U.S. patent application number 11/618472 was filed with the patent office on 2007-09-20 for cd52 optimized fc variants and methods for their generation.
This patent application is currently assigned to Xencor, Inc.. Invention is credited to Arthur J. Chirino, Wei Dang, John R. Desjarlais, Stephen Kohl Doberstein, Robert J. Hayes, Sher Bahadur Karki, Gregory Alan Lazar, Omid Vafa.
Application Number | 20070219133 11/618472 |
Document ID | / |
Family ID | 46301929 |
Filed Date | 2007-09-20 |
United States Patent
Application |
20070219133 |
Kind Code |
A1 |
Lazar; Gregory Alan ; et
al. |
September 20, 2007 |
CD52 OPTIMIZED Fc VARIANTS AND METHODS FOR THEIR GENERATION
Abstract
The present invention relates to CD52 optimized Fc variants,
methods for their generation, and antibodies and Fc fusions
comprising optimized Fc variants.
Inventors: |
Lazar; Gregory Alan;
(Alhambra, CA) ; Chirino; Arthur J.; (Camarillo,
CA) ; Dang; Wei; (Pasadena, CA) ; Desjarlais;
John R.; (Pasadena, CA) ; Doberstein; Stephen
Kohl; (San Francisco, CA) ; Hayes; Robert J.;
(Radnor, PA) ; Karki; Sher Bahadur; (Pasadena,
CA) ; Vafa; Omid; (Monrovia, CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS, LLP
ONE MARKET SPEAR STREET TOWER
SAN FRANCISCO
CA
94105
US
|
Assignee: |
Xencor, Inc.
Monrovia
CA
|
Family ID: |
46301929 |
Appl. No.: |
11/618472 |
Filed: |
December 29, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10822231 |
Mar 26, 2004 |
|
|
|
11618472 |
Dec 29, 2006 |
|
|
|
10672280 |
Sep 26, 2003 |
|
|
|
10822231 |
Mar 26, 2004 |
|
|
|
10379392 |
Mar 3, 2003 |
|
|
|
10822231 |
Mar 26, 2004 |
|
|
|
60477839 |
Jun 12, 2003 |
|
|
|
60467606 |
May 2, 2003 |
|
|
|
60414433 |
Sep 27, 2002 |
|
|
|
60442301 |
Jan 23, 2003 |
|
|
|
60384197 |
May 29, 2002 |
|
|
|
60360843 |
Mar 1, 2002 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
514/1.4; 514/16.9; 514/19.4; 514/19.6; 514/20.9; 514/3.8;
530/387.1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 37/02 20180101; C07K 16/283 20130101; C07K 16/30 20130101;
C07K 2317/71 20130101; A61P 9/04 20180101; A61P 35/02 20180101;
C07K 16/2896 20130101; C07K 2317/52 20130101; C07K 2317/92
20130101; C07K 16/22 20130101; C07K 2317/34 20130101; A61P 3/10
20180101; C07K 2317/732 20130101; C07K 16/32 20130101; C07K 2317/72
20130101; C07K 16/2863 20130101; A61P 35/00 20180101; C07K 16/3015
20130101; A61P 29/00 20180101; A61K 2039/505 20130101; A61P 31/00
20180101; A61P 37/00 20180101; C07K 16/00 20130101; C07K 2317/77
20130101; C07K 16/2887 20130101; A61P 43/00 20180101; C07K 16/2893
20130101; C07K 2317/734 20130101 |
Class at
Publication: |
514/012 ;
530/387.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 16/00 20060101 C07K016/00 |
Claims
1. A protein comprising an Fc variant of a parent Fc polypeptide
and an IgG polypeptide that has specificity for CD52, said Fc
variant comprising at least one amino acid modification in the Sc
region of said IgG polypeptide said modification occurring at a
position selected from the group consisting of: 228, 230, 231, 232,
240, 244, 245, 247, 262, 263, 266, 271, 273, 275, 281, 284, 291,
299, 302, 304, 313, 323, 325, 328, 332, wherein said Fc variant
protein exhibits altered effector function, wherein numbering is
according to the EU index.
2. A protein according to claim 1, wherein said Fc variant
comprises at least one substitution selected from the group
consisting of: 234, 235, 239, 240, 243, 264, 266, 272, 274, 278,
325, 328, 330, and 332.
3. A protein according to claim 1, wherein said altered effector
function is an increase in affinity of said Fc variant to
Fc.gamma.R.
4. A protein according to claim 3, wherein said Fc variant binds
with greater affinity to a mouse Fc.gamma.R.
5. A protein according to claim 3, wherein said Fc.gamma.R is a
human Fc receptor selected from the group consisting of
Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIc, and
Fc.gamma.RIIIa.
6. A protein according to claim 3, wherein said Fc variant binds
with greater affinity to human Fc.gamma.RI and Fc.gamma.RIIIa, but
exhibits unaltered affinity to a human receptor selected from the
group consisting of Fc.gamma.RIIa, Fc.gamma.RIIb, and
Fc.gamma.RIIc.
7. A protein according to claim 6, wherein said Fc variant exhibits
unaltered affinity to Fc.gamma.RIIa, Fc.gamma.RIIb, and
Fc.gamma.RIIc.
8. A protein according to claim 3, wherein the affinity increase
for binding to one or more human Fc.gamma.RII's is greater than the
affinity increase for binding to human Fc.gamma.RI or
F.gamma.RIIIa.
9. A protein according to claim 3, wherein the affinity increase is
the same for binding to Fc.gamma.RIIa, Fc.gamma.RIIb, and
Fc.gamma.RIIc.
10. A protein according to claim 3, wherein the affinity increase
for binding to Fc.gamma.RIIc is greater than the affinity increase
for binding to Fc.gamma.RIIb.
11. A protein according to claim 3, wherein said Fc variant binds
with greater affinity to human Fc.gamma.RIIa, but exhibits
unaltered affinity to a human receptor selected from the group
consisting of Fc.gamma.RI, Fc.gamma.RIIb, Fc.gamma.RIIc, and
Fc.gamma.RIIIa.
12. A protein according to claim 11, wherein said Fc variant
exhibits unaltered affinity to Fc.gamma.RI, Fc.gamma.RIIb,
Fc.gamma.RIIc, and Fc.gamma.RIIIa.
13. A protein according to any of claims 1-12, wherein said Fc
variant binds with greater affinity to FcRn.
14. A protein according to any of claims 1-12, wherein said Fc
variant binds with unaltered affinity to FcRn.
15. A protein according to any of claims 1-12, wherein said Fc
variant binds with reduced affinity to FcRn.
16. A protein according to any of claims 1-12, wherein said Fc
variant binds with greater affinity to C1q or enhances CDC.
17. A protein according to any of claims 1-12, wherein said Fc
variant binds with unaltered affinity to C1q or enhances CDC.
18. A protein according to claim 1, wherein said Fc variant further
comprises an engineered glycoform.
19. A protein according to claim 18 wherein said engineered
glycoform comprises an altered level of fucosylation or bisecting
oligosaccharides as compared to the parent Fc polypeptide.
20. A protein according to claim 18 wherein said engineered
glycoform improves effector function.
21. A protein according to claim 1, wherein said Fc variant
modulates effector function as compared to the parent Fc
polypeptide.
22. A protein according to claim 21, wherein said effector function
is ADCC.
23. A protein according to claim 22, wherein said Fc variant
improves ADCC in the presence of human effector cells as compared
to said parent Fc polypeptide.
24. A protein according to claim 22 wherein said ADCC improvement
is an enhancement in potency such that the EC50 of said Fc variant
is approximately 5-fold greater than that of said parent Fc
polypeptide.
25. A protein according to claim 23, wherein said ADCC improvement
is an enhancement in potency such that the EC50 of said Fc variant
is between approximately 5-fold and 1000-fold greater than that of
said parent Fc polypeptide.
26. A protein according to claim 23, wherein said ADCC improvement
is an enhancement in efficacy such that the maximal ADCC is
approximately 2-fold greater than that of said parent Fc
polypeptide.
27. A protein according to claim 26, wherein said Fc variant
improves ADCC in the presence of mouse effector cells as compared
to said parent Fc polypeptide.
28. A protein according to claim 21, wherein said effector function
is ADCP.
29. A protein according to claim 28, wherein said Fc variant
improves ADCP as compared to said parent Fc polypeptide.
30. A protein according to claim 25 or 29, wherein CDC is
unaffected.
31. A protein according to claim 25 or 29, wherein CDC is
ablated.
32. A protein according to claim 1 or 21 comprising a modification
selected from the group consisting of 228E, 228K, 228Y, 228G, 230E,
230Y, 230G, 231E, 231K, 231Y, 231P, 231G, 232E, 232K, 232Y, 232G,
262E, 262F, 271D, 271E, 271N, 271Q, 271K, 271R, 271S, 271T, 271H,
271A, 271V, 271L, 271I, 271F, 271M, 271Y, 271W, 271G, 275L, 281D,
281K, 281Y, 281P, 284E, 284N, 284T, 284L, 284Y, 291D, 291E, 291Q,
291T, 291H, 291I, 291G, 299D, 299E, 299N, 299Q, 299K, 299R, 299L,
299F, 299M, 299Y, 299W, 299P, 299G, 304D, 304N, 304T, 304H, 304L,
325K, 325R, 325S, 325F, 325M, 325Y, 325W, 325P, 325G, 328D, 328D,
328K, 328R, 328S, 328T, 328V, 328I, 328Y, 328W, 328P, 328G, 332K,
332R, 332S, 332V, 332L, 332F, 332M, 332W, 332P, 332G, 336E, 336K,
336Y, 230A, 240A, 240I, 240M, 240T, 244H, 245A, 247G, 247V, 262A,
262E, 262I, 262T, 263A, 263I, 263M, 263T, 266A, 266I, 266M, 266T,
273I, 275W, 299A, 299E, 299F, 299H, 299I, 299L, 299S, 299V, 302I,
313F, 323I, 325A, 325D, 325E, 325H, 325I, 325L, 3250, 325T, 325V,
328A, 3280, 328E, 328F, 328H, 328I, 328M, 328N, 328Q, 328T, 328V,
332A, 332Q, 332E, 332H, 332N, 332Q, 332T, and 332Y.
33. A protein according to claim 1 wherein said parent Fc
polypeptide is an antibody comprising said Fc variant.
34. A protein according to claim 1 wherein said Fc variant is an Fc
fusion protein comprising said Fc variant.
35. A pharmaceutical composition comprising a protein according to
claim 1 and a pharmaceutically acceptable carrier.
36. A method of treating a mammal in need of said treatment,
comprising administering a variant protein of claim 1.
37. A protein according to claims 1 or 33 comprising at least one
modification selected from the group consisting of A330L/I332E,
D265F/N297E/I332, D265Y/N297D/I332E, D265Y/N297D/T299L/I332E,
F241E/F243Q/V262T/V264F, F241E/F243Q/V262T/V264E/I332E,
F241E/F243R/V262E/V264R, F241E/F243R/V262E/V264R/I332E,
F241E/F243Y/V262T/V264R, F241E/F243Y/V262T/V264R/I332E,
F241L/F243L/V262I/V264I, F241L/V262I, F241R/F243Q/V262T/V264R,
F241R/F243Q/V262T/V264R/I332E, F241W/F243W/V262A/V264A,
F241Y/F243Y/V262T/V264T, F241Y/F243Y/V262T/V264T/N297D/I332E,
F243L/V262I/V264W, P243L/V264I, L328D/I332E, L328E/I332E,
L328H/I332E, L328I/I332E, L328M/I332E, L328N/I332E, L328Q/I332E,
L328T/I332E, L328V/I332E, N297D/A330Y/I332E, N297D/I332E,
N297D/I332E/S239D/A330L, N297D/S298A/A330Y/I332E,
N297D/T299E/I332E, N297D/T299F/I332E, N297D/T299H/I332E,
N297D/T299I/I332E, N297D/T299L/I332E, N297D/T299V/I332E,
N297E/I332E, N297S/I332E, P230A/E233D/I332, P244H/P245A/P247V,
S239D/A330L/I332E, S239D/A330Y/I332E, S239D/A330Y/I332E/K326E,
S239D/A330Y/I332E/K326T, S239D/A330Y/I332E/L234I,
S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I,
S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/V266I,
S239D/D265F/N297D/I332E, S239D/D265H/N297D/I332E,
S239D/D265I/N297D/I332E, S239D/D265L/N297D/I332E,
S239D/D265T/N297D/I332E, S239D/D0265V/N297D/I332E,
S239D/D265Y/N297D/I332E, S239D/I332D, S2390/I332E,
S239D/I332E/A330I, S239D/I332N, S239D/I332Q, S239D/N297D01332E,
S239D/N297D/I332E/A330Y,
S239D/N297D/I332E/A330Y/F241S/F243H/V262T/V264T,
S239D/N297D/I332E/K326E, S239D/N297D/I332E/L235D,
S239D/S298A/I332E, S239D/V264I/A330L/I332E, S239D/V264I/I332E,
S239D/V264I/S298A/I332E, S239E/D265N, S239E/D265Q, S239E/I332D,
S239E/I332E, S239E/I332N, S239E/I332Q, S239E/N297D/I332E,
S239E/V264I/A330Y/I332E, S239E/V264I/I 332E,
S239E/V264I/S298A/A330Y/I332E, S239N/A330L/I332E,
S239N/A330Y/I332E, S239N/I332D, S239N/I332E, S239N/I332N,
S239N/I332Q, S239N/S298A/I332E, S239Q/I332D, S239Q/I332E,
S239Q/I332N, S239Q/I332Q, S239Q/V264I/I332E, S298A/I332E,
V264E/N297D/I332E, V264I/A330L/I332E, V264I/A330Y/I332E,
V264I/I332E, V264I/S298A/I332E, Y296D/N297D/I332E,
Y296E/N297D/I332E, Y296H/N297D/I332E, Y296N/N297D/I332E,
Y296Q/N297D/I332E, and Y296T/N297D/I332E.
