mice with tumor sizes at the end of study being either
non-palpable or measuring less than D 0 (start of dosing) tumor
volume.
[0322] To determine the effects of a single dose of the xmPD1-IL-15
M2, the following experiment was conducted. A B16F10 tumor model
study was established as previously described herein, with the
exception that instead of previously giving 3 doses of each
compound, only a single dose was administered in the experiment
described below. This study included groups of n=10 animals, as
summarized in Table 17 below. Table 17. Treatment groups for B16
tumor efficacy study.
TABLE-US-00022 TABLE 17 Treatment groups for B16 tumor efficacy
study. Group Treatment 1 PBS 2 xmPD1-IL15 M2 5 mg/kg 3 xmPD1-IL15
M2 1 mg/kg 4 xmPD1-IL15 M2 0.3 mg/kg 5 xmPD1-IL15 M2 0.1 mg/kg
[0323] FIGS. 11E and 11F depict the results of a study wherein
tumor-bearing mice were singly-dosed with either concentrations of
xmPD1-IL-15 M2 listed in table 17, or PBS as a vehicle control.
FIG. 11E demonstrates xPD1-IL15 M2 reduces B16F10 tumor growth in a
dose-dependent manner. All xmPD1-IL15 M2 doses tested (0.1-5 mg/kg)
demonstrated significant inhibition of tumor growth relative to
control. Table 18 below, summarizes the number of mice at the end
of the study that were determined to be tumor-free relative to each
treatment. 5 mg/kg of xmPD1-IL15 M2 yielded the highest number of
tumor free mice (4/10) by the end of the study. FIG. 11F plots the
averaged body weight changes (from baseline, at day 0) of animals
in each treatment group throughout the study. Mice treated with a
single dose of xmPD1-IL15 M2 at 5 mg/kg exhibited average body
weight loss of 2.9% from baseline at day 6. This weight loss was
transient, with body weights returning back to baseline by the next
measurement 3 days later. No other treatment groups exhibited any
weight loss for the duration of the study.
TABLE-US-00023 TABLE 18 Number of Tumor-free mice at the end of
study. Treatment group Tumor-free mice* PBS 0/10 xmPD1-IL15 M2 5
mg/kg 1/10 xmPD1-IL15 M2 1 mg/kg 1/10 xmPD1-IL15 M2 0.3 mg/kg 3/10
xmPD1-IL15 M2 0.1 mg/kg 4/10 *Tumor-free mice were determined based
upon mice with tumor sizes at the end of study being either
non-palpable or measuring less than D 0 (start of dosing) tumor
volume.
[0324] To compare the effects of a single dose of the xmPD1-IL-15
M1 to xmPD1 alone, the following experiment was conducted. A B16F10
tumor model study was established as previously described herein,
with the exception that instead of previously giving 3 doses of
each compound, only a single dose was administered in the
experiment described below. The study included groups of n=10
animals, as summarized in Table 19, below.
TABLE-US-00024 TABLE 19 Treatment groups for B16 tumor efficacy
study. Group Treatment 1 PBS 2 xmPD1 1 mg/kg 3 xmPD1-IL15 M1 1
mg/kg
[0325] As demonstrated previously, 1 mg/kg of xmPD1-IL15 M1 given
as a single dose to B16F10 tumor-bearing mice demonstrated
significant tumor inhibition without any body weight loss. FIG. 11G
demonstrates that while a single dose of 1 mg/kg of xmPD1 can
impair B16F10 tumor growth, xmPD1-IL15 M1, demonstrates a
significant improvement greater to that of xmPD1 alone. FIG. 11H
plots the averaged body weight changes (from baseline, at day 0) of
animals in each treatment group throughout the study. Neither, mice
treated with a single dose of xmPD1-IL15 M1 at 1 mg/kg, nor 1 mg/kg
of xmPD1 exhibited any body weight loss from baseline.
Example 9. Anti-Tumor Efficacy of Targeted IL-15 Molecules in the
Murine Syngeneic MC38 Colon Adenocarcinoma Tumor Model
[0326] This Example demonstrates the effects of various compounds,
including 2 PD-1-targeted IL-15 muteins, on tumor growth and body
weight changes of mice in the murine syngeneic MC38 colon
adenocarcinoma tumor model. To evaluate the effects of
PD-1-targeted IL-15 muteins in this tumor model, the following
experiment was carried out.
[0327] Female C57/B16 mice were subcutaneously implanted in the
upper thigh with approximately 500,000 MC38 cells, which had been
freshly thawed from a single, low-passage vial (of 1*10{circumflex
over ( )}7 cells) and cultured for the minimum time required to
establish sufficient cells for implantation. When sufficient
animals with visible tumor masses of 60-90 mm.sup.3 (as defined by
(length.times.width.sup.2)/2) was obtained, mice were randomized
into groups (of n=10 animals) immediately prior to dosing. Each
compound of interest was injected subcutaneously (neck scruff),
once (day 0, defined as the day of first dosing), into each animal
of a given group. Tumor volumes and body weight were tracked
throughout the course of the experiment. Animals were sacrificed
once their tumor volume measured at least 2000 mm.sup.3.
[0328] FIGS. 11I and 11J depict the results of a study wherein
tumor-bearing mice were dosed with xmPD1-IL15 M1 at concentrations
of 5, 1, 0.3, or 0.1 mg/kg; or, with PBS as a vehicle control. FIG.
11I plots the size of implanted tumors starting at day 0 (first day
of drug injection). Animals in groups receiving 5, 1 and 0.3 mg/kg
of anti-mouse PD-1-IL15 M1 experienced a dose-dependent inhibition
in average tumor volume and in tumor volume per individual animal.
Table 20 below, summarizes the number of mice at the end of the
study that were determined to be tumor-free relative to each
treatment. 5 mg/kg of xmPD1-IL15 M1 yielded the highest number of
tumor free mice (9/10) by the end of the study. FIG. 11J plots the
averaged body weight changes (from baseline, at day 0) of animals
in each treatment group throughout the study. Animals receiving 5
mg/kg of xmPD1-IL15 M1 yielded a transient 13.2% body weight loss
from DO, that returned to baseline levels by the end of study.
TABLE-US-00025 TABLE 20 Number of Tumor-free mice at the end of
study. Treatment group Tumor-free mice* PBS 0/10 xmPD1-IL15 M1 0.1
mg/kg 0/10 xmPD1-IL15 M1 0.3 mg/kg 2/10 xmPD1-IL15 M1 1 mg/kg 4/10
xmPD1-IL15 M1 5 mg/kg 9/10 *Tumor-free mice were determined based
upon mice with tumor sizes at the end of study being either
non-palpable or measuring less than D 0 (start of dosing) tumor
volume.
[0329] FIGS. 11K and 11L depict the results of a study wherein
tumor-bearing mice were dosed with xmPD1-1L15 M2 at concentrations
of 5, 1, 0.3, or 0.1 mg/kg; or, with PBS as a vehicle control. FIG.
11K plots the size of implanted tumors starting at day 0 (first day
of drug injection). Animals in groups receiving 5, 1, and 0.3 mg/kg
of anti-mouse PD-1-IL15 M2 experienced a dose-dependent inhibition
in average tumor volume and in tumor volume per individual animal.
Table 21 below, summarizes the number of mice at the end of the
study that were determined to be tumor-free relative to each
treatment. 5 mg/kg of xmPD1-IL15 M2 yielded the highest number of
tumor free mice (4/10) by the end of the study. FIG. 11L plots the
averaged body weight changes (from baseline, at day 0) of animals
in each treatment group throughout the study. xmPD1-IL15 M2 at
doses described above, did not yield any body weight loss
throughout the study.
TABLE-US-00026 TABLE 21 Number of Tumor-free mice at the end of
study. Treatment group Tumor-free mice* PBS 0/10 xmPD1-IL15 M2 0.1
mg/kg 0/10 xmPD1-IL15 M2 0.3 mg/kg 0/10 xmPD1-IL15 M2 1 mg/kg 3/10
xmPD1-IL15 M2 5 mg/kg 4/10 *Tumor-free mice were determined based
upon mice with tumor sizes at the end of study being either
non-palpable or measuring less than D 0 (start of dosing) tumor
volume.
Example 10. Characterizing the Effects of Targeted IL-15 on Murine
Immune Cell Populations
[0330] This Example describes a method to assess the functional
consequences of administering a PD-1-targeted IL-15 mutein to a
mouse, using immune cell phenotyping of splenic and
tumor-infiltrating lymphocytes.
