U.S. patent application number 14/490328 was filed with the patent office on 2015-03-12 for human monoclonal antibodies against hendra and nipah viruses.
The applicant listed for this patent is The Government of the United States of America, as represented by the Secretary, Department of Healt, The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., The Government of the United States of America, as represented by the Secretary, Department of Healt. Invention is credited to Christopher C. Broder, Dimiter S. Dimitrov, Zhongyu Zhu.
Application Number | 20150071854 14/490328 |
Document ID | / |
Family ID | 37570906 |
Filed Date | 2015-03-12 |
United States Patent
Application |
20150071854 |
Kind Code |
A1 |
Dimitrov; Dimiter S. ; et
al. |
March 12, 2015 |
Human Monoclonal Antibodies Against Hendra and Nipah Viruses
Abstract
The present invention relates to monoclonal antibodies that bind
or neutralize Hendra or Nipah virus. The invention provides such
antibodies, fragments of such antibodies retaining Hendra or Nipah
virus-binding ability, fully human antibodies retaining Hendra or
Nipah virus-binding ability, and pharmaceutical compositions
including such antibodies. The invention further provides for
isolated nucleic acids encoding the antibodies of the invention and
host cells transformed therewith. Additionally, the invention
provides for prophylactic, therapeutic, and diagnostic methods
employing the antibodies and nucleic acids of the invention.
Inventors: |
Dimitrov; Dimiter S.;
(Rockville, MD) ; Zhu; Zhongyu; (Frederick,
MD) ; Broder; Christopher C.; (Silver Spring,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Henry M. Jackson Foundation for the Advancement of Military
Medicine, Inc.
The Government of the United States of America, as represented by
the Secretary, Department of Healt |
Bethesda
Rockville |
MD
MD |
US
US |
|
|
Family ID: |
37570906 |
Appl. No.: |
14/490328 |
Filed: |
September 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14026142 |
Sep 13, 2013 |
8858938 |
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14490328 |
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13671721 |
Nov 8, 2012 |
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14026142 |
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13195366 |
Aug 1, 2011 |
8313746 |
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13671721 |
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11886307 |
Jan 16, 2009 |
7988971 |
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PCT/US05/40050 |
Nov 4, 2005 |
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13195366 |
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60661766 |
Mar 14, 2005 |
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60678547 |
May 5, 2005 |
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60718902 |
Sep 20, 2005 |
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Current U.S.
Class: |
424/1.53 ;
424/1.49; 424/137.1; 435/252.3; 435/252.33; 435/5; 530/387.5;
536/23.53 |
Current CPC
Class: |
G01N 2333/115 20130101;
C07K 2317/51 20130101; C07K 2317/76 20130101; C07K 16/1027
20130101; G01N 33/6893 20130101; A61P 31/14 20180101; G01N 2469/10
20130101; A61K 51/1009 20130101; A61K 2039/505 20130101; C07K
2317/92 20130101; C07K 2317/55 20130101; C07K 2317/565 20130101;
C07K 16/1203 20130101; G01N 33/56983 20130101; C07K 2317/21
20130101 |
Class at
Publication: |
424/1.53 ;
530/387.5; 536/23.53; 424/137.1; 424/1.49; 435/252.33; 435/5;
435/252.3 |
International
Class: |
C07K 16/12 20060101
C07K016/12; G01N 33/569 20060101 G01N033/569; A61K 51/10 20060101
A61K051/10 |
Claims
1. A substantially pure polypeptide comprising an antibody
selectively binding a Hendra or Nipah G glycoprotein epitope,
wherein said antibody includes a heavy chain CDR3 region having the
amino acid sequence selected from the group consisting of SEQ ID
NO: 7, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 71,
SEQ ID NO: 87, SEQ ID NO: 103, SEQ ID NO: 119, SEQ ID NO: 135, SEQ
ID NO: 151, SEQ ID NO: 167, SEQ ID NO: 183, SEQ ID NO: 199, SEQ ID
NO: 215, SEQ ID NO: 231, SEQ ID NO: 247, SEQ ID NO: 263, SEQ ID NO:
279, SEQ ID NO: 295, SEQ TD NO: 311, SEQ ED NO: 327, SEQ ED NO:
343, SEQ ID NO: 359, SEQ ID NO: 375, SEQ ID NO: 391 and SEQ ID NO:
407.
2. The substantially pure polypeptide of claim 1 wherein said
antibody comprises an Fd fragment.
3. The substantially pure polypeptide of claim 1 wherein said
antibody comprises an Fab fragment.
4. The substantially pure polypeptide of claim 1 wherein said
antibody includes a heavy chain CDR2 region having the amino acid
sequence of: a) SEQ DD NO: 5 (when heavy chain CDR3 region is SEQ
ID NO: 7), b) SEQ ID NO: 21 (when heavy chain CDR3 region is SEQ ID
NO: 23), c) SEQ ID NO: 37 (when heavy chain CDR3 region is SEQ ID
NO: 39), d) SEQ ID NO: 53 (when heavy chain CDR3 region is SEQ ID
NO: 55), e) SEQ DD NO: 69 (when heavy chain CDR3 region is SEQ ID
NO: 71), f) SEQ ID NO: 85 (when heavy chain CDR3 region is SEQ ID
NO: 87), g) SEQ ID NO: 101 (when heavy chain CDR3 region is SEQ ID
NO: 103), h) SEQ ID NO: 117 (when heavy chain CDR3 region is SEQ ID
NO: 119), i) SEQ DD NO: 133 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ JD NO: 149 (when heavy chain CDR3 region is SEQ ID
NO: 151), k) SEQ ID NO: 165 (when heavy chain CDR3 region is SEQ ID
NO: 167), l) SEQ ID NO: 181 (when heavy chain CDR3 region is SEQ DD
NO: 183), m) SEQ DD NO: 197 (when heavy chain CDR3 region is SEQ DD
NO: 199), n) SEQ DD NO: 213 (when heavy chain CDR3 region is SEQ DD
NO: 215), o) SEQ DD NO: 229 (when heavy chain CDR3 region is SEQ DD
NO: 231), p) SEQ ID NO: 245 (when heavy chain CDR3 region is SEQ DD
NO: 247), q) SEQ DD NO: 261 (when heavy chain CDR3 region is SEQ DD
NO: 263), r) SEQ E) NO: 277 (when heavy chain CDR3 region is SEQ ID
NO: 279), s) SEQ TD NO: 293 (when heavy chain CDR3 region is SEQ ID
NO: 295), t) SEQ E) NO: 309 (when heavy chain CDR3 region is SEQ E)
NO: 311), u) SEQ E) NO: 325 (when heavy chain CDR3 region is SEQ E)
NO: 327), v) SEQ E) NO: 341 (when heavy chain CDR3 region is SEQ ID
NO: 343), w) SEQ E) NO: 357 (when heavy chain CDR3 region is SEQ E)
NO: 359), x) SEQ E) NO: 373 (when heavy chain CDR3 region is SEQ E)
NO: 375), y) SEQ E) NO: 389 (when heavy chain CDR3 region is SEQ E)
NO: 391), or z) SEQ ID NO: 405 (when heavy chain CDR3 region is SEQ
ID NO: 407).
5. The substantially pure polypeptide of claim 4 wherein said
antibody includes a heavy chain CDR1 region having the amino acid
sequence of: a) SEQ E) NO: 3 (when heavy chain CDR3 region is SEQ
E) NO: 7), b) SEQ E) NO: 19 (when heavy chain CDR3 region is SEQ ID
NO: 23), c) SEQ E) NO: 35 (when heavy chain CDR3 region is SEQ ID
NO: 39), d) SEQ E) NO: 51 (when heavy chain CDR3 region is SEQ E)
NO: 55), e) SEQ E) NO: 61 (when heavy chain CDR3 region is SEQ E)
NO: 71), f) SEQ E) NO: 83 (when heavy chain CDR3 region is SEQ E)
NO: 87), g) SEQ E) NO: 99 (when heavy chain CDR3 region is SEQ E)
NO: 103), h) SEQ E) NO: 115 (when heavy chain CDR3 region is SEQ E)
NO: 119), i) SEQ E) NO: 131 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ E) NO: 147 (when heavy chain CDR3 region is SEQ ID
NO: 151), k) SEQ E) NO: 163 (when heavy chain CDR3 region is SEQ E)
NO: 167), l) SEQ E) NO: 179 (when heavy chain CDR3 region is SEQ ID
NO: 183), m) SEQ E) NO: 195 (when heavy chain CDR3 region is SEQ E)
NO: 199), n) SEQ E) NO: 211 (when heavy chain CDR3 region is SEQ E)
NO: 215), o) SEQ E) NO: 227 (when heavy chain CDR3 region is SEQ E)
NO: 231), p) SEQ E) NO: 243 (when heavy chain CDR3 region is SEQ ID
NO: 247), q) SEQ E) NO: 259 (when heavy chain CDR3 region is SEQ E)
NO: 263), r) SEQ E) NO: 275 (when heavy chain CDR3 region is SEQ E)
NO: 279), s) SEQ E) NO: 291 (when heavy chain CDR3 region is SEQ TD
NO: 295), t) SEQ E) NO: 307 (when heavy chain CDR3 region is SEQ TD
NO: 311), u) SEQ E) NO: 323 (when heavy chain CDR3 region is SEQ TD
NO: 327), v) SEQ ID NO: 339 (when heavy chain CDR3 region is SEQ ID
NO: 343), w) SEQ ID NO: 355 (when heavy chain CDR3 region is SEQ ID
NO: 359), x) SEQ ID NO: 371 (when heavy chain CDR3 region is SEQ ID
NO: 375), y) SEQ ID NO: 387 (when heavy chain CDR3 region is SEQ ID
NO: 391), or z) SEQ ID NO: 403 (when heavy chain CDR3 region is SEQ
ID NO: 407).
6. The substantially pure polypeptide of claim 1 wherein said
antibody includes a heavy chain Fd region including the amino acid
sequence of: a) SEQ ID NO: 1 (when heavy chain CDR3 region is SEQ
ID NO: 7), b) SEQ ED NO: 17 (when heavy chain CDR3 region is SEQ ID
NO: 23), c) SEQ ID NO: 33 (when heavy chain CDR3 region is SEQ ID
NO: 39), d) SEQ ID NO: 49 (when heavy chain CDR3 region is SEQ ID
NO: 55), e) SEQ ID NO: 65 (when heavy chain CDR3 region is SEQ ID
NO: 71), f) SEQ ID NO: 81 (when heavy chain CDR3 region is SEQ ID
NO: 87), g) SEQ ID NO: 97 (when heavy chain CDR3 region is SEQ ID
NO: 103), h) SEQ ID NO: 113 (when heavy chain CDR3 region is SEQ ID
NO: 119), i) SEQ ID NO: 129 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ ID NO: 145 (when heavy chain CDR3 region is SEQ ID
NO: 151), k) SEQ ID NO: 161 (when heavy chain CDR3 region is SEQ ID
NO: 167), l) SEQ ID NO: 177 (when heavy chain CDR3 region is SEQ ID
NO: 183), m) SEQ ID NO: 193 (when heavy chain CDR3 region is SEQ ID
NO: 199), n) SEQ ID NO: 209 (when heavy chain CDR3 region is SEQ ID
NO: 215), o) SEQ ID NO: 225 (when heavy chain CDR3 region is SEQ ID
NO: 231), p) SEQ ID NO: 241 (when heavy chain CDR3 region is SEQ ID
NO: 247), q) SEQ ID NO: 257 (when heavy chain CDR3 region is SEQ ID
NO: 263), r) SEQ ID NO: 273 (when heavy chain CDR3 region is SEQ ID
NO: 279), s) SEQ ID NO: 289 (when heavy chain CDR3 region is SEQ ID
NO: 295), t) SEQ ID NO: 305 (when heavy chain CDR3 region is SEQ ID
NO: 311), u) SEQ ID NO: 321 (when heavy chain CDR3 region is SEQ ID
NO: 327), v) SEQ ID NO: 337 (when heavy chain CDR3 region is SEQ ID
NO: 343), w) SEQ ID NO: 353 (when heavy chain CDR3 region is SEQ ID
NO: 359), x) SEQ ID NO: 369 (when heavy chain CDR3 region is SEQ ID
NO: 375), y) SEQ ID NO: 385 (when heavy chain CDR3 region is SEQ ID
NO: 391), or z) SEQ ID NO: 401 (when heavy chain CDR3 region is SEQ
ID NO: 407).
7. The substantially pure polypeptide of claim 1 wherein said
antibody includes a light chain CDR3 region having the amino acid
sequence of: a) SEQ ID NO: 15 (when heavy chain CDR3 region is SEQ
ID NO: 7), b) SEQ ID NO: 31 (when heavy chain CDR3 region is SEQ ID
NO: 23), c) SEQ DD NO: 47 (when heavy chain CDR3 region is SEQ ID
NO: 39), d) SEQ ID NO: 63 (when heavy chain CDR3 region is SEQ ID
NO: 55), e) SEQ ID NO: 79 (when heavy chain CDR3 region is SEQ ID
NO: 71), f) SEQ ID NO: 95 (when heavy chain CDR3 region is SEQ ID
NO: 87), g) SEQ ID NO: 111 (when heavy chain CDR3 region is SEQ ID
NO: 103), h) SEQ ID NO: 127 (when heavy chain CDR3 region is SEQ ID
NO: 119), i) SEQ ID NO: 143 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ ID NO: 159 (when heavy chain CDR3 region is SEQ ID
NO: 151), k) SEQ ID NO: 175 (when heavy chain CDR3 region is SEQ ID
NO: 167), l) SEQ ID NO: 191 (when heavy chain CDR3 region is SEQ ID
NO: 183), m) SEQ ID NO: 207 (when heavy chain CDR3 region is SEQ ID
NO: 199), n) SEQ ID NO: 223 (when heavy chain CDR3 region is SEQ ID
NO: 215), o) SEQ ID NO: 239 (when heavy chain CDR3 region is SEQ ID
NO: 231), p) SEQ ID NO: 255 (when heavy chain CDR3 region is SEQ ID
NO: 247), q) SEQ ID NO: 271 (when heavy chain CDR3 region is SEQ ID
NO: 263), r) SEQ ID NO: 287 (when heavy chain CDR3 region is SEQ ID
NO: 279), s) SEQ ID NO: 303 (when heavy chain CDR3 region is SEQ ID
NO: 295), t) SEQ ID NO: 319 (when heavy chain CDR3 region is SEQ ID
NO: 311), u) SEQ ID NO: 335 (when heavy chain CDR3 region is SEQ ID
NO: 327), v) SEQ ID NO: 351 (when heavy chain CDR3 region is SEQ ID
NO: 343), w) SEQ ID NO: 367 (when heavy chain CDR3 region is SEQ ID
NO: 359), x) SEQ ID NO: 383 (when heavy chain CDR3 region is SEQ ID
NO: 375), y) SEQ ID NO: 399 (when heavy chain CDR3 region is SEQ ID
NO: 391), or z) SEQ ID NO: 415 (when heavy chain CDR3 region is SEQ
ID NO: 407).
8. The substantially pure polypeptide of claim 7 wherein said
antibody includes a light chain CDR2 region having the amino acid
sequence of: a) SEQ ID NO: 13 (when heavy chain CDR3 region is SEQ
ID NO: 7), b) SEQ ID NO: 29 (when heavy chain CDR3 region is SEQ ID
NO: 23), c) SEQ ID NO: 45 (when heavy chain CDR3 region is SEQ ID
NO: 39), d) SEQ ID NO: 61 (when heavy chain CDR3 region is SEQ ID
NO: 55), e) SEQ ID NO: 77 (when heavy chain CDR3 region is SEQ ID
NO: 71), f) SEQ E) NO: 93 (when heavy chain CDR3 region is SEQ E)
NO: 87), g) SEQ ID NO: 109 (when heavy chain CDR3 region is SEQ ID
NO: 103), h) SEQ ID NO: 125 (when heavy chain CDR3 region is SEQ ID
NO: 119), i) SEQ ID NO: 141 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ ID NO: 157 (when heavy chain CDR3 region is SEQ ID
NO: 151), k) SEQ E) NO: 173 (when heavy chain CDR3 region is SEQ ID
NO: 167), l) SEQ ID NO: 189 (when heavy chain CDR3 region is SEQ ID
NO: 183), m) SEQ E) NO: 205 (when heavy chain CDR3 region is SEQ E)
NO: 199), n) SEQ E) NO: 221 (when heavy chain CDR3 region is SEQ ID
NO: 215), o) SEQ E) NO: 237 (when heavy chain CDR3 region is SEQ ID
NO: 231), p) SEQ E) NO: 253 (when heavy chain CDR3 region is SEQ E)
NO: 247), q) SEQ E) NO: 269 (when heavy chain CDR3 region is SEQ E)
NO: 263), r) SEQ E) NO: 285 (when heavy chain CDR3 region is SEQ ID
NO: 279), s) SEQ E) NO: 301 (when heavy chain CDR3 region is SEQ E)
NO: 295), t) SEQ E) NO: 317 (when heavy chain CDR3 region is SEQ E)
NO: 311), u) SEQ E) NO: 333 (when heavy chain CDR3 region is SEQ E)
NO: 327), v) SEQ E) NO: 349 (when heavy chain CDR3 region is SEQ E)
NO: 343), w) SEQ E) NO: 365 (when heavy chain CDR3 region is SEQ TD
NO: 359), x) SEQ E) NO: 381 (when heavy chain CDR3 region is SEQ ID
NO: 375), y) SEQ E) NO: 397 (when heavy chain CDR3 region is SEQ E)
NO: 391), or z) SEQ E) NO: 413 (when heavy chain CDR3 region is SEQ
E) NO: 407).
9. The substantially pure polypeptide of claim 8 wherein said
antibody includes a light chain CDR1 region having the amino acid
sequence of: a) SEQ E) NO: 11 (when heavy chain CDR3 region is SEQ
E) NO: 7), b) SEQ E) NO: 27 (when heavy chain CDR3 region is SEQ E)
NO: 23), c) SEQ ID NO: 43 (when heavy chain CDR3 region is SEQ ID
NO: 39), d) SEQ ID NO: 59 (when heavy chain CDR3 region is SEQ ID
NO: 55), e) SEQ ID NO: 75 (when heavy chain CDR3 region is SEQ ID
NO: 71), f) SEQ ID NO: 91 (when heavy chain CDR3 region is SEQ ID
NO: 87), g) SEQ ID NO: 107 (when heavy chain CDR3 region is SEQ ID
NO: 103), h) SEQ ID NO: 123 (when heavy chain CDR3 region is SEQ ID
NO: 119), i) SEQ ID NO: 139 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ ID NO: 155 (when heavy chain CDR3 region is SEQ ID
NO: 151), k) SEQ ID NO: 171 (when heavy chain CDR3 region is SEQ ID
NO: 167), l) SEQ ID NO: 187 (when heavy chain CDR3 region is SEQ ID
NO: 183), m) SEQ ID NO: 203 (when heavy chain CDR3 region is SEQ ID
NO: 199), n) SEQ ID NO: 219 (when heavy chain CDR3 region is SEQ ID
NO: 215), o) SEQ ID NO: 235 (when heavy chain CDR3 region is SEQ ID
NO: 231), p) SEQ ID NO: 251 (when heavy chain CDR3 region is SEQ ID
NO: 247), q) SEQ ID NO: 267 (when heavy chain CDR3 region is SEQ ID
NO: 263), r) SEQ ID NO: 283 (when heavy chain CDR3 region is SEQ ID
NO: 279), s) SEQ ID NO: 299 (when heavy chain CDR3 region is SEQ ID
NO: 295), t) SEQ ID NO: 315 (when heavy chain CDR3 region is SEQ ID
NO: 311), u) SEQ ID NO: 331 (when heavy chain CDR3 region is SEQ ID
NO: 327), v) SEQ ID NO: 347 (when heavy chain CDR3 region is SEQ ID
NO: 343), w) SEQ ID NO: 363 (when heavy chain CDR3 region is SEQ ID
NO: 359), x) SEQ ID NO: 379 (when heavy chain CDR3 region is SEQ ID
NO: 375), y) SEQ ID NO: 395 (when heavy chain CDR3 region is SEQ ID
NO: 391), or z) SEQ ID NO: 411 (when heavy chain CDR3 region is SEQ
ID NO: 407).
10. The substantially pure polypeptide of claim 1 wherein said
antibody includes a light chain region including the amino acid
sequence of: a) SEQ ID NO: 9 (when heavy chain CDR3 region is SEQ
ID NO: 7), b) SEQ ID NO: 25 (when heavy chain CDR3 region is SEQ ID
NO: 23), c) SEQ ID NO: 41 (when heavy chain CDR3 region is SEQ ID
NO: 39), d) SEQ ID NO: 57 (when heavy chain CDR3 region is SEQ ID
NO: 55), e) SEQ ID NO: 73 (when heavy chain CDR3 region is SEQ ID
NO: 71), f) SEQ ID NO: 89 (when heavy chain CDR3 region is SEQ ID
NO: 87), g) SEQ ED NO: 105 (when heavy chain CDR3 region is SEQ ID
NO: 103), h) SEQ ID NO: 121 (when heavy chain CDR3 region is SEQ ID
NO: 119), i) SEQ ID NO: 137 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ ID NO: 153 (when heavy chain CDR3 region is SEQ ID
NO: 151), k) SEQ ID NO: 169 (when heavy chain CDR3 region is SEQ ID
NO: 167), l) SEQ ID NO: 185 (when heavy chain CDR3 region is SEQ ID
NO: 183), m) SEQ ID NO: 201 (when heavy chain CDR3 region is SEQ ID
NO: 199), n) SEQ ID NO: 217 (when heavy chain CDR3 region is SEQ ID
NO: 215), o) SEQ ID NO: 233 (when heavy chain CDR3 region is SEQ ID
NO: 231), p) SEQ ID NO: 249 (when heavy chain CDR3 region is SEQ ID
NO: 247), q) SEQ ID NO: 265 (when heavy chain CDR3 region is SEQ ID
NO: 263), r) SEQ ID NO: 281 (when heavy chain CDR3 region is SEQ ID
NO: 279), s) SEQ ID NO: 297 (when heavy chain CDR3 region is SEQ ID
NO: 295), t) SEQ ID NO: 313 (when heavy chain CDR3 region is SEQ ED
NO: 311), u) SEQ ID NO: 329 (when heavy chain CDR3 region is SEQ TD
NO: 327), v) SEQ ID NO: 345 (when heavy chain CDR3 region is SEQ ID
NO: 343), w) SEQ ID NO: 361 (when heavy chain CDR3 region is SEQ ID
NO: 359), x) SEQ ID NO: 377 (when heavy chain CDR3 region is SEQ ID
NO: 375), y) SEQ ID NO: 393 (when heavy chain CDR3 region is SEQ ID
NO: 391), or z) SEQ ID NO: 409 (when heavy chain CDR3 region is SEQ
ID NO: 407).