38. A protein according to claims 1, 33 or 37 further comprising a
modification selected from the group consisting of 227E, 227K,
227Y, 227G, 233N, 233Q, 233K, 233R, 233S, 233T, 233H, 233A, 233V,
233L, 233I, 233F, 233M, 233Y, 233W, 233G, 234K, 234R, 234S, 234A,
234M, 234W, 234P, 234G, 235E, 235K, 235R, 235A, 235M, 235W, 235P,
235G, 236D, 236E, 236N, 236Q, 236K, 236R, 236S, 236T, 236H, 236A,
236V, 236L, 236I, 236F, 236M, 236Y, 236W, 236P, 237D, 237E, 237N,
237Q, 237K, 237R, 237S, 237T, 237H, 237V, 237L, 237I, 237F, 237M,
237Y, 237W, 237P, 238D, 238E, 238N, 238Q, 238K, 238R, 238S, 238T,
238H, 233V, 238L, 238I, 238F, 238M, 238Y, 238W, 238G, 239Q, 239K,
239R, 239V, 239L, 239I, 239M, 239W, 239P, 239G, 241D, 241E, 241Y,
243E, 246D, 246E, 246H, 246Y, 249Q, 249H, 249Y, 255E, 255Y 258S,
258H, 258Y, 260D, 260E, 260H, 260Y, 264D, 264E, 264N, 264Q, 264K,
264R, 264S, 264H, 264W, 264P, 264G, 265Q, 265K, 265R, 265S, 265T,
265H, 265V, 265L, 265I, 265F, 265M, 265Y, 265W, 265P, 267E, 267Q,
267K, 267R, 267V, 267L, 267I, 267F, 267M, 267Y, 267W, 267P, 268D,
268E, 268Q, 268K, 268R, 268T, 268V, 268L, 268I, 268F, 268M, 268W,
26FP, 268G, 269K, 269S, 269V, 269I, 269M, 269W, 269P, 269G, 270R,
270S, 270L, 270I, 270F, 270M, 270Y, 270W, 270P, 270G, 272D, 272R,
272T, 272H, 272V, 272L, 272F, 272M, 272W, 272P, 272G, 274D, 274N,
274S, 274H, 274V, 274I, 274F, 274M, 274W, 274P, 274G, 276D, 276T,
276H, 276V, 276I, 276F, 276M, 276W, 276P, 276G, 278D, 278N, 278Q,
278R, 278S, 278H, 278V, 278L, 278I, 278M, 278P, 278G, 280K, 280L,
280W, 280P, 280G, 282E, 282K, 282Y, 282P, 282G, 283K, 283H, 283L,
283Y, 283P, 283G, 285D, 285E, 285Q, 285K, 285Y, 285W, 286E, 286Y,
286P, 286G, 288D, 288E 288Y, 290D, 290N, 290H, 290L, 290W, 292D,
292E, 292T, 292Y, 293N, 293R, 293S, 293T, 293H, 293V, 293L, 293I,
293F, 293M, 293Y, 293W, 293P, 293G, 294K, 294R, 294S, 294T, 294K,
294V, 294L, 294I, 294F, 294M, 294Y, 294W, 294P, 294G, 295D, 295E,
295N, 295R, 295S, 295T, 295H, 295V, 295I, 295F, 295M, 295Y, 295W,
295P, 295G, 296K, 296R, 296A, 296V, 296M, 296G, 297Q, 297K, 297R,
297T, 297H, 297V, 297L, 297I, 297F, 297M, 297Y, 297W, 297P, 297G,
298D, 298E, 298Q, 298K, 298R, 298I, 298F, 298M, 298Y, 298W, 300D,
300E, 300N, 300Q, 300K, 300R, 300S, 300T, 300H, 300A, 300V, 300M,
300W, 300P, 300G, 301D, 301E, 301H, 301Y, 303D, 303E, 303Y, 305E,
305T, 305Y, 317E, 317Q, 318Q, 318H, 318L, 318Y, 320N, 320S, 320H,
320V, 320L, 320F, 320Y, 320W, 320P, 320G, 322D, 322S, 322V, 322I,
322F, 322Y, 322W, 322P, 322G, 324H, 324F, 324M, 324W, 324P, 324G,
326P, 327E, 327K, 327R, 327H, 327V, 327I, 327F, 327M, 327Y, 327W,
327P, 329D, 329E, 329N, 329Q, 329K, 329R, 329S, 329T, 329H, 329V,
329L, 329I, 329M, 329Y, 329W, 329G, 330E, 330N, 330T, 330P, 330G,
331D, 331Q, 331R, 331T, 331L, 331I, 331F, 331M, 331Y, 331W, 333L,
333F, 333M, 333P, 334P, 335N, 335S, 335H, 335V, 335L, 335I, 335F,
335M, 335W, 335P, 335G, 337E, 337N, 337H, 233D, 234D, 234E, 234F,
234H, 234I, 234N, 234Q, 234T, 234V, 234Y, 235D, 235F, 235H, 235I,
235N, 235Q, 235S, 235T, 235V, 235Y, 239D, 239E, 239F, 239H, 239N,
239Q, 239T, 239Y, 241E, 241L, 241R, 241S, 241W, 241Y, 243H, 243L,
243Q, 243R, 243W, 243Y, 264A, 264E, 264F, 264I, 264L, 264M, 264R,
264T, 264W, 264Y, 266F, 265G, 265K, 265I, 265L, 265N, 265Q, 265T,
265V, 265Y, 267D, 267H, 267L, 267N, 267Q, 267T, 269F, 269H, 269L,
269N, 269R, 269T, 269Y, 270H, 270Q, 270T, 272I, 272K, 272S, 272Y,
274E, 274L, 274R, 274T, 274Y, 276E, 276L, 276R, 276S, 276Y, 278E,
278K, 273T, 278W, 283R, 296D, 296E, 296H, 296I, 296L, 296N, 296Q,
296S, 296T, 297D, 297E, 297S, 298A, 298H, 298N, 298T, 318R, 320D,
320I, 320T, 322H, 322T, 324D, 324I, 324L, 324R, 324T, 324V, 324Y,
326E, 326I, 326L, 326T, 327D, 327L, 327N, 327S, 327T, 329F, 330F,
330H, 330I, 330L, 330M, 330R, 330S, 330V, 330W, 330Y, 331H, 331V,
333A, 333H, 333I, 333T, 333Y, 334A, 334F, 334I, 334T, 335D, 335R,
and 335Y.
39. A protein according to claim 1 wherein said Fc variant further
comprises a set of modifications selected from 241W/243W,
267Q/327S, 243L/264I, 234I/235D, 264E/297D/332E, 239E/265N,
239E/265Q, 239E/265G, 267L/327S and 240I/266I.
40. A protein according to claim 33 wherein said antibody is a full
length antibody.
41. A protein according to claim 33 wherein said antibody is a
human antibody.
42. A protein according to claim 33 wherein said antibody is an
antibody fragment.
Description
[0001] This application is a continuation application of U.S. Ser.
No. 10/822,231, filed Mar. 26, 2004, pending, which is a
continuation-in-part of U.S. Ser. No. 10/672,280, filed Sep. 26,
2003, pending, which claims the benefit under 35 U.S.C.
.sctn.119(e) to U.S. Ser. No. 60/477,839, filed Jun. 12, 2003, U.S.
Ser. No. 60/467,606, filed May 2, 2003, U.S. Ser. No. 60/442,301,
filed Jan. 23, 2003, and U.S. Ser. No. 60/414,433, filed Sep. 27,
2002, and which is a continuation-in-part of U.S. Ser. No.
10/379,392, filed Mar. 3, 2003, now abandoned, which claims the
benefit under 35 U.S.C. .sctn.119(e) to U.S. Ser. No. 60/384,197,
filed May 29, 2002, and U.S. Ser. No. 60/360,843, filed Mar. 1,
2002, all of which are expressly incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel optimized Fc
variants, engineering methods for their generation, and their
application, particularly for therapeutic purposes.
BACKGROUND OF THE INVENTION
[0003] Antibodies are immunological proteins that bind a specific
antigen. In most mammals, including humans and mice, antibodies are
constructed from paired heavy and light polypeptide chains. Each
chain is made up of individual immunoglobulin (Ig) domains, and
thus the generic term immunoglobulin is used for such proteins.
Each chain is made up of two distinct regions, referred to as the
variable and constant regions. The light and heavy chain variable
regions show significant sequence diversity between antibodies, and
are responsible for binding the target antigen. The constant
regions show less sequence diversity, and are responsible for
binding a number of natural proteins to elicit important
biochemical events. In humans there are five different classes of
antibodies including IgA (which includes subclasses IgA1 and IgA2),
IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and
IgG4), and IgM. The distinguishing features between these antibody
classes are their constant regions, although subtler differences
may exist in the V region. FIG. 1 shows an IgG1 antibody, used here
as an example to describe the general structural features of
immunoglobulins. IgG antibodies are tetrameric proteins composed of
two heavy chains and two light chains. The IgG heavy chain is
composed of four immunoglobulin domains linked from N- to
C-terminus in the order V.sub.H-C.gamma.1-C.gamma.2-C.gamma.3,
referring to the heavy chain variable domain, constant gamma 1
domain, constant gamma 2 domain, and constant gamma 3 domain
respectively. The IgG light chain is composed of two immunoglobulin
domains linked from N- to C-terminus in the order V.sub.L-C.sub.L,
referring to the light chain variable domain and the light chain
constant domain respectively.
[0004] The variable region of an antibody contains the antigen
binding determinants of the molecule, and thus determines the
specificity of an antibody for its target antigen. The variable
region is so named because it is the most distinct in sequence from
other antibodies within the same class. The majority of sequence
variability occurs in the complementarity determining regions
(CDRs). There are 6 CDRs total, three each per heavy and light
chain, designated V.sub.H CDR1, V.sub.H CDR2, V.sub.H CDR3, V.sub.L
CDR1, V.sub.L CDR2, and V.sub.L CDR3. The variable region outside
of the CDRs is referred to as the framework (FR) region. Although
not as diverse as the CDRs, sequence variability does occur in the
FR region between different antibodies. Overall, this
characteristic architecture of antibodies provides a stable
scaffold (the FR region) upon which substantial antigen binding
diversity (the CDRs) can be explored by the immune system to obtain
specificity for a broad array of antigens. A number of
high-resolution structures are available for a variety of variable
region fragments from different organisms, some unbound and some in
complex with antigen. The sequence and structural features of
antibody variable regions are well characterized (Morea et al.,
1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods
20:267-279), and the conserved features of antibodies have enabled
the development of a wealth of antibody engineering techniques
(Maynard et al., 2000, Annu Rev Biomed Eng 2:339-376). For example,
it is possible to graft the CDRs from one antibody, for example a
murine antibody, onto the framework region of another antibody, for
example a human antibody. This process, referred to in the art as
"humanization", enables generation of less immunogenic antibody
therapeutics from nonhuman antibodies. Fragments comprising the
variable region can exist in the absence of other regions of the
antibody, including for example the antigen binding fragment (Fab)
comprising V.sub.H-C.gamma.1 and V.sub.H-C.sub.L, the variable
fragment (Fv) comprising V.sub.H and V.sub.L, the single chain
variable fragment (scFv) comprising V.sub.H and V.sub.L linked
together in the same chain, as well as a variety of other variable
region fragments (Little et al., 2000, Immunol Today
21:364-370).
[0005] The Fc region of an antibody interacts with a number of Fc
receptors and ligands, imparting an array of important functional
capabilities referred to as effector functions. For IgG the Fc
region, as shown in FIG. 1, comprises Ig domains C.gamma.2 and
C.gamma.3 and the N-terminal hinge leading into C.gamma.2. An
important family of Fc receptors for the IgG class are the Fc gamma
receptors (Fc.gamma.Rs). These receptors mediate communication
between antibodies and the cellular arm of the immune system
(Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch
et al., 2001, Annu Rev Immunol 19:275-290). In humans this protein
family includes Fc.gamma.RI (CD64), including isoforms
Fc.gamma.RIa, Fc.gamma.RIb, and Fc.gamma.RIc; Fc.gamma.RII (CD32),
including isoforms Fc.gamma.RIIa (including allotypes H131 and
R131), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and
Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and Fc.gamma.RII (CD16),
including isoforms Fc.gamma.RIIIa (including allotypes V158 and
F158) and Fc.gamma.RIIb (including allotypes Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65).
These receptors typically have an extracellular domain that
mediates binding to Fc, a membrane spanning region, and an
intracellular domain that may mediate some signaling event within
the cell. These receptors are expressed in a variety of immune
cells including monocytes, macrophages, neutrophils, dendritic
cells, eosinophils, mast cells, platelets, B cells, large granular
lymphocytes, Langerhans' cells, natural killer (NK) cells, and
.gamma..gamma. T cells. Formation of the Fc/Fc.gamma.R complex
recruits these effector cells to sites of bound antigen, typically
resulting in signaling events within the cells and important
subsequent immune responses such as release of inflammation
mediators, B cell activation, endocytosis, phagocytosis, and
cytotoxic attack. The ability to mediate cytotoxic and phagocytic
effector functions is a potential mechanism by which antibodies
destroy targeted cells. The cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell is referred to as antibody dependent cell-mediated
cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol
12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766;
Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The
cell-mediated reaction wherein nonspecific cytotoxic cells that
express Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause phagocytosis of the target cell is referred to
as antibody dependent cell-mediated phagocytosis (ADCP). A number
of structures have been solved of the extracellular domains of
human Fc.gamma.Rs, including Fc.gamma.RIIa (pdb accession code
1H9V)(Sondermann et al., 2001, J Mol Biol 309:737-749) (pdb
accession code 1FCG)(Maxwell et al., 1999, Nat Struct Biol
6:437-442), Fc.gamma.RIIb (pdb accession code 2FCB)(Sondermann et
al., 1999, Embo J 18:1095-1103); and Fc.gamma.RIIb (pdb accession
code 1E4J)(Sondermann et al., 2000, Nature 406:267-273.). All
Fc.gamma.Rs bind the same region on Fc, at the N-terminal end of
the C.gamma.2 domain and the preceding hinge, shown in FIG. 2. This
interaction is well characterized structurally (Sondermann et al.,
2001, J Mol Biol 309:737-749), and several structures of the human
Fc bound to the extracellular domain of human Fc.gamma.RIIIb have
been solved (pdb accession code 1E4K)(Sondermann et al., 2000,
Nature 406:267-273.) (pdb accession codes 1IIS and 1IIX)(Radaev et
al., 2001, J Biol Chem 276:16469-16477), as well as has the
structure of the human IgE Fc/Fc.epsilon.RI.alpha. complex (pdb
accession code 1F6A)(Garman et al., 2000, Nature 406:259-266).
[0006] The different IgG subclasses have different affinities for
the Fc.gamma.Rs, with IgG1 and IgG3 typically binding substantially
better to the receptors than IgG2 and IgG4 (Jefferis et al., 2002,
Immunol Lett 82:57-65). All Fc.gamma.Rs bind the same region on IgG
Fc, yet with different affinities: the high affinity binder
Fc.gamma.RI has a Kd for IgG1 of 10.sup.-8 M.sup.-1, whereas the
low affinity receptors Fc.gamma.RII and Fc.gamma.RII generally bind
at 10.sup.-6 and 10.sup.-5 respectively. The extracellular domains
of Fc.gamma.RIIIa and Fc.gamma.RIIIb are 96% identical, however
Fc.gamma.RIIIb does not have a intracellular signaling domain.
Furthermore, whereas Fc.gamma.RI, Fc.gamma.RIIa/c, and
Fc.gamma.RIIIa are positive regulators of immune complex-triggered
activation, characterized by having an intracellular domain that
has an immunoreceptor tyrosine-based activation motif (ITAM),
Fc.gamma.RIIb has an immunoreceptor tyrosine-based inhibition motif
(ITIM) and is therefore inhibitory. Thus the former are referred to
as activation receptors, and Fc.gamma.RIIb is referred to as an
inhibitory receptor. The receptors also differ in expression
pattern and levels on different immune cells. Yet another level of
complexity is the existence of a number of Fc.gamma.R polymorphisms
in the human proteome. A particularly relevant polymorphism with
clinical significance is V158/F158 Fc.gamma.RIIa. Human IgG1 binds
with greater affinity to the V158 allotype than to the F158
allotype. This difference in affinity, and presumably its effect on
ADCC and/or ADCP, has been shown to be a significant determinant of
the efficacy of the anti-CD20 antibody rituximab (Rituxan.RTM., a
registered trademark of IDEC Pharmaceuticals Corporation). Patients
with the V158 allotype respond favorably to rituximab treatment;
however, patients with the lower affinity F158 allotype respond
poorly (Cartron et al., 2002, Blood 99:754-758). Approximately
10-20% of humans are V158N158 homozygous, 45% are V158/F158
heterozygous, and 35-45% of humans are F158/F158 homozygous
(Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al.,
2002, Blood 99:754-758). Thus 80-90% of humans are poor responders,
that is they have at least one allele of the F158
Fc.gamma.RIIIa.
[0007] An overlapping but separate site on Fc, shown in FIG. 1,
serves as the interface for the complement protein C1q. In the same
way that Fc/Fc.gamma.R binding mediates ADCC, Fc/C1q binding
mediates complement dependent cytotoxicity (CDC). C1q forms a
complex with the serine proteases C1r and C1s to form the C1
complex. C1q is capable of binding six antibodies, although binding
to two IgGs is sufficient to activate the complement cascade.
Similar to Fc interaction with Fc.gamma.Rs, different IgG
subclasses have different affinity for C1q, with IgG1 and IgG3
typically binding substantially better to the Fc.gamma.Rs than IgG2
and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65). There is
currently no structure available for the Fc/C1q complex; however,
mutagenesis studies have mapped the binding site on human IgG for
C1q to a region involving residues D270, K322, K326, P329, and
P331, and E333 (Idusogie et al., 2000, J Immunol 164:4178-4184;
Idusogie et al., 2001, J Immunol 166:2571-2575).
[0008] A site on Fc between the C.gamma.2 and C.gamma.3 domains,
shown in FIG. 1, mediates interaction with the neonatal receptor
FcRn, the binding of which recycles endocytosed antibody from the
endosome back to the bloodstream (Raghavan et al., 1996, Annu Rev
Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol
18:739-766). This process, coupled with preclusion of kidney
filtration due to the large size of the full length molecule,
results in favorable antibody serum half-lives ranging from one to
three weeks. Binding of Fc to FcRn also plays a key role in
antibody transport. The binding site for FcRn on Fc is also the
site at which the bacterial proteins A and G bind. The tight
binding by these proteins is typically exploited as a means to
purify antibodies by employing protein A or protein G affinity
chromatography during protein purification. Thus the fidelity of
this region on Fc is important for both the clinical properties of
antibodies and their purification. Available structures of the rat
Fc/FcRn complex (Martin et al., 2001, Mol Cell 7:867-877), and of
the complexes of Fc with proteins A and G (Deisenhofer, 1981,
Biochemistry 20:2361-2370; Sauer-Eriksson et al., 1995, Structure
3:265-278; Tashiro et al., 1995, Curr Opin Struct Biol 5:471-481)
provide insight into the interaction of Fc with these proteins.