[0331] B16F10 tumor-bearing mice were established essentially as
described in Example 8, Table 14, with n=4 or 5 mice per treatment
group. When Table 22 lists the compounds received by each group and
the number of animals per group:
TABLE-US-00027 TABLE 22 Treatment groups for immune cell profiling
study. Group Treatment 1 PBS, n = 5 2 xmPD1-IL15 NQ, 0.3 mg/kg, n =
4 3 xmPD1, 0.3 mg/kg, n = 5 4 Ab8.8-IL15 NQ, 0.3 mg/kg, n = 5
[0332] Splenocytes and tumor cell suspensions were obtained from
each animal as described in Example 7. The resulting cell
suspensions were then stained with the following cocktail of
fluorescently-labeled antibodies or viability dyes, as indicated
below in Table 23:
TABLE-US-00028 TABLE 23 Antibody and live/dead dye staining panel
for immune cell profiling study. Target Label Clone Manufacturer
Dilution L/D Blue Life Tech 1:100 CD45 A700 30-F11 Biolegend 1:100
CD90.2 BUV395 30-H12 Biolegend 1:100 CD4 PerCPCy5.5 GK1.5 Biolegend
1:100 CD8 BV785 53-6.7 Biolegend 1:100 FoxP3 Pac Blue FJK-16s
eBiosciences 1:50 xhFc APC H2 Southern Biotech 1:500 CD16 APCe780
ebioCB16 Invitrogen Perforin PE dG9 Biolegend
[0333] Staining, washing, and analysis of cells by flow cytometry
were carried out as described in Example 7.
[0334] FIG. 12A shows the results from an experiment designed to
detect either Ab8.8-IL15 NQ, xmPD1-IL15 NQ, or xPD-1, on isolated
splenocytes or tumor-infiltrating lymphocytes. T cells (viable,
singlet, CD90.2+) from either tumor (histograms, shown on top row)
or spleen (shown on bottom row) were further subdivided into
CD8+(left column) or CD4+(right column). Overlaid histograms for
each cell population show the results of staining those cells with
anti-human Fc (to detect the common human IgG Fc portion present in
each of the recombinant molecules) from animals treated with the
compounds as listed next to each plot. The xmPD1-NQ molecule was
detected on CD8+ TILs and to a lesser extent on CD4+ TILs,
mirroring the detection of xmPD1 molecule, albeit with a lower
intensity. Ab8.8-IL15 NQ molecule was not detected above background
levels on CD8+ TILs. Very little signal above background staining
was observed for all molecules from CD4+ or CD8+ splenocytes at the
time of analysis. Table 24 summarizes the results obtained from
staining splenocytes and TILs from all animals including in this
experiment.
TABLE-US-00029 TABLE 24 Summary of anti-human Fc+ splenocytes or
TILs from mice of various treatment groups. Tumor Spleen Treatment
% xFc+ of % xFc+ of Treatment % xFc+ of % xFc+ of Sample # group
CD8+ CD4+ Sample # group CD8+ CD4+ T1 xPD1 85.9 70.3 S1 xPD1 3.84
4.75 T2 xPD1 68.6 87.1 S2 xPD1 1.64 15.5 T3 xPD1 87.4 57.4 S3 xPD1
1.98 7.64 T4 xPD1 81.1 69.7 S7 XPD1-IL15 0.25 1.39 NQ T5 xPD1 88
66.7 S8 XPD1-IL15 0.48 0.91 NQ T7 XPD1-IL15 63.2 22.5 S9 XPD1-IL15
0.27 0.84 NQ NQ T8 XPD1-IL15 39.8 12.8 S10 XPD1-IL15 0.23 2.17 NQ
NQ T9 XPD1-IL15 51.7 22.5 S11 Ab8.8-IL15 0.049 0.28 NQ NQ T10
XPD1-IL15 23.8 12.9 S12 Ab8.8-IL15 0.074 0.2 NQ NQ T11 Ab8.8-IL15
0.68 1 S13 Ab8.8-IL15 0.44 0.32 NQ NQ T12 Ab8.8-IL15 2.06 5.88 S14
Ab8.8-IL15 13.1 0.23 NQ NQ T13 Ab8.8-IL15 1.1 3.5 S15 Ab8.8-IL15
0.84 0.14 NQ NQ T14 Ab8.8-IL15 2.09 3.56 S16 PBS 0.17 0.096 NQ T15
Ab8.8-IL15 1.72 6.18 S17 PBS 0.07 0.089 NQ T16 PBS 0.56 0.93 S18
PBS 0.092 0.14 T17 PBS 0.38 0.44 S19 PBS 0.089 0.14 T18 PBS 0.33
0.44 S20 PBS 0.031 0.12 T19 PBS 2.2 1.02 T20 PBS 0.25 0.21
[0335] In summary, anti-PD1 and xmPD1-IL15 NQ show similar patterns
of targeting: specifically, they are enriched on tumor-resident
CD8+ T cells.
[0336] In addition, these same stained splenocytes and TILs were
analyzed to assess changes in immune cell frequencies due to the
aforementioned treatments. FIG. 12B and FIG. 120 depict the changes
in immune cell subsets due to various treatments in splenocytes or
TILs, respectively. FIG. 12D plots the CD8:CD4 ratios of T cells in
the spleen and the tumor. Of note, the CD8:CD4 T cell ratio in the
tumor increased with xmPD1-IL15 NQ treatment, but not with
Ab8.8-IL15 NQ or xmPD1 treatments. There were no changes in splenic
CD8:CD4 ratios in any of the treatment groups. Thus, using a
PD-1-targeted version of the IL-15 NQ mutein resulted in
significant changes in the CD8:CD4 ratio, as well as significant
differences in anti-tumor efficacy (FIG. 10A).
[0337] FIG. 13 describes a method to assess the pharmacodynamic
effect of administering a PD-1-targeted IL-15 muteins M1 and M2 to
a mouse, using immune cell phenotyping of peripheral blood and
tumor-infiltrating lymphocytes.
[0338] B16F10 tumor-bearing mice were established essentially as
described in Example 8 Table 15, with n=5 mice per treatment group.
When Table 25 lists the compounds received by each group and the
number of animals per group:
TABLE-US-00030 TABLE 25 Treatment groups for immune cell profiling
study. Group Treatment 1 PBS, n = 5 2 xmPD1-IL15 M1, 0.3 mg/kg, n =
5 3 xmPD1-IL15 M1, 1 mg/kg, n = 5 4 xmPD1-IL15 M2, 1 mg/kg, n = 5 5
xmPD1-IL15 M2, 3 mg/kg, n = 5
[0339] Peripheral blood and tumor cell suspensions were obtained
from each animal on Day 3, 6 or 9 post-xmPD1-IL15 treatment as
described in Example 7. The resulting cell suspensions were then
stained with a cocktail of fluorescently-labeled antibodies or
viability dyes, as indicated in Table 23. Staining, washing, and
analysis of cells by flow cytometry were carried out as described
in Example 7.
[0340] FIGS. 13A and 13B show the absolute counts of NK, CD8+T and
Treg cells in peripheral blood or tumor-infiltrating lymphocytes at
Day 6, which showed to have the maximum effect by xmPD1-IL15 M1 and
xmPD1-IL15 m2, respectively. Both xmPD1-IL15 muteins did not expand
Treg cells in peripheral blood and tumor. NK cells expanded more
than CD8+ T cells in peripheral blood, especially at the higher
dose. This is due to the fact that NK cells have higher expression
level of IL-2 receptors, while CD8+ T cells have no or low
expression of PD-1. But CD8+ T cells expanded more than NK cells in
the tumor, suggesting that both xmPD1-IL15 muteins are targeted to
the tumor via PD-1 on CD8+ TILs over NK cells.
[0341] Three studies were performed to determine which immune cell
type is required for anti-tumor efficacy of xmPD1-M1, and whether T
cell migration was required for efficacy: NK1.1+ depletion, CD8 T
cell depletion, and treatment with FTY720, an inhibitor of T cell
migration (shown in FIGS. 13C, 13D and 13E). CD8 T cells were
depleted with aCD8b antibody (clone 53-5.8, 350 mg dosed i.p., 3
and 1 day prior to M1 injection and weekly thereafter). NK cells
were depleted with aNK1.1 antibody (clone PK136, 200 .quadrature.g
i.p., 2 days prior to 1 mg/kg M1 injection and weekly thereafter).