11. The substantially pure polypeptide of claim 1 wherein said
antibody includes a heavy chain Fd region including the CDR amino
acid sequences of: a) SEQ ID NO: 1 (when heavy chain CDR3 region is
SEQ ID NO: 7), b) SEQ ID NO: 17 (when heavy chain CDR3 region is
SEQ ID NO: 23), c) SEQ ID NO: 33 (when heavy chain CDR3 region is
SEQ ID NO: 39), d) SEQ DD NO: 49 (when heavy chain CDR3 region is
SEQ DD NO: 55), e) SEQ DD NO: 65 (when heavy chain CDR3 region is
SEQ DD NO: 71), f) SEQ DD NO: 81 (when heavy chain CDR3 region is
SEQ DD NO: 87), g) SEQ DD NO: 97 (when heavy chain CDR3 region is
SEQ DD NO: 103), h) SEQ DD NO: 113 (when heavy chain CDR3 region is
SEQ DD NO: 119), l) SEQ DD NO: 129 (when heavy chain CDR3 region is
SEQ DD NO: 135), j) SEQ DD NO: 145 (when heavy chain CDR3 region is
SEQ ID NO: 151), k) SEQ ID NO: 161 (when heavy chain CDR3 region is
SEQ E) NO: 167), l) SEQ ID NO: 177 (when heavy chain CDR3 region is
SEQ ID NO: 183), m) SEQ ID NO: 193 (when heavy chain CDR3 region is
SEQ ID NO: 199), n) SEQ ID NO: 209 (when heavy chain CDR3 region is
SEQ ID NO: 215), o) SEQ ID NO: 225 (when heavy chain CDR3 region is
SEQ ID NO: 231), p) SEQ ID NO: 241 (when heavy chain CDR3 region is
SEQ ID NO: 247), q) SEQ ID NO: 257 (when heavy chain CDR3 region is
SEQ ID NO: 263), r) SEQ ID NO: 273 (when heavy chain CDR3 region is
SEQ ID NO: 279), s) SEQ ID NO: 289 (when heavy chain CDR3 region is
SEQ ID NO: 295), t) SEQ ID NO: 305 (when heavy chain CDR3 region is
SEQ ID NO: 311), u) SEQ ID NO: 321 (when heavy chain CDR3 region is
SEQ ID NO: 327), v) SEQ ID NO: 337 (when heavy chain CDR3 region is
SEQ ID NO: 343), w) SEQ ID NO: 353 (when heavy chain CDR3 region is
SEQ ID NO: 359), x) SEQ ID NO: 369 (when heavy chain CDR3 region is
SEQ ID NO: 375), y) SEQ ID NO: 385 (when heavy chain CDR3 region is
SEQ ID NO: 391), or z) SEQ ID NO: 401 (when heavy chain CDR3 region
is SEQ ID NO: 407).
12. The substantially pure polypeptide of claim 11 wherein said
antibody includes a light chain region including the CDR amino acid
sequences of: a) SEQ ID NO: 9 (when heavy chain CDR3 region is SEQ
E) NO: 7), b) SEQ E) NO: 25 (when heavy chain CDR3 region is SEQ E)
NO: 23), c) SEQ E) NO: 41 (when heavy chain CDR3 region is SEQ E)
NO: 39), d) SEQ E) NO: 57 (when heavy chain CDR3 region is SEQ ID
NO: 55), e) SEQ E) NO: 73 (when heavy chain CDR3 region is SEQ E)
NO: 71), f) SEQ E) NO: 89 (when heavy chain CDR3 region is SEQ E)
NO: 87), g) SEQ E) NO: 105 (when heavy chain CDR3 region is SEQ E)
NO: 103), h) SEQ E) NO: 121 (when heavy chain CDR3 region is SEQ E)
NO: 119), i) SEQ E) NO: 137 (when heavy chain CDR3 region is SEQ ID
NO: 135), j) SEQ E) NO: 153 (when heavy chain CDR3 region is SEQ E)
NO: 151), k) SEQ E) NO: 169 (when heavy chain CDR3 region is SEQ E)
NO: 167), l) SEQ E) NO: 185 (when heavy chain CDR3 region is SEQ E)
NO: 183), m) SEQ E) NO: 201 (when heavy chain CDR3 region is SEQ E)
NO: 199), n) SEQ E) NO: 217 (when heavy chain CDR3 region is SEQ E)
NO: 215), o) SEQ DD NO: 233 (when heavy chain CDR3 region is SEQ ID
NO: 231), p) SEQ ID NO: 249 (when heavy chain CDR3 region is SEQ ID
NO: 247), q) SEQ ID NO: 265 (when heavy chain CDR3 region is SEQ ID
NO: 263), r) SEQ ID NO: 281 (when heavy chain CDR3 region is SEQ ID
NO: 279), s) SEQ ID NO: 297 (when heavy chain CDR3 region is SEQ ID
NO: 295), t) SEQ ID NO: 313 (when heavy chain CDR3 region is SEQ ID
NO: 311), u) SEQ ID NO: 329 (when heavy chain CDR3 region is SEQ ID
NO: 327), v) SEQ ID NO: 345 (when heavy chain CDR3 region is SEQ ID
NO: 343), w) SEQ ID NO: 361 (when heavy chain CDR3 region is SEQ ID
NO: 359), x) SEQ ID NO: 377 (when heavy chain CDR3 region is SEQ ID
NO: 375), y) SEQ ID NO: 393 (when heavy chain CDR3 region is SEQ ID
NO: 391), or z) SEQ ID NO: 409 (when heavy chain CDR3 region is SEQ
ID NO: 407).
13. An isolated nucleic acid comprising a nucleotide sequence
encoding the polypeptide of claim 1.
14. An isolated nucleic acid as in claim 13 wherein said nucleic
acid comprises a vector including a regulatory sequence operably
joined to said nucleic acid.
15. A host cell including a vector comprising a nucleic acid of
claim 13.
16. A pharmaceutical preparation comprising a pharmaceutically
acceptable carrier and the polypeptide of claim 1.
17. A diagnostic preparation comprising a pharmaceutically
acceptable carrier and the polypeptide of claim 1.
18. A method for the treatment of Hendra Virus Disease or Nipah
Virus Disease comprising administering to a patient a
therapeutically effective amount of the pharmaceutical preparation
of claim 16.
19. A method for prophylaxis against Hendra Virus Disease or Nipah
Virus Disease comprising administering to a patient a
prophylactically effective amount of the pharmaceutical preparation
of claim 16.
20. A substantially pure polypeptide comprising a monoclonal
antibody that binds the antigen to which an antibody selectively
binding a Hendra or Nipah G glycoprotein epitope binds, wherein
said antibody includes a heavy chain CDR3 region having the amino
acid sequence selected from the group consisting of SEQ ID NO: 7,
SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 71, SEQ ID
NO: 87, SEQ ID NO: 103, SEQ ID NO: 119, SEQ ID NO: 135, SEQ ID NO:
151, SEQ ID NO: 167, SEQ ID NO: 183, SEQ ID NO: 199, SEQ ID NO:
215, SEQ ID NO: 231, SEQ ID NO: 247, SEQ ID NO: 263, SEQ ID NO:
279, SEQ ID NO: 295, SEQ ID NO: 311, SEQ ID NO: 327, SEQ ID NO:
343, SEQ ID NO: 359, SEQ ID NO: 375, SEQ ID NO: 391 and SEQ ID NO:
407.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No: 60/661,766, filed Mar. 14, 2005, U.S.
Provisional Patent Application No. 60/678,547, filed May 5, 2005,
and U.S. Provisional Patent Application No. 60/718,902, filed Sep.
20, 2005, the disclosures of all of which are hereby expressly
incorporated by reference in their entireties.
REFERENCE TO SEQUENCE LISTING
[0002] A computer readable text file, entitled
"044508-5017-04-SequenceListing.txt" created on or about 18 Sep.
2014 with a file size of about 130 kb contains the sequence listing
for this application and is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] This invention relates generally to the field of immunology
and specifically to monoclonal antibodies that bind or neutralize
Hendra and Nipah viruses.
BACKGROUND OF THE INVENTION
[0004] Nipah virus (NiV) and Hendra virus (HeV) are closely related
emerging paramyxoviruses that comprise the Henipavirus genus
(Anonymous 1999 MMWR Morb Mortal Wkly Rep Ward, J. W. ed.
48:335-337; Chew, M. H. et al. 2000 J Infect Dis 181:1760-1763;
Chua, K. B. et al. 2000 Ann Neurol 48:802-805; Eaton, B. T. 2001
Microbes Infect 3:277-278; Goh, K. J. et al. 2000 N Engl J Med
342:1229-1235; Lee, K. E. et al. 1999 Ann Neurol 46:428-432; Lim,
C. C. et al. 2000 Am J Neuroradiol 21:455-461; Murray, K. et al.
1995 Science 268:94-97). Paramyxoviruses are negative-sense RNA
containing enveloped viruses and contain two major
membrane-anchored envelope glycoproteins that are required for
infection of a receptive host cell. All members contain an F
glycoprotein which mediates pH-independent membrane fusion between
the virus and its host cell, while the second attachment
glycoprotein can be either a hemagglutinin-neuraminidase protein
(HN), a hemagglutinin protein (H), or a G protein depending on the
particular virus (reviewed in Lamb, R. A. and Kolakofsky, D. 2001
in Fields Virology, eds. Knippe, D. M. & Howley, P. M.,
Lippincott Williams & Wilkins, Philadelphia, pp. 1305-1340). As
with all paramyxoviruses, these glycoproteins are also the
principal antigens to which virtually all neutralizing antibodies
are directed.
[0005] The broad species tropisms and the ability to cause fatal
disease in both animals and humans distinguish HeV and NiV from all
other known paramyxoviruses (reviewed in Eaton, B. T. 2001 Microbes
Infect 3:277-278). They are Biological Safety Level-4 (BSL-4)
pathogens, and are on the NIAID Biodefense research agenda as
zoonotic emerging category C priority pathogens that could be used
as bioterror agents. The henipaviruses can be amplified and cause
disease in large animals and be aerosol transmitted to humans where
disease can be a severe respiratory illness and febrile
encephalitis. They can be readily grown in cell culture or
embryonated chicken eggs, produce high un-concentrated titers
(.about.10.sup.8 TCID.sub.50/ml; Crameri, G. et al. 2002 J Virol
Methods 99:41-51), and are highly infectious (Field, H. et al. 2001
Microbes Infect 3:307-314; Hooper, P. et al. 2001 Microbes Infect
3:315-322).
[0006] NiV has recently re-emerged in Bangladesh. Two outbreaks of
NiV in 2004 have been confirmed, and yet another one occurred in
January of 2005 (Anonymous 2005 Communicable Disease Report Weekly
(CDR Weekly) Vol. 15 No. 16). Several important observations in
these most recent outbreaks have been made, including a higher
incidence of acute respiratory distress syndrome, person-to-person
transmission, and significantly higher case fatality rates (60-75%)
than in Malaysia (about 40%) where the virus was discovered or
suspected to have originated (Anonymous 2004 Wkly Epidemiol Rec
79:168-171; Anonymous 2004 Health and Science Bulletin (ICDDR,B)
2:5-9; Butler, D. 2004 Nature 429:7; Enserink, M. 2004 Science
303:1121; Hsu, V. P. et al. 2004 Emerg Infect Dis 10:2082-2087).
Currently, no therapeutics for NiV or HeV-infected individuals are
available, and a vaccine for prevention of disease in human or
livestock populations does not exist. Although antibody responses
were detected in infections caused by these viruses, human
monoclonal antibodies (hmAbs) have not been identified against
either virus. A number of studies have shown the importance of
neutralizing antibodies in recovery and protection from viral
infections (Dimitrov, D. S. 2004 Nat Rev Microbiol 2:109-122).
Therefore, the development of neutralizing hmAbs against NiV and
HeV could have important implications for prophylaxis and passive
immunotherapy. In addition, the characterization of the epitopes of
the neutralizing antibodies could provide helpful information for
development of candidate vaccines and drugs. Finally, such
antibodies could be used for diagnosis and as research
reagents.
Segue to the Invention
[0007] Here, we report the identification of potent neutralizing
hmAbs targeting the viral envelope glycoprotein G by using a highly
purified, oligomeric, soluble HeV G (sG) glycoprotein as the
antigen for screening of a large naive human phage-display library.
One of these antibodies exhibited exceptional potency against
infectious HeV, and another one neutralized both HeV and NiV.
Because these antibodies are fully human antibodies, they serve as
the basis for prophylaxis and treatment of humans infected with HeV
or NiV.
SUMMARY OF THE INVENTION
[0008] The present invention relates to monoclonal antibodies that
bind or neutralize Hendra or Nipah virus. The invention provides
such antibodies, fragments of such antibodies retaining Hendra or
Nipah virus-binding ability, fully human antibodies retaining
Hendra or Nipah virus-binding ability, and pharmaceutical
compositions including such antibodies. The invention further
provides for isolated nucleic acids encoding the antibodies of the
invention and host cells transformed therewith. Additionally, the
invention provides for prophylactic, therapeutic, and diagnostic
methods employing the antibodies and nucleic acids of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1A and 1B: Significantly higher inhibitory activity of
IgG1 m101 than Fab m101 in HeV Env-mediated cell fusion. HeLa-USU
cells were infected with vaccinia recombinants encoding HeV F and G
glycoproteins, and with a vaccinia recombinant encoding T7 RNA
polymerase (effector cells). Each designated target cell type was
infected with the E. coli LacZ-encoding reporter vaccinia virus
vCB21R. IgG1 m101 and Fab m101 were pre-incubated with effector
cells and then mixed with target cells. The cell fusion assay was
performed for 2.5 hr at 37.degree. C. Fusion was measured as
described in Example 1. Inhibition of HeV Env-mediated fusion by
IgG1 m101 and Fab m101 in HeLa-ATCC cells is shown in FIG. 1A, and
of PCI-13 cells--in FIG. 1B. Percentage of fusion is shown as
function of the antibody concentration. The curves represent the
best fit of the experimental data from which IC.sub.50s were
calculated using Prism GraphPad software.
[0010] FIG. 2: Inhibition of HeV Env-mediated syncytia formation by
m101. The effector cells, prepared as described in FIG. 1 legend
were pre-incubated with IgG1 m101, Fab m101, or the control
irrelevant antibody (X5 specific for HIV (Moulard, M. et al. 2002
Proc Natl Acad Sci USA 99:6913-6918)) for 20 min at room
temperature, then 2.times.10.sup.5 cells in 200 .mu.l were overlaid
on 80% confluent monolayers of PCI-13 cells plated in a 48-well
plate, and incubated for 3 h at 37.degree. C. in a humidified 5%
CO.sub.2 atmosphere. Photographs were taken using phase contrast
microscope with a 10.times. objective. Shown are illustrative
portions of the original photographs that are electronically
amplified for clarity. The top and bottom pictures of the left
panel show formation of syncytia in the absence of antibody or in
the presence of a control antibody; there were 17 or 20 giant fused
cells (syncytia) counted per complete photograph view,
respectively. The top and bottom pictures of the right panel show
complete inhibition of syncytia formation by IgG1 m101 or reduction
in the number of syncytia by Fab m101 at 10 .mu.g/ml, there were 0
or 9 syncytia per complete view, respectively.
[0011] FIG. 3. Immunoprecipitation of HeV and NiV G glycoproteins
by anti-G Fabs. HeLa cells were infected with WR, a control
vaccinia virus, or recombinant vaccinia virus expressing myc-tagged
HeV G or NiV G, and beginning at 6 h postinfection, labeled with
[.sup.35S] methionine-cysteine at 37.degree. C. overnight. Lysates
were made in buffer containing Triton-X100 and incubated with
various Fabs or mouse anti-myc 9E10 for at least 1 h at 4.degree.
C., then precipitated with Protein G Sepharose. Immunoprecipitated
proteins were analyzed by 10% SDS-PAGE followed by autoradiography.
WR denotes a control where the cells were infected with wild type
vaccinia virus, X5 is a control antibody specific for gp120 of HIV,
and 9E10 is an anti-c-Myc antibody serving as a positive control.
Gels for m108 and m109 are also shown. The arrows next to G denote
the position of the bands corresponding to the monomeric G.
[0012] FIG. 4. Competition between anti-G antibodies and ephrin-B2
for binding to Hendra G. Serially diluted Fab m101, IgG1 m101 and
Fab m106 were mixed with Hendra G, and added to the virus receptor
ephrin-B2 coated on the bottom of a 96-well plate, and the amount
of bound G measured as described in Example 1. An Fab specific for
the SARS-CoV S protein was used as control.
[0013] FIG. 5. Binding of m101 and m102 to alanine mutants of HeV
G. HeLa cells transfected with wild type HeV G, various alanine
mutants of HeV G, or pMC02 (empty vector) were infected with WR
vaccinia virus to drive expression, radiolabeled with .sup.[35]S
methionine-cysteine overnight, lysed in buffer containing Triton
X-100, and subjected to immunoprecipitation by m101, m102, or
rabbit polyclonal G antisera. Lysates were then precipitated with
Protein G Sepharose and analyzed by 10% SDS-PAGE followed by
autoradiography.
[0014] FIG. 6. Comparison of the inhibitory activity of m101,
m102.4 Fab and IgG1, and m102 Fab in HeV Env-mediated cell fusion.
HeLa-USU cells were infected with vaccinia recombinants encoding
HeV F and G glycoproteins, and with a vaccinia recombinant encoding
T7 RNA polymerase (effecter cells). Target cell U373 was infected
with the E. coli LacZ-encoding reporter vaccinia virus vCB21R.
Serial diluted antibodies were pre-incubated with effecter cells
for 0.5 hr and then mixed with target cells. The cell fusion assay
was performed for 2.5 hr at 37.degree. C. Fusion was measured as
described in Example 1. Antibodies concentrations were plotted
against Beta-Gal assay reading at 595 nm.
[0015] FIG. 7. Significantly higher inhibitory activity of m102.4
Fab and IgG1 than m102 Fab in NiV-Env-mediated cell fusion.
HeLa-USU cells were infected with vaccinia recombinants encoding
NiV F and G glycoproteins, and with a vaccinia recombinant encoding
T7 RNA polymerase (effecter cells). Target cell U373 was infected
with the E. coli LacZ-encoding reporter vaccinia virus vCB21R.
Antibodies were pre-incubated with effecter cells for 0.5 hr and
then mixed with target cells. The cell fusion assay was performed
for 2.5 hr at 37.degree. C. Fusion was measured as described in
Example 1. Antibodies concentrations were plotted against Beta-Gal
assay reading at 595 nm.
[0016] FIG. 8. CDR1-3s and FR1-4s for m101-117 and m102
mutants.
TABLE-US-00001 [0017] TABLE A Brief Description of m101-m117 SEQ ID
NOs. Heavy Chain SEQ ID NOs Light Chain SEQ ID NOs Fab/mab V.sub.H
FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 V.sub.L FR1 CDR1 FR2 CDR2 FR3 CDR3
FR4 m101 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m102 17 18 19 20 21
22 23 24 25 26 27 28 29 30 31 32 m103 33 34 35 36 37 38 39 40 41 42
43 44 45 46 47 48 m104 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
64 m105 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 m106 81 82
83 84 85 86 87 88 89 90 91 92 93 94 95 96 m107 97 98 99 100 101 102
103 104 105 106 107 108 109 110 111 112 m108 113 114 115 116 117
118 119 120 121 122 123 124 125 126 127 128 m109 129 130 131 132
133 134 135 136 137 138 139 140 141 142 143 144 m110 145 146 147
148 149 150 151 152 153 154 155 156 157 158 159 160 m111 161 162
163 164 165 166 167 168 169 170 171 172 173 174 175 176 m112 177
178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 m113
193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208
m114 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223
224 m115 225 226 227 228 229 230 231 232 233 234 235 236 237 238
239 240 m116 241 242 243 244 245 246 247 248 249 250 251 252 253
254 255 256 m117 257 258 259 260 261 262 263 264 265 266 267 268
269 270 271 272
TABLE-US-00002 TABLE B Brief Description of Mutant m102 SEQ ID NOs.