[0009] A key feature of the Fc region is the conserved N-linked
glycosylation that occurs at N297, shown in FIG. 1. This
carbohydrate, or oligosaccharide as it is sometimes referred, plays
a critical structural and functional role for the antibody, and is
one of the principle reasons that antibodies must be produced using
mammalian expression systems. While not wanting to be limited to
one theory, it is believed that the structural purpose of this
carbohydrate may be to stabilize or solubilize Fc, determine a
specific angle or level of flexibility between the C.gamma.3 and
C.gamma.2 domains, keep the two C.gamma.2 domains from aggregating
with one another across the central axis, or a combination of
these. Efficient Fc binding to Fc.gamma.R and C1q requires this
modification, and alterations in the composition of the N297
carbohydrate or its elimination affect binding to these proteins
(Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,
2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J Biol
Chem 276:45539-45547; Radaev et al., 2001, J Biol Chem
276:16478-16483; Shields et al., 2001, J Biol Chem 276:6591-6604;
Shields et al., 2002, J Biol Chem 277:26733-26740; Simmons et al.,
2002, J Immunol Methods 263:133-147). Yet the carbohydrate makes
little if any specific contact with Fc.gamma.Rs (Radaev et al.,
2001, J Biol Chem 276:16469-16477), indicating that the functional
role of the N297 carbohydrate in mediating Fc/Fc.gamma.R binding
may be via the structural role it plays in determining the Fc
conformation. This is supported by a collection of crystal
structures of four different Fc glycoforms, which show that the
composition of the oligosaccharide impacts the conformation of
C.gamma.2 and as a result the Fc/Fc.gamma.R interface (Krapp et
al., 2003, J Mol Biol 325:979-989).
[0010] The features of antibodies discussed above--specificity for
target, ability to mediate immune effector mechanisms, and long
half-life in serum--make antibodies powerful therapeutics.
Monoclonal antibodies are used therapeutically for the treatment of
a variety of conditions including cancer, inflammation, and
cardiovascular disease. There are currently over ten antibody
products on the market and hundreds in development. In addition to
antibodies, an antibody-like protein that is finding an expanding
role in research and therapy is the Fc fusion (Chamow et al., 1996,
Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin
Immunol 9:195-200). An Fc fusion is a protein wherein one or more
polypeptides is operably linked to Fc. An Fc fusion combines the Fc
region of an antibody, and thus its favorable effector functions
and pharmacokinetics, with the target-binding region of a receptor,
ligand, or some other protein or protein domain. The role of the
latter is to mediate target recognition, and thus it is
functionally analogous to the antibody variable region. Because of
the structural and functional overlap of Fc fusions with
antibodies, the discussion on antibodies in the present invention
extends directly to Fc fusions.
[0011] Despite such widespread use, antibodies are not optimized
for clinical use. Two significant deficiencies of antibodies are
their suboptimal anticancer potency and their demanding production
requirements. These deficiencies are addressed by the present
invention
[0012] There are a number of possible mechanisms by which
antibodies destroy tumor cells, including anti-proliferation via
blockage of needed growth pathways, intracellular signaling leading
to apoptosis, enhanced down regulation and/or turnover of
receptors, CDC, ADCC, ADCP, and promotion of an adaptive immune
response (Cragg et al., 1999, Curr Opin Immunol 11:541-547; Glennie
et al., 2000, Immunol Today 21:403-410). Anti-tumor efficacy may be
due to a combination of these mechanisms, and their relative
importance in clinical therapy appears to be cancer dependent.
Despite this arsenal of anti-tumor weapons, the potency of
antibodies as anti-cancer agents is unsatisfactory, particularly
given their high cost. Patient tumor response data show that
monoclonal antibodies provide only a small improvement in
therapeutic success over normal single-agent cytotoxic
chemotherapeutics. For example, just half of all relapsed low-grade
non-Hodgkin's lymphoma patients respond to the anti-CD20 antibody
rituximab (McLaughlin et al., 1998, J Clin Oncol 16:2825-2833). Of
166 clinical patients, 6% showed a complete response and 42% showed
a partial response, with median response duration of approximately
12 months. Trastuzumab (Herceptin.RTM., a registered trademark of
Genentech), an anti-HER2/neu antibody for treatment of metastatic
breast cancer, has less efficacy. The overall response rate using
trastuzumab for the 222 patients tested was only 15%, with 8
complete and 26 partial responses and a median response duration
and survival of 9 to 13 months (Cobleigh et al., 1999, J Clin Oncol
17:2639-2648). Currently for anticancer therapy, any small
improvement in mortality rate defines success. Thus there is a
significant need to enhance the capacity of antibodies to destroy
targeted cancer cells.
[0013] A promising means for enhancing the anti-tumor potency of
antibodies is via enhancement of their ability to mediate cytotoxic
effector functions such as ADCC, ADCP, and CDC. The importance of
Fc.gamma.R-mediated effector functions for the anti-cancer activity
of antibodies has been demonstrated in mice (Clynes et al., 1998,
Proc Natl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med
6:443-446), and the affinity of interaction between Fc and certain
Fc.gamma.Rs correlates with targeted cytotoxicity in cell-based
assays (Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et
al., 2002, Biochem Soc Trans 30:487-490; Shields et al., 2002, J
Biol Chem 277:26733-26740). Additionally, a correlation has been
observed between clinical efficacy in humans and their allotype of
high (V158) or low (F158) affinity polymorphic forms of
Fc.gamma.RIIIa (Cartron et al., 2002, Blood 99:754-758). Together
these data suggest that an antibody with an Fc region optimized for
binding to certain Fc.gamma.Rs may better mediate effector
functions and thereby destroy cancer cells more effectively in
patients. The balance between activating and inhibiting receptors
is an important consideration, and optimal effector function may
result from an Fc with enhanced affinity for activation receptors,
for example Fc.gamma.RI, Fc.gamma.RIIa/c, and Fc.gamma.RIIIa, yet
reduced affinity for the inhibitory receptor Fc.gamma.RIIb.
Furthermore, because Fc.gamma.Rs can mediate antigen uptake and
processing by antigen presenting cells, enhanced Fc/Fc.gamma.R
affinity may also improve the capacity of antibody therapeutics to
elicit an adaptive immune response.
[0014] Mutagenesis studies have been carried out on Fc towards
various goals, with substitutions typically made to alanine
(referred to as alanine scanning) or guided by sequence homology
substitutions (Duncan et al., 1988, Nature 332:563-564; Lund et
al., 1991, J Immunol 147:2657-2662; Lund et al., 1992, Mol Immunol
29:53-59; Jefferis et al., 1995, Immunol Lett 44:111-117; Lund et
al., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett
54:101-104; Lund et al., 1996, J Immunol 157:4963-4969; Armour et
al., 1999, Eur J Immunol 29:2613-2624; Shields et al., 2001, J Biol
Chem 276:6591-6604; Jefferis et al., 2002, Immunol Lett 82:57-65)
(U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573; PCT WO 00/42072;
PCT WO 99/58572). The majority of substitutions reduce or ablate
binding with Fc.gamma.Rs. However some success has been achieved at
obtaining Fc variants with higher Fc.gamma.R affinity. (See for
example U.S. Pat. No. 5,624,821, and PCT WO 00/42072). For example,
Winter and colleagues substituted the human amino acid at position
235 of mouse IgG2b antibody (a glutamic acid to leucine mutation)
that increased binding of the mouse antibody to human Fc.gamma.RI
by 100-fold (Duncan et al., 1988, Nature 332:563-564) (U.S. Pat.
No. 5,624,821). Shields et al. used alanine scanning mutagenesis to
map Fc residues important to Fc.gamma.R binding, followed by
substitution of select residues with non-alanine mutations (Shields
et al., 2001, J Biol Chem 276:6591-6604; Presta et al., 2002,
Biochem Soc Trans 30:487-490) (PCT WO 00/42072). Several mutations
disclosed in this study, including S298A, E333A, and K334A, show
enhanced binding to the activating receptor Fc.gamma.RIIIa and
reduced binding to the inhibitory receptor Fc.gamma.RIIb. These
mutations were combined to obtain double and triple mutation
variants that show additive improvements in binding. The best
variant disclosed in this study is a S298A/E333A/K334A triple
mutant with approximately a 1.7-fold increase in binding to F158
Fc.gamma.RIIIa, a 5-fold decrease in binding to Fc.gamma.RIIb, and
a 2.1-fold enhancement in ADCC.
[0015] Enhanced affinity of Fc for Fc.gamma.R has also been
achieved using engineered glycoforms generated by expression of
antibodies in engineered or variant cell lines (Umana et al., 1999,
Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng
74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740;
Shinkawa et al., 2003, J Biol Chem 278:3466-3473). This approach
has generated substantial enhancements of the capacity of
antibodies to bind Fc.gamma.RIIIa and to mediate ADCC. Although
there are practical limitations such as the growth efficiency of
the expression strains under large scale production conditions,
this approach for enhancing Fc/Fc.gamma.R affinity and effector
function is promising. Indeed, coupling of these alternate
glycoform technologies with the Fc variants of the present
invention may provide additive or synergistic effects for optimal
effector function.
[0016] Although there is a need for greater effector function, for
some antibody therapeutics reduced or eliminated effector function
may be desired. This is often the case for therapeutic antibodies
whose mechanism of action involves blocking or antagonism but not
killing of the cells bearing target antigen. In these cases
depletion of target cells is undesirable and can be considered a
side effect. For example, the ability of anti-CD4 antibodies to
block CD4 receptors on T cells makes them effective
anti-inflammatories, yet their ability to recruit Fc.gamma.R
receptors also directs immune attack against the target cells,
resulting in T cell depletion (Reddy et al., 2000, J Immunol
164:1925-1933). Effector function can also be a problem for
radiolabeled antibodies, referred to as radioconjugates, and
antibodies conjugated to toxins, referred to as immunotoxins. These
drugs can be used to destroy cancer cells, but the recruitment of
immune cells via Fc interaction with Fc.gamma.Rs brings healthy
immune cells in proximity to the deadly payload (radiation or
toxin), resulting in depletion of normal lymphoid tissue along with
targeted cancer cells (Hutchins et al., 1995, Proc Natl Acad Sci
USA 92:11980-11984; White et al., 2001, Annu Rev Med 52:125-145).
This problem can potentially be circumvented by using IgG isotypes
that poorly recruit complement or effector cells, for example IgG2
and IgG4. An alternate solution is to develop Fc variants that
reduce or ablate binding (Alegre et al., 1994, Transplantation
57:1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci USA
92:11980-11984; Armour et al., 1999, Eur J Immunol 29:2613-2624;
Reddy et al., 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell
Immunol 200:16-26; Shields et al., 2001, J Biol Chem 276:6591-6604)
(U.S. Pat. No. 6,194,551; U.S. Pat. No. 5,885,573; PCT WO
99/58572). A critical consideration for the reduction or
elimination of effector function is that other important antibody
properties not be perturbed. Fc variants should be engineered that
not only ablate binding to Fc.gamma.Rs and/or C1q, but also
maintain antibody stability, solubility, and structural integrity,
as well as ability to interact with other important Fc ligands such
as FcRn and proteins A and G.
[0017] The present invention addresses another major shortcoming of
antibodies, namely their demanding production requirements (Garber,
2001, Nat Biotechnol 19:184-185; Dove, 2002, Nat Biotechnol
20:777-779). Antibodies must be expressed in mammalian cells, and
the currently marketed antibodies together with other high-demand
biotherapeutics consume essentially all of the available
manufacturing capacity. With hundreds of biologics in development,
the majority of which are antibodies, there is an urgent need for
more efficient and cheaper methods of production. The downstream
effects of insufficient antibody manufacturing capacity are
three-fold. First, it dramatically raises the cost of goods to the
producer, a cost that is passed on to the patient. Second, it
hinders industrial production of approved antibody products,
limiting availability of high demand therapeutics to patients.
Finally, because clinical trials require large amounts of a protein
that is not yet profitable, the insufficient supply impedes
progress of the growing antibody pipeline to market.
[0018] Alternative production methods have been explored in
attempts at alleviating this problem. Transgenic plants and animals
are being pursued as potentially cheaper and higher capacity
production systems (Chadd et al., 2001, Curr Opin Biotechnol
12:188-194). Such expression systems, however, can generate
glycosylation patterns significantly different from human
glycoproteins. This may result in reduced or even lack of effector
function because, as discussed above, the carbohydrate structure
can significantly impact Fc.gamma.R and complement binding. A
potentially greater problem with nonhuman glycoforms may be
immunogenicity; carbohydrates are a key source of antigenicity for
the immune system, and the presence of nonhuman glycoforms has a
significant chance of eliciting antibodies that neutralize the
therapeutic, or worse cause adverse immune reactions. Thus the
efficacy and safety of antibodies produced by transgenic plants and
animals remains uncertain. Bacterial expression is another
attractive solution to the antibody production problem. Expression
in bacteria, for example E. coli, provides a cost-effective and
high capacity method for producing proteins. For complex proteins
such as antibodies there are a number of obstacles to bacterial
expression, including folding and assembly of these complex
molecules, proper disulfide formation, and solubility, stability,
and functionality in the absence of glycosylation because proteins
expressed in bacteria are not glycosylated. Full length
unglycosylated antibodies that bind antigen have been successfully
expressed in E. coli (Simmons et al., 2002, J Immunol Methods
263:133-147), and thus, folding, assembly, and proper disulfide
formation of bacterially expressed antibodies are possible in the
absence of the eukaryotic chaperone machinery. However the ultimate
utility of bacterially expressed antibodies as therapeutics remains
hindered by the lack of glycosylation, which results in lack
effector function and may result in poor stability and solubility.
This will likely be more problematic for formulation at the high
concentrations for the prolonged periods demanded by clinical
use.
[0019] An aglycosylated Fc with favorable solution properties and
the capacity to mediate effector functions would be significantly
enabling for the alternate production methods described above. By
overcoming the structural and functional shortcomings of
aglycosylated Fc, antibodies can be produced in bacteria and
transgenic plants and animals with reduced risk of immunogenicity,
and with effector function for clinical applications in which
cytotoxicity is desired such as cancer. The present invention
describes the utilization of protein engineering methods to develop
stable, soluble Fc variants with effector function. Currently, such
Fc variants do not exist in the art.
[0020] In summary, there is a need for antibodies with enhanced
therapeutic properties. Engineering of optimized or enhanced Fc
variants is a promising approach to meeting this need. Yet a
substantial obstacle to engineering Fc variants with the desired
properties is the difficulty in predicting what amino acid
modifications, out of the enormous number of possibilities, will
achieve the desired goals, coupled with the inefficient production
and screening methods for antibodies. Indeed one of the principle
reasons for the incomplete success of the prior art is that
approaches to Fc engineering have thus far involved hit-or-miss
methods such as alanine scans or production of glycoforms using
different expression strains. In these studies, the Fc
modifications that were made were fully or partly random in hopes
of obtaining variants with favorable properties. The present
invention provides a variety of engineering methods, many of which
are based on more sophisticated and efficient techniques, which may
be used to overcome these obstacles in order to develop Fc variants
that are optimized for the desired properties. The described
engineering methods provide design strategies to guide Fc
modification, computational screening methods to design favorable
Fc variants, library generation approaches for determining
promising variants for experimental investigation, and an array of
experimental production and screening methods for determining the
Fc variants with favorable properties.
SUMMARY OF THE INVENTION
[0021] The present invention provides Fc variants that are
optimized for a number of therapeutically relevant properties.
These Fc variants are generally contained within a variant protein,
that preferably comprises an antibody or a Fc fusion protein.
[0022] It is an object of the present invention to provide novel Fc
positions at which amino acid modifications may be made to generate
optimized Fc variants. Said Fc positions include 230, 240, 244,
245, 247, 262, 263, 266, 273, 275, 299, 302, 313, 323, 325, 328,
and 332, wherein the numbering of the residues in the Fc region is
that of the EU index as in Kabat. The present invention describes
any amino acid modification at any of said novel Fc positions in
order to generate an optimized Fc variant.
[0023] It is a further object of the present invention to provide
Fc variants that have been screened computationally. A
computationally screened Fc variant is one that is predicted by the
computational screening calculations described herein as having a
significantly greater potential than random for being optimized for
a desired property. In this way, computational screening serves as
a prelude to or surrogate for experimental screening, and thus said
computationally screened Fc variants are considered novel.