Control groups included IgG2a isotype (clone C1.18.4) and IgG1
isotype (clone TNP6A7). FTY720 compound was dosed i.p. 1 mg/kg 3
and 1 days prior to 1 mg/kg M1 injection and every 3 days
thereafter.
[0342] FIGS. 13C and 13D show the respective effects of depleting
CD8 T and NK1.1+ cells on anti-tumor efficacy in B16 model after
treatment of 1 mg/kg xmPD1-IL15 M1. NK1.1+ cells contain both NK
and NK T cells. These studies suggested that efficacy of xmPD1-M1
was lost in the absence of CD8 T cells but not NK1.1+ cells.
[0343] FIG. 13E shows the effect of FTY420 treatment, which
inhibits T cell egress, on anti-tumor efficacy in B16 model after
treatment of 1 mg/kg xmPD1-IL15 M1. Results show that T cell
migration was not required for efficacy.
Example 11. Characterizing the Effects of Targeted IL-15 on
Cytokine Production In Vivo
[0344] This Example describes a method to assess the functional
consequences of administering a PD-1-targeted IL-15 mutein to a
mouse, by assessing the cytokines present in peripheral blood.
[0345] Female (8 week old) C57B6/6J mice (Jackson Laboratories,
Sacramento, Calif.) were dosed subcutaneously with the compounds
and doses as listed in Table 26, below:
TABLE-US-00031 TABLE 26 Treatment groups for in vivo cytokine
study. Group Treatment 1 xmPD1-IL15 NQ, 1 mg/kg 2 xmPD1-IL15 NQ,
0.3 mg/kg 3 xmPD1-IL15RaSu, 1 mg/kg 4 xmPD1-IL15RaSu, 0.3 mg/kg 5
Ab8.8-IL15 NQ, 1 mg/kg 6 Ab8.8-IL15 NQ, 0.3 mg/kg
[0346] Blood was collected at 0 hr, 2 hr, 4 hr, 6 hr, 24 hr, 48 hr,
72 hr, 96 hr and 120 hr post-dose (n=3 mice per time point) by
submandibular vein puncture (in-life) or by cardiac puncture
immediately (after euthanasia with CO.sub.2) and processed for
serum in micro 1.1 ml Z-gel tubes (Sarstedt, Numbrecht, Germany).
Appropriate volumes for blood draws in each mouse was followed
according to the NIH Guidelines for Survival Bleeding of Mice and
Rats http://oacu.od.nih.gov/ARAC/survival.pdf. All animals were
maintained in an AAALAC-accredited facility and used in accordance
with recommendations as described in the Guide for the Care and Use
of Laboratory Animals (Institute on Laboratory Animal Resources,
Rockville, Md.).
[0347] Multiple serum cytokines including IFNgamma and IL-6 were
measured using the multi-spot electrochemiluminescence U-plex assay
(Meso Scale Discovery, Rockville, Md.). A 1:5 dilution of total
serum from each mouse sample was made in assay buffer and added to
plates for an overnight incubation at 4.degree. C. The remaining
steps were followed as described by the manufacturer. Data were
plotted as groups with SEM and statistics were done by t-tests when
comparing 2 groups or 2-way ANOVA followed by Tukey's multiple
comparison tests (GraphPad Prism, La Jolla, Calif.).
[0348] FIG. 14 shows the results obtained from analyzing serum
cytokine levels in the various treatment group animals. FIG. 14A
shows the measured levels of IFN.gamma.. Of note,
xmPD1-IL15RaSu-IL15 treatment group animals had significantly
higher IFN.gamma. than did animals from the other treatment groups,
through 48 hrs post-dose. At 72 hrs, the xmPD1-IL15 NQ treated
animals exhibited slightly higher IFN.gamma. than animals from the
xmPD1-IL15RaSu-IL15 0.3 mg/kg group; and, at 96 hrs, animals from
both xmPD1-IL15 NQ groups had slightly higher IFN.gamma. than mice
from any other group. This difference persisted through the end of
the study (120 hrs post-dose). In contrast, animals receiving
untargeted, NQ-mutant IL15 (i.e., Ab8.8-IL15 NQ animals) produced
significantly lower IFNg at the 48 and 72 hr time points. Thus, the
kinetics and maximal response of IFN.gamma. generation differ
between the tested molecules, with NQ variants generating reduced
levels of IFNg compared to wildtype IL-15-containing molecules at
equivalent doses. Furthermore, the presence of an anti-PD-1
targeting arm led to significant differences in IFNg production,
highlighting the value of using a target-driven cytokine
modality.
[0349] FIG. 14B shows the levels of the cytokine IL-6 as measured
in the various treatment groups. In comparing all three tested
groups at 1 mg/kg, mPD1-IL15RaSu-IL15 treatment group animals had
significantly higher IL-6 than did animals from the other treatment
groups, through 48 hrs post-dose. In the 72-120 hour time point
samples, group xmPD1-IL15 NQ generated IL-6 levels that were
comparable to those from the xmPD1-IL15RaSu-IL15 1 mg/kg treatment
group. In contrast, animals from the Ab8.8-IL15 NQ group generated
a consistent amount of IL-6 which was always equivalent to or below
the levels generated by the PD-1 targeted versions. A similar
pattern of IL-6 production was seen in the 0.3 mg/kg groups, with
xmPD1-IL15RaSu-IL15 producing an earlier, higher peak, followed by
xmPD1-IL15 NQ producing a later-emerging and longer-lasting peak.
Thus, the NQ variant differs from its wildtype IL-15 moiety
counterpart in the kinetics and maximal output of two inflammatory
cytokines produced in vivo at comparable doses, and, targeted NQ
produced more IFN.gamma. and IL-6 across multiple time points than
did untargeted.
Example 12: In Vitro Functional Assay of Anti-Human PD-1-IL15 M1
and M2 Molecules Using Human Peripheral Blood Mononuclear Cells
(hPBMCs)
[0350] In this example, the effects of the targeted anti-human
PD-1-IL15 on the induction of pSTAT5 is compared to that of the
untargeted isotype control-antibody-IL15 chimeric molecules on
human NK cells, CD8.sup.+ effector memory (em) T cells and
CD8.sup.+ central memory (cm) T cells from hPBMCs.
[0351] Blood from healthy volunteers was taken and hPBMCs were
isolated using a ficoll-paque (GE Healthcare) gradient, washed with
PBS to remove platelets, and cleared of red blood cells using ACK
lysis buffer (Gibco). Cells were then plated at 2*10e6 cells/well
in 50 ul serum free RPMI 1640 media (Gibco), and allowed to rest
for 2-4 hours at 37.degree. C. After resting, cells were treated
with the listed molecules (Table 18) at the given concentrations
for 30 min at 37.degree. C. Cells were then immediately fixed with
2% PFA (Electron Microscopy Sciences) for 10 min at room
temperature, after which they were stained for surface markers for
30 min at 4.degree. C. Cells were then fixed again with 2% PFA for
10 min at room temperature, then resuspended in cold Perm Buffer
III (BD), and stored at -20.degree. C. overnight. The following
day, cells were stained for intracellular markers using the
antibodies (as described in Table 27, below) in permeabilization
buffer (ebioscience) for 45 min, then analyzed by flow
cytometry.