Mutant Heavy Chain SEQ ID NOs Light Chain SEQ ID NOs m102 V.sub.H
FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 V.sub.L FR1 CDR1 FR2 CDR2 FR3 CDR3
FR4 m102.2 273 274 275 276 277 278 279 280 281 282 283 284 285 286
287 288 m102.3 289 290 291 292 293 294 295 296 297 298 299 300 301
302 303 304 m102.4 305 306 307 308 309 310 311 312 313 314 315 316
317 318 319 320 m102.5 321 322 323 324 325 326 327 328 329 330 331
332 333 334 335 336 m102.11 337 338 339 340 341 342 343 344 345 346
347 348 349 350 351 352 m102.12 353 354 355 356 357 358 359 360 361
362 363 364 365 366 367 368 m102.13 369 370 371 372 373 374 375 376
377 378 379 380 381 382 383 384 m102.15 385 386 387 388 389 390 391
392 393 394 395 396 397 398 399 400 m102.16 401 402 403 404 405 406
407 408 409 410 411 412 413 414 415 416
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0018] Hendra virus (HeV) and Nipah virus (NiV) are closely related
emerging viruses comprising the Henipavirus genus of the
Paramyxovirinae. Each has a broad species tropism and can cause
high mortality disease in both animal and human hosts. These
viruses infect cells by a pH-independent membrane fusion event
mediated by their attachment (G) and fusion (F) envelope
glycoproteins (Envs). Seven Fabs, m101-7, were selected for their
significant binding to a soluble form of Hendra G (sG) which was
used as the antigen for panning of a large naive human antibody
library. The selected Fabs inhibited to various degrees cell fusion
mediated by the HeV or NiV Envs and virus infection. The conversion
of the most potent neutralizer of infectious HeV, Fab m101, to IgG1
significantly increased its cell fusion inhibitory activity--the
IC.sub.50 was decreased more than 10-fold to approximately 1
.mu.g/ml. The IgG1 m101 was also exceptionally potent in
neutralizing infectious HeV; complete (100%) neutralization was
achieved with 12.5 .mu.g/ml and 98% neutralization required only
1.6 .mu.g/ml. The inhibition of fusion and infection correlated
with binding of the Fabs to full-length G as measured by
immunoprecipitation, and less with binding to sG as measured by
ELISA and Biacore. M101 and m102 competed with the ephrin-B2, which
we recently identified as a functional receptor for HeV and NiV,
indicating a possible mechanism of neutralization by these
antibodies. The m101, m102 and m103 antibodies competed with each
other indicating that they bind to overlapping epitopes which are
distinct from the epitopes of m106 and m107. In an initial attempt
to localize the epitopes of m101 and m102 we measured their binding
to a panel of 10 G alanine scanning mutants, and identified one
residue, G183, which decreases binding of both m101 and m102 to G;
it is localized at the base of the globular head of the G protein
according to a model structure, and could be part of the antibody
epitope that does not overlap with the receptor binding site on G.
These results indicate that m101-7 are specific for HeV or NiV or
both, and exhibit various neutralizing activity; they are the first
human monoclonal antibodies identified against these viruses and
are contemplated for use in treatment, prophylaxis and diagnosis,
and as research reagents and serving as the basis for vaccines.
DEFINITIONS
[0019] Unless defined otherwise, 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. See,
e.g., Singleton P and Sainsbury D., Dictionary of Microbiology and
Molecular Biology 3.sup.rd ed., J. Wiley & Sons, Chichester,
N.Y., 2001, and Fields Virology 4.sup.th ed., Knipe D. M. and
Howley P. M. eds, Lippincott Williams & Wilkins, Philadelphia
2001.
[0020] As used herein, the term "antibody" means an immunoglobulin
molecule or a fragment of an immunoglobulin molecule having the
ability to specifically bind to a particular antigen. Antibodies
are well known to those of ordinary skill in the science of
immunology. As used herein, the term "antibody" means not only
full-length antibody molecules but also fragments of antibody
molecules retaining antigen binding ability. Such fragments are
also well known in the art and are regularly employed both in vitro
and in vivo. In particular, as used herein, the term "antibody"
means not only full-length immunoglobulin molecules but also
antigen binding active fragments such as the well-known active
fragments F(ab')2, Fab, Fv, and Fd.
[0021] As used herein, the terms "Hendra Virus Disease" and "Nipah
Virus Disease" refer to diseases caused, directly or indirectly, by
infection with Hendra or Nipah virus. The broad species tropisms
and the ability to cause fatal disease in both animals and humans
have distinguished Hendra virus (HeV) and Nipah virus (NiV) from
all other known paramyxoviruses (Eaton B. T. 2001 Microbes Infect
3:277-278). These viruses can be amplified and cause disease in
large animals and can be transmitted to humans where infection is
manifested as a severe respiratory illness and/or febrile
encephalitis.
[0022] As used herein with respect to polypeptides, the term
"substantially pure" means that the polypeptides are essentially
free of other substances with which they may be found in nature or
in vivo systems to an extent practical and appropriate for their
intended use. In particular, the polypeptides are sufficiently pure
and are sufficiently free from other biological constituents of
their host cells so as to be useful in, for example, generating
antibodies, sequencing, or producing pharmaceutical preparations.
By techniques well known in the art, substantially pure
polypeptides may be produced in light of the nucleic acid and amino
acid sequences disclosed herein. Because a substantially purified
polypeptide of the invention may be admixed with a pharmaceutically
acceptable carrier in a pharmaceutical preparation, the polypeptide
may comprise only a certain percentage by weight of the
preparation. The polypeptide is nonetheless substantially pure in
that it has been substantially separated from the substances with
which it may be associated in living systems.
[0023] As used herein with respect to nucleic acids, the term
"isolated" means: (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which polymerase chain reaction (PCR) primer sequences
have been disclosed is considered isolated but a nucleic acid
sequence existing in its native state in its natural host is not.
An isolated nucleic acid may be substantially purified, but need
not be. For example, a nucleic acid that is isolated within a
cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein because it is readily manipulable by standard
techniques known to those of ordinary skill in the art.
[0024] As used herein, a coding sequence and regulatory sequences
are said to be "operably joined" when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
sequences. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operably joined if induction of a promoter in the 5' regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide.
[0025] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribing and 5'
non-translating sequences involved with initiation of transcription
and translation respectively, such as a TATA box, capping sequence,
CAAT sequence, and the like. Especially, such 5' non-transcribing
regulatory sequences will include a promoter region which includes
a promoter sequence for transcriptional control of the operably
joined gene. Regulatory sequences may also include enhancer
sequences or upstream activator sequences, as desired.
[0026] As used herein, a "vector" may be any of a number of nucleic
acids into which a desired sequence may be inserted by restriction
and ligation for transport between different genetic environments
or for expression in a host cell. Vectors are typically composed of
DNA although RNA vectors are also available. Vectors include, but
are not limited to, plasmids and phagemids. A cloning vector is one
which is able to replicate in a host cell, and which is further
characterized by one or more endonuclease restriction sites at
which the vector may be cut in a determinable fashion and into
which a desired DNA sequence may be ligated such that the new
recombinant vector retains its ability to replicate in the host
cell. In the case of plasmids, replication of the desired sequence
may occur many times as the plasmid increases in copy number within
the host bacterium or just a single time per host before the host
reproduces by mitosis. In the case of phage, replication may occur
actively during a lytic phase or passively during a lysogenic
phase. An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. Vectors may further contain one or more marker
sequences suitable for use in the identification and selection of
cells which have been transformed or transfected with the vector.
Markers include, for example, genes encoding proteins which
increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art
(e.g., .beta.-galactosidase or alkaline phosphatase), and genes
which visibly affect the phenotype of transformed or transfected
cells, hosts, colonies or plaques. Preferred vectors are those
capable of autonomous replication and expression of the structural
gene products present in the DNA segments to which they are
operably joined.
Novel Anti-HeV and NiV G Glycoprotein Monoclonal Antibodies
[0027] The present invention derives, in part, from the isolation
and characterization of novel, fully human monoclonal antibodies
that selectively bind to and neutralize Hendra and Nipah viruses.
As described more fully below, these monoclonal antibodies have
been shown to bind the G glycoprotein and to neutralize Hendra and
Nipah viruses. The paratope of the anti-HeV and NiV Fab fragments
associated with the neutralization epitope on the HeV and NiV
glycoprotein G are defined by the amino acid (aa) sequences of the
immunoglobulin heavy and light chain V-regions described in Tables
A and B and SEQ ID NO: 1 through SEQ ID NO: 416.
[0028] In one set of embodiments, the present invention provides
the full-length, fully human Hendra and Nipah monoclonal antibodies
in isolated form and in pharmaceutical preparations. Similarly, as
described below, the present invention provides isolated nucleic
acids, host cells transformed with nucleic acids, and
pharmaceutical preparations including isolated nucleic acids,
encoding the full-length, fully human Hendra and Nipah monoclonal
antibodies. Finally, the present invention provides methods, as
described more fully below, employing these antibodies and nucleic
acids in the in vitro and in vivo diagnosis, prevention and therapy
of Hendra Virus Disease or Nipah Virus Disease.
[0029] Significantly, as is well-known in the art, only a small
portion of an antibody molecule, the paratope, is involved in the
binding of the antibody to its epitope (see, in general, Clark, W.
R. 1986 in The Experimental Foundations of Modern Immunology Wiley
& Sons, Inc., New York; Roitt, I. 1991 in Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and
Fc regions, for example, are effectors of the complement cascade
but are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab')2 fragment,
retains both of the antigen binding sites of a full-length
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of a full-length antibody molecule. Proceeding
further, Fab fragments consist of a covalently bound antibody light
chain and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0030] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, 1986, supra;
Roitt, 1991, supra). In both the heavy chain Fd fragment and the
light chain of IgG immunoglobulins, there are four framework
regions (FR1 through FR4) separated respectively by three
complementarity determining regions (CDR1 through CDR3). The CDRs,
and in particular the CDR3 regions, and more particularly the heavy
chain CDR3, are largely responsible for antibody specificity.
[0031] The complete amino acid sequences of the antigen-binding Fab
portions of the Hendra and Nipah monoclonal antibodies as well as
the relevant FR and CDR regions are disclosed herein. SEQ ID NOs:
1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225,
241, 257, 273, 289, 305, 321, 337, 353, 369, 385, and 401 disclose
the amino acid sequences of the Fd fragment of the Hendra and Nipah
monoclonal antibodies. The amino acid sequences of the heavy chain
FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions are disclosed as
(FR1, SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,
178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370,
386, and 402); (CDR1, SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115,
131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323,
339, 355, 371, 387, and 403); (FR2, SEQ ID NOs: 4, 20, 36, 52, 68,
84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276,
292, 308, 324, 340, 356, 372, 388, and 404); (CDR2, SEQ ID NOs: 5,
21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229,
245, 261, 277, 293, 309, 325, 341, 357, 373, 389, and 405); (FR3,
SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182,
198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390,
and 406); (CDR3, SEQ ID NOs: 7, 23, 39, 55, 71, 87, 103, 119, 135,
151, 167, 183, 199, 215, 231, 247, 263, 279, 295, 311, 327, 343,
359, 375, 391, and 407); and (FR4, SEQ ID NOs: 8, 24, 40, 56, 72,
88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280,
296, 312, 328, 344, 360, 376, 392 and 408). SEQ ID NOs: 9, 25, 41,
57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265,
281, 297, 313, 329, 345, 361, 377, 393 and 409 disclose the amino
acid sequences of the light chains of the Hendra and Nipah
monoclonal antibodies. The amino acid sequences of the light chain
FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions are disclosed as
(FR1, SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170,
186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378,
394, and 410); (CDR1, SEQ ID NOs: 11, 27, 43, 59, 75, 91, 107, 123,
139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331,
347, 363, 379, 395, and 411); (FR2, SEQ ID NOs: 12, 28, 44, 60, 76,
92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284,
300, 316, 332, 348, 364, 380, 396, and 412); (CDR2, SEQ ID NOs: 13,
29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237,
253, 269, 285, 301, 317, 333, 349, 365, 381, 397, and 413); (FR3,
SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190,
206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 398,
and 414); (CDR3, SEQ ID NOs: 15, 31, 47, 63, 79, 95, 111, 127, 143,
159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319, 335, 351,
367, 383, 399, and 415); (FR4, SEQ ID NOs: 16, 32, 48, 64, 80, 96,
112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304,
320, 336, 352, 368, 384, 400 and 416).
[0032] It is now well-established in the art that the non-CDR
regions of a mammalian antibody may be replaced with similar
regions of conspecific or heterospecific antibodies while retaining
the epitopic specificity of the original antibody. This is most
clearly manifested in the development and use of "humanized"
antibodies in which non-human CDRs are covalently joined to human
FR and/or Fc/pFc' regions to produce a functional antibody. Thus,
for example, PCT International Publication Number WO 92/04381
teaches the production and use of humanized murine RSV antibodies
in which at least a portion of the murine FR regions have been
replaced by FR regions of human origin. Such antibodies, including
fragments of full-length antibodies with antigen-binding ability,
are often referred to as "chimeric" antibodies.
[0033] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab')2, Fab, Fv and
Fd fragments of Hendra and Nipah monoclonal antibodies; chimeric
antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or
light chain CDR3 regions of the Hendra and Nipah monoclonal
antibodies have been replaced by homologous human or non-human
sequences; chimeric F(ab')2 fragment antibodies in which the FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions of the
Hendra and Nipah monoclonal antibodies have been replaced by
homologous human or non-human sequences; chimeric Fab fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light
chain CDR3 regions have been replaced by homologous human or
non-human sequences; and chimeric Fd fragment antibodies in which
the FR and/or CDR1 and/or CDR2 regions have been replaced by
homologous human or non-human sequences. Thus, those skilled in the
art may alter the Hendra and Nipah monoclonal antibodies by the
construction of CDR grafted or chimeric antibodies or antibody
fragments containing all, or part thereof, of the disclosed heavy
and light chain V-region CDR aa sequences (Jones, P. T. et al. 1986
Nature 321:522-525; Verhoeyen, M. et al. 1988 Science 39:1534-1536;
and Tempest, P. R. et al. 1991 Biotechnology 9:266-271), without
destroying the specificity of the antibodies for the G glycoprotein
epitope. Such CDR grafted or chimeric antibodies or antibody
fragments can be effective in prevention and treatment of Hendra or
Nipah virus infection in animals (e.g., horses) and man.
[0034] In preferred embodiments, the chimeric antibodies of the
invention are fully human monoclonal antibodies including at least
the heavy chain CDR3 region of the Hendra and Nipah monoclonal
antibodies. As noted above, such chimeric antibodies may be
produced in which some or all of the FR regions of the Hendra and
Nipah monoclonal antibodies have been replaced by other homologous
human FR regions. In addition, the Fc portions may be replaced so
as to produce IgA or IgM as well as IgG antibodies bearing some or
all of the CDRs of the Hendra and Nipah monoclonal antibodies. Of
particular importance is the inclusion of the Hendra and Nipah
monoclonal antibodies heavy chain CDR3 region and, to a lesser
extent, the other CDRs of the Hendra and Nipah monoclonal
antibodies. Such fully human or chimeric antibodies will have
particular utility in that they will not evoke an immune response
against the antibody itself.
[0035] It is also possible, in accordance with the present
invention, to produce chimeric antibodies including non-human
sequences. Thus, one may use, for example, murine, ovine, equine,
bovine or other mammalian Fc or FR sequences to replace some or all
of the Fc or FR regions of the Hendra and Nipah monoclonal
antibodies. Some of the CDRs may be replaced as well. Again,
however, it is preferred that at least the heavy chain CDR3 of the
Hendra and Nipah monoclonal antibodies, be included in such
chimeric antibodies and, to a lesser extent, it is also preferred
that some or all of the other CDRs of the Hendra and Nipah
monoclonal antibodies be included. Such chimeric antibodies bearing
non-human immunoglobulin sequences admixed with the CDRs of the
human Hendra and Nipah monoclonal antibodies are not preferred for
use in humans and are particularly not preferred for extended use
because they may evoke an immune response against the non-human
sequences. They may, of course, be used for brief periods or in
immunosuppressed individuals but, again, fully human monoclonal
antibodies are preferred. Because, however, Hendra and Nipah
viruses also infect animals and because such antibodies may be used
for brief periods or in immunosuppressed subjects, chimeric
antibodies bearing non-human mammalian Fc and FR sequences but
including at least the heavy chain CDR3 of the Hendra and Nipah
monoclonal antibodies are contemplated as alternative embodiments
of the present invention.
[0036] For inoculation or prophylactic uses, the antibodies of the
present invention are preferably full-length antibody molecules
including the Fc region. Such full-length antibodies will have
longer half-lives than smaller fragment antibodies (e.g., Fab) and
are more suitable for intravenous, intraperitoneal, intramuscular,
intracavity, subcutaneous, or transdermal administration.
[0037] In some embodiments, Fab fragments, including chimeric Fab
fragments, are preferred. Fabs offer several advantages over
F(ab').sub.2 and whole immunoglobulin molecules for this
therapeutic modality. First, because Fabs have only one binding
site for their cognate antigen, the formation of immune complexes
is precluded whereas such complexes can be generated when bivalent
F(ab').sub.2 s and whole immunoglobulin molecules encounter their
target antigen. This is of some importance because immune complex
deposition in tissues can produce adverse inflammatory reactions.
Second, because Fabs lack an Fc region they cannot trigger adverse
inflammatory reactions that are activated by Fc, such as activation
of the complement cascade. Third, the tissue penetration of the
small Fab molecule is likely to be much better than that of the
larger whole antibody. Fourth, Fabs can be produced easily and
inexpensively in bacteria, such as E. coli, whereas whole
immunoglobulin antibody molecules require mammalian cells for their
production in useful amounts. The latter entails transfection of
immunoglobulin sequences into mammalian cells with resultant
transformation. Amplification of these sequences must then be
achieved by rigorous selective procedures and stable transformants
must be identified and maintained. The whole immunoglobulin
molecules must be produced by stably transformed, high expression
mammalian cells in culture with the attendant problems of
serum-containing culture medium. In contrast, production of Fabs in
E. coli eliminates these difficulties and makes it possible to
produce these antibody fragments in large fermenters which are less
expensive than cell culture-derived products.
[0038] In addition to Fabs, smaller antibody fragments and
epitope-binding peptides having binding specificity for the
epitopes defined by the Hendra and Nipah monoclonal antibodies are
also contemplated by the present invention and can also be used to
bind or neutralize the virus. For example, single chain antibodies
can be constructed according to the method of U.S. Pat. No.
4,946,778, to Ladner et al. Single chain antibodies comprise the
variable regions of the light and heavy chains joined by a flexible
linker moiety. Yet smaller is the antibody fragment known as the
single domain antibody or Fd, which comprises an isolated V.sub.H
single domain. Techniques for obtaining a single domain antibody
with at least some of the binding specificity of the full-length
antibody from which they are derived are known in the art.
[0039] It is possible to determine, without undue experimentation,
if an altered or chimeric antibody has the same specificity as the
Hendra and Nipah monoclonal antibodies by ascertaining whether the
former blocks the latter from binding to G glycoprotein. If the
monoclonal antibody being tested competes with the Hendra or Nipah
monoclonal antibody as shown by a decrease in binding of the Hendra
or Nipah monoclonal antibody, then it is likely that the two
monoclonal antibodies bind to the same, or a closely spaced,
epitope. Still another way to determine whether a monoclonal
antibody has the specificity of the Hendra and Nipah monoclonal
antibodies is to pre-incubate the Hendra or Nipah monoclonal
antibody with G glycoprotein with which it is normally reactive,
and then add the monoclonal antibody being tested to determine if
the monoclonal antibody being tested is inhibited in its ability to
bind G glycoprotein. If the monoclonal antibody being tested is
inhibited then, in all likelihood, it has the same, or a
functionally equivalent, epitope and specificity as the Hendra and
Nipah monoclonal antibodies of the invention. Screening of Hendra
and Nipah monoclonal antibodies also can be carried out by
utilizing Hendra or Nipah viruses and determining whether the mAb
neutralizes the virus.
[0040] By using the antibodies of the invention, it is now possible
to produce anti-idiotypic antibodies which can be used to screen
other monoclonal antibodies to identify whether the antibody has
the same binding specificity as an antibody of the invention. In
addition, such antiidiotypic antibodies can be used for active
immunization (Herlyn, D. et al. 1986 Science 232:100-102). Such
anti-idiotypic antibodies can be produced using well-known
hybridoma techniques (Kohler, G. and Milstein, C. 1975 Nature
256:495-497). An anti-idiotypic antibody is an antibody which
recognizes unique determinants present on the monoclonal antibody
produced by the cell line of interest. These determinants are
located in the hypervariable region of the antibody. It is this
region which binds to a given epitope and, thus, is responsible for
the specificity of the antibody. An anti-idiotypic antibody can be
prepared by immunizing an animal with the monoclonal antibody of
interest. The immunized animal will recognize and respond to the
idiotypic determinants of the immunizing antibody and produce an
antibody to these idiotypic determinants. By using the
anti-idiotypic antibodies of the immunized animal, which are
specific for the monoclonal antibodies of the invention, it is
possible to identify other clones with the same idiotype as the
antibody of the hybridoma used for immunization. Idiotypic identity
between monoclonal antibodies of two cell lines demonstrates that
the two monoclonal antibodies are the same with respect to their
recognition of the same epitopic determinant. Thus, by using
anti-idiotypic antibodies, it is possible to identify other
hybridomas expressing monoclonal antibodies having the same
epitopic specificity.
[0041] It is also possible to use the anti-idiotype technology to
produce monoclonal antibodies which mimic an epitope. For example,
an anti-idiotypic monoclonal antibody made to a first monoclonal
antibody will have a binding domain in the hypervariable region
which is the image of the epitope bound by the first monoclonal
antibody. Thus, the anti-idiotypic monoclonal antibody can be used
for immunization, since the anti-idiotype monoclonal antibody
binding domain effectively acts as an antigen.
Nucleic Acids Encoding Anti-HeV and NiV G Glycoprotein
Antibodies
[0042] Given the disclosure herein of the amino acid sequences of
the heavy chain Fd and light chain variable domains of the Hendra
and Nipah monoclonal antibodies, one of ordinary skill in the art
is now enabled to produce nucleic acids which encode this antibody
or which encode the various fragment antibodies or chimeric
antibodies described above. It is contemplated that such nucleic
acids will be operably joined to other nucleic acids forming a
recombinant vector for cloning or for expression of the antibodies
of the invention. The present invention includes any recombinant
vector containing the coding sequences, or part thereof, whether
for prokaryotic or eukaryotic transformation, transfection or gene
therapy. Such vectors may be prepared using conventional molecular
biology techniques, known to those with skill in the art, and would
comprise DNA coding sequences for the immunoglobulin V-regions of
the Hendra and Nipah monoclonal antibodies, including framework and
CDRs or parts thereof, and a suitable promoter either with
(Whittle, N. et al. 1987 Protein Eng 1:499-505 and Burton, D. R. et
al. 1994 Science 266:1024-1027) or without (Marasco, W. A. et al.