[0024] It is a further object of the present invention to provide
Fc variants that have been characterized using one or more of the
experimental methods described herein. In one embodiment, said Fc
variants comprise at least one amino acid substitution at a
position selected from the group consisting of: 230, 233, 234, 235,
239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267,
269, 270, 272, 273, 274, 275, 276, 278, 283, 296, 297, 298, 299,
302, 313, 318, 320, 323, 324, 325, 326, 327, 328, 329, 330, 331,
332, 333, 334, and 335, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat. In one
embodiment, said Fc variants comprise at least one amino acid
substitution at a position selected from the group consisting of:
221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237,
238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262,
263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,
276, 278, 280, 281, 283, 285, 286, 288, 290, 291, 293, 294, 295,
296, 297, 298, 299, 300, 302, 313, 317, 318, 320, 322, 323, 324,
325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 336 and 428,
wherein the numbering of the residues in the Fc region is that of
the EU index as in Kabat. In a preferred embodiment, said Fc
variants comprise at least one substitution selected from the group
consisting of P230A, E233D, L234D, L234E, L234N, L234Q, L234T,
L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q,
L235T, L235H, L235Y, L235I, L235V, L235F, S239D, S239E, S239N,
S239Q, S239F, S239T, S239H, S239Y, V240I, V240A, V240T, V240M,
F241W, F241L, F241Y, F241E, F241R, F243W, F243L F243Y, F243R,
F243Q, P244H, P245A, P247V, P247G, V262I, V262A, V262T, V262E,
V263I, V263A, V263T, V263M, V264L, V264I, V264W, V264T, V264R,
V264F, V264M, V264Y, V264E, D265G, D265N, D265Q, D265Y, D265F,
D265V, D265I, D265L, D265H, D265T, V266I, V266A, V266T, V266M,
S267Q, S267L, S267T, S267H, S267D, S267N, E269H, E269Y, E269F,
E269R, E269T, E269L, E269N, D270Q, D270T, D270H, E272S, E272K,
E272I, E272Y, V273I, K274T, K274E, K274R, K274L, K274Y, F275W,
N276S, N276E, N276R, N276L, N276Y, Y278T, Y278E, Y278K, Y278W,
E283R, Y296E, Y296Q, Y296D, Y296N, Y296S, Y296T, Y296L, Y296I,
Y296H, N297S, N297D, N297E, A298H, T299I, T299L, T299A, T299S,
T299V, T299H, T299F, T299E, V302I, W313F, E318R, K320T, K320D,
K320I, K322T, K322H, V323I, S324T, S324D, S324R, S324I, S324V,
S324L, S324Y, N325Q, N325L, N325I, N325D, N325E, N325A, N325T,
N325V, N325H, K326L, K326I, K326T, A327N, A327L, A327D, A327T,
L328M, L328D, L328E, L328N, L328Q, L328F, L328I, L328V, L328T,
L328H, L328A, P329F, A330L, A330Y, A330V, A330I, A330F, A330R,
A330H, A330S, A330W, A330M, P331V, P331H, I332D, I332E, I332N,
I332Q, I332T, I332H, I332Y, I332A, E333T, E333H, E333I, E333Y,
K334I, K334T, K334F, T335D, T335R, and T335Y, wherein the numbering
of the residues in the Fc region is that of the EU index as in
Kabat. In a mostly preferred embodiment, said Fc variants are
selected from the group consisting of V264L, V264I, F241W, F241L,
F243W, F243L, F241L/F243L/V262I/V264I, F241W/F243W,
F241W/F243W/V262A/V264A, F241L/V262I, F243L/V264I,
F243L/V262I/V264W, F241Y/F243Y/V262T/V264T,
F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,
F241R/F243Q/V262T/V264R, F241E/F243Y/V262T/V264R, L328M, L328E,
L328F, I332E, L328M/I332E, P244H, P245A, P247V, W313F,
P244H/P245A/P247V, P247G, V264I/I332E,
F241E/F243R/V262E/V264R/I332E, F241E/F243Q/V262T/V264E/I332E,
F241R/F243Q/V262T/V264R/I332E, F241E/F243Y/V262T/V264R/I332E,
S298A/I332E, S239E/I332E, S239Q/I332E, S239E, D265G, D265N,
S239E/D265G, S239E/D265N, S239E/D265Q, Y296E, Y296Q, T299I, A327N,
S267Q/A327S, S267L/A327S, A327L, P329F, A330L, A330Y, I332D, N297S,
N297D, N297S/I332E, N297D/I332E, N297E/I332E, D265Y/N297D/I332E,
D265Y/N297D/T299L/I332E, D265F/N297E/I332E, L328I/I332E,
L328Q/I332E, I332N, I332Q, V264T, V264F, V240I, V263I, V266I,
T299A, T299S, T299V, N325Q, N325L, N325I, S239D, S239N, S239F,
S239D/I332D, S239D/I332E, S239D/I332N, S239D/I332Q, S239E/I332D,
S239E/I332N, S239E/I332Q, S239N/I332D, S239N/I332E, S239N/I332N,
S239N/I332Q, S239Q/I332D, S239Q/I332N, S239Q/I332Q, Y296D, Y296N,
F241Y/F243Y/V262T/V264T/N297D/I332E, A330Y/I332E,
V264I/A330Y/I332E, A330L/I332E, V264I/A330L/I332E, L234D, L234E,
L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D,
L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F,
S239T, S239H, S239Y, V240A, V240T, V240M, V263A, V263T, V263M,
V264M, V264Y, V266A, V266T, V266M, E269H, E269Y, E269F, E269R,
Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V, A330I, A330F,
A330R, A330H, N325D, N325E, N325A, N325T, N325V, N325H,
L328D/I332E, L328E/I332E, L328N/I332E, L328Q/I332E, L328V/I332E,
L328T/I332E, L328H/I332E, L328I/I332E, L328A, I332T, I332H, I332Y,
I332A, S239E/V264I/I332E, S239Q/V264I/I332E,
S239E/V264I/A330Y/I332E, S239E/V264I/S298A/A330Y/I332E,
S239D/N297D/I332E, S239E/N297D/I332E, S239D/D265V/N297D/I332E,
S239D/D265I/N297D/I332E, S239D/D265L/N297D/I332E,
S239D/D265F/N297D/I332E, S239D/D265Y/N297D/I332E,
S239D/D265H/N297D/I332E, S239D/D265T/N297D/I332E,
V264E/N297D/I332E, Y296D/N297D/I332E, Y296E/N297D/I332E,
Y296N/N297D/I332E, Y296Q/N297D/I332E, Y296H/N297D/I332E,
Y296T/N297D/I332E, N297D/T299V/I332E, N297D/T299I/I332E,
N297D/T299L/I332E, N297D/T299F/I332E, N297D/T299H/I332E,
N297D/T299E/I332E, N297D/A330Y/I332E, N297D/S298A/A330Y/I332E,
S239D/A330Y/I332E, S239N/A330Y/I332E, S239D/A330L/I332E,
S239N/A330L/I332E, V264I/S298A/I332E, S239D/S298A/I332E,
S239N/S298A/I332E, S239D/V264I/I332E, S239D/V264I/S298A/I332E,
S239D/V264I/A330L/I332E, L328N, L328H, S239D/I332E/A330I,
N297D/I332E/S239D/A330L, P230A, E233D, P230A/E233D,
P230A/E233D/I332E, S267T, S267H, S267D, S267N, E269T, E269L, E269N,
D270Q, D270T, D270H, E272S, E272K, E272I, E272Y, V273I, K274T,
K274E, K274R, K274L, K274Y, F275W, N276S, N276E, N276R, N276L,
N276Y, Y278T, Y278E, Y278K, Y278W, E283R, V302I, E318R, K320T,
K320D, K320I, K322T, K322H, V323I, S324T, S324D, S324R, S324I,
S324V, S324L, S324Y, K326L, K326I, K326T, A327D, A327T, A330S,
A330W, A330M, P331V, P331H, E333T, E333H, E333I, E333Y, K334I,
K334T, K334F, T335D, T335R, T335Y, L234I/L235D, V240I/V266I,
S239D/A330Y/I332E/L234I, S239D/A330Y/I332E/L235D,
S239D/A330Y/I332E/V240I, S239D/A330Y/I332E/V264T,
S239D/A330Y/I332E/V266I, S239D/A330Y/I332E/K326E,
S239D/A330Y/I332E/K326T, S239D/N297D/I332E/A330Y,
S239D/N297D/I332E/A330Y/F241S/F243H/V262T/V264T,
S239D/N297D/I332E/L235D, and S239D/N297D/I332E/K326E, wherein the
numbering of the residues in the Fc region is that of the EU index
as in Kabat.
[0025] It is a further object of the present invention to provide
Fc variants that are selected from the group consisting of D221K,
D221Y, K222E, K222Y, T223E, T223K, H224E, H224Y, T225E, T225,
T225K, T225W, P227E, P227K, P227Y, P227G, P228E, P228K, P228Y,
P228G, P230E, P230Y, P230G, A231E, A231K, A231Y, A231P, A231G,
P232E, P232K, P232Y, P232G, E233N, E233Q, E233K, E233R, E233S,
E233T, E233H, E233A, E233V, E233L, E233I, E233F, E233M, E233Y,
E233W, E233G, L234K, L234R, L234S, L234A, L234M, L234W, L234P,
L234G, L235E, L235K, L235R, L235A, L235M, L235W, L235P, L235G,
G236D, G236E, G236N, G236Q, G236K, G236R, G236S, G236T, G236H,
G236A, G236V, G236L, G236I, G236F, G236M, G236Y, G236W, G236P,
G237D, G237E, G237N, G237Q, G237K, G237R, G237S, G237T, G237H,
G237V, G237L, G237I, G237F, G237M, G237Y, G237W, G237P, P238D,
P238E, P238N, P238Q, P238K, P238R, P238S, P238T, P238H, P238V,
P238L, P238I, P238F, P238M, P238Y, P238W, P238G, S239Q, S239K,
S239R, S239V, S239L, S239I, S239M, S239W, S239P, S239G, F241D,
F241E, F241Y, F243E, K246D, K246E, K246H, K246Y, D249Q, D249H,
D249Y, R255E, R255Y, E258S, E258H, E258Y, T260D, T260E, T260H,
T260Y, V262E, V262F, V264D, V264E, V264N, V264Q, V264K, V264R,
V264S, V264H, V264W, V264P, V264G, D265Q, D265K, D265R, D265S,
D265T, D265H, D265V, D265L, D265I, D265F, D265M, D265Y, D265W,
D265P, S267E, S267Q, S267K, S267R, S267V, S267L, S267I, S267F,
S267M, S267Y, S267W, S267P, H268D, H268E, H268Q, H268K, H268R,
H268T, H268V, H268L, H268I, H268F, H268M, H268W, H268P, H268G,
E269K, E269S, E269V, E269I, E269M, E269W, E269P, E269G, D270R,
D270S, D270L, D270I, D270F, D270M, D270Y, D270W, D270P, D270G,
P271D, P271E, P271N, P271Q, P271K, P271R, P271S, P271T, P271H,
P271A, P271V, P271L, P271I, P271F, P271M, P271Y, P271W, P271G,
E272D, E272R, E272T, E272H, E272V, E272L, E272F, E272M, E272W,
E272P, E272G, K274D, K274N, K274S, K274H, K274V, K274I, K274F,
K274M, K274W, K274P, K274G, F275L, N276D, N276T, N276H, N276V,
N276I, N276F, N276M, N276W, N276P, N276G, Y278D, Y278N, Y278Q,
Y278R, Y278S, Y278H, Y278V, Y278L, Y278I, Y278M, Y278P, Y278G,
D280K, D280L, D280W, D280P, D280G, G281D, G281K, G281Y, G281P,
V282E, V282K, V282Y, V282P, V282G, E283K, E283H, E283L, E283Y,
E283P, E283G, V284E, V284N, V284T, V284L, V284Y, H285D, H285E,
H285Q, H285K, H285Y, H285W, N286E, N286Y, N286P, N286G, K288D,
K288E, K288Y, K290D, K290N, K290H, K290L, K290W, P291D, P291E,
P291Q, P291T, P291H, P291I, P291G, R292D, R292E, R292T, R292Y,
E293N, E293R, E293S, E293T, E293H, E293V, E293L, E293I, E293F,
E293M, E293Y, E293W, E293P, E293G, E294K, E294R, E294S, E294T,
E294H, E294V, E294L, E294I, E294F, E294M, E294Y, E294W, E294P,
E294G, Q295D, Q295E, Q295N, Q295R, Q295S, Q295T, Q295H, Q295V,
Q295I, Q295F, Q295M, Q295Y, Q295W, Q295P, Q295G, Y296K, Y296R,
Y296A, Y296V, Y296M, Y296G, N297Q, N297K, N297R, N297T, N297H,
N297V, N297L, N297I, N297F, N297M, N297Y, N297W, N297P, N297G,
S298D, S298E, S298Q, S298K, S298R, S298I, S298F, S298M, S298Y,
S298W, T299D, T299E, T299N, T299Q, T299K, T299R, T299L, T299F,
T299M, T299Y, T299W, T299P, T299G, Y300D, Y300E, Y300N, Y300Q,
Y300K, Y300R, Y300S, Y300T, Y300H, Y300A, Y300V, Y300M, Y300W,
Y300P, Y300G, R301D, R301E, R301H, R301Y, V303D, V303E, V303Y,
S304D, S304N, S304T, S304H, S304L, V305E, V305T, V305Y, K317E,
K317Q, E318Q, E318H, E318L, E318Y, K320N, K320S, K320H, K320V,
K320L, K320F, K320Y, K320W, K320P, K320G, K322D, K322S, K322V,
K322I, K322F, K322Y, K322W, K322P, K322G, S324H, S324F, S324M,
S324W, S324P, S324G, N325K, N325R, N325S, N325F, N325M, N325Y,
N325W, N325P, N325G, K326P, A327E, A327K, A327R, A327H, A327V,
A327I, A327F, A327M, A327Y, A327W, A327P, L328D, L328Q, L328K,
L328R, L328S, L328T, L328V, L328I, L328Y, L328W, L328P, L328G,
P329D, P329E, P329N, P329Q, P329K, P329R, P329S, P329T, P329H,
P329V, P329L, P329I, P329M, P329Y, P329W, P329G, A330E, A330N,
A330T, A330P, A330G, P331D, P331Q, P331R, P331T, P331L, P331I,
P331F, P331M, P331Y, P331W, I332K, I332R, I332S, I332V, I332L,
I332F, I332M, I332W, I332P, I332G, E333L, E333F, E333M, E333P,
K334P, T335N, T335S, T335H, T335V, T335L, T335I, T335F, T335M,
T335W, T335P, T335G, I336E, I336K, I336Y, S337E, S337N, and S337H,
wherein the numbering of the residues in the Fc region is that of
the EU index as in Kabat.
[0026] It is a further object of the present invention to provide
an Fc variant that binds with greater affinity to one or more
Fc.gamma.Rs. In one embodiment, said Fc variants have affinity for
an Fc.gamma.R that is more than 1-fold greater than that of the
parent Fc polypeptide. In an alternate embodiment, said Fc variants
have affinity for an Fc.gamma.R that is more than 5-fold greater
than that of the parent Fc polypeptide. In a preferred embodiment,
said Fc variants have affinity for an Fc.gamma.R that is between
5-fold and 300-fold greater than that of the parent Fc polypeptide.
In one embodiment, said Fc variants comprise at least one amino
acid substitution at a position selected from the group consisting
of: 230, 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275,
276, 278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, wherein
the numbering of the residues in the Fc region is that of the EU
index as in Kabat. In a preferred embodiment, said Fc variants
comprise at least one amino acid substitution selected from the
group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D,
L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I,
V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E,
K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D,
S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D,
L328V, L328T, A330Y, A330L, A3301, 1332D, I332E, I332N, I332Q,
T335D, T335R, and T335Y, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat. In a mostly
preferred embodiment, said Fc variants are selected from the group
consisting of V264I, F243L/V264I, L328M, I332E, L328M/I332E,
V264I/I332E, S298A/I332E, S239E/I332E, S239Q/I332E, S239E, A330Y,
I332D, L328I/I332E, L328Q/I332E, V264T, V240I, V266I, S239D,
S239D/I332D, S239D/I332E, S239D/I332N, S239D/I332Q, S239E/I332D,
S239E/I332N, S239E/I332Q, S239N/I332D, S239N/I332E, S239Q/I332D,
A330Y/I332E, V264I/A330Y/I332E, A330L/I332E, V264I/A330L/I332E,
L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M,
V264Y, A330I, N325T, L328D/I332E, L328V/I332E, L328T/I332E,
L328I/I332E, S239E/V264I/I332E, S239Q/V264I/I332E,
S239E/V264I/A330Y/I332E, S239D/A330Y/I332E, S239N/A330Y/I332E,
S239D/A330L/I332E, S239N/A330L/I332E, V264I/S298A/I332E,
S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264I/I332E,
S239D/V264I/S298A/I332E, S239D/V264I/A330L/I332E,
S239D/I332E/A330I, P230A, P230A/E233D/I332E, E272Y, K274T, K274E,
K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D,
S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I/V266I,
S239D/A330Y/I332E/L234I, S239D/A330Y/I332E/L235D, S239D/A330Y/I332
E/V240I, S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, and
S239D/A330Y/I332E/K326T, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat.