TABLE-US-00032 TABLE 27 Antibodies used for staining different
lymphocytes in hPBMC and pSTAT5 activity for flow cytometry. Target
Label Clone Vendor CD45 BV605 HI30 Biolegend CD3 BUV395 SK7 BD CD4
BV711 RPA-T4 Biolegend CD8 BV510 SK1 Biolegend CD25 BV421 BC96
Biolegend CD62L BV786 DREG-56 Biolegend CD45RO FITC UCHL1 Biolegend
CD16 APCe780 ebioCB16 Invitrogen Perforin PE dG9 Biolegend PSTAT5
A647 47/STAT5(p694) BD
[0352] FIGS. 15A-15F depict the effects of adding targeted
anti-human PD-1 (xhPD1)-IL15 and untargeted isotype
control-antibody-IL15 chimeric molecules to the hPBMCs, where they
cause different pSTAT5 activation on human NK cells, CD8.sup.+ em T
cells and CD8.sup.+ cm T cells. In NK cells, there is no
significant targeting activity for xhPD1-IL15 compared to that of
the isotype control-IL15 (M1 in FIG. 15A and M2 in FIG. 15D), as NK
cells have no or low PD-1 expression (Table 28). In CD8.sup.+ em T
cells and CD8.sup.+ cm T cells, there are significant targeting
activities for xhPD1-IL15 compared to that of the isotype
control-IL15 (M1 in FIGS. 15B-15C and M2 in FIG. 15E-15F), as
CD8.sup.+ em T cells and CD8.sup.+ cm T cells have PD-1 expression
(Table 28). The EC50 values for the isotype control-IL15 on NK
cells are 3-4-fold lower as compared to that on CD8+ em T cells and
CD8+cm T cells, confirming that NK cells are more sensitive to IL15
activation in general due to the fact that they have higher level
of CD122/CD132 expression. However, the CD8.sup.+ cm T cells and
CD8.sup.+ cm T cells are more sensitive to the xPD1-IL15 activation
because of the targeting activity driven by the PD-1 expression on
those cell types. This example shows that xPD1-IL15 can also
preferentially activates PD-1+ cells over PD-1(low) cells in a
human system as well as the mouse system as shown in the previous
Examples. Table 28, below, summarizes the calculated EC50 values
for the aforementioned molecules, and also displays the fold change
as the ratio of EC50 between isotype control-IL15 and
xhPD1-IL15.
TABLE-US-00033 TABLE 28 Average EC50 (nM) of 3 Isotype Isotype
healthy FMO/ FMO/ control-- xhPD1-- control-- xhPD1-- Fold change*
donors % PD-1 MFI PD-1 IL15 M1 IL15 M1 IL15 M2 IL15 M2 M1 M2 NK
1.4/ 495/ 0.9455 0.4746 11.22 8.266 1.99 1.36 cells 6.55 491
CD8.sup.+em 2.53/ -3.76/ T cells 70.4 897 CD8.sup.+cm 2.89/ -10.1/
3.665 0.07804 37.63 0.4916 46.96 76.55 T cells 61.3 742 *Fold
change = EC50 (Isotype control--IL15)/EC50 (xhPD1--IL15)
Example 13: In Vivo Efficacy Study of Anti-Mouse PD-1-IL15 M1 and
Anti-EDA-IL-10 Fusion Protein in MC38 Model
[0353] This Example describes a method to assess the anti-tumor
efficacy of administering a PD-1-targeted IL-15 molecule (xPD1-IL15
M1) in combination with an anti-EDA-IL-10 fusion protein (xEDA-IL
10). Mice were dosed as follows: xPD1-IL15 M1 was dosed on day Oat
0.3 mg/kg or 1 mg/kg and anti-EDA-IL-10 fusion protein (xEDA-IL 10)
was dosed on day 0 and 2 times/week thereafter for a total of 5
doses at 5 mg/kg each dose. As shown in FIGS. 16A and 16B, a
synergistic effect was observed in both doses of xPD1-IL15 M1
administered in combination with anti-EDA-IL-10 fusion protein
(xEDA-IL 10), and in particular, the lower dose (0.3 mg/kg)
xPD1-IL15 M1.
[0354] FIG. 16C plots the averaged body weight changes (from
baseline, at day 0) of animals in each treatment group throughout
the study. Mice treated with a single dose of xPD1-IL15 M1 at 1
mg/kg in combination with 5 mg/kg of anti-EDA-IL-10 fusion protein
(xEDA-IL 10) exhibited average body weight loss of 12% from
baseline at day 6. This weight loss was transient, with body
weights returning back to baseline by the next measurement 3 days
later. No other treatment groups exhibited any weight loss for the
duration of the study.
[0355] Although the disclosed teachings have been described with
reference to various applications, methods, kits, and compositions,
it will be appreciated that various changes and modifications can
be made without departing from the teachings herein and the claimed
invention below. The foregoing examples are provided to better
illustrate the disclosed teachings and are not intended to limit
the scope of the teachings presented herein. While the present
teachings have been described in terms of these exemplary
embodiments, the skilled artisan will readily understand that
numerous variations and modifications of these exemplary
embodiments are possible without undue experimentation. All such
variations and modifications are within the scope of the current
teachings.
[0356] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials
differs from or contradicts this application, including but not
limited to defined terms, term usage, described techniques, or the
like, this application controls.
[0357] The foregoing description and Examples detail certain
specific embodiments of the invention and describes the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
Sequence CWU 1
1
991114PRTHomo sapiens 1Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys
Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr
Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys
Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp
Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn
Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys
Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu
Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr
Ser2448PRTArtificial SequenceSynthetic Construct 2Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
4453444PRTArtificial SequenceSynthetic construct 3Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
210 215 220Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
4404445PRTArtificial SequenceSynthetic construct 4Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220Pro Ser Cys Pro Ala Pro Glu Phe 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
Gln Glu Asp Pro Glu Val Gln 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
Phe 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 Gly Leu Pro Ser Ser 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 350Gln Glu Glu Met 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 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu 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 Leu Gly Lys 435 440
4455162PRTHomo sapiens 5Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile
Ser Ile Gln Cys Tyr1 5 10 15Leu Cys Leu Leu Leu Asn Ser His Phe Leu
Thr Glu Ala Gly Ile His 20 25 30Val Phe Ile Leu Gly Cys Phe Ser Ala
Gly Leu Pro Lys Thr Glu Ala 35 40 45Asn Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His65 70 75 80Pro Ser Cys Lys Val
Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95Val Ile Ser Leu
Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110Asn Leu
Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120
125Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
130 135 140Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe
Ile Asn145 150 155 160Thr Ser614PRTArtificial SequenceSynthetic
Construct 6Gly Gly Gly Gly Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala1
5 10715PRTArtificial SequenceSynthetic Construct 7Gly Leu Asn Asp
Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu1 5 10
158241PRTArtificial SequenceSynthetic Construct 8Ile Thr Cys Pro
Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr
Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys
Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys
Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55
60Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly65
70 75 80Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly 85 90 95Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu
Asp Leu 100 105 110Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr
Glu Ser Asp Val 115 120 125His Pro Ser Cys Lys Val Thr Ala Met Lys
Cys Phe Leu Leu Glu Leu 130 135 140Gln Val Ile Ser Leu Glu Ser Gly
Asp Ala Ser Ile His Asp Thr Val145 150 155 160Glu Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn 165 170 175Val Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn 180 185 190Ile
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile 195 200
205Asn Thr Gly Gly Gly Gly Ser Gly His His His His His His His His
210 215 220Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu
Trp His225 230 235 240Glu9144PRTArtificial SequenceSynthetic
Construct 9Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp
Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser
Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
Leu Glu Leu Gln 35 40 45Arg Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile
His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu
Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys
Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe
Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr Gly Gly Gly Gly
Ser Gly His His His His His His His His Gly 115 120 125Gly Gly Leu
Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 130 135
14010144PRTArtificial SequenceSynthetic Construct 10Asn Trp Val Asn
Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met
His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser
Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Asn
Ala Thr Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55
60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65
70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe
Ile Asn 100 105 110Thr Gly Gly Gly Gly Ser Gly His His His His His
His His His Gly 115 120 125Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln
Lys Ile Glu Trp His Glu 130 135 14011144PRTArtificial
SequenceSynthetic Construct 11Asn Trp Val Asn Val Ile Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr
Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala
Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Asn Ala Thr Leu Glu Ser
Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu
Ala Asn Asn Ser Leu Ser Ser Asn Gly Gln Val65 70 75 80Thr Glu Ser
Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu
Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Gly Gly Gly Gly Ser Gly His His His His His His His His Gly
115 120 125Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp
His Glu 130 135 14012118PRTArtificial SequenceSynthetic Construct
12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr
Asp Leu
Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11513107PRTArtificial SequenceSynthetic Construct 13Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly1 5 10 15Asp Lys Val
Thr Met Ser Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Trp Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr
Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala65
70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro
Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105147PRTArtificial SequenceSynthetic Construct 14Gly Phe Thr Phe
Ser Ser Tyr1 5155PRTArtificial SequenceSynthetic Construct 15Ser
Tyr Trp Met Ser1 5166PRTArtificial SequenceSynthetic Construct
16Ser Pro Ser Gly Gly Ser1 51717PRTArtificial SequenceSynthetic
Construct 17Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly189PRTArtificial SequenceSynthetic Construct
18Glu Ser Trp Gly Ala Tyr Tyr Asp Leu1 51911PRTArtificial
SequenceSynthetic Construct 19Arg Ala Ser Gln Gly Ile Ser Ser Trp
Leu Ala1 5 10207PRTArtificial SequenceSynthetic Construct 20Lys Ala
Ser Thr Leu Glu Ser1 5219PRTArtificial SequenceSynthetic Construct
21Gln Gln Ser Tyr Ser Thr Pro Trp Thr1 522668PRTArtificial
SequenceSynthetic Construct 22Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln
Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Pro Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met
Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Lys
Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn Phe Gly Thr Gln
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys 210 215 220Pro
Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe225 230
235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val 245 250 255Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu
Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg Val Val Ser Val Leu Thr 290 295 300Val Val His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr 325 330 335Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345
350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly
355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440 445Gly Thr Ser
Ala Thr Ala Thr Pro Gly Ala Ile Thr Cys Pro Pro Pro 450 455 460Met
Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr465 470
475 480Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala
Gly 485 490 495Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr
Asn Val Ala 500 505 510His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg
Asp Pro Ala Leu Val 515 520 525His Gln Arg Pro Ala Pro Pro Ser Gly
Gly Ser Gly Gly Gly Gly Ser 530 535 540Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Asn Trp Val Asn Val545 550 555 560Ile Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile 565 570 575Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val 580 585
590Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu
595 600 605Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile
Ile Leu 610 615 620Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
Glu Ser Gly Cys625 630 635 640Lys Glu Cys Glu Glu Leu Glu Glu Lys
Asn Ile Lys Glu Phe Leu Gln 645 650 655Ser Phe Val His Ile Val Gln
Met Phe Ile Asn Thr 660 665239PRTArtificial SequenceSynthetic
Construct 23Gly Gly Gly Gly Ser Gly Ser Gly Gly1 52414PRTArtificial
SequenceSynthetic Construct 24Gly Gly Gly Gly Gly Thr Ser Ala Thr
Ala Thr Pro Gly Ala1 5 102519PRTArtificial SequenceSynthetic
Construct 25Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Gly1 5 10 15Ser Gly Gly268PRTArtificial SequenceSynthetic
Construct 26His His His His His His His His1 5276PRTArtificial
SequenceSynthetic Construct 27His His His His His His1
528461PRTArtificial SequenceSynthetic Construct 28His His His His
His His His His Gly Gly Gly Gly Ser Gly Ser Gly1 5 10 15Gly Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly 20 25 30Gly Ser
Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 35 40 45Tyr
Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp 50 55
60Val Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser65
70 75 80Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu 85 90 95Phe Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr Tyr 100 105 110Cys Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu
Trp Gly Gln Gly 115 120 125Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 130 135 140Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu145 150 155 160Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 165 170 175Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 180 185 190Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 195 200
205Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
210 215 220Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Arg
Val Arg225 230 235 240Cys Pro Arg Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu 245 250 255Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 260 265 270Val Thr Cys Val Val Val Ala
Val Ser His Glu Asp Pro Glu Val Gln 275 280 285Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300Pro Arg Glu
Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu305 310 315
320Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys 340 345 350Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 355 360 365Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 370 375 380Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln385 390 395 400Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 405 410 415Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440
445His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455
46029570PRTArtificial SequenceSynthetic Construct 29Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile Ser
450 455 460Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
Asn Ala465 470 475 480Thr Leu Phe Thr Glu Ser Asp Val His Pro Ser
Cys Lys Val Thr Ala 485 490 495Met Lys Cys Phe Leu Leu Gln Leu Gln
Val Ile Ser Leu Gln Ser Gly 500 505 510Asp Ala Ser Ile His Asp Thr
Val Glu Asn Leu Ile Ile Leu Ala Asn 515 520 525Asn Ser Leu Ser Ser
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu 530 535 540Cys Gln Glu
Leu Glu Gln Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe545 550 555
560Val His Ile Val Gln Met Phe Ile Asn Thr 565
57030214PRTArtificial SequenceSynthetic Construct 30Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly1 5 10 15Asp Lys Val
Thr Met Ser Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Trp Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr
Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala65
70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro
Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 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 2103117PRTArtificial SequenceSynthetic
Construct 31Lys Ser Ser Gln Ser Leu Trp Asp Ser Gly Asn Gln Lys Asn
Phe Leu1 5 10 15Thr327PRTArtificial SequenceSynthetic Construct
32Trp Thr Ser Tyr Arg Glu Ser1 5339PRTArtificial SequenceSynthetic
Construct 33Gln Asn Asp Tyr Phe Tyr Pro Leu Thr1
534117PRTArtificial SequenceSynthetic Construct 34Gln 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 Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Asn Glu Lys Phe 50 55
60Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11535113PRTArtificial
SequenceSynthetic Construct 35Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Leu Trp Asp Ser 20
25 30Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly
Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr Arg Glu Ser
Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr Phe Tyr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile 100 105 110Lys36117PRTArtificial
SequenceSynthetic Construct 36Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Tyr Pro Gly
Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser 11537113PRTArtificial SequenceSynthetic
Construct 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 Lys Ser Ser Gln Ser Leu
Trp Asp Ser 20 25 30Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln
Lys Pro Gly Lys 35 40 45Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr
Arg Glu Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr Phe Tyr Pro Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105
110Lys38571PRTArtificial SequenceSynthetic Construct 38Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp
Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val
Glu Cys 210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val
Ser His Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310
315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425
430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly
435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn
Val Ile 450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
Met His Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asp Val
His Pro Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu
Gly Leu Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile
His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550
555 560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57039571PRTArtificial SequenceSynthetic Construct 39Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Gly Leu
Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Gln Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57040571PRTArtificial SequenceSynthetic Construct 40Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Gly Leu
Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Gln Asn Leu Ile Ser Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57041571PRTArtificial SequenceSynthetic Construct 41Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser
Gly Gly Gly Gly 435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala
Asn Trp Val Lys Val Ile 450 455 460Ser Asp Leu Lys Lys Ile Glu Asp
Leu Ile Gln Ser Met His Ile Asp465 470 475 480Ala Thr Leu Tyr Thr
Glu Ser Asn Val His Pro Ser Cys Lys Val Thr 485 490 495Ala Met Lys
Cys Phe Leu Leu Gly Leu Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly
Asp Ala Ser Ile His Asp Thr Val Gln Asn Leu Ile Ile Leu Ala 515 520
525Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys
530 535 540Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu
Gln Ser545 550 555 560Phe Val His Ile Val Gln Met Phe Ile Asn Thr
565 57042571PRTArtificial SequenceSynthetic Construct 42Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp
Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val
Glu Cys 210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val
Ser His Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310
315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425
430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly
435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn
Val Ile 450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
Met His Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asn Val
His Pro Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu
Gly Leu Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile
His Asp Thr Val Gln Asn Leu Ile Ser Leu Ala 515 520 525Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550
555 560Phe Val His Ile Val Gln Ala Phe Ile Asn Thr 565
57043571PRTArtificial SequenceSynthetic Construct 43Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Lys Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Gly Leu
Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Gln Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Ala Phe Ile Asn Thr 565
57044571PRTArtificial SequenceSynthetic Construct 44Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Gly Leu
Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Gln Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Ala Phe Ile Asn Thr 565
57045571PRTArtificial SequenceSynthetic Construct 45Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Lys Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Glu Leu
Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57046571PRTArtificial SequenceSynthetic Construct 46Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser
Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu
Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Glu Val Glu Cys 210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val
Ala Val Ser His Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295
300Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410
415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly
Gly Gly 435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp
Val Asn Val Ile 450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
Gln Ser Met His Ile Asp465 470 475 480Ala Thr Leu Lys Thr Glu Ser
Asn Val His Pro Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe
Leu Leu Glu Leu Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala
Ser Ile His Asp Thr Val Gln Asn Leu Ile Ile Leu Ala 515 520 525Asn
Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535
540Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln
Ser545 550 555 560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57047571PRTArtificial SequenceSynthetic Construct 47Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Lys Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Glu Leu