1993 Proc Natl Acad Sci USA 90:7889-7893 and Duan, L. et al. 1994
Proc Natl Acad Sci USA 91:5075-5079) a signal sequence for export
or secretion. Such vectors may be transformed or transfected into
prokaryotic (Huse, W. D. et al. 1989 Science 246:1275-1281; Ward,
S. et al. 1989 Nature 341:544-546; Marks, J. D. et al. 1991 J Mol
Biol 222:581-597; and Barbas, C. F. et al. 1991 Proc Natl Acad Sci
USA 88:7978-7982) or eukaryotic (Whittle, N. et al. 1987 Protein
Eng 1:499-505 and Burton, D. R. et al. 1994 Science 266:1024-1027)
cells or used for gene therapy (Marasco, W. A. et al. 1993 Proc
Natl Acad Sci USA 90:7889-7893 and Duan, L. et al. 1994 Proc Natl
Acad Sci USA 91:5075-5079) by conventional techniques, known to
those with skill in the art.
[0043] The expression vectors of the present invention include
regulatory sequences operably joined to a nucleotide sequence
encoding one of the antibodies of the invention. As used herein,
the term "regulatory sequences" means nucleotide sequences which
are necessary for or conducive to the transcription of a nucleotide
sequence which encodes a desired polypeptide and/or which are
necessary for or conducive to the translation of the resulting
transcript into the desired polypeptide. Regulatory sequences
include, but are not limited to, 5' sequences such as operators,
promoters and ribosome binding sequences, and 3' sequences such as
polyadenylation signals. The vectors of the invention may
optionally include 5' leader or signal sequences, 5' or 3'
sequences encoding fusion products to aid in protein purification,
and various markers which aid in the identification or selection of
transformants. The choice and design of an appropriate vector is
within the ability and discretion of one of ordinary skill in the
art. The subsequent purification of the antibodies may be
accomplished by any of a variety of standard means known in the
art.
[0044] A preferred vector for screening monoclonal antibodies, but
not necessarily preferred for the mass production of the antibodies
of the invention, is a recombinant DNA molecule containing a
nucleotide sequence that codes for and is capable of expressing a
fusion polypeptide containing, in the direction of amino- to
carboxy-terminus, (1) a prokaryotic secretion signal domain, (2) a
polypeptide of the invention, and, optionally, (3) a fusion protein
domain. The vector includes DNA regulatory sequences for expressing
the fusion polypeptide, preferably prokaryotic, regulatory
sequences. Such vectors can be constructed by those with skill in
the art and have been described by Smith, G. P. et al. (1985
Science 228:13151317); Clackson, T. et al. (1991 Nature
352:624-628); Kang et al. (1991 in Methods: A Companion to Methods
in Enzymology, vol. 2, R. A. Lerner and D. R. Burton, ed. Academic
Press, NY, pp 111-118); Barbas, C. F. et al. (1991 Proc Natl Acad
Sci USA 88:7978-7982); Roberts, B. L. et al. (1992 Proc Natl Acad
Sci USA 89:2429-2433).
[0045] A fusion polypeptide may be useful for purification of the
antibodies of the invention. The fusion domain may, for example,
include a poly-His tail which allows for purification on Ni.sup.+
columns or the maltose binding protein of the commercially
available vector pMAL (New England BioLabs, Beverly, Mass.). A
currently preferred, but by no means necessary, fusion domain is a
filamentous phage membrane anchor. This domain is particularly
useful for screening phage display libraries of monoclonal
antibodies but may be of less utility for the mass production of
antibodies. The filamentous phage membrane anchor is preferably a
domain of the cpIII or cpVIII coat protein capable of associating
with the matrix of a filamentous phage particle, thereby
incorporating the fusion polypeptide onto the phage surface, to
enable solid phase binding to specific antigens or epitopes and
thereby allow enrichment and selection of the specific antibodies
or fragments encoded by the phagemid vector.
[0046] The secretion signal is a leader peptide domain of a protein
that targets the protein to the membrane of the host cell, such as
the periplasmic membrane of Gram-negative bacteria. A preferred
secretion signal for E. coli is a pelB secretion signal. The leader
sequence of the pelB protein has previously been used as a
secretion signal for fusion proteins (Better, M. et al. 1988
Science 240:1041-1043; Sastry, L. et al. 1989 Proc Natl Acad Sci
USA 86:5728-5732; and Mullinax, R. L. et al., 1990 Proc Natl Acad
Sci USA 87:8095-8099). Amino acid residue sequences for other
secretion signal polypeptide domains from E. coli useful in this
invention can be found in Neidhard, F. C. (ed.), 1987 in
Escherichia coli and Salmonella Typhimurium: Typhimurium Cellular
and Molecular Biology, American Society for Microbiology,
Washington, D.C.
[0047] To achieve high levels of gene expression in E. coli, it is
necessary to use not only strong promoters to generate large
quantities of mRNA, but also ribosome binding sites to ensure that
the mRNA is efficiently translated. In E. coli, the ribosome
binding site includes an initiation codon (AUG) and a sequence 3-9
nucleotides long located 3-11 nucleotides upstream from the
initiation codon (Shine J. and Dalgarno L. 1975 Nature 254:34-38).
The sequence, which is called the Shine-Dalgarno (SD) sequence, is
complementary to the 3' end of E. coli 16S rRNA. Binding of the
ribosome to mRNA and the sequence at the 3' end of the mRNA can be
affected by several factors: the degree of complementarity between
the SD sequence and 3' end of the 16S rRNA; the spacing lying
between the SD sequence and the AUG; and the nucleotide sequence
following the AUG, which affects ribosome binding. The 3'
regulatory sequences define at least one termination (stop) codon
in frame with and operably joined to the heterologous fusion
polypeptide.
[0048] In preferred embodiments with a prokaryotic expression host,
the vector utilized includes a prokaryotic origin of replication or
replicon, i.e., a DNA sequence having the ability to direct
autonomous replication and maintenance of the recombinant DNA
molecule extrachromosomally in a prokaryotic host cell, such as a
bacterial host cell, transformed therewith. Such origins of
replication are well known in the art. Preferred origins of
replication are those that are efficient in the host organism. A
preferred host cell is E. coli. For use of a vector in E. coli, a
preferred origin of replication is ColEI found in pBR322 and a
variety of other common plasmids. Also preferred is the p15A origin
of replication found on pACYC and its derivatives. The ColEI and
p15A replicons have been extensively utilized in molecular biology,
are available on a variety of plasmids and are described by
Sambrook et al., 1989, in Molecular Cloning: A Laboratory Manual,
2nd edition, Cold Spring Harbor Laboratory Press.
[0049] In addition, those embodiments that include a prokaryotic
replicon preferably also include a gene whose expression confers a
selective advantage, such as drug resistance, to a bacterial host
transformed therewith. Typical bacterial drug resistance genes are
those that confer resistance to ampicillin, tetracycline,
neomycin/kanamycin or chloramphenicol. Vectors typically also
contain convenient restriction sites for insertion of translatable
DNA sequences. Exemplary vectors are the plasmids pUC18 and pUC19
and derived vectors such as those commercially available from
suppliers such as Invitrogen (San Diego, Calif.).
[0050] When the antibodies of the invention include both heavy
chain and light chain sequences, these sequences may be encoded on
separate vectors or, more conveniently, may be expressed by a
single vector. The heavy and light chain may, after translation or
after secretion, form the heterodimeric structure of natural
antibody molecules. Such a heterodimeric antibody may or may not be
stabilized by disulfide bonds between the heavy and light
chains.
[0051] A vector for expression of heterodimeric antibodies, such as
the full-length antibodies of the invention or the F(ab').sub.2,
Fab or Fv fragment antibodies of the invention, is a recombinant
DNA molecule adapted for receiving and expressing translatable
first and second DNA sequences. That is, a DNA expression vector
for expressing a heterodimeric antibody provides a system for
independently cloning (inserting) the two translatable DNA
sequences into two separate cassettes present in the vector, to
form two separate cistrons for expressing the first and second
polypeptides of a heterodimeric antibody. The DNA expression vector
for expressing two cistrons is referred to as a dicistronic
expression vector.
[0052] Preferably, the vector comprises a first cassette that
includes upstream and downstream DNA regulatory sequences operably
joined via a sequence of nucleotides adapted for directional
ligation to an insert DNA. The upstream translatable sequence
preferably encodes the secretion signal as described above. The
cassette includes DNA regulatory sequences for expressing the first
antibody polypeptide that is produced when an insert translatable
DNA sequence (insert DNA) is directionally inserted into the
cassette via the sequence of nucleotides adapted for directional
ligation.
[0053] The dicistronic expression vector also contains a second
cassette for expressing the second antibody polypeptide. The second
cassette includes a second translatable DNA sequence that
preferably encodes a secretion signal, as described above, operably
joined at its 3' terminus via a sequence of nucleotides adapted for
directional ligation to a downstream DNA sequence of the vector
that typically defines at least one stop codon in the reading frame
of the cassette. The second translatable DNA sequence is operably
joined at its 5' terminus to DNA regulatory sequences forming the
5' elements. The second cassette is capable, upon insertion of a
translatable DNA sequence (insert DNA), of expressing the second
fusion polypeptide comprising a secretion signal with a polypeptide
coded by the insert DNA.
[0054] The antibodies of the present invention may additionally, of
course, be produced by eukaryotic cells such as CHO cells, human or
mouse hybridomas, immortalized B-lymphoblastoid cells, and the
like. In this case, a vector is constructed in which eukaryotic
regulatory sequences are operably joined to the nucleotide
sequences encoding the antibody polypeptide or polypeptides. The
design and selection of an appropriate eukaryotic vector is within
the ability and discretion of one of ordinary skill in the art. The
subsequent purification of the antibodies may be accomplished by
any of a variety of standard means known in the art.
[0055] The antibodies of the present invention may furthermore, of
course, be produced in plants. In 1989, Hiatt A. et al. 1989 Nature
342:76-78 first demonstrated that functional antibodies could be
produced in transgenic plants. Since then, a considerable amount of
effort has been invested in developing plants for antibody (or
"plantibody") production (for reviews see Giddings, G. et al. 2000
Nat Biotechnol 18:1151-1155; Fischer, R. and Emans, N. 2000
Transgenic Res 9:279-299). Recombinant antibodies can be targeted
to seeds, tubers, or fruits, making administration of antibodies in
such plant tissues advantageous for immunization programs in
developing countries and worldwide.
[0056] In another embodiment, the present invention provides host
cells, both prokaryotic and eukaryotic, transformed or transfected
with, and therefore including, the vectors of the present
invention.
Diagnostic and Pharmaceutical Anti-HeV and NiV G Glycoprotein
Antibody Preparations
[0057] The invention also relates to a method for preparing
diagnostic or pharmaceutical compositions comprising the monoclonal
antibodies of the invention or polynucleotide sequences encoding
the antibodies of the invention or part thereof, the pharmaceutical
compositions being used for immunoprophylaxis or immunotherapy of
Hendra Virus Disease or Nipah Virus Disease. The pharmaceutical
preparation includes a pharmaceutically acceptable carrier. Such
carriers, as used herein, means a non-toxic material that does not
interfere with the effectiveness of the biological activity of the
active ingredients. The term "physiologically acceptable" refers to
a non-toxic material that is compatible with a biological system
such as a cell, cell culture, tissue, or organism. The
characteristics of the carrier will depend on the route of
administration. Physiologically and pharmaceutically acceptable
carriers include diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other materials which are well known in the
art.
[0058] A preferred embodiment of the invention relates to
monoclonal antibodies whose heavy chains comprise in CDR3 the
polypeptide having SEQ ID NO: 7, and/or whose light chains comprise
in CDR3 the polypeptide having SEQ ID NO: 15; whose heavy chains
comprise in CDR3 the polypeptide having SEQ ID NO: 23, and/or whose
light chains comprise in CDR3 the polypeptide having SEQ ID NO: 31;
whose heavy chains comprise in CDR3 the polypeptide having SEQ ID
NO: 39, and/or whose light chains comprise in CDR3 the polypeptide
having SEQ ID NO: 47; whose heavy chains comprise in CDR3 the
polypeptide having SEQ ID NO: 55, and/or whose light chains
comprise in CDR3 the polypeptide having SEQ ID NO: 63; whose heavy
chains comprise in CDR3 the polypeptide having SEQ ID NO: 71,
and/or whose light chains comprise in CDR3 the polypeptide having
SEQ ID NO: 79; whose heavy chains comprise in CDR3 the polypeptide
having SEQ ID NO: 87, and/or whose light chains comprise in CDR3
the polypeptide having SEQ ID NO: 95; whose heavy chains comprise
in CDR3 the polypeptide having SEQ ID NO: 103, and/or whose light
chains comprise in CDR3 the polypeptide having SEQ ID NO: 111;
whose heavy chains comprise in CDR3 the polypeptide having SEQ ID
NO: 119, and/or whose light chains comprise in CDR3 the polypeptide
having SEQ ID NO: 127; whose heavy chains comprise in CDR3 the
polypeptide having SEQ ID NO: 135, and/or whose light chains
comprise in CDR3 the polypeptide having SEQ ID NO: 143; whose heavy
chains comprise in CDR3 the polypeptide having SEQ ID NO: 151,
and/or whose light chains comprise in CDR3 the polypeptide having
SEQ ID NO: 159; whose heavy chains comprise in CDR3 the polypeptide
having SEQ ID NO: 167, and/or whose light chains comprise in CDR3
the polypeptide having SEQ ID NO: 175; whose heavy chains comprise
in CDR3 the polypeptide having SEQ ID NO: 183, and/or whose light
chains comprise in CDR3 the polypeptide having SEQ ID NO: 191;
whose heavy chains comprise in CDR3 the polypeptide having SEQ ID
NO: 199, and/or whose light chains comprise in CDR3 the polypeptide
having SEQ ID NO: 207; whose heavy chains comprise in CDR3 the
polypeptide having SEQ ID NO: 215, and/or whose light chains
comprise in CDR3 the polypeptide having SEQ ID NO: 223; whose heavy
chains comprise in CDR3 the polypeptide having SEQ ID NO: 231,
and/or whose light chains comprise in CDR3 the polypeptide having
SEQ ID NO: 239; whose heavy chains comprise in CDR3 the polypeptide
having SEQ ID NO: 247, and/or whose light chains comprise in CDR3
the polypeptide having SEQ ID NO: 255; whose heavy chains comprise
in CDR3 the polypeptide having SEQ ID NO: 263, and/or whose light
chains comprise in CDR3 the polypeptide having SEQ ID NO: 271;
whose heavy chains comprise in CDR3 the polypeptide having SEQ ID
NO: 279, and/or whose light chains comprise in CDR3 the polypeptide
having SEQ ID NO: 287; whose heavy chains comprise in CDR3 the
polypeptide having SEQ ID NO: 295, and/or whose light chains
comprise in CDR3 the polypeptide having SEQ ID NO: 303; whose heavy
chains comprise in CDR3 the polypeptide having SEQ ID NO: 311,
and/or whose light chains comprise in CDR3 the polypeptide having
SEQ ID NO: 319; whose heavy chains comprise in CDR3 the polypeptide
having SEQ ID NO: 327, and/or whose light chains comprise in CDR3
the polypeptide having SEQ ID NO: 335; whose heavy chains comprise
in CDR3 the polypeptide having SEQ ID NO: 343, and/or whose light
chains comprise in CDR3 the polypeptide having SEQ ID NO: 351;
whose heavy chains comprise in CDR3 the polypeptide having SEQ ID
NO: 359, and/or whose light chains comprise in CDR3 the polypeptide
having SEQ ID NO: 367; whose heavy chains comprise in CDR3 the
polypeptide having SEQ ID NO: 375, and/or whose light chains
comprise in CDR3 the polypeptide having SEQ ID NO: 383; whose heavy
chains comprise in CDR3 the polypeptide having SEQ ID NO: 391,
and/or whose light chains comprise in CDR3 the polypeptide having
SEQ ID NO: 399; whose heavy chains comprise in CDR3 the polypeptide
having SEQ ID NO: 407, and/or whose light chains comprise in CDR3
the polypeptide having SEQ ID NO: 415; and conservative variations
of these peptides. The term "conservative variation" as used herein
denotes the replacement of an amino acid residue by another,
biologically similar residue. Examples of conservative variations
include the substitution of one hydrophobic residue such as
isoleucine, valine, leucine or methionine for another, or the
substitution of one polar residue for another, such as the
substitution of arginine for lysine, glutamic for aspartic acids,
or glutamine for asparagine, and the like. The term "conservative
variation" also includes the use of a substituted amino acid in
place of an unsubstituted parent amino acid provided that
antibodies having the substituted polypeptide also bind or
neutralize Hendra or Nipah virus. Analogously, another preferred
embodiment of the invention relates to polynucleotides which encode
the above noted heavy chain polypeptides and to polynucleotide
sequences which are complementary to these polynucleotide
sequences. Complementary polynucleotide sequences include those
sequences that hybridize to the polynucleotide sequences of the
invention under stringent hybridization conditions.
[0059] The anti-Hendra and Nipah antibodies of the invention may be
labeled by a variety of means for use in diagnostic and/or
pharmaceutical applications. There are many different labels and
methods of labeling known to those of ordinary skill in the art.
Examples of the types of labels which can be used in the present
invention include enzymes, radioisotopes, fluorescent compounds,
colloidal metals, chemiluminescent compounds, and bioluminescent
compounds. Those of ordinary skill in the art will know of other
suitable labels for binding to the monoclonal antibodies of the
invention, or will be able to ascertain such, using routine
experimentation. Furthermore, the binding of these labels to the
monoclonal antibodies of the invention can be done using standard
techniques common to those of ordinary skill in the art.
[0060] Another labeling technique which may result in greater
sensitivity consists of coupling the antibodies to low molecular
weight haptens. These haptens can then be specifically altered by
means of a second reaction. For example, it is common to use
haptens such as biotin, which reacts with avidin, or dinitrophenol,
pyridoxal, or fluorescein, which can react with specific
anti-hapten antibodies.
[0061] The materials for use in the assay of the invention are
ideally suited for the preparation of a kit. Such a kit may
comprise a carrier means being compartmentalized to receive in
close confinement one or more container means such as vials, tubes,
and the like, each of the container means comprising one of the
separate elements to be used in the method. For example, one of the
container means may comprise a monoclonal antibody of the invention
that is, or can be, detectably labeled. The kit may also have
containers containing buffer(s) and/or a container comprising a
reporter-means, such as a biotin-binding protein, such as avidin or
streptavidin, bound to a reporter molecule, such as an enzymatic or
fluorescent label.
In Vitro Detection and Diagnostics
[0062] The monoclonal antibodies of the invention are suited for in
vitro use, for example, in immunoassays in which they can be
utilized in liquid phase or bound to a solid phase carrier. In
addition, the monoclonal antibodies in these immunoassays can be
detectably labeled in various ways. Examples of types of
immunoassays which can utilize the monoclonal antibodies of the
invention are competitive and non-competitive immunoassays in
either a direct or indirect format. Examples of such immunoassays
are the radioimmunoassay (RIA) and the sandwich (immunometric)
assay. Detection of antigens using the monoclonal antibodies of the
invention can be done utilizing immunoassays which are run in
either the forward, reverse, or simultaneous modes, including
immunohistochemical assays on physiological samples. Those of skill
in the art will know, or can readily discern, other immunoassay
formats without undue experimentation.
[0063] The monoclonal antibodies of the invention can be bound to
many different carriers and used to detect the presence of Hendra
or Nipah virus. Examples of well-known carriers include glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylase,
natural and modified cellulose, polyacrylamide, agarose and
magnetite. The nature of the carrier can be either soluble or
insoluble for purposes of the invention. Those skilled in the art
will know of other suitable carriers for binding monoclonal
antibodies, or will be able to ascertain such, using routine
experimentation.
[0064] For purposes of the invention, Hendra or Nipah virus may be
detected by the monoclonal antibodies of the invention when present
in biological fluids and tissues. Any sample containing a
detectable amount of Hendra or Nipah virus can be used. A sample
can be a liquid such as urine, saliva, cerebrospinal fluid, blood,
serum or the like; a solid or semi-solid such as tissues, feces, or
the like; or, alternatively, a solid tissue such as those commonly
used in histological diagnosis.
In Vivo Detection of Hendra or Nipah Virus
[0065] In using the monoclonal antibodies of the invention for the
in vivo detection of antigen, the detectably labeled monoclonal
antibody is given in a dose which is diagnostically effective. The
term "diagnostically effective" means that the amount of detectably
labeled monoclonal antibody is administered in sufficient quantity
to enable detection of the site having the Hendra or Nipah virus
antigen for which the monoclonal antibodies are specific.
[0066] The concentration of detectably labeled monoclonal antibody
which is administered should be sufficient such that the binding to
Hendra or Nipah virus is detectable compared to the background.
Further, it is desirable that the detectably labeled monoclonal
antibody be rapidly cleared from the circulatory system in order to
give the best target-to-background signal ratio.
[0067] As a rule, the dosage of detectably labeled monoclonal
antibody for in vivo diagnosis will vary depending on such factors
as age, sex, and extent of disease of the individual. The dosage of
monoclonal antibody can vary from about 0.01 mg/kg to about 50
mg/kg, preferably 0.1 mg/kg to about 20 mg/kg, most preferably
about 0.1 mg/kg to about 2 mg/kg. Such dosages may vary, for
example, depending on whether multiple injections are given, on the
tissue being assayed, and other factors known to those of skill in
the art.
[0068] For in vivo diagnostic imaging, the type of detection
instrument available is a major factor in selecting an appropriate
radioisotope. The radioisotope chosen must have a type of decay
which is detectable for the given type of instrument. Still another
important factor in selecting a radioisotope for in vivo diagnosis
is that the half-life of the radioisotope be long enough such that
it is still detectable at the time of maximum uptake by the target,
but short enough such that deleterious radiation with respect to
the host is acceptable. Ideally, a radioisotope used for in vivo
imaging will lack a particle emission but produce a large number of
photons in the 140-250 keV range, which may be readily detected by
conventional gamma cameras.
[0069] For in vivo diagnosis, radioisotopes may be bound to
immunoglobulin either directly or indirectly by using an
intermediate functional group. Intermediate functional groups which
often are used to bind radioisotopes which exist as metallic ions
are the bifunctional chelating agents such as
diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetra-acetic acid (EDTA) and similar molecules.
Typical examples of metallic ions which can be bound to the
monoclonal antibodies of the invention are .sup.111In, .sup.97Ru,
.sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr and .sup.201Tl.