[0027] It is a further object of the present invention to provide
Fc variant that have a Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold ratio
greater than 1:1. In one embodiment, said Fc variants have a
Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold ratio greater than 11:1. In
a preferred embodiment, said Fc variants have a
Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold ratio between 11:1 and 86:1.
In one embodiment, said Fc variants comprise at least one amino
acid substitution at a position selected from the group consisting
of: 234, 235, 239, 240, 264, 296, 330, and 1332, wherein the
numbering of the residues in the Fc region is that of the EU index
as in Kabat. In a preferred embodiment, said Fc variants comprise
at least one amino acid substitution selected from the group
consisting of: L234Y, L234I, L235I, S239D, S239E, S239N, S239Q,
V240A, V240M, V264I, V264Y, Y296Q, A330L, A330Y, A330I, I332D, and
I332E, wherein the numbering of the residues in the Fc region is
that of the EU index as in Kabat. In a mostly preferred embodiment,
said Fc variants are selected from the group consisting of: I332E,
V264I/I332E, S239E/I332E, S239Q/I332E, Y296Q, A330L, A330Y, I332D,
S239D, S239D/I332E, A330Y/I332E, V264I/A330Y/I332E, A330L/I332E,
V264I/A330L/I332E, L234Y, L234I, L235I, V240A, V240M, V264Y, A330I,
S239D/A330L/I332E, S239D/S298A/I332E, S239N/S298A/I332E,
S239D/V264I/I332E, S239D/V264I/S298A/I332E, and
S239D/V264I/A330L/I332E, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat.
[0028] It is a further object of the present invention to provide
Fc variants that mediate effector function more effectively in the
presence of effector cells. In one embodiment, said Fc variants
mediate ADCC that is greater than that mediated by the parent Fc
polypeptide. In a preferred embodiment, said Fc variants mediate
ADCC that is more than 5-fold greater than that mediated by the
parent Fc polypeptide. In a mostly preferred embodiment, said Fc
variants mediate ADCC that is between 5-fold and 1000-fold greater
than that mediated by the parent Fc polypeptide. In one embodiment,
said Fc variants comprise at least one amino acid substitution at a
position selected from the group consisting of: 230, 233, 234, 235,
239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324,
325, 326, 328, 330, 332, and 335, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat. In a
preferred embodiment, said Fc variants comprise at least one amino
acid substitutions selected from the group consisting of: P230A,
E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D,
S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T,
V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W,
N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I,
K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L,
A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y, wherein
the numbering of the residues in the Fc region is that of the EU
index as in Kabat. In a mostly preferred embodiment, said Fc
variants are selected from the group consisting of: V264I,
F243L/V264I, L328M, I332E, L328M/I332E, V264I/I332E, S298A/I332E,
S239E/I332E, S239Q/I332E, S239E, A330Y, I332D, L328I/I332E,
L328Q/I332E, V264T, V240I, V266I, S239D, S239D/I332D, S239D/I332E,
S239D/I332N, S239D/I332Q, S239E/I332D, S239E/I332N, S239E/I332Q,
S239N/I332D, S239N/I332E, S239Q/I332D, A330Y/I332E,
V264I/A330Y/I332E, A330L/I332E, V264I/A330L/I332E, L234E, L234Y,
L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I,
N325T, L328D/I332E, L328V/I332E, L328T/I332E, L328I/I332E,
S239E/V264I/I332E, S239Q/V264I/I332E, S239E/V264I/A330Y/I332E,
S239D/A330Y/I332E, S239N/A330Y/I332E, S239D/A330L/I332E,
S239N/A330L/I332E, V264I/S298A/I332E, S239D/S298A/I332E,
S239N/S298A/I332E, S239D/V264I/I332E, S239D/V264I/S298A/1332 E,
S239D/V264I/A330L/I332 E, S239D/I332E/A330I, P230A,
P230A/E233D/I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W,
N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T,
T335D, T335R, T335Y, V240I/V266I, S239D/A330Y/I332E/L234I,
S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I,
S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, and
S239D/A330Y/I332E/K326T, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat.
[0029] It is a further object of the present invention to provide
Fc variants that bind with weaker affinity to one or more
Fc.gamma.Rs. In one embodiment, said Fc variants comprise at least
one amino acid substitution at a position selected from the group
consisting of: 230, 233, 234, 235, 239, 240, 241, 243, 244, 245,
247, 262, 263, 264, 265, 266, 267, 269, 270, 273, 276, 278, 283,
296, 297, 298, 299, 313, 323, 324, 325, 327, 328, 329, 330, 332,
and 333, wherein the numbering of the residues in the Fc region is
that of the EU index as in Kabat. In a preferred embodiment, said
Fc variants comprise an amino acid substitution at a position
selected from the group consisting of: P230A, E233D, L234D, L234N,
L234Q, L234T, L234H, L234V, L234F, L234I, L235N, L235Q, L235T,
L235H, L235V, L235F, L235D, S239E, S239N, S239Q, S239F, S239H,
S239Y, V240A, V240T, F241W, F241L, F241Y, F241E, F241R, F243W,
F243L F243Y, F243R, F243Q, P244H, P245A, P247V, P247G, V262I,
V262A, V262T, V262E, V263I, V263A, V263T, V263M, V264L, V264I,
V264W, V264T, V264R, V264F, V264M, V264E, D265G, D265N, D265Q,
D265Y, D265F, D265V, D265I, D265L, D265H, D265T, V266A, V266T,
V266M, S267Q, S267L, E269H, E269Y, E269F, E269R, E269T, E269L,
E269N, D270Q, D270T, D270H, V273I, N276S, N276E, N276R, N276Y,
Y278E, Y278W, E283R, Y296E, Y296Q, Y296D, Y296N, Y296S, Y296T,
Y296L, Y296I, Y296H, N297S, N297D, N297E, A298H, T299I, T299L,
T299A, T299S, T299V, T299H, T299F, T299E, W313F, V323I, S324R,
S324L, S324Y, N325Q, N325L, N325I, N325D, N325E, N325A, N325V,
N325H, A327N, A327L, L328M, 328E, L328N, L328Q, A327D, A327T,
L328F, L328H, L328A, L328N, L328H, P329F, A330L, A330V, A330F,
A330R, A330H, I332N, I332Q, I332T, I332H, I332Y, I332A, E333T, and
E333H, wherein the numbering of the residues in the Fc region is
that of the EU index as in Kabat. In a mostly preferred embodiment,
said Fc variants are selected from the group consisting of: V264L,
F241W, F241L, F243W, F243L, F241L/F243L/V262I/V264I, F241W/F243W,
F241W/F243W/V262A/V264A, F241L/V262I, F243L/V262I/V264W,
F241Y/F243Y/V262T/V264T, F241E/F243R/V262E/V264R,
F241E/F243Q/V262T/V264E, F241R/F243Q/V262T/V264R,
F241E/F243Y/V262T/V264R, L328M, L328E, L328F, P244H, P245A, P247V,
W313F, P244H/P245A/P247V, P247G, F241E/F243R/V262E/V264R/I332E,
F241E/F243Y/V262T/V264R/I332E, D265G, D265N, S239E/D265G,
S239E/D265N, S239E/D265Q, Y296E, Y296Q, T299I, A327N, S267Q/A327S,
S267L/A327S, A327L, P329F, A330L, N297S, N297D, N297S/I332E, I332N,
I332Q, V264F, V263I, T299A, T299S, T299V, N325Q, N325L, N325I,
S239N, S239F, S239N/I332N, S239N/I332Q, S239Q/I332N, S239Q/I332Q,
Y296D, Y296N, L234D, L234N, L234Q, L234T, L234H, L234V, L234F,
L235N, L235Q, L235T, L235H, L235V, L235F, S239H, S239Y, V240A,
V263T, V263M, V264M, V266A, V266T, V266M, E269H, E269Y, E269F,
E269R, Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V, A330F,
A330R, A330H, N325D, N325E, N325A, N325V, N325H, L328E/I332E,
L328N/I332E, L328Q/I332E, L328H/I332E, L328A, I332T, I332H, I332Y,
I332A, L328N, L328H, E233D, P230A/E233D, E269T, E269L, E269N,
D270Q, D270T, D270H, V273I, N276S, N276E, N276R, N276Y, Y278E,
Y278W, E283R, V323I, S324R, S324L, S324Y, A327D, A327T, E333T,
E333H, and L234I/L235D, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat.
[0030] It is a further object of the present invention to provide
Fc variants that mediate ADCC in the presence of effector cells
less effectively. In one embodiment, said Fc variants comprise at
least one amino acid substitution at a position selected from the
group consisting of: 230, 233, 234, 235, 239, 240, 241, 243, 244,
245, 247, 262, 263, 264, 265, 266, 267, 269, 270, 273, 276, 278,
283, 296, 297, 298, 299, 313, 323, 324, 325, 327, 328, 329, 330,
332, and 333, wherein the numbering of the residues in the Fc
region is that of the EU index as in Kabat. In a preferred
embodiment, said Fc variants comprise at least one amino acid
substitution at a position selected from the group consisting of:
P230A, E233D, L234D, L234N, L234Q, L234T, L234H, L234V, L234F,
L234I, L235N, L235Q, L235T, L235H, L235V, L235F, L235D, S239E,
S239N, S239Q, S239F, S239H, S239Y, V240A, V240T, F241W, F241L,
F241Y, F241E, F241R, F243W, F243L F243Y, F243R, F243Q, P244H,
P245A, P247V, P247G, V262I, V262A, V262T, V262E, V263I, V263A,
V263T, V263M, V264L, V264I, V264W, V264T, V264R, V264F, V264M,
V264E, D265G, D265N, D265Q, D265Y, D265F, D265V, D265I, D265L,
D265H, D265T, V266A, V266T, V266M, S267Q, S267L, E269H, E269Y,
E269F, E269R, E269T, E269L, E269N, D270Q, D270T, D270H, V273I,
N276S, N276E, N276R, N276Y, Y278E, Y278W, E283R, Y296E, Y296Q,
Y296D, Y296N, Y296S, Y296T, Y296L, Y296I, Y296H, N297S, N297D,
N297E, A298H, T299I, T299L, T299A, T299S, T299V, T299H, T299F,
T299E, W313F, V323I, S324R, S324L, S324Y, N325Q, N325L, N325I,
N325D, N325E, N325A, N325V, N325H, A327N, A327L, L328M, 328E,
L328N, L328Q, A327D, A327T, L328F, L328H, L328A, L328N, L328H,
P329F, A330L, A330V, A330F, A330R, A330H, I332N, I332Q, I332T,
I332H, I332Y, I332A, E333T, and E333H, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat. In a
mostly preferred embodiment, said Fc variants are selected from the
group consisting of: V264L, F241W, F241L, F243W, F243L,
F241L/F243L/V262I/V264I, F241W/F243W, F241W/F243W/V262A/V264A,
F241L/V262I, F243L/V262I/V264W, F241Y/F243Y/V262T/V264T,
F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,
F241R/F243Q/V262T/V264R, F241E/F243Y/V262T/V264R, L328M, L328E,
L328F, P244H, P245A, P247V, W313F, P244H/P245A/P247V, P247G,
F241E/F243R/V262E/V264R/I332E, F241E/F243Y/V262T/V264R/I332E,
D265G, D265N, S239E/D265G, S239E/D265N, S239E/D265Q, Y296E, Y296Q,
T299I, A327N, S267Q/A327S, S267L/A327S, A327L, P329F, A330L, N297S,
N297D, N297S/I332E, I332N, I332Q, V264F, V263I, T299A, T299S,
T299V, N325Q, N325L, N325I, S239N, S239F, S239N/I332N, S239N/I332Q,
S239Q/I332N, S239Q/I332Q, Y296D, Y296N, L234D, L234N, L234Q, L234T,
L234H, L234V, L234F, L235N, L235Q, L235T, L235H, L235V, L235F,
S239H, S239Y, V240A, V263T, V263M, V264M, V266A, V266T, V266M,
E269H, E269Y, E269F, E269R, Y296S, Y296T, Y296L, Y296I, A298H,
T299H, A330V, A330F, A330R, A330H, N325D, N325E, N325A, N325V,
N325H, L328E/I332E, L328N/I332E, L328Q/I332E, L328H/I332E, L328A,
I332T, I332H, I332Y, I332A, L328N, L328H, E233D, P230A/E233D,
E269T, E269L, E269N, D270Q, D270T, D270H, V273I, N276S, N276E,
N276R, N276Y, Y278E, Y278W, E283R, V323I, S324R, S324L, S324Y,
A327D, A327T, E333T, E333H, and L234I/L235D, wherein the numbering
of the residues in the Fc region is that of the EU index as in
Kabat.
[0031] It is a further object of the present invention to provide
Fc variants that have improved function and/or solution properties
as compared to the aglycosylated form of the parent Fc polypeptide.
Improved functionality herein includes but is not limited to
binding affinity to an Fc ligand. Improved solution properties
herein includes but is not limited to stability and solubility. In
one embodiment, said aglycosylated Fc variants bind to an
Fc.gamma.R with an affinity that is comparable to or better than
the glycosylated parent Fc polypeptide. In an alternate embodiment,
said Fc variants bind to an Fc.gamma.R with an affinity that is
within 0.4-fold of the glycosylated form of the parent Fc
polypeptide. In one embodiment, said Fc variants comprise at least
one amino acid substitution at a position selected from the group
consisting of: 239, 241, 243, 262, 264, 265, 296, 297, 330, and
332, wherein the numbering of the residues in the Fc region is that
of the EU index as in Kabat. In a preferred embodiment, said Fc
variants comprise an amino acid substitution selected from the
group consisting of: S239D, S239E, F241Y, F243Y, V262T, V264T,
V264E, D265Y, D265H, D265V, D265I, Y296N, N297D, A330Y, and I332E,
wherein the numbering of the residues in the Fc region is that of
the EU index as in Kabat. In a mostly preferred embodiment, said Fc
variants are selected from the group consisting of: N297D/I332E,
F241Y/F243Y/V262T/V264T/N297D/I332E, S239D/N297D/I332E,
S239E/N297D/I332E, S239D/D265Y/N297D/I332E,
S239D/D265H/N297D/I332E, V264E/N297D/I332E, Y296N/N297D/I332E,
N297D/A330Y/I332E, S239D/D265V/N297D/I332E,
S239D/D265I/N297D/I332E, and N297D/S298A/A330Y/I332E, wherein the
numbering of the residues in the Fc region is that of the EU index
as in Kabat.
[0032] The present invention also provides methods for engineering
optimized Fc variants. It is an object of the present invention to
provide design strategies that may be used to guide Fc
optimization. It is a further object of the present invention to
provide computational screening methods that may be used to design
Fc variants. It is a further object of the present invention to
provide methods for generating libraries for experimental testing.
It is a further object of the present invention to provide
experimental production and screening methods for obtaining
optimized Fc variants.
[0033] The present invention provides isolated nucleic acids
encoding the Fc variants described herein. The present invention
provides vectors comprising said nucleic acids, optionally,
operably linked to control sequences. The present invention
provides host cells containing the vectors, and methods for
producing and optionally recovering the Fc variants.
[0034] The present invention provides novel antibodies and Fc
fusions that comprise the Fc variants disclosed herein. Said novel
antibodies and Fc fusions may find use in a therapeutic
product.
[0035] The present invention provides compositions comprising
antibodies and Fc fusions that comprise the Fc variants described
herein, and a physiologically or pharmaceutically acceptable
carrier or diluent.