Gln Lys Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57048571PRTArtificial SequenceSynthetic Construct 48Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Gly Leu
Gln Lys Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57049668PRTArtificial SequenceSynthetic Construct 49Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Ile Thr Cys Pro Pro Pro
450 455 460Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser
Leu Tyr465 470 475 480Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe
Lys Arg Lys Ala Gly 485 490 495Thr Ser Ser Leu Thr Glu Cys Val Leu
Asn Lys Ala Thr Asn Val Ala 500 505 510His Trp Thr Thr Pro Ser Leu
Lys Cys Ile Arg Asp Pro Ala Leu Val 515 520 525His Gln Arg Pro Ala
Pro Pro Ser Gly Gly Ser Gly Gly Gly Gly Ser 530 535 540Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Asn Trp Val Asn Val545 550 555
560Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
565 570 575Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys
Lys Val 580 585 590Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu 595 600 605Ser Gly Asp Ala Ser Ile His Asp Thr Val
Gln Asn Leu Ile Ile Leu 610 615 620Ala Asn Asn Ser Leu Ser Ser Asn
Gly Asn Val Thr Glu Ser Gly Cys625 630 635 640Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln 645 650 655Ser Phe Val
His Ile Val Gln Met Phe Ile Asn Thr 660 66550668PRTArtificial
SequenceSynthetic Construct 50Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln
Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Pro Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Ile Thr Cys Pro Pro Pro
450 455 460Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser
Leu Tyr465 470 475 480Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe
Lys Arg Lys Ala Gly 485 490 495Thr Ser Ser Leu Thr Glu Cys Val Leu
Asn Lys Ala Thr Asn Val Ala 500 505 510His Trp Thr Thr Pro Ser Leu
Lys Cys Ile Arg Asp Pro Ala Leu Val 515 520 525His Gln Arg Pro Ala
Pro Pro Ser Gly Gly Ser Gly Gly Gly Gly Ser 530 535 540Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Asn Trp Val Asn Val545 550 555
560Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
565 570 575Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys
Lys Val 580 585 590Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu 595 600 605Ser Gly Asp Ala Ser Ile His Asp Thr Val
Gln Asn Leu Ile Ser Leu 610 615 620Ala Asn Asn Ser Leu Ser Ser Asn
Gly Asn Val Thr Glu Ser Gly Cys625 630 635 640Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln 645 650 655Ser Phe Val
His Ile Val Gln Met Phe Ile Asn Thr 660 66551668PRTArtificial
SequenceSynthetic Construct 51Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln
Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Pro Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met
Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Lys
Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn Phe Gly Thr Gln
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys 210 215 220Pro
Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe225 230
235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val 245 250 255Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu
Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg Val Val Ser Val Leu Thr 290 295 300Val Val His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr 325 330 335Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345
350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly
355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440 445Gly Thr Ser
Ala Thr Ala Thr Pro Gly Ala Ile Thr Cys Pro Pro Pro 450 455 460Met
Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr465 470
475 480Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala
Gly 485 490 495Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr
Asn Val Ala 500 505 510His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg
Asp Pro Ala Leu Val 515 520 525His Gln Arg Pro Ala Pro Pro Ser Gly
Gly Ser Gly Gly Gly Gly Ser 530 535 540Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Asn Trp Val Lys Val545 550 555 560Ile Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile 565 570 575Asp Ala
Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val 580 585
590Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu
595 600 605Ser Gly Asp Ala Ser Ile His Asp Thr Val Gln Asn Leu Ile
Ile Leu 610 615 620Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
Glu Ser Gly Cys625 630 635 640Lys Glu Cys Glu Glu Leu Glu Glu Lys
Asn Ile Lys Glu Phe Leu Gln 645 650 655Ser Phe Val His Ile Val Gln
Met Phe Ile Asn Thr 660 66552668PRTArtificial SequenceSynthetic
Construct 52Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly1 5 10 15Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly
Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala
Tyr Tyr Asp Leu Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser 180 185 190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Glu Val Glu Cys 210 215 220Pro Glu Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys
Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Gln Phe 260 265
270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val
Leu Thr 290 295 300Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly 355 360 365Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser385 390
395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Ser Gly Gly Gly Gly 435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly
Ala Ile Thr Cys Pro Pro Pro 450 455 460Met Ser Val Glu His Ala Asp
Ile Trp Val Lys Ser Tyr Ser Leu Tyr465 470 475 480Ser Arg Glu Arg
Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly 485 490 495Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala 500 505
510His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val
515 520 525His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly Gly Gly
Gly Ser 530 535 540Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn
Trp Val Asn Val545 550 555 560Ile Ser Asp Leu Lys Lys Ile Glu Asp
Leu Ile Gln Ser Met His Ile 565 570 575Asp Ala Thr Leu Tyr Thr Glu
Ser Asn Val His Pro Ser Cys Lys Val 580 585 590Thr Ala Met Lys Cys
Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu 595 600 605Ser Gly Asp
Ala Ser Ile His Asp Thr Val Gln Asn Leu Ile Ile Leu 610 615 620Ala
Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys625 630
635 640Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu
Gln 645 650 655Ser Phe Val His Ile Val Gln Ala Phe Ile Asn Thr 660
66553668PRTArtificial SequenceSynthetic Construct 53Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Ile Thr Cys Pro Pro Pro
450 455 460Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser
Leu Tyr465 470 475 480Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe
Lys Arg Lys Ala Gly 485 490 495Thr Ser Ser Leu Thr Glu Cys Val Leu
Asn Lys Ala Thr Asn Val Ala 500 505 510His Trp Thr Thr Pro Ser Leu
Lys Cys Ile Arg Asp Pro Ala
Leu Val 515 520 525His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly
Gly Gly Gly Ser 530 535 540Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Asn Trp Val Asn Val545 550 555 560Ile Ser Asp Leu Lys Lys Ile
Glu Asp Leu Ile Gln Ser Met His Ile 565 570 575Asp Ala Thr Leu Tyr
Thr Glu Ser Asn Val His Pro Ser Cys Lys Val 580 585 590Thr Ala Met
Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu 595 600 605Ser
Gly Asp Ala Ser Ile His Asp Thr Val Gln Asn Leu Ile Ser Leu 610 615
620Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly
Cys625 630 635 640Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys
Glu Phe Leu Gln 645 650 655Ser Phe Val His Ile Val Gln Ala Phe Ile
Asn Thr 660 66554668PRTArtificial SequenceSynthetic Construct 54Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp
Val 35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp
Leu Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Glu Val Glu Cys 210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val
Ala Val Ser His Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295
300Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410
415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly
Gly Gly 435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Ile Thr
Cys Pro Pro Pro 450 455 460Met Ser Val Glu His Ala Asp Ile Trp Val
Lys Ser Tyr Ser Leu Tyr465 470 475 480Ser Arg Glu Arg Tyr Ile Cys
Asn Ser Gly Phe Lys Arg Lys Ala Gly 485 490 495Thr Ser Ser Leu Thr
Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala 500 505 510His Trp Thr
Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val 515 520 525His
Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly Gly Gly Gly Ser 530 535
540Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Trp Val Lys
Val545 550 555 560Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln
Ser Met His Ile 565 570 575Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His Pro Ser Cys Lys Val 580 585 590Thr Ala Met Lys Cys Phe Leu Leu
Glu Leu Gln Val Ile Ser Leu Glu 595 600 605Ser Gly Asp Ala Ser Ile
His Asp Thr Val Gln Asn Leu Ile Ile Leu 610 615 620Ala Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys625 630 635 640Lys
Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln 645 650
655Ser Phe Val His Ile Val Gln Ala Phe Ile Asn Thr 660
66555571PRTArtificial SequenceSynthetic Construct 55Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Gln Leu
Gln Arg Ile Ser Leu Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57056571PRTArtificial SequenceSynthetic Construct 56Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Glu Leu
Gln Arg Ile Ser Leu Gln Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57057571PRTArtificial SequenceSynthetic Construct 57Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr
485 490 495Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Arg Ile Ser Leu
Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu
Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu Glu Leu Glu Gln Lys
Asn Ile Lys Glu Phe Leu Gln Ser545 550 555 560Phe Val His Ile Val
Gln Met Phe Ile Asn Thr 565 57058571PRTArtificial SequenceSynthetic
Construct 58Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly1 5 10 15Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly
Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala
Tyr Tyr Asp Leu Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser 180 185 190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Glu Val Glu Cys 210 215 220Pro Glu Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys
Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Gln Phe 260 265
270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val
Leu Thr 290 295 300Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly 355 360 365Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser385 390
395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Ser Gly Gly Gly Gly 435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly
Ala Asn Trp Val Asn Val Ile 450 455 460Ser Asp Leu Lys Lys Ile Glu
Asp Leu Ile Gln Ser Met His Ile Asp465 470 475 480Ala Thr Leu Tyr
Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr 485 490 495Ala Met
Lys Cys Phe Leu Leu Gln Leu Gln Val Ile Ser Leu Glu Ser 500 505
510Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala
515 520 525Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly
Cys Lys 530 535 540Glu Cys Glu Glu Leu Glu Gln Lys Asn Ile Lys Glu
Phe Leu Gln Ser545 550 555 560Phe Val His Ile Val Gln Met Phe Ile
Asn Thr 565 57059571PRTArtificial SequenceSynthetic Construct 59Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp
Val 35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp
Leu Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Glu Val Glu Cys 210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val