[0070] The monoclonal antibodies of the invention can also be
labeled with a paramagnetic isotope for purposes of in vivo
diagnosis, as in magnetic resonance imaging (MRI) or electron spin
resonance (ESR). In general, any conventional method for
visualizing diagnostic imaging can be utilized. Usually gamma and
positron emitting radioisotopes are used for camera imaging and
paramagnetic isotopes for MRI. Elements which are particularly
useful in such techniques include .sup.157Gd, .sup.55Mn,
.sup.162Dy, .sup.52Cr and .sup.56Fe.
[0071] The monoclonal antibodies of the invention can be used in
vitro and in vivo to monitor the course of Hendra Virus Disease or
Nipah Virus Disease therapy. Thus, for example, by measuring the
increase or decrease in the number of cells infected with Hendra or
Nipah virus or changes in the concentration of Hendra or Nipah
virus present in the body or in various body fluids, it would be
possible to determine whether a particular therapeutic regimen
aimed at ameliorating Hendra Virus Disease or Nipah Virus Disease
is effective.
Prophylaxis and Therapy of Hendra Virus Disease and Nipah Virus
Disease
[0072] The monoclonal antibodies can also be used in prophylaxis
and as therapy for Hendra Virus Disease and Nipah Virus Disease in
both humans and other animals. The terms, "prophylaxis" and
"therapy" as used herein in conjunction with the monoclonal
antibodies of the invention denote both prophylactic as well as
therapeutic administration and both passive immunization with
substantially purified polypeptide products, as well as gene
therapy by transfer of polynucleotide sequences encoding the
product or part thereof. Thus, the monoclonal antibodies can be
administered to high-risk subjects in order to lessen the
likelihood and/or severity of Hendra Virus Disease and Nipah Virus
Disease or administered to subjects already evidencing active
Hendra or Nipah virus infection. In the present invention, Fab
fragments also bind or neutralize Hendra or Nipah virus and
therefore may be used to treat infection but full-length antibody
molecules are otherwise preferred.
[0073] As used herein, a "prophylactically effective amount" of the
monoclonal antibodies of the invention is a dosage large enough to
produce the desired effect in the protection of individuals against
Hendra or Nipah virus infection for a reasonable period of time,
such as one to two months or longer following administration. A
prophylactically effective amount is not, however, a dosage so
large as to cause adverse side effects, such as hyperviscosity
syndromes, pulmonary edema, congestive heart failure, and the like.
Generally, a prophylactically effective amount may vary with the
subject's age, condition, and sex, as well as the extent of the
disease in the subject and can be determined by one of skill in the
art. The dosage of the prophylactically effective amount may be
adjusted by the individual physician or veterinarian in the event
of any complication. A prophylactically effective amount may vary
from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1
mg/kg to about 20 mg/kg, most preferably from about 0.2 mg/kg to
about 2 mg/kg, in one or more administrations (priming and
boosting).
[0074] As used herein, a "therapeutically effective amount" of the
monoclonal antibodies of the invention is a dosage large enough to
produce the desired effect in which the symptoms of Hendra Virus
Disease or Nipah Virus Disease are ameliorated or the likelihood of
infection is decreased. A therapeutically effective amount is not,
however, a dosage so large as to cause adverse side effects, such
as hyperviscosity syndromes, pulmonary edema, congestive heart
failure, and the like. Generally, a therapeutically effective
amount may vary with the subject's age, condition, and sex, as well
as the extent of the disease in the subject and can be determined
by one of skill in the art. The dosage of the therapeutically
effective amount may be adjusted by the individual physician or
veterinarian in the event of any complication. A therapeutically
effective amount may vary from about 0.01 mg/kg to about 50 mg/kg,
preferably from about 0.1 mg/kg to about 20 mg/kg, most preferably
from about 0.2 mg/kg to about 2 mg/kg, in one or more dose
administrations daily, for one or several days. Preferred is
administration of the antibody for 2 to 5 or more consecutive days
in order to avoid "rebound" of virus replication from
occurring.
[0075] The monoclonal antibodies of the invention can be
administered by injection or by gradual infusion over time. The
administration of the monoclonal antibodies of the invention may,
for example, be intravenous, intraperitoneal, intramuscular,
intracavity, subcutaneous, or transdermal. Techniques for preparing
injectate or infusate delivery systems containing antibodies are
well known to those of skill in the art. Generally, such systems
should utilize components which will not significantly impair the
biological properties of the antibodies, such as the paratope
binding capacity (see, for example, Remington's Pharmaceutical
Sciences, 18th edition, 1990, Mack Publishing). Those of skill in
the art can readily determine the various parameters and conditions
for producing antibody injectates or infusates without resort to
undue experimentation.
[0076] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and the like.
[0077] Potent Neutralization of Hendra and Nipah Viruses by Human
Monoclonal Antibodies
Selection of Phage-Displayed Fabs (m101-7) Specific for Hendra
Virus Soluble G Glycoprotein (sG)
[0078] Our initial efforts to develop G-specific human monoclonal
antibodies (hmAbs) by using cell-associated G and synthetic
antibody libraries as well as an immune library constructed from
frozen lymphocytes of a survivor from Nipah infection have not been
successful. To develop hmAbs against the G envelope glycoprotein of
HeV and NiV we used a large naive human Fab library containing
about 10.sup.10 different phage-displayed Fabs we have recently
developed. Here, we have made use of a unique soluble and secreted
form of the attachment (G) glycoprotein of HeV (sG) which we have
recently produced and characterized (Bossart, K. N. et al. 2005 J
Virol 79:6690-6702). This protein was used as an antigen for
screening of the antibody library. After four rounds of panning,
screening of 380 individual phage clones was performed in phage
ELISA with sG as described in Example 1. Of those, 71 clones that
exhibited significant binding to sG were sequenced. Seventeen Fabs
had unique sequences (Table 1). They were expressed in bacteria,
purified and tested for binding activity. Seven Fabs, designated
m101 through m107, exhibited significant (A450>0.5) binding to
sG in ELISA (Table 1). Notably, on average, the heavy chain CDR3s
(H3) of the binders (m101-7) were significantly longer than those
of Fabs that bind weaker (m108-17) (Table 1). Interestingly, the
heavy chains of m101 and m102 (the most potent HeV and NiV
neutralizers--see below) were the most divergent from the germ line
heavy chains indicating a certain level of maturation although they
are IgM specific. The light chains were from all Ig classes and
show greater variation as compared to the germ line light chains
(Table 2).
Inhibition of HeV Env-Mediated Fusion by the Selected Fabs
[0079] To test the neutralizing activity of the antibodies we first
measured their ability to inhibit fusion mediated by HeV envelope
glycoprotein (Env) expressing cells with cells that we had
previously identified as fusion-competent. Fusion was measured by
two assays--a reporter gene assay and a syncytia formation assay.
The seven Fabs that bound strongly to sG (Table 1) also exhibited
measurable inhibitory activity in the reporter gene assay (Table 3)
and were selected for further characterization. They also inhibited
syncytia formation to various degrees in general correlation with
the inhibitory activity measured by the reporter gene assay.
Interestingly, six of the seven Fabs also inhibited to various
degrees NiV Env-mediated fusion (Table 3). One antibody, m101, was
most active against HeV Env-mediated fusion, while another one,
m102, exhibited the highest cross-inhibitory activity against both
HeV- and NiV Env-mediated fusion.
Neutralization of HeV and NiV by Fabs
[0080] The inhibitory activity of these Fabs was further tested by
using infectious HeV and NiV in a neutralization assay as described
in the Example 1. When tested at concentrations above 80 .mu.g/ml,
Fab m101 showed neutralizing activity against HeV but not against
NiV, and Fab m102 exhibited weaker neutralizing activity against
HeV as compared to m101. Interestingly, as in the cell fusion
assay, m102 exhibited cross-neutralizing activity for both HeV and
NiV. The other tested Fabs did not show measurable neutralizing
activity when tested at concentrations up to 100 .mu.g/ml. These
results indicated that two of the selected Fabs could neutralize
infectious HeV and NiV.
Potent Inhibitory Activity of IgG1 m101 Against HeV Env-Mediated
Fusion and Live Virus
[0081] In most cases, but not always, whole antibodies are better
neutralizers than Fabs. Thus the most potent HeV-fusion inhibiting
Fab, m101, was converted to a whole antibody format (IgG1) and
tested in a cell fusion assay. The IgG1 m101 inhibitory activity
was much higher than the activity of the Fab m101 (FIGS. 1 and 2).
The conversion of Fab m101 to IgG1 dramatically decreased its
IC50s. For HeLa-ATCC cells which exhibit lower fusion rates, the
IC50 decreased from 4.2 .mu.g/ml to 0.5 .mu.g/ml (FIG. 1A). For the
highly fusogenic PCI-13 cells, the IC50 decreased from 38 .mu.g/ml
to 1.2 .mu.g/ml (FIG. 1B). In another experiment the IgG1 m101
inhibited 95% of fusion at 3 .mu.g/ml. IgG1 m101 also potently
inhibited syncytia formation in correlation with its inhibitory
activity measured by the reporter gene assay. An example using the
highly fusogenic PCI-13 cells is shown in FIG. 2. Here IgG1 m101
completely inhibited formation of syncytia at 10 .mu.g/ml, whereas
at the same concentration, Fab m101 inhibited approximately 50% of
syncytia formation.
[0082] IgG1 m101 was also exceptionally potent in neutralizing
infectious HeV. Complete (100%) neutralization was achieved at 12.5
.mu.g/ml, more than 99% at 6 .mu.g/ml, and 98% at 1.6 .mu.g/ml
(Table 4). These results demonstrated that IgG1 m101 is a very
potent neutralizer of infectious HeV.
Mechanism of Virus Entry Inhibition by the Antibodies: Correlation
with Binding to Native G
[0083] To begin to elucidate the mechanisms of the inhibitory
activity of the selected antibodies we measured their binding rate
constants and affinities to sG in a Biacore assay. The antibodies
bound with high (1 to 10.sup.3 nM range) affinity to sG as measured
by Biacore (Table 5). The on rate constants varied significantly
but there was no significant variation in the off rate constants
except the very low dissociation rate constant of m102. The best
inhibitors of HeV G-mediated fusion and infection, m101 and m102,
exhibited the highest affinity. In this context there was
correlation between binding to sG and fusion inhibition by groups
of antibodies divided into good (m101 and m102) and poor (the rest)
neutralizers although direct mathematically calculated correlation
between the Biacore measured affinity of each antibody to sG and
fusion inhibitory activity was not found.
[0084] To find other possible correlations between binding and
inhibition we measured binding to native G which was
immunoprecipitated from lysates of cells infected with recombinant
vaccinia viruses. The extent of immunoprecipitation, which is
proportional to the antibody binding affinity to native full-length
G glycoprotein, was highest for m101 binding to HeV G (FIG. 3). Two
of these antibodies, m102 and m106 demonstrated significant
cross-reactivity to both HeV and NiV G (FIG. 3). The levels of
immunoprecipitation correlated with cell fusion (Table 3)
indicating that binding to native G is a better correlate of fusion
inhibitory activity than binding to soluble G.
Outcompeting the Receptor Ephrin-B2 as a Mechanism of Virus Entry
Inhibition by m101 and m102
[0085] To further define the mechanism of virus entry inhibition by
the most potent neutralizer m101 we measured its competition with
the recently identified receptor for Hendra and Nipah viruses,
ephrin-B2 (Bonaparte M. I. et al. 2005 Proc Natl Acad Sci USA
102:10652-10657). M101 competed with ephrin-B2 for binding to sG;
IgG m101 was a much better competitor than Fab m101 (FIG. 4), which
correlates with their inhibitory activity and is likely due to the
multivalent nature of their interaction. Similar results were
obtained with m102 and by using Biacore (supplemental FIG. 2 in
Bonaparte M. I. et al. 2005 Proc Natl Acad Sci USA
102:10652-10657). These data indicate that m101 and m102 inhibit
entry of Hendra virus and likely Nipah virus by preventing the
access of these viruses to their receptor. They also indicate that
the epitopes of m101 and m102 overlap with the receptor binding
site on G. Interestingly, m106 competed with ephrin-B2 much weaker
than m101 and only at very high concentrations (FIG. 4).
Further Characterization of the Epitopes of the Selected Anti-G
Antibodies
[0086] To further characterize the epitopes of the newly identified
antibodies we measured the competition of m101, m102, m103, m106
and m107 with one another by ELISA (presently, there are no
anti-Hendra G antibodies with known epitopes). The m101, m102, and
m103 antibodies competed with each other indicating that they bind
to overlapping epitopes that are distinct from the epitopes of m106
and m107. Interestingly, m103 appears to synergize with m106
leading to increased binding of one in the presence of the other.
These results indicate that m101-3 may neutralize the virus by a
different mechanism from m106 and m107 but further studies with
ephrin competition are needed to definitely elucidate the mechanism
of their neutralizing activity.
[0087] In an initial attempt to localize the epitopes of m101 and
m102 we measured their binding to a panel of 10 G alanine scanning
mutants, selected to represent different portions of the protein:
G183A, L184A, P185A, (Q191, K192A), S195A, D289A, K324A, (I385,
H386A), L517A, N570A, where the two double mutants are in
parentheses. Of these mutants only one, G183A, decreases binding of
both m101 and m102 to G; this mutant bound strongly to anti-G
rabbit polyclonal antibodies and to the receptor ephrin-B2. (FIG.
5). The G183 residue is localized at the base of the globular head
of the G protein according to a model structure (Yu M. et al. 1998
Virology 251:227-233), and could be a part of the antibody epitope
that does not overlap with the receptor binding site on G. Another
residue, N570, appears to decrease binding of m102 to G but not the
binding of m101 and the receptor (FIG. 5). This residue could be a
part of the m102 epitope that does not overlap with the epitope of
m101 and the receptor binding site on G.
TABLE-US-00003 TABLE 1 Selection of phage clones with unique
sequences that exhibit significant binding to sG Fab H3 Sequence
A.sub.450 m101 D P G G Y S Y G P Y Y Y Y Y G M D V 1.0 m102 G W G R
E Q L A P H P S Q Y Y Y Y Y Y G M 1.4 D V m103 D S R Y H D A F D I
0.8 m104 E S S W L D A F D I 0.7 m105 V G G I T G T A D A F D I 0.9
m106 D Q L A G Y Y Y D S S G Y H Y Y Y Y G 1.6 M D V m107 D H V H G
P D A F D I 0.6 m108 V G G A F D I 0.5 m109 G W F R D W Y F D L 0.0
m110 E G L P E T D D A F D I 0.0 m111 E G A D Y 0.0 m112 D G A D Y
0.4 m113 Y K L Q S D A F D I 0.1 m114 A G P V G A T T G T F D Y 0.0
m115 G S Q S Y D H Y Y Y Y 0.4 m116 D S A G L G A 0.3 m117 R E S G
P E F F Q H 0.0
Screening of 380 individual phage clones was performed in phage
ELISA with sG as described in Example 1. The sequences of the HC
CDR3s (H3s) of phage-displayed Fabs that exhibited significant
binding to sG in phage ELISA are shown as identified according to
the IMGT database (http://imgt.cines.fr). Soluble Fabs were
expressed, purified, and tested in ELISA for binding to sG. The
solution absorbance at 450 nm (A.sub.450) is shown as a measure of
the strength of binding.
TABLE-US-00004 TABLE 2 V-gene families and number of amino acids
changed compared to the germ line Antibody VH family VL family VH
changes VL changes m101 VH1 Vk1 2 6 m102 VH1 Vk3 5 8 m103 VH3 Vk2 0
0 m104 VH1 Vk2 0 13 m105 VH3 Vk1 0 0 m106 VH1 Vk1 0 0 m107 VH1
V.lamda.1 1 3
Shown are the gene families for the V.sub.H genes, which are IgM
specific, and for the V.sub.L genes, which are from all Ig classes,
and their variations compared to germline sequences.
TABLE-US-00005 TABLE 3 Inhibition of HeV Env-mediated cell fusion
by the selected Fabs Fab HeV NiV m101 +++ + m102 ++ ++ m103 + 0
m104 + + m105 0 + m106 + ++ m107 0 + m108-17 0 0 X5 0 0
Anti-HeV G Fabs were used for inhibition of fusion as described in
Example 1. A summary of four different experiments are shown where
each + is a measure of increased inhibitory activity, and 0 means
no measurable fusion activity compared to the background. Fab X5 is
a control antibody specific for the HIV-1 gp120 (Moulard, M. et al.
2002 Proc Natl Acad Sci USA 99:6913-6918).
TABLE-US-00006 TABLE 4 Neutralization of infectious HeV by IgG1
m101 Antibody Average number concentration Number of foci per of
foci (.mu.g/ml) well in each replica (% neutralization) 25 0 0 0 0
0 (100) 12.5 0 0 0 0 0 (100) 6.2 0 0 1 0 0.25 (99) 3.1 1 0 1 0 0.5
(98) 1.6 1 1 0 0 0.5 (98) 0.8 5 2 2 2 2.75 (91) 0 30 37 34 30 33
(0)
IgG1 m101 was incubated with infectious HeV and the mixture added
to plated Vero cells. After the 30 minute incubation,
antibody-virus mixtures were removed, cells were washed and fresh
EMEM-10 containing fresh antibody was added to the cells and
incubated overnight. The average number of foci in the absence of
antibody was 33. The percentage of neutralization shown below in
parentheses was calculated by subtracting the number of foci in the
wells with antibodies from the number of foci without antibodies
and dividing the resulting number by the number of foci without
antibodies, and multiplying by 100.
TABLE-US-00007 TABLE 5 Binding rate constants and affinities of
selected Fabs Antibody k.sub.a (.times.10.sup.4) M.sup.-1 s.sup.-1
k.sub.d (.times.10.sup.-3) s.sup.-1 K.sub.d, nM m101 13 3.5 28 m102
5.7 0.068 1.2 m103 * * 1800 m104 0.3 1.6 600 m105 7.4 5.1 69 m106
6.2 3.3 54 m107 30 2.2 78
Interaction between various Fabs and sG was analyzed at 25.degree.
C. by surface plasmon resonance technology. Fabs at different
concentrations were injected at flow rate of 30 .mu.l/min, and the
association and dissociation phase data were fitted simultaneously
to a 1:1 Langmuir global model by using the nonlinear data analysis
program BIAevaluation 3.2. Individual association rate constant,
k.sub.a, dissociation rate constant, k.sub.d, and equilibrium
dissociation constant, K.sub.d, were obtained from at least three
separate experiments. The standard deviation was on average about
20%. * denotes that only steady state affinity was calculated due
to fast kinetics.
Discussion
[0088] The major finding of this study is the identification of an
antibody, m101, with exceptional potency against infectious HeV.
Six other antibodies were also identified that are specific for HeV
G and two of them significantly cross-reacted with NiV G. To our
knowledge these antibodies are the first human monoclonal
antibodies (hmAbs) identified against HeV and NiV. Interestingly,
the only monoclonal antibody, Synagis (Pollack, P. and Groothuis,
J. R. 2002 J Infect Chemotheri 8:201-206) (palivizumab, MEDI-493),
approved by FDA for clinical use against a viral disease (Dimitrov,
D. S. 2004 Nat Rev Microbiol 2:109-122), is also specific for a
paramyxovirus, respiratory syncytial virus (RSV); it is a humanized
version of a mouse antibody and is used for prevention of RSV
infections in neonates and immune-compromised individuals. Synagis
inhibits virus entry and cell fusion in vitro very potently, it
appears that its efficacy in vivo is correlated to its potency in
vitro, and it was proposed that its fusion-inhibiting activity
could be a major determinant of its potency in vivo (Johnson, S. et
al. 1999 J Infect Dis 180:35-40). Because RSV and henipaviruses
enter cells by the same pathway, fusion at the cell surface and not
through endocytosis as most other enveloped viruses do (Dimitrov,
D. S. 2004 Nat Rev Microbiol 2:109-122), and are members of the
same virus family, one predicts that m101 might be equally if not
more effective against HeV as palivizumab is against RSV.
[0089] Each of the newly identified Fabs were examined in HeV and
NiV-mediated cell fusion assays to evaluate their potential in
blocking binding and/or the subsequent membrane fusion process. The
Fab m101 demonstrated the most potent cell-fusion inhibitory
activity, and m102 exhibited cross-reactive activity against both
HeV and NiV. The mechanism by which m101 and m102 inhibit HeV
fusion and infection is by blocking the interaction of G with
ephrin-B2, which we recently identified as a functional receptor
for HeV and NiV (Bonaparte et al. 2005 Proc Natl Acad Sci USA
102:10652-10657) (the receptor function of ephrin-B2 for NiV was
also independently identified by using a different approach
(Negrete O. A. et al. 2005 Nature 436:401-405)). However, an
alternative possibility is that these antibodies bind to G and can
also prevent its required interaction with the F glycoprotein to
trigger the fusion process. There was a general correlation between
their inhibitory activity and binding affinity of the Fabs to the G
glycoprotein especially to the native membrane-associated protein
as measured by immunoprecipitation.
[0090] Conversion of Fab m101 to IgG1 led to potent neutralization
of infectious HeV where it neutralized more than 90% at
concentration less than 1 .mu.g/ml. One could speculate that this
may be due to increased stability and avidity of the antibody
and/or its ability to cross-link the oligomeric G glycoprotein on
the surfaces of virus and infected cells. Nonetheless, the extreme
potency of IgG1 m101 in infectious HeV neutralization assays
suggests that it may be important to convert other Fabs to IgG1 for
evaluation as potent neutralizing hmAbs.
[0091] There is considerable amino acid homology between the F and
G Envs of HeV and NiV (Harcourt, B. H. et al. 2000 Virology
271:334-349; Wang, L. et al. 2001 Microbes Infect 3:279-287).
Previous studies have demonstrated that HeV and NiV antisera do
cross neutralize, with each serum being slightly less effective
against the heterotypic virus (Crameri, G. et al. 2002 J Virol
Methods 99:41-51; Tamin, A. 2002 Virology 296:190-200). Further,
the HeV and NiV glycoproteins can functionally complement one
another in mediating membrane fusion with wild-type efficiency
(Bossart, K. N. and Broder, C. C. 2004 Methods Mol Biol
269:309-332; Bossart, K. N. et al. 2002 J Virol 76:11186-11198).