[0036] The present invention contemplates therapeutic and
diagnostic uses for antibodies and Fc fusions that comprise the Fc
variants disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1. Antibody structure and function. Shown is a model of
a full length human IgG1 antibody, modeled using a humanized Fab
structure from pdb accession code 1CE1 (James et al., 1999, J Mol
Biol 289:293-301) and a human IgG1 Fc structure from pdb accession
code 1DN2 (DeLano et al., 2000, Science 287:1279-1283). The
flexible hinge that links the Fab and Fc regions is not shown. IgG1
is a homodimer of heterodimers, made up of two light chains and two
heavy chains. The Ig domains that comprise the antibody are
labeled, and include V.sub.L and C.sub.L for the light chain, and
V.sub.H, Cgamma1 (C.gamma.1), Cgamma2 (C.gamma.2), and Cgamma3
(C.gamma.3) for the heavy chain. The Fc region is labeled. Binding
sites for relevant proteins are labeled, including the antigen
binding site in the variable region, and the binding sites for
Fc.gamma.Rs, FcRn, C1q, and proteins A and G in the Fc region.
[0038] FIG. 2. The Fc/Fc.gamma.RIIb complex structure 1IIS. Fc is
shown as a gray ribbon diagram, and Fc.gamma.RIIIb is shown as a
black ribbon. The N297 carbohydrate is shown as black sticks.
[0039] FIG. 3. The amino acid sequence of the heavy chain of the
antibody alemtuzumab (Campath.RTM., a registered trademark of Ilex
Pharmaceuticals LP) (SEQ ID NO:1), illustrating positions numbered
sequentially (2 lines above the amino acid sequence) and positions
numbered according to the EU index as in Kabat (2 lines below the
amino acid sequence. The approximate beginnings of Ig domains VH1,
C.gamma.1, the hinge, C.gamma.2, and C.gamma.3 are also labeled
above the sequential numbering. Polymorphisms have been observed at
a number of Fc positions, including but not limited to Kabat 270,
272, 312, 315, 356, and 358, and thus slight differences between
the presented sequence and sequences in the prior art may
exist.
[0040] FIG. 4. Experimental library residues mapped onto the
Fc/Fc.gamma.RIIIb complex structure 1IIS. Fc is shown as a gray
ribbon diagram, and Fc.gamma.RIIIb is shown as a black ribbon.
Experimental library residues are shown in black, the N297
carbohydrate is shown in grey.
[0041] FIG. 5. The human IgG1 Fc sequence (SEQ ID NO:2) showing
positions relevant to the design of the Fc variant experimental
library. The sequence includes the hinge region, domain C.gamma.2,
and domain C.gamma.3. Residue numbers are according to the EU index
as in Kabat. Positions relevant to the experimental library are
underlined. Because of observed polymorphic mutations at a number
of Fc positions, slight differences between the presented sequence
and sequences in the literature may exist.
[0042] FIG. 6. Expression of Fc variant and wild type (WT) proteins
of alemtuzumab in 293T cells. Plasmids containing alemtuzumab heavy
chain genes (WT or variants) were co-transfected with plasmid
containing the alemtuzumab light chain gene. Media were harvested 5
days after transfection. For each transfected sample, 10 ul medium
was loaded on a SDS-PAGE gel for Western analysis. The probe for
Western was peroxidase-conjugated goat-anti human IgG (Jackson
Immuno-Research, catalog #109-035-088). WT: wild type alemtuzumab;
1-10: alemtuzumab variants. H and L indicate antibody heavy chain
and light chain, respectively.
[0043] FIG. 7. Purification of alemtuzumab using protein A
chromatography. WT alemtuzumab proteins was expressed in 293T cells
and the media was harvested 5 days after transfection. The media
were diluted 1:1 with PBS and purified with protein A (Pierce,
Catalog #20334). O: original sample before purification; FT: flow
through; E: elution; C: concentrated final sample. The left picture
shows a Simple Blue-stained SDS-PAGE gel, and the right shows a
western blot labeled using peroxidase-conjugated goat-anti human
IgG.
[0044] FIG. 8. Production of deglycosylated antibodies. Wild type
and variants of alemtuzumab were expressed in 293T cells and
purified with protein A chromatography. Antibodies were incubated
with peptide-N-glycosidase (PNGase F) at 37.degree. C. for 24 h.
For each antibody, a mock treated sample (-PNGase F) was done in
parallel. WT: wild-type alemtuzumab; #15, #16, #17, #18, #22:
alemtuzumab variants F241E/F243R/V262E/V264R,
F241E/F243Q/V262T/V264E, F241R/F243Q/V262T/V264R,
F241E/F243Y/V262T/V264R, and I332E respectively. The faster
migration of the PNGase F treated versus the mock treated samples
represents the deglycosylated heavy chains.
[0045] FIG. 9. Alemtuzumab expressed from 293T cells binds its
antigen. The antigenic CD52 peptide, fused to GST, was expressed in
E. coli BL21 (DE3) under IPTG induction. Both uninduced and induced
samples were run on a SDS-PAGE gel, and transferred to PVDF
membrane. For western analysis, either alemtuzumab from Sotec
(.alpha.-CD52, Sotec) (final concentration 2.5 ng/ul) or media of
transfected 293T cells (Campath, Xencor) (final alemtuzumab
concentration approximately 0.1-0.2 ng/ul) were used as primary
antibody, and peroxidase-conjugated goat-anti human IgG was used as
secondary antibody. M: pre-stained marker; U: un-induced sample for
GST-CD52; I: induced sample for GST-CD52.
[0046] FIG. 10. Expression and purification of extracellular region
of human V158 Fc.gamma.RIIIa. Tagged Fc.gamma.RIIIa was transfected
in 293T cells, and media containing secreted Fc.gamma.RIIIa were
harvested 3 days later and purified using affinity chromatography.
1: media; 2: flow through; 3: wash; 4-8: serial elutions. Both
simple blue-stained SDS-PAGE gel and western result are shown. For
the western blot, membrane was probed with anti-GST antibody.
[0047] FIG. 11. Binding to human V158 Fc.gamma.RIIIa by select
alemtuzumab Fc variants from the experimental library as determined
by the AlphaScreen.TM. assay, described in Example 2. In the
presence of competitor antibody (Fc variant or WT alemtuzumab) a
characteristic inhibition curve is observed as a decrease in
luminescence signal. Phosphate buffer saline (PBS) alone was used
as the negative control. The binding data were normalized to the
maximum and minimum luminescence signal for each particular curve,
provided by the baselines at low and high antibody concentrations
respectively. The curves represent the fits of the data to a one
site competition model using nonlinear regression. These fits
provide IC50s for each antibody, illustrated for WT and S239D by
the dotted lines.
[0048] FIG. 12. AlphaScreen.TM. assay showing binding of select
alemtuzumab Fc variants to human Fc.gamma.RIIb. The binding data
were normalized to the upper and lower baselines for each
particular antibody, and the curves represent the fits of the data
to a one site competition model. PBS was used as a negative
control.
[0049] FIGS. 13a and 13b. AlphaScreen.TM. assay showing binding of
select alemtuzumab (FIG. 13a) and trastuzumab (FIG. 13b) Fc
variants to human Val158 Fc.gamma.RIIIa. The binding data were
normalized to the upper and lower baselines for each particular
antibody, and the curves represent the fits of the data to a one
site competition model. PBS was used as a negative control.
[0050] FIGS. 14a and 14b. AlphaScreen.TM. assay measuring binding
to human V158 Fc.gamma.RIIIa by select Fc variants in the context
of trastuzumab. The binding data were normalized to the upper and
lower baselines for each particular antibody, and the curves
represent the fits of the data to a one site competition model. PBS
was used as a negative control.
[0051] FIGS. 15a and 15b. AlphaScreen.TM. assay measuring binding
to human V158 Fc.gamma.RIIIa by select Fc variants in the context
of rituximab (FIG. 15a) and cetuximab (FIG. 15b). The binding data
were normalized to the upper and lower baselines for each
particular antibody, and the curves represent the fits of the data
to a one site competition model. PBS was used as a negative
control.
[0052] FIGS. 16a-16b. AlphaScreen.TM. assay comparing binding of
select alemtuzumab Fc variants to human V158 Fc.gamma.RIIIa (FIG.
16a) and human Fc.gamma.RIIb (FIG. 16b). The binding data were
normalized to the upper and lower baselines for each particular
antibody, and the curves represent the fits of the data to a one
site competition model. PBS was used as a negative control.
[0053] FIG. 17. AlphaScreen.TM. assay measuring binding to human
V158 Fc.gamma.RIIIa by select Fc variants in the context of
trastuzumab. The binding data were normalized to the upper and
lower baselines for each particular antibody, and the curves
represent the fits of the data to a one site competition model. PBS
was used as a negative control.
[0054] FIG. 18. AlphaScreen.TM. assay showing binding of select
alemtuzumab Fc variants to human R131Fc.gamma.RIIa. The binding
data were normalized to the upper and lower baselines for each
particular antibody, and the curves represent the fits of the data
to a one site competition model.
[0055] FIGS. 19a and 19b. AlphaScreen.TM. assay showing binding of
select alemtuzumab Fc variants to human V158 Fc.gamma.RIIIa. The
binding data were normalized to the upper and lower baselines for
each particular antibody, and the curves represent the fits of the
data to a one site competition model. PBS was used as a negative
control.
[0056] FIG. 20. AlphaScreen.TM. assay showing binding of
aglycosylated alemtuzumab Fc variants to human V158 Fc.gamma.RIIIa.
The binding data were normalized to the upper and lower baselines
for each particular antibody, and the curves represent the fits of
the data to a one site competition model. PBS was used as a
negative control.
[0057] FIG. 21. AlphaScreen.TM. assay comparing human V158
Fc.gamma.RIIIa binding by select alemtuzumab Fc variants in
glycosylated (solid symbols, solid lines) and deglycosylated (open
symbols, dotted lines). The binding data were normalized to the
upper and lower baselines for each particular antibody, and the
curves represent the fits of the data to a one site competition
model.
[0058] FIGS. 22a-22b. AlphaScreen.TM. assay showing binding of
select alemtuzumab Fc variants to the V158 (FIG. 22a) and F158
(FIG. 22b) allotypes of human Fc.gamma.RIIIa. The binding data were
normalized to the upper and lower baselines for each particular
antibody, and the curves represent the fits of the data to a one
site competition model. PBS was used as a negative control.
[0059] FIGS. 23a-23d. FIGS. 23a and 23b show the correlation
between SPR Kd's and AlphaScreen.TM. IC50's from binding of select
alemtuzumab Fc variants to V158 Fc.gamma.RIIIa (FIG. 23a) and F158
Fc.gamma.RIIIa (FIG. 23b). FIGS. 23c and 23d show the correlation
between SPR and AlphaScreen.TM. fold-improvements over WT for
binding of select alemtuzumab Fc variants to V158 Fc.gamma.RIIIa
(FIG. 23c) and F158 Fc.gamma.RIIIa (FIG. 23d). Binding data are
presented in Table 63. The lines through the data represent the
linear fits of the data, and the r.sup.2 values indicate the
significance of these fits.
[0060] FIGS. 24a-24b. Cell-based ADCC assays of select Fc variants
in the context of alemtuzumab. ADCC was measured using the
DELFIA.RTM. EuTDA-based cytotoxicity assay (Perkin Elmer, MA), as
described in Example 7, using DoHH-2 lymphoma target cells and
50-fold excess human PBMCs. FIG. 24a is a bar graph showing the raw
fluorescence data for the indicated alemtuzumab antibodies at 10
ng/ml. The PBMC bar indicates basal levels of cytotoxicity in the
absence of antibody. FIG. 24b shows the dose-dependence of ADCC on
antibody concentration for the indicated alemtuzumab antibodies,
normalized to the minimum and maximum fluorescence signal for each
particular curve, provided by the baselines at low and high
antibody concentrations respectively. The curves represent the fits
of the data to a sigmoidal dose-response model using nonlinear
regression.
[0061] FIGS. 25a-25c. Cell-based ADCC assays of select Fc variants
in the context of trastuzumab. ADCC was measured using the
DELFIA.RTM. EuTDA-based cytotoxicity assay, as described in Example
7, using BT474 and Sk-Br-3 breast carcinoma target cells and
50-fold excess human PBMCs. FIG. 25a is a bar graph showing the raw
fluorescence data for the indicated trastuzumab antibodies at 1
ng/ml. The PBMC bar indicates basal levels of cytotoxicity in the
absence of antibody. FIGS. 25b and 25c show the dose-dependence of
ADCC on antibody concentration for the indicated trastuzumab
antibodies, normalized to the minimum and maximum fluorescence
signal for each particular curve, provided by the baselines at low
and high antibody concentrations respectively. The curves represent
the fits of the data to a sigmoidal dose-response model using
nonlinear regression.
[0062] FIGS. 26a-26c. Cell-based ADCC assays of select Fc variants
in the context of rituximab. ADCC was measured using the
DELFIA.RTM. EuTDA-based cytotoxicity assay, as described in Example
7, using WIL2-S lymphoma target cells and 50-fold excess human
PBMCs. FIG. 26a is a bar graph showing the raw fluorescence data
for the indicated rituximab antibodies at 1 ng/ml. The PBMC bar
indicates basal levels of cytotoxicity in the absence of antibody.
FIGS. 26b and 26c show the dose-dependence of ADCC on antibody
concentration for the indicated rituximab antibodies, normalized to
the minimum and maximum fluorescence signal for each particular
curve, provided by the baselines at low and high antibody
concentrations respectively. The curves represent the fits of the
data to a sigmoidal dose-response model using nonlinear
regression.
[0063] FIGS. 27a-27b. Cell-based ADCC assay of select trastuzumab
(FIG. 27a) and rituximab (FIG. 27b) Fc variants showing
enhancements in potency and efficacy. Both assays used homozygous
F158/F158 Fc.gamma.RIIIa PBMCs as effector cells at a 25-fold
excess to target cells, which were Sk-Br-3 for the trastuzumab
assay and WIL2-S for the rituximab assay. Data were normalized
according to the absolute minimal lysis for the assay, provided by
the fluorescence signal of target cells in the presence of PBMCs
alone (no antibody), and the absolute maximal lysis for the assay,
provided by the fluorescence signal of target cells in the presence
of Triton X1000, as described in Example 7.
[0064] FIG. 28. Cell-based ADCC assay of select trastuzumab Fc
variants against different cell lines expressing varying levels of
the Her2/neu target antigen. ADCC assays were run as described in
Example 7, with various cell lines expressing amplified to low
levels of Her2/neu receptor, including Sk-Br-3 (1.times.10.sup.6
copies), SkOV3 (.about.1.times.10.sup.5),
OVCAR3(.about.1.times.10.sup.4), and MCF-7 (.about.3.times.10.sup.3
copies). Human PBMCs allotyped as homozygous F158/F158
Fc.gamma.RIIIa were used at 25-fold excess to target cells. The bar
graph provides ADCC data for WT and Fc variant against the
indicated cell lines, normalized to the minimum and maximum
fluorescence signal provided by minimal lysis (PBMCs alone) and
maximal lysis (Triton X1000).
[0065] FIG. 29. Cell-based ADCC assays of select Fc variants in the
context of trastuzumab using natural killer (NK) cells as effector
cells and measuring LDH release to monitor cell lysis. NK cells,
allotyped as heterozygous V158/F158 Fc.gamma.RIIIa, were at an
8-fold excess to Sk-Br-3 breast carcinoma target cells, and the
level of cytotoxicity was measured using the LDH Cytotoxicity
Detection Kit, according to the manufacturer's protocol (Roche
Diagnostics GmbH, Penzberg, Germany). The graph shows the
dose-dependence of ADCC on antibody concentration for the indicated
trastuzumab antibodies, normalized to the minimum and maximum
fluorescence signal for each particular curve, provided by the
baselines at low and high antibody concentrations respectively. The
curves represent the fits of the data to a sigmoidal dose-response
model using nonlinear regression.
[0066] FIG. 30. Cell-based ADCP assay of select variants. The ADCP
assay was carried out as described in Example 8, using a
co-labeling strategy coupled with flow cytometry. Differentiated
macrophages were used as effector cells, and Sk-Br-3 cells were
used as target cells. Percent phagocytosis represents the number of
co-labeled cells (macrophage+Sk-Br-3) over the total number of
Sk-Br-3 in the population (phagocytosed+non-phagocytosed).
[0067] FIGS. 31a-31c. Capacity of select Fc variants to mediate
binding and activation of complement. FIG. 31a shows an
AlphaScreen.TM. assay measuring binding of select alemtuzumab Fc
variants to C1q. The binding data were normalized to the upper and
lower baselines for each particular antibody, and the curves
represent the fits of the data to a one site competition model.