Ala Val Ser His Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295
300Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410
415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly
Gly Gly 435 440 445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp
Val Asn Val Ile 450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
Gln Ser Met His Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser
Asp Val His Pro Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe
Leu Leu Gln Leu Gln Val Ile Ser Leu Gln Ser 500 505 510Gly Asp Ala
Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn
Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535
540Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln
Ser545 550 555 560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57060571PRTArtificial SequenceSynthetic Construct 60Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Glu Leu
Gln Val Ile Ser Leu Gln Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Gln Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
57061571PRTArtificial SequenceSynthetic Construct 61Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys
210 215 220Pro Glu Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Ala Val Ser His
Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Glu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 435 440
445Gly Thr Ser Ala Thr Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile
450 455 460Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His
Ile Asp465 470 475 480Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
Ser Cys Lys Val Thr 485 490 495Ala Met Lys Cys Phe Leu Leu Gln Leu
Gln Val Ile Ser Leu Gln Ser 500 505 510Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu
Glu Leu Glu Gln Lys Asn Ile Lys Glu Phe Leu Gln Ser545 550 555
560Phe Val His Ile Val Gln Met Phe Ile Asn Thr 565
570628PRTArtificial SequenceSynthetic Construct 62Leu Thr Thr Gly
Thr Phe Ala Tyr1 563571PRTArtificial SequenceSynthetic Construct
63Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Thr
Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Lys Glu
Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln Gly Thr 100 105 110Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Asn Phe Gly Thr Gln Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys 210 215 220Pro Glu Cys
Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val
Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr 290 295 300Val Val His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr 325 330 335Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly 355 360
365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys Gly Gly Gly Gly 435 440 445Arg Thr Ser Ala Thr
Ala Thr Pro Gly Ala Asn Trp Val Asn Val Ile 450 455 460Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp465 470 475
480Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr
485 490 495Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Arg Ile Ser Leu
Glu Ser 500 505 510Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu
Ile Ile Leu Ala 515 520 525Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
Thr Glu Ser Gly Cys Lys 530 535 540Glu Cys Glu Glu Leu Glu Glu Lys
Asn Ile Lys Glu Phe Leu Gln Ser545 550 555 560Phe Val His Ile Val
Gln Met Phe Ile Asn Thr 565 57064326PRTArtificial SequenceSynthetic
Construct 64Ala 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 Val Glu Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala 130 135 140Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150
155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro 195 200 205Ser Ser Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265
270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro Gly Lys
32565220PRTArtificial SequenceSynthetic Construct 65Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Trp Asp Ser 20 25 30Gly Asn
Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala
Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr Arg Glu Ser Gly Val 50 55
60Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65
70 75 80Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Asn 85 90 95Asp Tyr Phe Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 115 120 125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200
205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
22066447PRTArtificial SequenceSynthetic Construct 66Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Arg Lys Thr His
210 215 220Thr Cys Pro Arg Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44567671PRTArtificial SequenceSynthetic Construct 67Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Glu Lys Thr His
210 215 220Thr Cys Pro Glu Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Glu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 435 440
445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ile Thr Cys Pro
450 455 460Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser
Tyr Ser465 470 475 480Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly Phe Lys Arg Lys 485 490 495Ala Gly Thr Ser Ser Leu Thr Glu Cys
Val Leu Asn Lys Ala Thr Asn 500 505 510Val Ala His Trp Thr Thr Pro
Ser Leu Lys Cys Ile Arg Asp Pro Ala 515 520 525Leu Val His Gln Arg
Pro Ala Pro Pro Ser Gly Gly Ser Gly Gly Gly 530 535 540Gly Ser Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Trp Val545 550 555
560Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met
565 570 575His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
Ser Cys 580 585 590Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
Gln Val Ile Ser 595 600 605Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu Ile 610 615 620Ile Leu Ala Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu Ser625 630 635 640Gly Cys Lys Glu Cys
Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe 645 650 655Leu Gln Ser
Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 660 665
67068574PRTArtificial SequenceSynthetic Construct 68Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Glu Lys Thr His
210 215 220Thr Cys Pro Glu Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310
315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Glu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asn Trp
Val Asn 450 455 460Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
Gln Ser Met His465 470 475 480Ile Asn Ala Thr Leu Phe Thr Glu Ser
Asp Val His Pro Ser Cys Lys 485 490 495Val Thr Ala Met Lys Cys Phe
Leu Leu Gln Leu Gln Val Ile Ser Leu 500 505 510Gln Ser Gly Asp Ala
Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile 515 520 525Leu Ala Asn
Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 530 535 540Cys
Lys Glu Cys Gln Glu Leu Glu Gln Lys Asn Ile Lys Glu Phe Leu545 550
555 560Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565
57069574PRTArtificial SequenceSynthetic Construct 69Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Glu Lys Thr His
210 215 220Thr Cys Pro Glu Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Glu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 435 440
445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asn Trp Val Asn
450 455 460Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
Met His465 470 475 480Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His Pro Ser Cys Lys 485 490 495Val Thr Ala Met Lys Cys Phe Leu Leu
Gly Leu Gln Arg Ile Ser Leu 500 505 510Glu Ser Gly Asp Ala Ser Ile
His Asp Thr Val Gln Asn Leu Ile Ile 515 520 525Leu Ala Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 530 535 540Cys Lys Glu
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu545 550 555
560Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565
57070574PRTArtificial SequenceSynthetic Construct 70Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Glu Lys Thr His
210 215 220Thr Cys Pro Glu Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Glu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 435 440
445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asn Trp Val Asn
450 455 460Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
Met His465 470 475 480Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His Pro Ser Cys Lys 485 490 495Val Thr Ala Met Lys Cys Phe Leu Leu
Gly Leu Gln Arg Ile Ser Leu 500 505 510Glu Ser Gly Asp Ala Ser Ile
His Asp Thr Val Gln Asn Leu Ile Ile 515 520 525Leu Ala Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 530 535 540Cys Lys Glu
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu545 550 555
560Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565
57071574PRTArtificial SequenceSynthetic Construct 71Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Glu Lys Thr His
210 215 220Thr Cys Pro Glu Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Glu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 435 440
445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asn Trp Val Gln
450 455 460Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
Met His465 470 475 480Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His Pro Ser Cys Lys 485 490 495Val Thr Ala Met Lys Cys Phe Leu Leu
Gly Leu Gln Arg Ile Ser Leu 500 505 510Glu Ser Gly Asp Ala Ser Ile
His Asp Thr Val Glu Asn Leu Ile Ile 515 520 525Leu Ala Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 530 535 540Cys Lys Glu
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu545 550 555
560Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565
57072574PRTArtificial SequenceSynthetic Construct 72Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Glu Lys Thr His
210 215 220Thr Cys Pro Glu Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Glu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Lys Trp
Val Asn 450 455 460Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
Gln Ser Met His465 470 475 480Ile Asp Ala Thr Leu Tyr Thr Glu Ser
Asn Val His Pro Ser Cys Lys 485 490 495Val Thr Ala Met Lys Cys Phe
Leu Leu Gly Leu Gln Arg Ile Ser Leu 500 505 510Glu Ser Gly Asp Ala
Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile 515 520 525Leu Ala Asn
Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 530 535 540Cys
Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu545 550
555 560Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565
57073574PRTArtificial SequenceSynthetic Construct 73Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Glu Lys Thr His
210 215 220Thr Cys Pro Glu Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Glu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 435 440
445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asn Trp Val Asn
450 455 460Val Ile Thr Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
Met His465 470 475 480Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His Pro Ser Cys Lys 485 490 495Val Thr Ala Met Lys Cys Phe Leu Leu
Gly Leu Gln Arg Ile Ser Leu 500 505 510Glu Ser Gly Asp Ala Ser Ile
His Asp Thr Val Glu Asn Leu Ile Ile 515 520 525Leu Ala Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 530 535 540Cys Lys Glu
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu545 550 555
560Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565
57074447PRTArtificial SequenceSynthetic Construct 74Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Trp Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44575574PRTArtificial SequenceSynthetic Construct 75Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Ser Cys Ala 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 435 440
445Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asn Trp Val Asn
450 455 460Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
Met His465 470 475 480Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His Pro Ser Cys Lys 485 490 495Val Thr Ala Met Lys Cys Phe Leu Leu
Gly Leu Gln Arg Ile Ser Leu 500 505 510Glu Ser Gly Asp Ala Ser Ile