Thus, we anticipated that if antibodies were identified using sG
from HeV some should display cross-reactive binding to shared
epitopes between HeV and NiV. Indeed, two of the seven Fabs were
capable of reacting equally well in immunoprecipitation of
recombinant membrane-associated HeV and NiV G. In addition, m102
was capable of inhibiting HeV and NiV-mediated fusion and may
reflect a conserved epitope between these viruses that could be
important not only for neutralization but also for the entry
mechanism. Upon conversion to an IgG1, m102 might be capable of
potently neutralizing both HeV and NiV.
[0092] Antibody binding competition assays revealed that the panel
of Fabs developed here, comprise two distinct groups and Fabs
within each group possess overlapping epitopes. Analysis of the
anti-G Fabs by Western-blotting revealed no reactivity to G,
indicating that the epitopes recognized by these Fabs are likely
conformation dependent. The most potent neutralizers, m101 and
m102, bound to most of the alanine mutants tested except one which
appears to be located at the base of the globular head of G
according to a model of its structure (Yu M. et al. 1998 Virology
251:227-233). Further studies are needed to precisely localize
their epitopes.
[0093] Taken together, our results demonstrate new
immuno-therapeutics against HeV and NiV. These human antibodies are
also expected to be useful for diagnosis, as research reagents and
serving as the basis for vaccines.
Example 1
Cells and Culture Conditions
[0094] HeLa-USU cells were provided by Anthony Maurelli, Uniformed
Services University (USU). HeLa-ATCC was obtained from the American
Tissue Culture Collection (ATCC #CCL 2). Vero cells were provided
by Alison O'Brien, USU. The human glioblastoma cell line U373-MG
was provided by Adam P. Geballe, Fred Hutchinson Cancer Research
Center (Harrington R. D. 1993 J Virol 67:5939-5947). The Human head
and neck carcinoma PCI 13 cell line was the gift of Ernest Smith,
Vaccinex, Inc. HeLa-USU, HeLa-ATCC, and U373 cells were maintained
in Dulbecco's modified Eagle's medium (Quality Biologicals,
Gaithersburg, Md.) supplemented with 10% cosmic calf serum (CCS)
(HyClone, Logan, Utah), and 2 mM L-glutamine (DMEM-10). PCI 13
cells were maintained in DMEM-10 supplemented with 1 mM HEPES
(Quality Bio.). Vero cells were maintained in Eagle's minimal
essential medium (EMEM) (Quality Bio.) supplemented with 10% cosmic
calf serum (CCS) (HyClone), and 2 mM L-glutamine (EMEM-10). All
cell cultures were maintained at 37.degree. C. in a humidified 5%
CO2 atmosphere.
Alanine G Mutants
[0095] Alanine mutations were made at specific residues in
myc-tagged HeV G using site-directed mutagenesis (Stratagene). All
mutants were sequenced and tested for expression. Plasmids
containing either mutant or wild type HeV Gmyc were transfected
into HeLa USU monolayers using Fugene (Roche), and incubated
overnight. The immunoprecipitation of the mutant G was performed as
described below in the section Immunoprecipitation except that 3.0
.mu.g of either m101 or m102 or 5 .mu.L of rabbit polyclonal
.alpha.-sHeV G sera was incubated with 80 .mu.L lysates overnight
at 4.degree. C., followed by precipitation at room temperature with
100 .mu.L 20% Protein G-Sepharose for 45 minutes.
Selection of G-Specific Phage-Displayed Fabs
[0096] A naive human Fab phage display library (a total of about
10.sup.10 members), constructed from peripheral blood B cells of 10
healthy donors, was used for selection of Fabs against purified,
soluble and oligomeric HeV G glycoprotein (sG) (Bossart, K. N. et
al. 2005 J Virol 79:6690-6702), conjugated to magnetic beads
(Dynabeads M-270 Epoxy, DYNAL Inc., New Hyde Park, N.Y.). Amplified
libraries of 10.sup.12 phage-displayed Fabs were incubated with 5,
3, 3 and 1 .mu.g of sG in 500 .mu.l volume for 2 hours at room
temperature during the 1st, 2nd, 3rd and 4th rounds of biopanning,
respectively. After each round of incubation the beads were washed
5 times for the first round and 15 times for the later rounds with
PBST (PBS containing 0.05% Tween-20) to remove non-specifically
bound phage, the bead-bound phage were mixed with TG1 cells for one
hour at 37.degree. C., the phage was amplified from the infected
cells and used in the next round of biopanning. After the 4th round
of biopanning 380 clones were randomly picked from the infected TG1
cells and each inoculated into 150 .mu.l 2YT medium containing 100
.mu.g/ml carbenicillin and 0.2% glucose in 96-well plates by using
the automated BioRobotics BioPick colony picking system (Genomic
Solutions, Ann Arbor, Mich.). After the bacterial cultures reached
optical density (OD) 0.5 at 600 nm, helper phage M13K07 at 10
M.O.I. and kanamycin at 50 .mu.g/ml final concentration were added
to the medium, and the plates were further incubated at 30.degree.
C. overnight in a shaker at 250 rpm. The phage supernatants were
mixed with 3% nonfat milk in PBS at a 4:1 volume ratio and used for
ELISA to identify clones of phage displaying Fabs with high
sG-binding affinity. The supernatants were incubated for 2 hours at
room temperature with sG protein coated at 50 ng per well in
96-well plates and washed 5 times with PBST. (sG was coated in 50
.mu.l coating buffer (50 mM NaHCO.sub.3 pH 9.6); after overnight
incubation at 40.degree. C. it was blocked with 3% nonfat milk in
PBS and washed 3 times with PBS containing 0.05% Tween-20). Soluble
G-bound phage were detected by using
horseradish-peroxidase-conjugated goat anti-M13 antibody. After
incubation with the antibody, the non-specifically bound antibody
was washed, the TMB substrate was added and the solution absorbance
at 450 nm (A.sub.450) was measured. Clones that bound to sG with
A.sub.450>1.0 were selected for further characterization.
Expression and Purification of Selected Soluble Fabs
[0097] The V.sub.H and V.sub.L of the selected clones were
sequenced, and the Fabs encoded by clones with unique sequences
were expressed and purified as described below. Plasmids extracted
from these clones were used for transformation of HB2151 cells. A
single colony was picked from the plate containing freshly
transformed cells, inoculated into 200 ml 2YT medium containing 100
.mu.g/ml ampicillin and 0.2% glucose, and incubated at 37.degree.
C. with shaking at 250 rpm. When the culture OD at 600 nm reached
0.90, IPTG at 0.5 mM final concentration was added, and the culture
was further incubated overnight at 30.degree. C. Bacterial pellet
was collected after centrifugation at 8000 g for 20 minutes and
resuspended in PBS buffer containing 0.5 mU Polymixin B
(Sigma-Aldrich, St. Louis, Mo.). After 30 min incubation with
rotation at 50 rpm at room temperature, it was centrifuged at 25000
g for 25 min at 4.degree. C., and the supernatant used for Fab
purification with protein G column (Sigma-Aldrich, St. Louis,
Mo.).
Conversion of Fab to IgG1, and IgG1 Expression and Purification
[0098] The Fab heavy and light chain were amplified and re-cloned
in the PDR12 vector (provided by D. Burton, the Scripps Research
Institute, La Jolla, Calif.) for whole IgG1 expression. The
resulting construct was transfected and the IgG1 expressed using
the FreeStyle.TM. 293 Expression Kit following the protocol from
the manufacturer (Invitrogen, Carlsbad, Calif.). The IgG1 was
purified from the culture medium with protein G column
(Sigma-Aldrich, St. Louis, Mo.).
Affinity Determination by Surface Plasmon Resonance
[0099] Interactions between various Fabs and G were analyzed by
surface plasmon resonance technology using a BIACORE 1000
instrument (Biacore, Pharmacia, Piscataway, N.J.). sG was
covalently immobilized onto a sensor chip (CMS) using carbodiimide
coupling chemistry. A control reference surface was prepared for
nonspecific binding and refractive index changes. For analysis of
the kinetics of interactions, varying concentrations of Fabs (300,
100, 33, 11, and 3.7 nM) were injected at flow rate of 30 .mu.l/min
using running buffer containing 150 mM NaCl, 3 mM EDTA, and 0.005%
P-20 (pH 7.4). The association and dissociation phase data were
fitted simultaneously to a 1:1 Langmuir global model by using the
nonlinear data analysis program BIAevaluation 3.2. All the
experiments were performed at 25.degree. C.
Competition ELISA
[0100] The Fabs m101, m102, m103, m106 and m107 were coated at 150,
50, 300, 300 and 100 ng per well, respectively in 50 .mu.l coating
buffer as described above for sG, blocked with nonfat milk and
washed. C-Myc tagged sG mixed with each of the Fabs in blocking
buffer at final concentration 5 .mu.g/ml and 20 .mu.g/ml,
respectively, were added to each of the Fab coated wells; sG (5
.mu.g/ml) without antibody was added to each of the coated Fabs as
a positive control. Bound c-myc tagged sG protein was detected by
an HRP conjugated anti-c-Myc antibody (Roche Diagnostics
Corporation, Indianapolis, Ind.); the TMB substrate (Sigma-Aldrich,
St. Louis, Mo.) was added and A.sub.450 measured.
Immunoprecipitation
[0101] HeLa-USU monolayers were infected with wild-type vaccinia
(WR) or recombinant vaccinia expressing myc-tagged HeV G or NiV G
at an MOI of 10 for 6 hours, then washed twice, and incubated
overnight in methionine and cysteine-free essential medium plus
2.5% dialyzed fetal calf serum (Invitrogen) and 100 .mu.Ci of
[.sup.35S] ProMix/ml (Amersham Pharmacia Biotech, Piscataway,
N.J.). Cells were lysed in 100 mM Tris-HCl (pH 8.0), 100 mM NaCl,
and 1% Triton X-100. Lysates were incubated with each Fab at a
concentration of 1 ug per 100 ul of lysate for at least one hour at
4.degree. C., followed by precipitation at room temperature with
100 ul 20% Protein G-Sepharose (Amersham) for 45 minutes. Anti-myc
antibody 9E10 (Roche Molecular Biochemicals) was used at a
concentration of 2 ug per 100 ul of lysate. Samples were washed
twice with lysis buffer followed by one wash with DOC buffer
containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1% sodium
deoxycholate, and 0.1% SDS. Samples were boiled in
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
with 2-mercaptoethanol and analyzed by SDS-PAGE and
autoradiography.
Cell-Fusion Assays
[0102] Fusion between HeV and NiV F and G envelope
glycoprotein-expressing (effector cells) and target cells was
measured by two assays: first, a reporter gene assay in which the
cytoplasm of one cell population contained vaccinia virus-encoded
T7 RNA polymerase and the cytoplasm of the other contained the E.
coli lacZ gene linked to the T7 promoter; .beta.-galactosidase
(.beta.-Gal) is synthesized only in fused cells (Bossart, K. N. and
Broder, C. C. 2004 Methods Mol Biol 269:309-332; Nussbaum, O. et
al. 1994 J Virol 68:5411-5422), and second, a syncitia assay.
Typically, the expression of HeV and NiV F and G is performed in a
HeV and NiV fusion and infection negative HeLa cell line derivative
(HeLa-USU). Cytogenetic analysis has confirmed that the HeLa-USU
cell line resistant to NiV and HeV mediated membrane fusion and
live virus infection is derived from the ATCC(CCL-2) HeLa cell
line. Vaccinia virus-encoded proteins (Bossart, K. N. et al. 2001
Virology 290:121-135) were produced by infecting cells at a MOI of
10 and incubating infected cells at 31.degree. C. overnight.
Cell-fusion reactions were conducted with the various cell mixtures
in 96-well plates at 37.degree. C. Typically, the ratio of envelope
glycoprotein-expressing cells to target cells was 1:1
(2.times.10.sup.5 total cells per well, 0.2-ml total volume).
Cytosine arabinoside (40 .mu.g/ml) was added to the fusion reaction
mixture to reduce nonspecific .beta.-Gal production. For
quantitative analyses, Nonidet P-40 was added (0.5% final) at 2.5 h
and aliquots of the lysates were assayed for .beta.-Gal at ambient
temperature with the substrate chlorophenol red-D-galactopyranoside
(CPRG; Roche Diagnostics Corp., Indianapolis, Ind.). For inhibition
by antibodies, serial antibody dilutions were made and added to
effector cell populations 30 min prior to the addition of target
cell populations. All assays were performed in duplicate and fusion
data were calculated and expressed as rates of .beta.-Gal activity
(change in OD at 570 nm per minute.times.1,000) (Nussbaum, O. et
al. 1994 J Virol 68:5411-5422). They were normalized with respect
to cell fusion in the absence of antibodies, and plotted as
function of the antibody concentration.
[0103] The syncytia assay was performed in 48-well plates. Target
PCI-13 cells were plated to reach 80% confluency at the time of the
experiment. Effector cells, HeLa USU, which are non-permissive to
HeV Env and NiV Env mediated fusion, were infected with recombinant
vaccinia virus to express HeV G and F proteins. Three wells of a
six-well plate 80% confluent HeLa-USU were incubated with both
recombinant vaccinia viruses, encoding HeV G and HeV F, MOI of 10
for each virus at 37.degree. C. for 3 h in DMEM-10 containing 2.5%
cosmic calf serum, 1 ml per well, then washed once and dissociated
from the plates by using 0.5 ml per well enzyme-free PBS-based cell
dissociation buffer (Invitrogen Corp., Carlsbad, Calif.). The cells
were gathered into 50 ml sterile centrifuge tube (Corning Inc.,
Corning, N.Y.) and 20 ml DMEM-10 was added. The suspension was
incubated 16 hours at 31.degree. C. in a humidified 5% CO.sub.2
atmosphere. Before the experiment, the cells were centrifuged at
1200 rpm for 5 min and the pellet was re-suspended in DMEM-10. The
cells were counted, centrifuged again and re-suspended at a
concentration of 2.times.10.sup.6 cells/ml. Cytosine arabinoside
was added to a concentration of 80 .mu.g/ml.
One-hundred-microliters of this cell suspension was mixed with the
same amount DMEM-10 containing the antibody and incubated for 20
min at room temperature. The mixtures were added to the freshly
washed (with DMEM-10) PCI-13 target cells in the 48-well plate and
incubated for 3 h at 37.degree. C. in a humidified 5% CO.sub.2
atmosphere. Photographs were taken by using phase contrast mode of
an Olympus IX81 microscope with a 10.times. objective lens then
electronically amplified whenever needed.
HeV and NiV Neutralization Assays
[0104] All live virus experiments were conducted under strict
bio-containment procedures in a BSL-4 laboratory. 2.times.10.sup.4
Vero cells were added to wells in 150 .mu.l EMEM-10 in a 96-well
plate and incubated at 37.degree. C. overnight in a humidified 5%
CO.sub.2 atmosphere. Antibodies were diluted in EMEM-10 by doubling
dilution and an equal volume of either HeV or NiV was added to each
dilution and incubated at 37.degree. C. for 30 min. The titer of
HeV was 1.0.times.10.sup.8 TCID.sub.50/ml and NiV was
3.0.times.10.sup.7 TCID.sub.50/ml. Virus dilutions were done in
EMEM-10 and chosen to generate 50 plaques following adsorption of
virus for 30 min at 37.degree. C. to Vero cell monolayers
(1.5.times.10.sup.3 TCID.sub.50/ml for HeV and 7.5.times.10.sup.2
TCID.sub.50/ml for NiV). Antibody-virus mixtures were added to Vero
cell monolayers in quadruplicate and incubated for 30 minutes at
37.degree. C. in a humidified 5% CO.sub.2 atmosphere. After 30
minute incubation, antibody-virus mixtures were removed and cells
were washed 3 times with Ca.sup.++/Mg.sup.++-free PBS. Two
different variations of this assay were conducted. In the first,
EMEM-10 was added to Vero cells after washing and incubated
overnight. In the second, the same antibody dilution as both the
pre-incubation and virus incubation was added to the respective
wells and incubated overnight. For both assays, the culture medium
was discarded the next day, and plates immersed in ice-cold
absolute methanol for 20 min prior to air-drying outside the
biohazard level 4 facility. Fixed chamber slides were either stored
overnight at 4.degree. C. or immunolabeled immediately with
anti-phosphoprotein (P) monospecific antiserum (Michalski, W. P. et
al. 2000 Virus Res 69:83-93). Wells were washed in 0.01 M PBS, pH
7.2 containing 1% BSA for 5 min. 40 .mu.l of anti-P antiserum
(1:200 in PBS-BSA) was applied to each well and incubated at
37.degree. C. for 30 min. Slides were rinsed with PBS containing
0.05% Tween 20 (PBS-T) and washed for 5 min in PBS-BSA. 40 .mu.l of
FITC labeled goat anti-rabbit antiserum (ICN Pharmaceuticals, Costa
Mesa, USA) diluted 1:200 in PBS-BSA containing DAPI (10 .mu.g/ml)
was then applied to each well and incubated at 37.degree. C. for 30
min. Wells were rinsed again with PBS containing 0.05% Tween 20
(PBS-T) and washed for 5 min in PBS-BSA. Wells were overlaid with
100 .mu.l Glycerol/PBS (9:1) containing DABCO (25 .mu.g/ml) and
stored in the dark prior to imaging. FITC immunofluorescence was
visualized using an Olympus IX71 inverted microscope (Olympus
Australia, Mt. Waverley, Australia). Percentage neutralization at a
given antibody concentration was calculated as the ratio of the
average number of foci per well due to cytopathic effect (CPE) to
the same number for the positive control multiplied by 100.
[0105] Neutralization of HeV and NiV by Fabs was performed as
follows. Fabs were diluted in EMEM-10 by doubling dilution and an
equal volume of EMEM-10 containing 200 TCID.sub.50 of either HeV or
NiV was added to each dilution and incubated at 37.degree. C. for
30 min. The titer of HeV was 1.0.times.10.sup.8 TCID.sub.50/ml and
NiV was 3.0.times.10.sup.7 TCID.sub.50/ml. 2.times.10.sup.4 Vero
cells were added to each Fab-virus mixture in six replicate wells
and incubated for 5 days. Fab neutralization was determined by the
level of cytopathic effect (CPE) in replicate wells at each Fab
concentration.
Example 2
Affinity Maturation of m102
[0106] The original human Fab phage display library from which the
antibodies m101-m107 were identified was used as the source of the
VL repertoire in the shuffled library. The phagemid preparation
from the original library was first digested with Nco I and Spe I
and followed by electrophoresis on an agarose gel to separate the
VH and CH1 gene fragments from the antibody light chain-containing
backbone vector to delete the entire VH repertoire. The gene
encoding the VH domain of clone m102 was amplified by error-prone
PCR kit from Stratagene to introduce random mutations and then
fused with CH1 gene fragment by SOE PCR. The fused fragment was
digested with NcoI and Spe I and purified from gel and was then
ligated into the purified backbone vector to create the VL-shuffled
Fab repertoire. E. coli TG1 cells were transformed with the
ligation mixtures via electroporation. The transformed TG1 cells
were plated on 2YT agar plates containing 100 .mu.g/ml ampicillin
and 2% glucose. After incubation overnight at 37.degree. C., all of
the colonies grown on the plates were scraped into 5 ml of 2YTAG
medium, mixed with 1.2 ml of 50% glycerol (final concentration
10%), aliquoted, and stored at -70.degree. C. as the library
stock.
[0107] The library stock (100 ul) was grown to log phase in 20 ml
of 2YT medium, rescued with M13K07 helper phage, and amplified
overnight in 2YT medium (2YT containing 100 .mu.g/ml of ampicillin
and 50 .mu.g/ml of kanamycin) at 30.degree. C. The phage
preparation was precipitated in 4% PEG, 0.5 M NaCl, resuspended in
1 ml of PBS as phage library stock. Two rounds of biopanning were
performed on Hendra G conjugated magnetic beads as described in the
original library panning. 9 clones were identified as affinity
maturated antibodies and m102.4 (produced by ATCC Deposit Number
PTA-13287) was selected for further characterization.
[0108] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
figures, tables, and appendices, as well as patents, applications,
and publications, referred to above, are hereby incorporated by
reference.