FIGS. 31b and 31c show a cell-based assay measuring capacity of
select rituximab Fc variants to mediate CDC. CDC assays were
performed using Alamar Blue to monitor lysis of Fc variant and WT
rituximab-opsonized WIL2-S lymphoma cells by human serum complement
(Quidel, San Diego, Calif.). The dose-dependence on antibody
concentration of complement-mediated lysis is shown for the
indicated rituximab antibodies, normalized to the minimum and
maximum fluorescence signal for each particular curve, provided by
the baselines at low and high antibody concentrations respectively.
The curves represent the fits of the data to a sigmoidal
dose-response model using nonlinear regression.
[0068] FIG. 32. AlphaScreen.TM. assay measuring binding of select
alemtuzumab Fc variants to bacterial protein A, as described in
Example 10. The binding data were normalized to the upper and lower
baselines for each particular antibody, and the curves represent
the fits of the data to a one site competition model. PBS was used
as a negative control.
[0069] FIG. 33. AlphaScreen.TM. assay measuring binding of select
alemtuzumab Fc variants to human FcRn, as described in Example 10.
The binding data were normalized to the upper and lower baselines
for each particular antibody, and the curves represent the fits of
the data to a one site competition model. PBS was used as a
negative control.
[0070] FIGS. 34a and 34b. AlphaScreen.TM. assay measuring binding
of select alemtuzumab (FIG. 34a) and trastuzumab (FIG. 34b) Fc
variants to mouse Fc.gamma.RIII, as described in Example 11. The
binding data were normalized to the upper and lower baselines for
each particular antibody, and the curves represent the fits of the
data to a one site competition model. PBS was used as a negative
control.
[0071] FIG. 35. Cell-based ADCC assays of select Fc variants in the
context of trastuzumab using mouse PBMCs as effector cells. ADCC
was measured using the DELFIA.RTM. EuTDA-based cytotoxicity assay
using Sk-Br-3 breast carcinoma target cells and 8-fold excess mouse
PBMCs. The bar graph shows the raw fluorescence data for the
indicated trastuzumab antibodies at 10 ng/ml. The PBMC bar
indicates basal levels of cytotoxicity in the absence of antibody,
and TX indicates complete cell lysis in the presence of Triton
X1000.
[0072] FIG. 36. AlphaScreen.TM. assay measuring binding to human
V158 Fc.gamma.RIIIa by select trastuzumab Fc variants expressed in
293T and CHO cells, as described in Example 12. The binding data
were normalized to the upper and lower baselines for each
particular antibody, and the curves represent the fits of the data
to a one site competition model. PBS was used as a negative
control.
[0073] FIGS. 37a-37b. Synergy of Fc variants and engineered
glycoforms. FIG. 37a presents an AlphaScreen.TM. assay showing V158
Fc.gamma.RIIIa binding by WT and Fc variant (V209,
S239/I332E/A330L) trastuzumab expressed in 293T, CHO, and Lec-13
CHO cells. The data were normalized to the upper and lower
baselines for each antibody, and the curves represent the fits of
the data to a one site competition model. PBS was used as a
negative control. FIG. 37b presents a cell-based ADCC assay showing
the ability of 239T, CHO, and Lec-13 CHO expressed WT and V209
trastuzumab to mediate ADCC. ADCC was measured using the
DELFIA.RTM. EuTDA-based cytotoxicity assay as described previously,
with Sk-Br-3 breast carcinoma target cells. The data show the
dose-dependence of ADCC on antibody concentration for the indicated
trastuzumab antibodies, normalized to the minimum and maximum
fluorescence signal for each particular curve, provided by the
baselines at low and high antibody concentrations respectively. The
curves represent the fits of the data to a sigmoidal dose-response
model using nonlinear regression.
[0074] FIGS. 38a-38c. Sequences showing improved anti-CD20
antibodies. The light and heavy chain sequences of rituximab (SEQ
ID NOs:3 and 4) are presented in FIG. 38a and FIG. 38b
respectively, and are taken from translated Sequence 3 of U.S. Pat.
No. 5,736,137. Relevant positions in FIG. 38b are bolded, including
S239, V240, V264I, E272, K274, N297, S298, K326, A330, and I332.
FIG. 38c shows the improved anti-CD20 antibody heavy chain
sequences, with variable positions designated in bold as X.sub.1,
X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, X.sub.8,
Z.sub.1, and Z.sub.2 (SEQ ID NO:5). The table below the sequence
provides possible substitutions for these positions. The improved
anti-CD20 antibody sequences comprise at least one non-WT amino
acid selected from the group of possible substitutions for X.sub.1,
X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, and X.sub.8.
These improved anti-CD20 antibody sequences may also comprise a
substitution Z.sub.1 and/or Z.sub.2. These positions are numbered
according to the EU index as in Kabat, and thus do not correspond
to the sequential order in the sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0075] In order that the invention may be more completely
understood, several definitions are set forth below. Such
definitions are meant to encompass grammatical equivalents.
[0076] By "ADCC" or "antibody dependent cell-mediated cytotoxicity"
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell.
[0077] By "ADCP" or antibody dependent cell-mediated phagocytosis
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause phagocytosis
of the target cell.
[0078] By "amino acid modification" herein is meant an amino acid
substitution, insertion, and/or deletion in a polypeptide sequence.
The preferred amino acid modification herein is a substitution. By
"amino acid substitution" or "substitution" herein is meant the
replacement of an amino acid at a particular position in a parent
polypeptide sequence with another amino acid. For example, the
substitution I332E refers to a variant polypeptide, in this case an
Fc variant, in which the isoleucine at position 332 is replaced
with a glutamic acid.
[0079] By "antibody" herein is meant a protein consisting of one or
more polypeptides substantially encoded by all or part of the
recognized immunoglobulin genes. The recognized immunoglobulin
genes, for example in humans, include the kappa (.kappa.), lambda
(.lamda.), and heavy chain genetic loci, which together comprise
the myriad variable region genes, and the constant region genes mu
(.nu.), delta (.delta.), gamma (.gamma.), sigma (.epsilon.), and
alpha (.alpha.) which encode the IgM, IgD, IgG, IgE, and IgA
isotypes respectively. Antibody herein is meant to include full
length antibodies and antibody fragments, and may refer to a
natural antibody from any organism, an engineered antibody, or an
antibody generated recombinantly for experimental, therapeutic, or
other purposes as further defined below. The term "antibody"
includes antibody fragments, as are known in the art, such as Fab,
Fab', F(ab').sub.2, Fv, scFv, or other antigen-binding subsequences
of antibodies, either produced by the modification of whole
antibodies or those synthesized de novo using recombinant DNA
technologies. Particularly preferred are full length antibodies
that comprise Fc variants as described herein. The term "antibody"
comprises monoclonal and polyclonal antibodies. Antibodies can be
antagonists, agonists, neutralizing, inhibitory, or
stimulatory.
[0080] The antibodies of the present invention may be nonhuman,
chimeric, humanized, or fully human. For a description of the
concepts of chimeric and humanized antibodies see Clark et al.,
2000 and references cited therein (Clark, 2000, Immunol Today
21:397-402). Chimeric antibodies comprise the variable region of a
nonhuman antibody, for example VH and VL domains of mouse or rat
origin, operably linked to the constant region of a human antibody
(see for example U.S. Pat. No. 4,816,567). In a preferred
embodiment, the antibodies of the present invention are humanized.
By "humanized" antibody as used herein is meant an antibody
comprising a human framework region (FR) and one or more
complementarity determining regions (CDR's) from a non-human
(usually mouse or rat) antibody. The non-human antibody providing
the CDR's is called the "donor" and the human immunoglobulin
providing the framework is called the "acceptor". Humanization
relies principally on the grafting of donor CDRs onto acceptor
(human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539). This
strategy is referred to as "CDR grafting". "Backmutation" of
selected acceptor framework residues to the corresponding donor
residues is often required to regain affinity that is lost in the
initial grafted construct (U.S. Pat. No. 5,530,101; U.S. Pat. No.
5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S.
Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No.
5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213). The
humanized antibody optimally also will comprise at least a portion
of an immunoglobulin constant region, typically that of a human
immunoglobulin, and thus will typically comprise a human Fc region.
Methods for humanizing non-human antibodies are well known in the
art, and can be essentially performed following the method of
Winter and co-workers (Jones et al., 1986, Nature 321:522-525;
Riechmann et al., 1988, Nature 332:323-329; Verhoeyen et al., 1988,
Science, 239:1534-1536). Additional examples of humanized murine
monoclonal antibodies are also known in the art, for example
antibodies binding human protein C(O'Connor et al., 1998, Protein
Eng 11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc
Natl Acad Sci, USA 86:10029-33), and human epidermal growth factor
receptor 2 (Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9).
In an alternate embodiment, the antibodies of the present invention
may be fully human, that is the sequences of the antibodies are
completely or substantially human. A number of methods are known in
the art for generating fully human antibodies, including the use of
transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol
8:455-458) or human antibody libraries coupled with selection
methods (Griffiths et al., 1998, Curr Opin Biotechnol
9:102-108).
[0081] Specifically included within the definition of "antibody"
are aglycosylated antibodies. By "aglycosylated antibody" as used
herein is meant an antibody that lacks carbohydrate attached at
position 297 of the Fc region, wherein numbering is according to
the EU system as in Kabat. The aglycosylated antibody may be a
deglycosylated antibody, that is an antibody for which the Fc
carbohydrate has been removed, for example chemically or
enzymatically. Alternatively, the aglycosylated antibody may be a
nonglycosylated or unglycosylated antibody, that is an antibody
that was expressed without Fc carbohydrate, for example by mutation
of one or residues that encode the glycosylation pattern or by
expression in an organism that does not attach carbohydrates to
proteins, for example bacteria.
[0082] Specifically included within the definition of "antibody"
are full-length antibodies that contain an Fc variant portion. By
"full length antibody" herein is meant the structure that
constitutes the natural biological form of an antibody, including
variable and constant regions. For example, in most mammals,
including humans and mice, the full length antibody of the IgG
class is a tetramer and consists of two identical pairs of two
immunoglobulin chains, each pair having one light and one heavy
chain, each light chain comprising immunoglobulin domains V.sub.L
and C.sub.L, and each heavy chain comprising immunoglobulin domains
V.sub.H, C.gamma.1, C.gamma.2, and C.gamma.3. In some mammals, for
example in camels and llamas, IgG antibodies may consist of only
two heavy chains, each heavy chain comprising a variable domain
attached to the Fc region. By "IgG" as used herein is meant a
polypeptide belonging to the class of antibodies that are
substantially encoded by a recognized immunoglobulin gamma gene. In
humans this class comprises IgG1, IgG2, IgG3, and IgG4. In mice
this class comprises IgG1, IgG2a, IgG2b, IgG3.
[0083] By "amino acid" and "amino acid identity" as used herein is
meant one of the 20 naturally occurring amino acids or any
non-natural analogues that may be present at a specific, defined
position. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures, i.e. "analogs", such as peptoids (see
Simon et al., 1992, Proc Nat Acad Sci USA 89(20):9367) particularly
when LC peptides are to be administered to a patient. Thus "amino
acid", or "peptide residue", as used herein means both naturally
occurring and synthetic amino acids. For example,
homophenylalanine, citrulline and noreleucine are considered amino
acids for the purposes of the invention. "Amino acid" also includes
imino acid residues such as proline and hydroxyproline. The side
chain may be in either the (R) or the (S) configuration. In the
preferred embodiment, the amino acids are in the (S) or
L-configuration. If non-naturally occurring side chains are used,
non-amino acid substituents may be used, for example to prevent or
retard in vivo degradation.
[0084] By "computational screening method" herein is meant any
method for designing one or more mutations in a protein, wherein
said method utilizes a computer to evaluate the energies of the
interactions of potential amino acid side chain substitutions with
each other and/or with the rest of the protein. As will be
appreciated by those skilled in the art, evaluation of energies,
referred to as energy calculation, refers to some method of scoring
one or more amino acid modifications. Said method may involve a
physical or chemical energy term, or may involve knowledge-,
statistical-, sequence-based energy terms, and the like. The
calculations that compose a computational screening method are
herein referred to as "computational screening calculations".
[0085] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include but are
not limited to ADCC, ADCP, and CDC. By "effector cell" as used
herein is meant a cell of the immune system that expresses one or
more Fc receptors and mediates one or more effector functions.
Effector cells include but are not limited to monocytes,
macrophages, neutrophils, dendritic cells, eosinophils, mast cells,
platelets, B cells, large granular lymphocytes, Langerhans' cells,
natural killer (NK) cells, and .gamma..gamma. T cells, and may be
from any organism including but not limited to humans, mice, rats,
rabbits, and monkeys. By "library" herein is meant a set of Fc
variants in any form, including but not limited to a list of
nucleic acid or amino acid sequences, a list of nucleic acid or
amino acid substitutions at variable positions, a physical library
comprising nucleic acids that encode the library sequences, or a
physical library comprising the Fc variant proteins, either in
purified or unpurified form.
[0086] By "Fc", "Fc region", FC polypeptide", etc. as used herein
is meant an antibody as defined herein that includes the
polypeptides comprising the constant region of an antibody
excluding the first constant region immunoglobulin domain. Thus Fc
refers to the last two constant region immunoglobulin domains of
IgA, IgD, and IgG, and the last three constant region
immunoglobulin domains of IgE and IgM, and the flexible hinge
N-terminal to these domains. For IgA and IgM Fc may include the J
chain. For IgG, as illustrated in FIG. 1, Fc comprises
immunoglobulin domains Cgamma2 and Cgamma3 (C.gamma.2 and
C.gamma.3) and the hinge between Cgamma1 (C.gamma.1) and Cgamma2
(C.gamma.2). Although the boundaries of the Fc region may vary, the
human IgG heavy chain Fc region is usually defined to comprise
residues C226 or P230 to its carboxyl-terminus, wherein the
numbering is according to the EU index as in Kabat. Fc may refer to
this region in isolation, or this region in the context of an
antibody, antibody fragment, or Fc fusion. An Fc may be an
antibody, Fc fusion, or an protein or protein domain that comprises
Fc. Particularly preferred are Fc variants, which are non-naturally
occurring variants of an Fc.
[0087] By "Fc fusion" as used herein is meant a protein wherein one
or more polypeptides is operably linked to an Fc region or a
derivative thereof. Fc fusion is herein meant to be synonymous with
the terms "immunoadhesin", "Ig fusion", "Ig chimera", and "receptor
globulin" (sometimes with dashes) as used in the prior art (Chamow
et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997,
Curr Opin Immunol 9:195-200). An Fc fusion combines the Fc region
of an immunoglobulin with a fusion Partner, which in general can be
any protein or small molecule. The role of the non-Fc part of an Fc
fusion, i.e. the fusion partner, is to mediate target binding, and
thus it is functionally analogous to the variable regions of an
antibody. Virtually any protein or small molecule may be linked to
Fc to generate an Fc fusion. Protein fusion partners may include,
but are not limited to, the target-binding region of a receptor, an
adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or
some other protein or protein domain. Small molecule fusion
partners may include any therapeutic agent that directs the Fc
fusion to a therapeutic target. Such targets may be any molecule,
preferrably an extracellular receptor, that is implicated in
disease. Two families of surface receptors that are targets of a
number of approved small molecule drugs are G-Protein Coupled
Receptors (GPCRs), and ion channels, including K+, Na+, Ca+
channels. Nearly 70% of all drugs currently marketed worldwide
target GPCRs. Thus the Fc variants of the present invention may be
fused to a small molecule that targets, for example, one or more
GABA receptors, purinergic receptors, adrenergic receptors,
histaminergic receptors, opiod receptors, chemokine receptors,
glutamate receptors, nicotinic receptors, the 5HT (serotonin)
receptor, and estrogen receptors. A fusion partner may be a
small-molecule mimetic of a protein that targets a therapeutically
useful target. Specific examples of particular drugs that may serve
as Fc fusion partners can be found in L. S. Goodman et al., Eds.,
Goodman and Gilman's The Pharmacological Basis of Therapeutics
(McGraw-Hill, New York, ed. 9, 1996). Fusion partners include not
only small molecules and proteins that bind known targets for
existing drugs, but orphan receptors that do not yet exist as drug
targets. The completion of the genome and proteome projects are
proving to be a driving force in drug discovery, and these projects
have yielded a trove of orphan receptors. There is enormous
potential to validate these new molecules as drug targets, and
develop protein and small molecule therapeutics that target them.