His Asp Thr Val Gln Asn Leu Ile Ile 515 520 525Leu Ala Asn Asn Ser
Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 530 535 540Cys Lys Glu
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu545 550 555
560Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565
57076114PRTArtificial SequenceSynthetic construct 76Asn Trp Val Asn
Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met
His Ile Asn Ala Thr Leu Phe Thr Glu Ser Asp Val His 20 25 30Pro Ser
Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Gln Leu Gln 35 40 45Val
Ile Ser Leu Gln Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55
60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65
70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Gln Glu Leu Glu Gln Lys Asn
Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe
Ile Asn 100 105 110Thr Ser7715PRTArtificial SequenceSynthetic
construct 77Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly1 5 10 1578117PRTArtificial SequenceSynthetic construct 78Gln
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 Ser Tyr
20 25 30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu
Lys Phe 50 55 60Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Thr Gly Thr Phe Ala Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11579113PRTArtificial SequenceSynthetic construct 79Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Trp Asp Ser 20 25 30Gly Asn
Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro
Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr Arg Glu Ser Gly Val 50 55
60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65
70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln
Asn 85 90 95Asp Tyr Phe Tyr Pro His Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile 100 105 110Lys805PRTArtificial SequenceSynthetic construct
80Ser Tyr Trp Ile Asn1 5817PRTArtificial SequenceSynthetic
construct 81Gly Tyr Thr Phe Thr Ser Tyr1 58217PRTArtificial
SequenceSynthetic construct 82Asn Ile Tyr Pro Gly Ser Ser Ile Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly836PRTArtificial
SequenceSynthetic construct 83Tyr Pro Gly Ser Ser Ile1
584114PRTArtificial SequenceSynthetic Construct 84Ala Trp Val Asn
Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met
His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val His 20 25 30Pro Ser
Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Gly Leu Gln 35 40 45Arg
Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55
60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65
70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe
Ile Asn 100 105 110Thr Ser85114PRTArtificial SequenceSynthetic
Construct 85Gly Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp
Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser
Asn Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
Leu Gly Leu Gln 35 40 45Arg Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile
His Asp Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu
Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys
Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe
Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr
Ser86576PRTArtificial SequenceSynthetic Construct 86Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln
Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Pro Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met
Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Lys
Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys
355 360 365Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Trp 450 455 460Val
Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser465 470
475 480Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro
Ser 485 490 495Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Gly Leu
Gln Arg Ile 500 505 510Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu Asn Leu 515 520 525Ile Ile Leu Ala Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val Thr Glu 530 535 540Ser Gly Cys Lys Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile Lys Glu545 550 555 560Phe Leu Gln Ser
Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 565 570
57587576PRTArtificial SequenceSynthetic Construct 87Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Glu
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala
Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65
70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Ser Cys 355 360 365Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440
445Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Trp
450 455 460Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
Gln Ser465 470 475 480Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser
Asn Val His Pro Ser 485 490 495Cys Lys Val Thr Ala Met Lys Cys Phe
Leu Leu Gly Leu Gln Arg Ile 500 505 510Ser Leu Glu Ser Gly Asp Ala
Ser Ile His Asp Thr Val Gln Asn Leu 515 520 525Ile Ile Leu Ala Asn
Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu 530 535 540Ser Gly Cys
Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu545 550 555
560Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
565 570 57588448PRTArtificial SequenceSynthetic Construct 88Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser
Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Ala Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Trp Gly Ala Tyr Tyr Asp Leu
Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Ala Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Cys Thr Leu 340 345 350Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Trp Cys 355 360 365Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 44589575PRTArtificial SequenceSynthetic Construct
89Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr Phe Ala
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155
160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Ala Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Cys Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Ser Cys Ala 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Ser Gly 435 440 445Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ala Trp Val 450 455 460Asn Val Ile Ser Asp Leu Lys Lys
Ile Glu Asp Leu Ile Gln Ser Met465 470 475 480His Ile Asp Ala Thr
Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys 485 490 495Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Gly Leu Gln Arg Ile Ser 500 505 510Leu
Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile 515 520
525Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser
530 535 540Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys
Glu Phe545 550 555 560Leu Gln Ser Phe Val His Ile Val Gln Met Phe
Ile Asn Thr Ser 565 570 57590575PRTArtificial SequenceSynthetic
Construct 90Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Asn Ile Tyr Pro Gly Ser Ser Ile Thr Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Thr Thr Gly Thr
Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150
155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly Ala Pro Ser Val225 230 235 240Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265
270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Cys Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 355 360 365Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Ser Gly 435 440 445Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Gly Trp Val 450 455 460Asn Val Ile Ser
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met465 470 475 480His
Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys 485 490
495Lys Val Thr Ala Met Lys Cys Phe Leu Leu Gly Leu Gln Arg Ile Ser
500 505 510Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Gln Asn
Leu Ile 515 520 525Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn
Val Thr Glu Ser 530 535 540Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu
Lys Asn Ile Lys Glu Phe545 550 555 560Leu Gln Ser Phe Val His Ile
Val Gln Met Phe Ile Asn Thr Ser 565 570 5759110PRTArtificial
SequenceSynthetic Construct 91Gly Tyr Thr Phe Thr Ser Tyr Trp Ile
Asn1 5 109210PRTArtificial SequenceSynthetic Construct 92Gly Phe
Thr Phe Ser Ser Tyr Trp Met Ser1 5 1093114PRTArtificial
SequenceSynthetic Construct 93Asn Trp Val Asn Val Ile Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asn Ala Thr
Leu Phe Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala
Met Lys Cys Phe Leu Leu Gln Leu Gln 35 40 45Val Ile Ser Leu Gln Ser
Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu
Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser
Gly Cys Lys Glu Cys Gln Glu Leu Glu Gln Lys Asn Ile 85 90 95Lys Glu
Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser94117PRTArtificial SequenceSynthetic Construct 94Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Phe 20 25
30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Trp Val Ser
35 40 45Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Lys Ser Thr His Leu Tyr Leu Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115955PRTArtificial SequenceSynthetic Construct 95Gly Gly Ser Gly
Gly1 596108PRTArtificial SequenceSynthetic Construct 96Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Met Pro 20 25 30Phe
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg
Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 1059715PRTArtificial SequenceSynthetic Construct 97Ser Ser Ser
Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly1 5 10
1598160PRTArtificial SequenceSynthetic Construct 98Ser Pro Gly Gln
Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro1 5 10 15Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30Val Lys
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45Lys
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55
60Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala65
70 75 80Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly
Glu 85 90 95Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg
Phe Leu 100 105 110Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val
Lys Asn Ala Phe 115 120 125Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met Ser Glu Phe Asp 130 135 140Ile Phe Ile Asn Tyr Ile Glu Ala
Tyr Met Thr Met Lys Ile Arg Asn145 150 155 16099406PRTArtificial
SequenceSynthetic Construct 99Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Leu Phe 20 25 30Thr Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys
Ser Thr His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Glu Ile Val Leu Thr
115 120 125Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala
Thr Leu 130 135 140Ser Cys Arg Ala Ser Gln Ser Val Ser Met Pro Phe
Leu Ala Trp Tyr145 150 155 160Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile Tyr Gly Ala Ser 165 170 175Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly 180 185 190Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 195 200 205Val Tyr Tyr
Cys Gln Gln Met Arg Gly Arg Pro Pro Thr Phe Gly Gln 210 215 220Gly
Thr Lys Val Glu Ile Lys Ser Ser Ser Ser Gly Ser Ser Ser Ser225 230
235 240Gly Ser Ser Ser Ser Gly Ser Pro Gly Gln Gly Thr Gln Ser Glu
Asn 245 250 255Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
Arg Asp Leu 260 265 270Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe
Gln Met Lys Asp Gln 275 280 285Leu Asp Asn Leu Leu Leu Lys Glu Ser
Leu Leu Glu Asp Phe Lys Gly 290 295 300Tyr Leu Gly Cys Gln Ala Leu
Ser Glu Met Ile Gln Phe Tyr Leu Glu305 310 315 320Glu Val Met Pro
Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His 325 330 335Val Asn
Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg 340 345
350Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu
355 360 365Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile
Tyr Lys 370 375 380Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile
Glu Ala Tyr Met385 390 395 400Thr Met Lys Ile Arg Asn 405
* * * * *
References