Sequence CWU 1
1
4331127PRTHomo sapiens 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Pro Gly Gly Tyr Ser Tyr Gly Pro Tyr Tyr Tyr Tyr Tyr
100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 125 225PRTHomo sapiens 2Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser 20 25 38PRTHomo sapiens 3Gly Gly Thr Phe Ser Ser Tyr
Ala1 5 418PRTHomo sapiens 4Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met Gly1 5 10 15 Gly Ile57PRTHomo sapiens 5Ile Pro
Ile Phe Gly Thr Ala1 5 638PRTHomo sapiens 6Asn Tyr Ala Gln Lys Phe
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu1 5 10 15 Ser Thr Ser Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Gly Asp 20 25 30 Thr Ala
Val Tyr Tyr Cys 35 720PRTHomo sapiens 7Ala Arg Asp Pro Gly Gly Tyr
Ser Tyr Gly Pro Tyr Tyr Tyr Tyr Tyr1 5 10 15 Gly Met Asp Val 20
811PRTHomo sapiens 8Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5
10 9111PRTHomo sapiens 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Gly Pro Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Arg Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Phe Pro Phe 85 90
95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100
105 110 1026PRTHomo sapiens 10Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser 20 25 116PRTHomo sapiens 11Gln Gly Ile Gly Pro Trp1 5
1217PRTHomo sapiens 12Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Phe Leu Ile1 5 10 15 Tyr133PRTHomo sapiens 13Arg Ala Ser1
1436PRTHomo sapiens 14Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 159PRTHomo
sapiens 15Gln Gln Ala His Ser Phe Pro Phe Thr1 5 1614PRTHomo
sapiens 16Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala1
5 10 17132PRTHomo sapiens 17Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser
Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile
Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro His Pro Ser Gln
Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly
Thr Thr Val 115 120 125 Thr Val Ser Ser 130 1825PRTHomo sapiens
18Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser 20 25 198PRTHomo
sapiens 19Gly Gly Thr Phe Ser Asn Tyr Ala1 5 2018PRTHomo sapiens
20Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly1
5 10 15 Gly Ile217PRTHomo sapiens 21Ile Pro Ile Leu Gly Ile Ala1 5
2238PRTHomo sapiens 22Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35
2325PRTHomo sapiens 23Ala Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro
His Pro Ser Gln Tyr1 5 10 15 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 20
25 2411PRTHomo sapiens 24Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser1 5 10 25111PRTHomo sapiens 25Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Ile Thr Asn Gly 20 25 30 Arg Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr
Gly Val Ser Ser Arg Ala Ser Gly Ile Pro Glu Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
Ser Val 85 90 95 Leu Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
Thr Val Ala 100 105 110 2626PRTHomo sapiens 26Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser 20 25 277PRTHomo sapiens 27Gln Ser Ile
Thr Asn Gly Arg1 5 2817PRTHomo sapiens 28Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile1 5 10 15 Tyr293PRTHomo
sapiens 29Gly Val Ser1 3036PRTHomo sapiens 30Ser Arg Ala Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 20 25 30 Val
Tyr Tyr Cys 35 318PRTHomo sapiens 31Gln Gln Tyr Gly Ser Ser Val
Leu1 5 3214PRTHomo sapiens 32Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys Arg Thr Val Ala1 5 10 33118PRTHomo sapiens 33Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Arg Tyr His Asp Ala Phe
Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115
3425PRTHomo sapiens 34Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Ile Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20
25 358PRTHomo sapiens 35Gly Phe Thr Val Ser Ser Asn Tyr1 5
3618PRTHomo sapiens 36Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser1 5 10 15 Val Ile376PRTHomo sapiens 37Tyr Ser
Gly Gly Ser Thr1 5 3838PRTHomo sapiens 38Tyr Tyr Ala Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn1 5 10 15 Ser Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala
Val Tyr Tyr Cys 35 3912PRTHomo sapiens 39Ala Arg Asp Ser Arg Tyr
His Asp Ala Phe Asp Ile1 5 10 4011PRTHomo sapiens 40Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser1 5 10 41116PRTHomo sapiens 41Asp Val
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20
25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Leu Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala 115
4226PRTHomo sapiens 42Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser 20 25 4311PRTHomo sapiens 43Gln Ser Leu Leu His Ser Asn Gly Tyr
Asn Tyr1 5 10 4417PRTHomo sapiens 44Leu Asp Trp Tyr Leu Gln Lys Pro
Gly Gln Ser Pro Gln Leu Leu Ile1 5 10 15 Tyr453PRTHomo sapiens
45Leu Gly Ser1 4636PRTHomo sapiens 46Asn Arg Ala Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly 20 25 30 Val Tyr Tyr
Cys 35 479PRTHomo sapiens 47Met Gln Ala Leu Gln Thr Leu Tyr Thr1 5
4814PRTHomo sapiens 48Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala1 5 10 49119PRTHomo sapiens 49Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Ser Trp Leu Asp Ala Phe Asp
Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115
5025PRTHomo sapiens 50Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20
25 518PRTHomo sapiens 51Gly Gly Thr Phe Ser Ser Tyr Ala1 5
5219PRTHomo sapiens 52Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile Ile536PRTHomo sapiens 53Pro
Ile Phe Gly Thr Ala1 5 5438PRTHomo sapiens 54Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 5512PRTHomo sapiens 55Ala Arg Glu Ser Ser
Trp Leu Asp Ala Phe Asp Ile1 5 10 5611PRTHomo sapiens 56Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser1 5 10 57116PRTHomo sapiens 57Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Ala Gly1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly His Ile Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Met Ala Ser Asn Arg Ala
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Arg Ile65 70 75 80 Asn Arg Val Glu Thr Glu Asp Val
Gly Ile Tyr Tyr Cys Met Gln Ser 85 90 95 Leu His Thr Thr Arg Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala
115 5826PRTHomo sapiens 58Asp Val Val Met Thr Gln Ser Pro Leu Ser
Leu Ser Val Thr Ala Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser 20 25 5911PRTHomo sapiens 59Gln Ser Leu Leu His Ser Asn Gly
His Ile Tyr1 5 10 6017PRTHomo sapiens 60Leu Asp Trp Tyr Leu Gln Lys
Pro Gly Gln Ser Pro Gln Leu Leu Ile1 5 10 15 Tyr613PRTHomo sapiens
61Met Ala Ser1 6236PRTHomo sapiens 62Asn Arg Ala Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu
Arg Ile Asn Arg Val Glu Thr Glu Asp Val Gly 20 25 30 Ile Tyr Tyr
Cys 35 639PRTHomo sapiens 63Met Gln Ser Leu His Thr Thr Arg Thr1 5
6414PRTHomo sapiens 64Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala1 5 10 65122PRTHomo sapiens 65Gln Val Gln Leu Val Gln
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Val Gly Gly Ile Thr Gly Thr Ala Asp
Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser
Ser 115 120 6624PRTHomo sapiens 66Gln Val Gln Leu Val Gln Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala 20 679PRTHomo sapiens 67Ser Gly Phe Thr Phe Ser Ser Tyr
Ala1 5 6818PRTHomo sapiens 68Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ala1 5 10 15 Val Ile697PRTHomo sapiens
69Ser Tyr Asp Gly Ser Asn Lys1 5 7040PRTHomo sapiens 70Tyr Tyr Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn1 5 10 15 Ser
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25
30 Thr Ala Val Tyr Tyr Cys Ala Arg 35 40 7113PRTHomo sapiens 71Val
Gly Gly Ile Thr Gly Thr Ala Asp Ala Phe Asp
Ile1 5 10 7211PRTHomo sapiens 72Trp Gly Gln Gly Thr Met Val Thr Val
Ser Ser1 5 10 73116PRTHomo sapiens 73Asp Val Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Asn
Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln Gly 85 90 95 Thr His Trp Pro Phe Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Lys 100 105 110 Arg Thr Val Ala 115 7426PRTHomo
sapiens 74Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Leu Gly1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser 20 25
7511PRTHomo sapiens 75Gln Ser Leu Val Tyr Ser Asp Gly Asn Thr Tyr1
5 10 7617PRTHomo sapiens 76Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln
Ser Pro Arg Arg Leu Ile1 5 10 15 Tyr773PRTHomo sapiens 77Lys Val
Ser1 7836PRTHomo sapiens 78Asn Arg Asp Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Val Gly 20 25 30 Val Tyr Tyr Cys 35
799PRTHomo sapiens 79Met Gln Gly Thr His Trp Pro Phe Thr1 5
8014PRTHomo sapiens 80Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
Thr Val Ala1 5 10 81131PRTHomo sapiens 81Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Leu Ala Gly Tyr Tyr Tyr Asp
Ser Ser Gly Tyr His 100 105 110 Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr 115 120 125 Val Ser Ser 130 8225PRTHomo
sapiens 82Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25
838PRTHomo sapiens 83Gly Gly Thr Phe Ser Ser Tyr Ala1 5 8418PRTHomo
sapiens 84Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met Gly1 5 10 15 Gly Ile857PRTHomo sapiens 85Ile Pro Ile Phe Gly
Thr Ala1 5 8638PRTHomo sapiens 86Asn Tyr Ala Gln Lys Phe Gln Gly
Arg Val Thr Ile Thr Ala Asp Lys1 5 10 15 Ser Thr Ser Thr Ala Tyr
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr
Tyr Cys 35 8724PRTHomo sapiens 87Ala Arg Asp Gln Leu Ala Gly Tyr
Tyr Tyr Asp Ser Ser Gly Tyr His1 5 10 15 Tyr Tyr Tyr Tyr Gly Met
Asp Val 20 8811PRTHomo sapiens 88Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser1 5 10 89111PRTHomo sapiens 89Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser
Thr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 9026PRTHomo sapiens 90Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser 20 25 916PRTHomo sapiens 91Gln Ser
Ile Ser Ser Tyr1 5 9217PRTHomo sapiens 92Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile1 5 10 15 Tyr933PRTHomo
sapiens 93Ala Ala Ser1 9436PRTHomo sapiens 94Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr
Tyr Tyr Cys 35 959PRTHomo sapiens 95Gln Gln Ser Tyr Ser Thr Pro Ile
Thr1 5 9614PRTHomo sapiens 96Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys Arg Thr Val Ala1 5 10 97120PRTHomo sapiens 97Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Val His Gly Pro Asp
Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser
Ser 115 120 9824PRTHomo sapiens 98Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala 20 999PRTHomo sapiens 99Ser Gly Tyr Thr Phe Thr Gly Tyr
Tyr1 5 10017PRTHomo sapiens 100Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met Gly1 5 10 15 Ile1018PRTHomo sapiens 101Ile
Asn Pro Ser Gly Gly Ser Thr1 5 10238PRTHomo sapiens 102Ser Tyr Ala
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr1 5 10 15 Ser
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25
30 Thr Ala Val Tyr Tyr Cys 35 10313PRTHomo sapiens 103Ala Arg Asp
His Val His Gly Pro Asp Ala Phe Asp Ile1 5 10 10411PRTHomo sapiens
104Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser1 5 10 105114PRTHomo
sapiens 105Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro
Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80 Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 His Val
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110
Lys Ala10625PRTHomo sapiens 106Ser Tyr Glu Leu Thr Gln Pro Pro Ser
Ala Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Ser
Gly Ser 20 25 1078PRTHomo sapiens 107Ser Ser Asn Ile Gly Ser Asn
Thr1 5 10817PRTHomo sapiens 108Val Asn Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu Ile1 5 10 15 Tyr1093PRTHomo sapiens 109Arg
Asn Asn1 11036PRTHomo sapiens 110Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser Gly Ser Lys Ser Gly1 5 10 15 Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35
11110PRTHomo sapiens 111Ala Ala Trp Asp Asp Ser Leu His Val Val1 5
10 11215PRTHomo sapiens 112Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln Pro Lys Ala1 5 10 15 113116PRTHomo sapiens 113Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Gly Gly Ala Phe Asp
Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115
11425PRTHomo sapiens 114Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20
25 1158PRTHomo sapiens 115Gly Phe Thr Phe Ser Asp Tyr Tyr1 5
11617PRTHomo sapiens 116Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser1 5 10 15 Tyr1178PRTHomo sapiens 117Ile Ser Ser
Ser Gly Ser Thr Ile1 5 11838PRTHomo sapiens 118Tyr Tyr Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn1 5 10 15 Ala Lys Asn
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 1199PRTHomo sapiens 119Ala Arg Val Gly Gly
Ala Phe Asp Ile1 5 12011PRTHomo sapiens 120Trp Gly Gln Gly Thr Met
Val Thr Val Ser Ser1 5 10 121112PRTHomo sapiens 121Asn Phe Met Leu
Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys1 5 10 15 Thr Val
Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30
Tyr Val Gln Trp Tyr Arg Gln Ser Pro Gly Ser Ala Pro Thr Thr Val 35
40 45 Ile Tyr Glu Gly Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe
Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr
Ile Ser Gly65 70 75 80 Leu Glu Thr Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Ala 85 90 95 Thr Asn His Gln Val Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 110 12226PRTHomo sapiens 122Asn
Phe Met Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys1 5 10
15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser 20 25 1237PRTHomo
sapiens 123Gly Ser Ile Ala Ser Asn Tyr1 5 12417PRTHomo sapiens
124Val Gln Trp Tyr Arg Gln Ser Pro Gly Ser Ala Pro Thr Thr Val Ile1
5 10 15 Tyr1253PRTHomo sapiens 125Glu Gly Tyr1 12638PRTHomo sapiens
126Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser1
5 10 15 Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu Glu Thr Glu
Asp 20 25 30 Glu Ala Asp Tyr Tyr Cys 35 12711PRTHomo sapiens 127Gln
Ser Tyr Asp Ala Thr Asn His Gln Val Val1 5 10 12810PRTHomo sapiens
128Phe Gly Gly Gly Thr Lys Leu Thr Val Leu1 5 10 129118PRTHomo
sapiens 129Gln Met Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser
Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80 Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly
Trp Phe Arg Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110
Leu Val Thr Val Ser Ser 115 13025PRTHomo sapiens 130Gln Met Gln Leu
Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15 Thr Leu
Ser Leu Thr Cys Ala Val Tyr 20 25 1318PRTHomo sapiens 131Gly Gly
Ser Phe Ser Gly Tyr Tyr1 5 13217PRTHomo sapiens 132Trp Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly1 5 10 15
Glu1337PRTHomo sapiens 133Ile Asn His Ser Gly Ser Thr1 5
13438PRTHomo sapiens 134Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr
Ile Ser Val Asp Thr1 5 10 15 Ser Lys Asn Gln Phe Ser Leu Lys Leu
Ser Ser Val Thr Ala Ala Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35
13512PRTHomo sapiens 135Ala Arg Gly Trp Phe Arg Asp Trp Tyr Phe Asp
Leu1 5 10 13611PRTHomo sapiens 136Trp Gly Arg Gly Thr Leu Val Thr
Val Ser Ser1 5 10 137108PRTHomo sapiens 137Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp 20 25 30 Leu Gly
Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Ser Ala Thr Tyr Phe Cys Leu Gln Asp Tyr Gln
Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 13826PRTHomo sapiens 138Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser 20 25 1396PRTHomo sapiens 139Gln Asp Ile Arg Asn
Asp1 5 14017PRTHomo sapiens 140Leu Gly Trp Tyr Gln Gln Arg Pro Gly
Lys Ala Pro Lys Leu Leu Ile1 5 10 15 Tyr1413PRTHomo sapiens 141Ala
Ala Ser1 14236PRTHomo sapiens 142Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Ser Ala 20 25 30 Thr Tyr Phe Cys 35
1439PRTHomo sapiens 143Leu Gln Asp Tyr Gln Tyr Pro Trp Thr1 5
14411PRTHomo sapiens 144Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg1 5 10 145121PRTHomo sapiens 145Glu Val Gln
Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25
30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Glu Gly Leu Pro Glu Thr
Asp Asp Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 14625PRTHomo sapiens 146Glu Val Gln Leu Val Gln
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser 20 25 1478PRTHomo sapiens 147Gly Phe Thr Phe
Asp Asp Tyr Ala1 5 14817PRTHomo sapiens 148Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser1 5 10 15 Ala1498PRTHomo
sapiens 149Ile Ser Gly Ser Gly Gly Ser Thr1 5 15038PRTHomo sapiens
150Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn1
5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 15114PRTHomo sapiens 151Ala
Ser Glu Gly Leu Pro Glu Thr Asp Asp Ala Phe Asp Ile1 5 10
15211PRTHomo sapiens 152Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser1 5 10 153113PRTHomo sapiens 153Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asp Gly Tyr Asn
Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Leu Gly Ser Arg Arg Ala Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65
70 75 80 Asn Thr Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
Gln Gly 85 90 95 Val Glu Ile Pro Phe Thr Phe Gly Pro Gly Thr Lys
Val Glu Ile Lys 100 105 110 Arg15426PRTHomo sapiens 154Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser 20 25 15511PRTHomo sapiens
155Gln Ser Leu Leu Tyr Ser Asp Gly Tyr Asn Tyr1 5 10 15617PRTHomo
sapiens 156Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu
Leu Ile1 5 10 15 Tyr1573PRTHomo sapiens 157Leu Gly Ser1
15836PRTHomo sapiens 158Arg Arg Ala Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Asn Thr
Val Glu Ala Glu Asp Val Gly 20 25 30 Val Tyr Tyr Cys 35 1599PRTHomo
sapiens 159Met Gln Gly Val Glu Ile Pro Phe Thr1 5 16011PRTHomo
sapiens 160Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg1 5 10
161114PRTHomo sapiens 161Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Leu Val Asp
Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Thr Glu Gly Ala Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val 100 105 110 Ser Ser16225PRTHomo sapiens 162Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Thr Val Lys
Ile Ser Cys Lys Val Ser 20 25 1638PRTHomo sapiens 163Gly Tyr Thr
Phe Thr Asp Tyr Tyr1 5 16417PRTHomo sapiens 164Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly1 5 10 15 Leu1658PRTHomo
sapiens 165Val Asp Pro Glu Asp Gly Glu Thr1 5 16638PRTHomo sapiens
166Ile Tyr Ala Glu Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr1
5 10 15 Ser Thr Asp Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 1677PRTHomo sapiens 167Ala
Thr Glu Gly Ala Asp Tyr1 5 16811PRTHomo sapiens 168Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser1 5 10 169114PRTHomo sapiens 169Asp Val
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ala Leu Gly1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln
Ser 35 40 45 Pro Arg Arg Leu Leu Tyr Lys Val Ser Asn Arg Glu Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp
Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Ile Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Pro Ile Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg17026PRTHomo
sapiens 170Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ala
Leu Gly1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser 20 25
17111PRTHomo sapiens 171Gln Ser Leu Val His Ser Asp Gly Asn Thr
Tyr1 5 10 17217PRTHomo sapiens 172Leu Asn Trp Phe Gln Gln Arg Pro
Gly Gln Ser Pro Arg Arg Leu Leu1 5 10 15 Tyr1733PRTHomo sapiens
173Lys Val Ser1 17436PRTHomo sapiens 174Asn Arg Glu Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Ser Asp Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly 20 25 30 Ile Tyr Tyr
Cys 35 17510PRTHomo sapiens 175Met Gln Gly Thr His Trp Pro Pro Ile
Thr1 5 10 17611PRTHomo sapiens 176Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys Arg1 5 10 177114PRTHomo sapiens 177Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Thr Val Lys Ile
Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met
His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45
Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Thr Asp Gly Ala Asp Tyr Trp Asp Gln Gly
Thr Leu Gly Thr Val 100 105 110 Ser Thr17825PRTHomo sapiens 178Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15 Thr Val Lys Ile Ser Cys Lys Val Ser 20 25 1798PRTHomo sapiens
179Gly Tyr Thr Phe Thr Asp Tyr Tyr1 5 18018PRTHomo sapiens 180Met
His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly1 5 10
15 Leu Val1818PRTHomo sapiens 181Asp Pro Glu Asp Gly Glu Thr Ile1 5
18237PRTHomo sapiens 182Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser1 5 10 15 Thr Asn Thr Ala Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr 20 25 30 Ala Val Tyr Tyr Cys 35
1837PRTHomo sapiens 183Ala Thr Asp Gly Ala Asp Tyr1 5 18411PRTHomo
sapiens 184Trp Asp Gln Gly Thr Leu Gly Thr Val Ser Thr1 5 10
185111PRTHomo sapiens 185Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Ser Ser Ser Asn Ile Gly Gly Asp 20 25 30 Ser Asp Val His Trp Tyr
Gln Gln Leu Pro Gly Ser Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly
Asn Arg Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Val Ile Gly Val65 70 75 80
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gln Cys Tyr Asp Ser Ser 85
90 95 Leu Asn Gly Tyr Val Phe Gly Pro Gly Thr Lys Val Ile Val Leu
100 105 110 18626PRTHomo sapiens 186Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15 Thr Val Thr Ile Ser Cys
Thr Gly Ser Ser 20 25 1878PRTHomo sapiens 187Ser Asn Ile Gly Gly
Asp Ser Asp1 5 18817PRTHomo sapiens 188Val His Trp Tyr Gln Gln Leu
Pro Gly Ser Ala Pro Lys Leu Leu Ile1 5 10 15 Tyr1893PRTHomo sapiens
189Gly Asn Arg1 19036PRTHomo sapiens 190Asn Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Arg Ser Gly1 5 10 15 Thr Ser Ala Ser Leu
Ala Val Ile Gly Val Gln Ala Asp Asp Glu Ala 20 25 30 Asp Tyr Tyr
Cys 35 19111PRTHomo sapiens 191Gln Cys Tyr Asp Ser Ser Leu Asn Gly
Tyr Val1 5 10 19210PRTHomo sapiens 192Phe Gly Pro Gly Thr Lys Val
Ile Val Leu1 5 10 193119PRTHomo sapiens 193Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Thr Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Leu 50
55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Asn Tyr Lys Leu Gln Ser Asp Ala Phe Asp
Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115
19425PRTHomo sapiens 194Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20
25 1956PRTHomo sapiens 195Gly Gly Thr Phe Ser Ser1 5 19620PRTHomo
sapiens 196Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp1 5 10 15 Met Gly Trp Thr 20 1977PRTHomo sapiens 197Asn Pro
Asn Ser Gly Gly Thr1 5 19838PRTHomo sapiens 198Asn Tyr Ala Gln Lys
Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 19912PRTHomo sapiens 199Ala Asn Tyr Lys Leu
Gln Ser Asp Ala Phe Asp Ile1 5 10 20011PRTHomo sapiens 200Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser1 5 10 201108PRTHomo sapiens
201Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn
Phe 20 25 30 Leu Val Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys
Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln His Tyr Lys Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg 100 105 20226PRTHomo sapiens 202Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 2037PRTHomo sapiens
203Gln Asp Ile Gly Asn Phe Leu1 5 20416PRTHomo sapiens 204Val Trp
Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr1 5 10 15
2053PRTHomo sapiens 205Ala Ala Ser1 20636PRTHomo sapiens 206Arg Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20
25 30 Thr Tyr Tyr Cys 35 2079PRTHomo sapiens 207Gln His Tyr Lys Ser
Tyr Pro Leu Thr1 5 20811PRTHomo sapiens 208Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg1 5 10 209122PRTHomo sapiens 209Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Pro Val Gly Ala Thr
Thr Gly Thr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 21025PRTHomo sapiens 210Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser 20 25 21110PRTHomo sapiens 211Gly Gly Thr Phe
Ser Ser Tyr Ala Ile Ser1 5 10 21216PRTHomo sapiens 212Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile1 5 10 15
2137PRTHomo sapiens 213Ile Pro Ile Phe Gly Thr Ala1 5 21438PRTHomo
sapiens 214Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala
Asp Glu1 5 10 15 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 21515PRTHomo
sapiens 215Ala Arg Ala Gly Pro Val Gly Ala Thr Thr Gly Thr Phe Asp
Tyr1 5 10 15 21611PRTHomo sapiens 216Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser1 5 10 217108PRTHomo sapiens 217Asp Ile Val Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Ser Ser Phe 85 90 95 Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Lys Arg 100 105 21826PRTHomo sapiens 218Asp Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25 2196PRTHomo sapiens
219Gln Ser Val Ser Ser Ser1 5 22018PRTHomo sapiens 220Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu1 5 10 15 Ile
Tyr2213PRTHomo sapiens 221Gly Ala Ser1 22236PRTHomo sapiens 222Ser
Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10
15 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala
20 25 30 Val Tyr Tyr Cys 35 2238PRTHomo sapiens 223Gln Gln Tyr Gly
Ser Ser Phe Thr1 5 22411PRTHomo sapiens 224Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys Arg1 5 10 225124PRTHomo sapiens 225Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Ser Tyr 20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Gln Ser Tyr Asp His
Tyr Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 22625PRTHomo sapiens 226Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser 20 25 2278PRTHomo sapiens 227Gly Gly
Thr Phe Arg Ser Tyr Ala1 5 22818PRTHomo sapiens 228Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly1 5 10 15 Gly
Ile2297PRTHomo sapiens 229Ile Pro Ile Phe Gly Thr Ala1 5
23038PRTHomo sapiens 230Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Ile Thr Ala Asp Glu1 5 10 15 Ser Thr Ser Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35
23117PRTHomo sapiens 231Ala Arg Gly Ser Gln Ser Tyr Asp His Tyr Tyr
Tyr Tyr Gly Met Asp1 5 10 15 Val23211PRTHomo sapiens 232Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 10 233109PRTHomo sapiens 233Gln
Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Phe Asp Val Ser Asn Arg Pro Ser Gly Val
Ser Asn Arg Leu 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Ser Ser Tyr Thr Ser Asn 85 90 95 Thr Val Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 23426PRTHomo sapiens 234Gln Ser
Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser 20 25 2358PRTHomo sapiens
235Ser Asp Val Gly Gly Tyr Asn Tyr1 5 23617PRTHomo sapiens 236Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile1 5 10
15 Phe2373PRTHomo sapiens 237Asp Val Ser1 23836PRTHomo sapiens
238Asn Arg Pro Ser Gly Val Ser Asn Arg Leu Ser Gly Ser Lys Ser Gly1
5 10 15 Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu
Ala 20 25 30 Asp Tyr Tyr Cys 35 2399PRTHomo sapiens 239Ser Ser Tyr
Thr Ser Asn Thr Val Val1 5 24010PRTHomo sapiens 240Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu1 5 10 241116PRTHomo sapiens 241Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ala Phe Ser Ser Tyr 20 25
30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr
Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ala Gly Leu Gly
Ala Trp Gly Gln Gly Thr Leu Val 100 105 110 Ala Val Ser Ser 115
24225PRTHomo sapiens 242Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20
25 2438PRTHomo sapiens 243Gly Gly Ala Phe Ser Ser Tyr Ala1 5
24418PRTHomo sapiens 244Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile2457PRTHomo sapiens 245Ile Pro
Ile Phe Gly Thr Ala1 5 24638PRTHomo sapiens 246Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 2479PRTHomo sapiens 247Ala Arg Asp Ser Ala
Gly Leu Gly Ala1 5 24811PRTHomo sapiens 248Trp Gly Gln Gly Thr Leu
Val Ala Val Ser Ser1 5 10 249108PRTHomo sapiens 249Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys Arg 100 105 25026PRTHomo sapiens 250Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser 20 25 2516PRTHomo sapiens 251Gln Gly Ile
Ser Ser Ala1 5 25217PRTHomo sapiens 252Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile1 5 10 15 Tyr2533PRTHomo sapiens
253Asp Ala Ser1 25436PRTHomo sapiens 254Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr
Cys 35 2559PRTHomo sapiens 255Gln Gln Phe Asn Ser Tyr Pro Leu Thr1
5 25611PRTHomo sapiens 256Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg1 5 10 257120PRTHomo sapiens 257Gln Ile Thr Leu Lys Glu Ser Gly
Pro Thr Leu Val Lys Pro Thr Gln1 5 10 15 Thr Leu Thr Leu Thr Cys
Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val
Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu
Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser 50 55 60
Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val65
70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
Tyr Tyr 85 90 95 Cys Ala His Arg Glu Ser Gly Pro Glu Phe Phe Gln
His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
25825PRTHomo sapiens 258Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu
Val Lys Pro Thr Gln1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser 20
25 25910PRTHomo sapiens 259Gly Phe Ser Leu Ser Thr Ser Gly Val Gly1
5 10 26017PRTHomo sapiens 260Val Gly Trp Ile Arg Gln Pro Pro Gly
Lys Ala Leu Glu Trp Leu Ala1 5 10 15 Leu2618PRTHomo sapiens 261Ile
Tyr Trp Asp Asp Asp Lys Arg1 5 26237PRTHomo sapiens 262Tyr Ser Pro
Ser Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser1 5 10 15 Lys
Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr 20 25
30 Ala Thr Tyr Tyr Cys 35 26312PRTHomo sapiens 263Ala His Arg Glu
Ser Gly Pro Glu Phe Phe Gln His1 5 10 26411PRTHomo sapiens 264Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 10 265113PRTHomo sapiens
265Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1
5 10 15 Gln Pro Ala Ser Ile Ser Cys Asn Ser Ser Gln Ser Leu Val Tyr
Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Asn Trp Phe His Gln Arg Pro
Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Gln Val Ser Asn Trp
Asp Ser Glu Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Ala
Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Asp Asp
Val Gly Ile Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Pro
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110
Arg26626PRTHomo sapiens 266Asp Val Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Leu Gly1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Asn
Ser Ser 20 25 26711PRTHomo sapiens 267Gln Ser Leu Val Tyr Ser Asn
Gly Ile Thr Tyr1 5 10 26817PRTHomo sapiens 268Leu Asn Trp Phe His
Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile1 5 10 15 Tyr2693PRTHomo
sapiens 269Gln Val Ser1 27036PRTHomo sapiens 270Asn Trp Asp Ser Glu
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Ala1 5 10 15 Thr Asp Phe
Thr Leu Lys Ile Ser Arg Val Glu Ala Asp Asp Val Gly 20 25 30 Ile
Tyr Tyr Cys 35 2719PRTHomo sapiens 271Met Gln Gly Thr His Trp Pro
Pro Thr1 5 27211PRTHomo sapiens 272Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys Arg1 5 10 273132PRTHomo sapiens 273Glu Val Gln Val Ile Gln
Ser Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys Tyr 20 25 30 Ala Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro
His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr Val Ser Ser 130
27425PRTHomo sapiens 274Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser 20
25 2758PRTHomo sapiens 275Gly Gly Thr Phe Ser Lys Tyr Ala1 5
27618PRTHomo sapiens 276Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile2777PRTHomo sapiens 277Ile Pro
Ile Leu Gly Ile Ala1 5 27838PRTHomo sapiens 278Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 27925PRTHomo sapiens 279Ala Arg Gly Trp Gly
Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr1 5 10 15 Tyr Tyr Tyr
Tyr Tyr Gly Met Asp Val 20 25 28011PRTHomo sapiens 280Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 10 281110PRTHomo sapiens 281Glu
Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ala Ser Arg
20 25 30 Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly Leu Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Arg Thr Pro 85 90 95 Ser Val Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 28226PRTHomo sapiens
282Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25 2837PRTHomo
sapiens 283Gln Ser Val Ala Ser Arg Tyr1 5 28417PRTHomo sapiens
284Leu Ala Trp Tyr Gln His Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile1
5 10 15 Tyr2853PRTHomo sapiens 285Gly Ala Ser1 28636PRTHomo sapiens
286Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1
5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe
Ala 20 25 30 Val Tyr Tyr Cys 35 28710PRTHomo sapiens 287Gln Gln Tyr
Gly Arg Thr Pro Ser Val Thr1 5 10 28811PRTHomo sapiens 288Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg1 5 10 289132PRTHomo sapiens
289Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ser1
5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys
Tyr 20
25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser
Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Arg Glu
Gln Leu Ala Pro His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr Val Ser
Ser 130 29025PRTHomo sapiens 290Glu Val Gln Val Ile Gln Ser Gly Ala
Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ser Ser 20 25 2918PRTHomo sapiens 291Gly Gly Thr Phe Ser Lys Tyr
Ala1 5 29218PRTHomo sapiens 292Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met Gly1 5 10 15 Gly Ile2937PRTHomo sapiens
293Ile Pro Ile Leu Gly Ile Ala1 5 29438PRTHomo sapiens 294Asn Tyr
Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1 5 10 15
Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20
25 30 Thr Ala Val Tyr Tyr Cys 35 29525PRTHomo sapiens 295Ala Arg
Gly Trp Gly Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr1 5 10 15
Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 20 25 29611PRTHomo sapiens
296Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5 10 297108PRTHomo
sapiens 297Glu Ile Val Met Thr Gln Ser Pro Gly Thr Pro Ser Leu Ser
Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Ile Arg Ser Thr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Arg Ser Pro 85 90 95 Ser Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 29826PRTHomo
sapiens 298Glu Ile Val Met Thr Gln Ser Pro Gly Thr Pro Ser Leu Ser
Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25
2997PRTHomo sapiens 299Gln Ser Ile Arg Ser Thr Tyr1 5 30017PRTHomo
sapiens 300Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile1 5 10 15 Tyr3013PRTHomo sapiens 301Gly Ala Ser1
30236PRTHomo sapiens 302Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu Pro Glu Asp Phe Ala 20 25 30 Val Tyr Tyr Cys 35 3038PRTHomo
sapiens 303Gln Gln Tyr Gly Arg Ser Pro Ser1 5 30411PRTHomo sapiens
304Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg1 5 10 305132PRTHomo
sapiens 305Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val Lys Lys Pro
Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser Gly Gly Thr
Phe Ser Lys Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Leu Gly
Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile
Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Trp Gly Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr 100 105 110
Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 115
120 125 Thr Val Ser Ser 130 30625PRTHomo sapiens 306Glu Val Gln Val
Ile Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ser Ser 20 25 3078PRTHomo sapiens 307Gly Gly
Thr Phe Ser Lys Tyr Ala1 5 30818PRTHomo sapiens 308Ile Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly1 5 10 15 Gly
Ile3097PRTHomo sapiens 309Ile Pro Ile Leu Gly Ile Ala1 5
31038PRTHomo sapiens 310Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35
31125PRTHomo sapiens 311Ala Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro
His Pro Ser Gln Tyr1 5 10 15 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 20
25 31211PRTHomo sapiens 312Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser1 5 10 313110PRTHomo sapiens 313Glu Ile Val Met Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ala Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Trp Ala Ser Gln Ser Val Arg Asn Asn 20 25 30 Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Val 35 40 45 Ile Tyr
Asn Gly Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Asp65
70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Asn
Ser Arg 85 90 95 Arg Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg 100 105 110 31426PRTHomo sapiens 314Glu Ile Val Met Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ala Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Trp Ala Ser 20 25 3157PRTHomo sapiens 315Gln Ser Val
Arg Asn Asn Tyr1 5 31617PRTHomo sapiens 316Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Val Ile1 5 10 15 Tyr3173PRTHomo
sapiens 317Asn Gly Ser1 31836PRTHomo sapiens 318Thr Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Asp Pro Glu Asp Phe Ala 20 25 30 Val
Tyr Tyr Cys 35 31910PRTHomo sapiens 319Gln Gln Tyr Gly Asn Ser Arg
Arg Val Thr1 5 10 32011PRTHomo sapiens 320Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg1 5 10 321132PRTHomo sapiens 321Glu Val Gln Val
Ile Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys Tyr 20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Arg Glu Gln Leu
Ala Pro His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr Val Ser Ser 130
32225PRTHomo sapiens 322Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser 20
25 3238PRTHomo sapiens 323Gly Gly Thr Phe Ser Lys Tyr Ala1 5
32418PRTHomo sapiens 324Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile3257PRTHomo sapiens 325Ile Pro
Ile Leu Gly Ile Ala1 5 32638PRTHomo sapiens 326Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 32725PRTHomo sapiens 327Ala Arg Gly Trp Gly
Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr1 5 10 15 Tyr Tyr Tyr
Tyr Tyr Gly Met Asp Val 20 25 32811PRTHomo sapiens 328Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 10 329108PRTHomo sapiens 329Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Thr Ser Thr Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Arg Tyr Gly Ser Ser Pro 85 90 95 Ala Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 33026PRTHomo sapiens 330Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25 3317PRTHomo sapiens
331Gln Ser Val Ser Ser Ser Tyr1 5 33217PRTHomo sapiens 332Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile1 5 10 15
Tyr3333PRTHomo sapiens 333Gly Thr Ser1 33436PRTHomo sapiens 334Thr
Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10
15 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala
20 25 30 Val Tyr Tyr Cys 35 3358PRTHomo sapiens 335Gln Arg Tyr Gly
Ser Ser Pro Ala1 5 33611PRTHomo sapiens 336Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg1 5 10 337132PRTHomo sapiens 337Glu Val Gln Val
Ile Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys Tyr 20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Arg Glu Gln Leu
Ala Pro His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr Val Ser Ser 130
33825PRTHomo sapiens 338Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser 20
25 3398PRTHomo sapiens 339Gly Gly Thr Phe Ser Lys Tyr Ala1 5
34018PRTHomo sapiens 340Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile3417PRTHomo sapiens 341Ile Pro
Ile Leu Gly Ile Ala1 5 34238PRTHomo sapiens 342Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 34325PRTHomo sapiens 343Ala Arg Gly Trp Gly
Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr1 5 10 15 Tyr Tyr Tyr
Tyr Tyr Gly Met Asp Val 20 25 34411PRTHomo sapiens 344Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 10 345107PRTHomo sapiens 345Asp
Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser Ile Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Lys Trp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Ile Asn Tyr Ala Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 34626PRTHomo sapiens 346Asp Ile Gln Met
Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser Ile Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser 20 25 3476PRTHomo sapiens 347Gln
Ser Ile Ser Lys Trp1 5 34817PRTHomo sapiens 348Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile1 5 10 15 Tyr3493PRTHomo
sapiens 349Lys Ala Ser1 35036PRTHomo sapiens 350Thr Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 20 25 30 Thr
Tyr Tyr Cys 35 3518PRTHomo sapiens 351Gln Gln Tyr Ile Asn Tyr Ala
Thr1 5 35211PRTHomo sapiens 352Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg1 5 10 353132PRTHomo sapiens 353Glu Val Gln Val Ile Gln Ser
Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys Tyr 20 25 30 Ala Ile Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro
His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr Val Ser Ser 130
35425PRTHomo sapiens 354Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser 20
25 3558PRTHomo sapiens 355Gly Gly Thr Phe Ser Lys Tyr Ala1 5
35618PRTHomo sapiens 356Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile3577PRTHomo sapiens 357Ile Pro
Ile Leu Gly Ile Ala1 5 35838PRTHomo sapiens 358Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 35925PRTHomo
sapiens 359Ala Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro His Pro Ser
Gln Tyr1 5 10 15 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 20 25
36011PRTHomo sapiens 360Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser1 5 10 361110PRTHomo sapiens 361Glu Ile Val Met Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ala Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Trp Ala Ser Gln Ser Val Arg Asn Asn 20 25 30 Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Val 35 40 45 Ile Tyr
Asn Gly Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Asp65
70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Asn
Ser Arg 85 90 95 Arg Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg 100 105 110 36226PRTHomo sapiens 362Glu Ile Val Met Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ala Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Trp Ala Ser 20 25 3637PRTHomo sapiens 363Gln Ser Val
Arg Asn Asn Tyr1 5 36417PRTHomo sapiens 364Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Val Ile1 5 10 15 Tyr3653PRTHomo
sapiens 365Asn Gly Ser1 36636PRTHomo sapiens 366Thr Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Asp Pro Glu Asp Phe Ala 20 25 30 Val
Tyr Tyr Cys 35 36710PRTHomo sapiens 367Gln Gln Tyr Gly Asn Ser Arg
Arg Val Thr1 5 10 36811PRTHomo sapiens 368Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg1 5 10 369132PRTHomo sapiens 369Glu Val Gln Val
Ile Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys Tyr 20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Arg Glu Gln Leu
Ala Pro His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr Val Ser Ser 130
37025PRTHomo sapiens 370Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser 20
25 3718PRTHomo sapiens 371Gly Gly Thr Phe Ser Lys Tyr Ala1 5
37218PRTHomo sapiens 372Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile3737PRTHomo sapiens 373Ile Pro
Ile Leu Gly Ile Ala1 5 37438PRTHomo sapiens 374Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 37525PRTHomo sapiens 375Ala Arg Gly Trp Gly
Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr1 5 10 15 Tyr Tyr Tyr
Tyr Tyr Gly Met Asp Val 20 25 37611PRTHomo sapiens 376Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 10 377107PRTHomo sapiens 377Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 37826PRTHomo sapiens 378Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser 20 25 3796PRTHomo sapiens 379Gln
Ser Val Ser Ser Tyr1 5 38017PRTHomo sapiens 380Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile1 5 10 15 Tyr3813PRTHomo
sapiens 381Asp Ala Ser1 38236PRTHomo sapiens 382Asn Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala 20 25 30 Val
Tyr Tyr Cys 35 3838PRTHomo sapiens 383Gln Gln Arg Ser Asn Trp Pro
Thr1 5 38411PRTHomo sapiens 384Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg1 5 10 385132PRTHomo sapiens 385Glu Val Gln Val Ile Gln Ser
Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys Tyr 20 25 30 Ala Ile Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro
His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr Val Ser Ser 130
38625PRTHomo sapiens 386Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val
Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser 20
25 3878PRTHomo sapiens 387Gly Gly Thr Phe Ser Lys Tyr Ala1 5
38818PRTHomo sapiens 388Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met Gly1 5 10 15 Gly Ile3897PRTHomo sapiens 389Ile Pro
Ile Leu Gly Ile Ala1 5 39038PRTHomo sapiens 390Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1 5 10 15 Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 39125PRTHomo sapiens 391Ala Arg Gly Trp Gly
Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr1 5 10 15 Tyr Tyr Tyr
Tyr Tyr Gly Met Asp Val 20 25 39211PRTHomo sapiens 392Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 10 393110PRTHomo sapiens 393Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Thr Ile Thr Phe Gly Gln
Gly Thr Arg Leu Glu Ile Lys Arg 100 105 110 39426PRTHomo sapiens
394Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25 3957PRTHomo
sapiens 395Gln Ser Val Ser Ser Ser Tyr1 5 39617PRTHomo sapiens
396Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile1
5 10 15 Tyr3973PRTHomo sapiens 397Gly Ala Ser1 39836PRTHomo sapiens
398Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1
5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe
Ala 20 25 30 Val Tyr Tyr Cys 35 39910PRTHomo sapiens 399Gln Gln Tyr
Gly Ser Ser Pro Thr Ile Thr1 5 10 40011PRTHomo sapiens 400Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys Arg1 5 10 401132PRTHomo sapiens
401Glu Val Gln Val Ile Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ser1
5 10 15 Ser Val Lys Val Ser Cys Lys Ser Ser Gly Gly Thr Phe Ser Lys
Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp
Glu Ser Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly
Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr 100 105 110 Tyr Tyr Tyr
Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 115 120 125 Thr
Val Ser Ser 130 40225PRTHomo sapiens 402Glu Val Gln Val Ile Gln Ser
Gly Ala Asp Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ser Ser 20 25 4038PRTHomo sapiens 403Gly Gly Thr Phe Ser
Lys Tyr Ala1 5 40418PRTHomo sapiens 404Ile Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met Gly1 5 10 15 Gly Ile4057PRTHomo
sapiens 405Ile Pro Ile Leu Gly Ile Ala1 5 40638PRTHomo sapiens
406Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Thr Asp Glu1
5 10 15 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 40725PRTHomo sapiens 407Ala
Arg Gly Trp Gly Arg Glu Gln Leu Ala Pro His Pro Ser Gln Tyr1 5 10
15 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 20 25 40811PRTHomo sapiens
408Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5 10 409108PRTHomo
sapiens 409Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Val Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 41026PRTHomo
sapiens 410Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25
4117PRTHomo sapiens 411Gln Ser Val Ser Ser Ser Tyr1 5 41217PRTHomo
sapiens 412Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile1 5 10 15 Tyr4133PRTHomo sapiens 413Gly Ala Ser1
41436PRTHomo sapiens 414Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu Pro Glu Asp Phe Ala 20 25 30 Val Tyr Tyr Cys 35 4158PRTHomo
sapiens 415Gln Gln Tyr Gly Ser Ser Pro Val1 5 41611PRTHomo sapiens
416Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg1 5 10 41718PRTHomo
sapiens 417Asp Pro Gly Gly Tyr Ser Tyr Gly Pro Tyr Tyr Tyr Tyr Tyr
Gly Met 1 5 10 15 Asp Val 41823PRTHomo sapiens 418Gly Trp Gly Arg
Glu Gln Leu Ala Pro His Pro Ser Gln Tyr Tyr Tyr 1 5 10 15 Tyr Tyr
Tyr Gly Met Asp Val 20 41910PRTHomo sapiens 419Asp Ser Arg Tyr His
Asp Ala Phe Asp Ile 1 5 10 42010PRTHomo sapiens 420Glu Ser Ser Trp
Leu Asp Ala Phe Asp Ile 1 5 10 42113PRTHomo sapiens 421Val Gly Gly
Ile Thr Gly Thr Ala Asp Ala Phe Asp Ile 1 5 10 42222PRTHomo sapiens
422Asp Gln Leu Ala Gly Tyr Tyr Tyr Asp Ser Ser Gly Tyr His Tyr Tyr
1 5 10 15 Tyr Tyr Gly Met Asp Val 20 42311PRTHomo sapiens 423Asp
His Val His Gly Pro Asp Ala Phe Asp Ile 1 5 10 4247PRTHomo sapiens
424Val Gly Gly Ala Phe Asp Ile 1 5 42510PRTHomo sapiens 425Gly Trp
Phe Arg Asp Trp Tyr Phe Asp Leu 1 5 10 42612PRTHomo sapiens 426Glu
Gly Leu Pro Glu Thr Asp Asp Ala Phe Asp Ile 1 5 10 4275PRTHomo
sapiens 427Glu Gly Ala Asp Tyr 1 5 4285PRTHomo sapiens 428Asp Gly
Ala Asp Tyr 1 5 42910PRTHomo sapiens 429Tyr Lys Leu Gln Ser Asp Ala
Phe Asp Ile 1 5 10 43013PRTHomo sapiens 430Ala Gly Pro Val Gly Ala
Thr Thr Gly Thr Phe Asp Tyr 1 5 10 43111PRTHomo sapiens 431Gly Ser
Gln Ser Tyr Asp His Tyr Tyr Tyr Tyr 1 5 10 4327PRTHomo sapiens
432Asp Ser Ala Gly Leu Gly Ala 1 5 43310PRTHomo sapiens 433Arg Glu
Ser Gly Pro Glu Phe Phe Gln His 1 5 10
* * * * *
References