Such protein and small molecule therapeutics are contemplated as Fc
fusion partners that employ the Fc variants of the present
invention. A variety of linkers, defined and described below, may
be used to covalently link Fc to a fusion partner to generate an Fc
fusion.
[0088] By "Fc gamma receptor" or "Fc.gamma.R" as used herein is
meant any member of the family of proteins that bind the IgG
antibody Fc region and are substantially encoded by the Fc.gamma.R
genes. In humans this family includes but is not limited to
Fc.gamma.RI (CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb,
and Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms
Fc.gamma.RIIa (including allotypes H131 and R131), Fc.gamma.RIIb
(including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and Fc.gamma.RIIc;
and Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIa
(including allotypes V158 and F158) and Fc.gamma.RIIIb (including
allotypes Fc.gamma.RIIIb-NA1 and Fc.gamma.RIIIb-NA2) (Jefferis et
al., 2002, Immunol Lett 82:57-65), as well as any undiscovered
human Fc.gamma.Rs or Fc.gamma.R isoforms or allotypes. An
Fc.gamma.R may be from any organism, including but not limited to
humans, mice, rats, rabbits, and monkeys. Mouse Fc.gamma.Rs include
but are not limited to Fc.gamma.RI (CD64), Fc.gamma.RII (CD32),
Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2 (CD16-2), as well as any
undiscovered mouse Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes.
[0089] By "Fc ligand" as used herein is meant a molecule,
preferably a polypeptide, from any organism that binds to the Fc
region of an antibody to form an Fc-ligand complex. Fc ligands
include but are not limited to Fc.gamma.Rs, Fc.gamma.Rs,
Fc.gamma.Rs, FcRn, C1q, C3, mannan binding lectin, mannose
receptor, staphylococcal protein A, streptococcal protein G, and
viral Fc.gamma.R. Fc ligands also include Fc receptor homologs
(FcRH), which are a family of Fc receptors that are homologous to
the Fc.gamma.Rs (Davis et al., 2002, Immunological Reviews
190:123-136). Fc ligands may include undiscovered molecules that
bind Fc.
[0090] By "IgG" as used herein is meant a polypeptide belonging to
the class of antibodies that are substantially encoded by a
recognized immunoglobulin gamma gene. In humans this class
comprises IgG1, IgG2, IgG3, and IgG4. In mice this class comprises
IgG1, IgG2a, IgG2b, IgG3. By "immunoglobulin (Ig)" herein is meant
a protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. Immunoglobulins include but are
not limited to antibodies. Immunoglobulins may have a number of
structural forms, including but not limited to full length
antibodies, antibody fragments, and individual immunoglobulin
domains. By "immunoglobulin (Ig) domain" herein is meant a region
of an immunoglobulin that exists as a distinct structural entity as
ascertained by one skilled in the art of protein structure. Ig
domains typically have a characteristic 1-sandwich folding
topology. The known Ig domains in the IgG class of antibodies are
V.sub.H, C.gamma.1, C.gamma.2, C.gamma.3, V.sub.L, and C.sub.L.
[0091] By "parent polypeptide" or "precursor polypeptide"
(including Fc parent or precursors) as used herein is meant a
polypeptide that is subsequently modified to generate a variant.
Said parent polypeptide may be a naturally occurring polypeptide,
or a variant or engineered version of a naturally occurring
polypeptide. Parent polypeptide may refer to the polypeptide
itself, compositions that comprise the parent polypeptide, or the
amino acid sequence that encodes it. Accordingly, by "parent Fc
polypeptide" as used herein is meant an unmodified Fc polypeptide
that is modified to generate a variant, and by "parent antibody" as
used herein is meant an unmodified antibody that is modified to
generate a variant antibody.
[0092] As outlined above, certain positions of the Fc molecule can
be altered. By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index as in
Kabat. For example, position 297 is a position in the human
antibody IgG1. Corresponding positions are determined as outlined
above, generally through alignment with other parent sequences.
[0093] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297, also referred to as N297) is a residue
in the human antibody IgG1.
[0094] By "target antigen" as used herein is meant the molecule
that is bound specifically by the variable region of a given
antibody. A target antigen may be a protein, carbohydrate, lipid,
or other chemical compound.
[0095] By "target cell" as used herein is meant a cell that
expresses a target antigen.
[0096] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the V.kappa., V.lamda., and/or
V.sub.H genes that make up the kappa, lambda, and heavy chain
immunoglobulin genetic loci respectively.
[0097] By "variant polypetide" as used herein is meant a
polypeptide sequence that differs from that of a parent polypeptide
sequence by virtue of at least one amino acid modification. Variant
polypeptide may refer to the polypeptide itself, a composition
comprising the polypeptide, or the amino sequence that encodes it.
Preferably, the variant polypeptide has at least one amino acid
modification compared to the parent polypeptide, e.g. from about
one to about ten amino acid modifications, and preferably from
about one to about five amino acid modifications compared to the
parent. The variant polypeptide sequence herein will preferably
possess at least about 80% homology with a parent polypeptide
sequence, and most preferably at least about 90% homology, more
preferably at least about 95% homology. Accordingly, by "Fc
variant" as used herein is meant an Fc sequence that differs from
that of a parent Fc sequence by virtue of at least one amino acid
modification. An Fc variant may only encompass an Fc region, or may
exist in the context of an antibody, Fc fusion, or other
polypeptide that is substantially encoded by Fc. Fc variant may
refer to the Fc polypeptide itself, compositions comprising the Fc
variant polypeptide, or the amino acid sequence that encodes it. In
a preferred embodiment, the variant proteins of the invention
comprise an Fc variant, as described herein, and as such, may
comprise an antibody (and the corresponding derivatives) with the
Fc variant, or an Fc fusion protein that comprises the Fc variant.
In addition, in some cases, the Fc is a variant as compared to a
wild-type Fc, or to a "parent" variant.
[0098] For all positions discussed in the present invention,
numbering of an immunoglobulin heavy chain is according to the EU
index (Kabat et al., 1991, Sequences of Proteins of Immunological
Interest, 5th Ed., United States Public Health Svice, National
Institutes of Health, Bethesda). The "EU index as in Kabat" refers
to the residue numbering of the human IgG1 EU antibody.
[0099] The Fc variants of the present invention may be optimized
for a variety of properties. Properties that may be optimized
include but are not limited to enhanced or reduced affinity for an
Fc.gamma.R. In a preferred embodiment, the Fc variants of the
present invention are optimized to possess enhanced affinity for a
human activating Fc.gamma.R, preferably Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIc, Fc.gamma.RIIIa, and Fc.gamma.RIIIb, most preferably
Fc.gamma.RIIa. In an alternately preferred embodiment, the Fc
variants are optimized to possess reduced affinity for the human
inhibitory receptor Fc.gamma.RIIb. These preferred embodiments are
anticipated to provide antibodies and Fc fusions with enhanced
therapeutic properties in humans, for example enhanced effector
function and greater anti-cancer potency. In an alternate
embodiment, the Fc variants of the present invention are optimized
to have reduced or ablated affinity for a human Fc.gamma.R,
including but not limited to Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIb, Fc.gamma.RIIc, Fc.gamma.RIIIa, and Fc.gamma.RIIIb.
These embodiments are anticipated to provide antibodies and Fc
fusions with enhanced therapeutic properties in humans, for example
reduced effector function and reduced toxicity. Preferred
embodiments comprise optimization of Fc binding to a human
Fc.gamma.R, however in alternate embodiments the Fc variants of the
present invention possess enhanced or reduced affinity for
Fc.gamma.Rs from nonhuman organisms, including but not limited to
mice, rats, rabbits, and monkeys. Fc variants that are optimized
for binding to a nonhuman Fc.gamma.R may find use in
experimentation. For example, mouse models are available for a
variety of diseases that enable testing of properties such as
efficacy, toxicity, and pharmacokinetics for a given drug
candidate. As is known in the art, cancer cells can be grafted or
injected into mice to mimic a human cancer, a process referred to
as xenografting. Testing of antibodies or Fc fusions that comprise
Fc variants that are optimized for one or more mouse Fc.gamma.Rs,
may provide valuable information with regard to the efficacy of the
antibody or Fc fusion, its mechanism of action, and the like. The
Fc variants of the present invention may also be optimized for
enhanced functionality and/or solution properties in aglycosylated
form. In a preferred embodiment, the aglycosylated Fc variants of
the present invention bind an Fc ligand with greater affinity than
the aglycosylated form of the parent Fc polypeptide. Said Fc
ligands include but are not limited to Fc.gamma.Rs, C1q, FcRn, and
proteins A and G, and may be from any source including but not
limited to human, mouse, rat, rabbit, or monkey, preferably human.
In an alternately preferred embodiment, the Fc variants are
optimized to be more stable and/or more soluble than the
aglycosylated form of the parent Fc polypeptide. An Fc variant that
is engineered or predicted to display any of the aforementioned
optimized properties is herein referred to as an "optimized Fc
variant".
[0100] The Fc variants of the present invention may be derived from
parent Fc polypeptides that are themselves from a wide range of
sources. The parent Fc polypeptide may be substantially encoded by
one or more Fc genes from any organism, including but not limited
to humans, mice, rats, rabbits, camels, llamas, dromedaries,
monkeys, preferably mammals and most preferably humans and mice. In
a preferred embodiment, the parent Fc polypeptide composes an
antibody, referred to as the parent antibody. The parent antibody
may be fully human, obtained for example using transgenic mice
(Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or human
antibody libraries coupled with selection methods (Griffiths et
al., 1998, Curr Opin Biotechnol 9:102-108). The parent antibody
need not be naturally occurring. For example, the parent antibody
may be an engineered antibody, including but not limited to
chimeric antibodies and humanized antibodies (Clark, 2000, Immunol
Today 21:397-402). The parent antibody may be an engineered variant
of an antibody that is substantially encoded by one or more natural
antibody genes. In one embodiment, the parent antibody has been
affinity matured, as is known in the art. Alternatively, the
antibody has been modified in some other way, for example as
described in U.S. Ser. No. 10/339,788, filed on Mar. 3, 2003.
[0101] The Fc variants of the present invention may be
substantially encoded by immunoglobulin genes belonging to any of
the antibody classes. In a preferred embodiment, the Fc variants of
the present invention find use in antibodies or Fc fusions that
comprise sequences belonging to the IgG class of antibodies,
including IgG1, IgG2, IgG3, or IgG4. In an alternate embodiment the
Fc variants of the present invention find use in antibodies or Fc
fusions that comprise sequences belonging to the IgA (including
subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes of
antibodies. The Fc variants of the present invention may comprise
more than one protein chain. That is, the present invention may
find use in an antibody or Fc fusion that is a monomer or an
oligomer, including a homo- or hetero-oligomer.
[0102] In a preferred embodiment, the antibodies of the invention
are based on human sequences, and thus human sequences are used as
the "base" sequences, against which other sequences, such as rat,
mouse, and monkey sequences are compared. In order to establish
homology to primary sequence or structure, the amino acid sequence
of a precursor or parent Fc polypeptide is directly compared to the
human Fc sequence outlined herein. After aligning the sequences,
using one or more of the homology alignment programs known in the
art (for example using conserved residues as between species),
allowing for necessary insertions and deletions in order to
maintain alignment (i.e., avoiding the elimination of conserved
residues through arbitrary deletion and insertion), the residues
equivalent to particular amino acids in the primary sequence of
human Fc are defined. Alignment of conserved residues preferably
should conserve 100% of such residues. However, alignment of
greater than 75% or as little as 50% of conserved residues is also
adequate to define equivalent residues (sometimes referred to as
"corresponding residues"). Equivalent residues may also be defined
by determining homology at the level of tertiary structure for an
Fc polypeptide whose tertiary structure has been determined.
Equivalent residues are defined as those for which the atomic
coordinates of two or more of the main chain atoms of a particular
amino acid residue of the parent or precursor (N on N, CA on CA, C
on C and O on O) are within 0.13 nm and preferably 0.1 nm after
alignment. Alignment is achieved after the best model has been
oriented and positioned to give the maximum overlap of atomic
coordinates of non-hydrogen protein atoms of the Fc
polypeptide.
[0103] The Fc variants of the present invention may be combined
with other Fc modifications, including but not limited to
modifications that alter effector function or interaction with one
or more Fc ligands. Such combination may provide additive,
synergistic, or novel properties in antibodies or Fc fusions. In
one embodiment, the Fc variants of the present invention may be
combined with other known Fc variants (Duncan et al., 1988, Nature
332:563-564; Lund et al., 1991, J Immunol 147:2657-2662; Lund et
al., 1992, Mol Immunol 29:53-59; Alegre et al., 1994,
Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl Acad
Sci USA 92:11980-11984; Jefferis et al., 1995, Immunol Lett
44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al.,
1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol
157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624;
Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000,
J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26;
Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al.,
2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol
Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490;
Hinton et al., 2004, J Biol Chem 279:6213-6216) (U.S. Pat. No.
5,624,821; U.S. Pat. No. 5,885,573; U.S. Pat. No. 6,194,551; PCT WO
00/42072; PCT WO 99/58572; US 2004/0002587 A1). In an alternate
embodiment, the Fc variants of the present invention are
incorporated into an antibody or Fc fusion that comprises one or
more engineered glycoforms. By "engineered glycoform" as used
herein is meant a carbohydrate composition that is covalently
attached to an Fc polypeptide, wherein said carbohydrate
composition differs chemically from that of a parent Fc
polypeptide. Engineered glycoforms may be useful for a variety of
purposes, including but not limited to enhancing or reducing
effector function. Engineered glycoforms may be generated by a
variety of methods known in the art (Umana et al., 1999, Nat
Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng
74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740;
Shinkawa et al., 2003, J Biol Chem 278:3466-3473); (U.S. Pat. No.
6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT
WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO
02/30954A1); (Potelligent.TM. technology [Biowa, Inc., Princeton,
N.J.]; GlycoMAb.TM. glycosylation engineering technology [GLYCART
biotechnology AG, Zurich, Switzerland]). Many of these techniques
are based on controlling the level of fucosylated and/or bisecting
oligosaccharides that are covalently attached to the Fc region, for
example by expressing an Fc polypeptide in various organisms or
cell lines, engineered or otherwise (for example Lec-13 CHO cells
or rat hybridoma YB2/0 cells), by regulating enzymes involved in
the glycosylation pathway (for example FUT8
[.alpha.1,6-fucosyltranserase] and/or
.beta.1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by
modifying carbohydrate(s) after the Fc polypeptide has been
expressed. Engineered glycoform typically refers to the different
carbohydrate or oligosaccharide; thus an Fc polypeptide, for
example an antibody or Fc fusion, may comprise an engineered
glycoform. Alternatively, engineered glycoform may refer to the Fc
polypeptide that comprises the different carbohydrate or
oligosaccharide. Thus combinations of the Fc variants of the
present invention with other Fc modifications, as well as
undiscovered Fc modifications, are contemplated with the goal of
generating novel antibodies or Fc fusions with optimized
properties.
[0104] The Fc variants of the present invention may find use in an
antibody. By "antibody of the present invention" as used herein is
meant an antibody that comprises an Fc variant of the present
invention. The present invention may, in fact, find use in any
protein that comprises Fc, and thus application of the Fc variants
of the present invention is not limited to antibodies. The Fc
variants of the present invention may find use in an Fc fusion. By
"Fc fusion of the present invention" as used herein refers to an Fc
fusion that comprises an Fc variant of the present invention. Fc
fusions may comprise an Fc variant of the present invention
operably linked to a cytokine, soluble receptor domain, adhesion
molecule, ligand, enzyme, peptide, or other protein or protein
domain, and include but are not limited to Fc fusions described in
U.S. Pat. No. 5,843,725; U.S. Pat. No. 6,018,026; U.S. Pat. No.
6,291,212; U.S. Pat. No. 6,291,646; U.S. Pat. No. 6,300,099; U.S.
Pat. No. 6,323,323; PCT WO 00/24782; and in (Chamow et al., 1996,
Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin
Immunol 9:195-200).
[0105] Virtually any antigen may be targeted by the antibodies and
fusions of the present invention, including but not limited to the
following list of proteins, subunits, domains, motifs, and epitopes
belonging to the following list of proteins: CD2; CD3, CD3E, CD4,
CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28,
CD29, CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52,
CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5,
IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon alpha,
interferon beta, interferon gamma; TNF-alpha, TNFbeta2, TNFc,
TNFalphabeta, TNF-RI, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL,
RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L, TRAIL Receptor-1, A1
Adenosine Receptor, Lymphotoxin Beta Receptor, TACI, BAFF-R, EPO;
LFA-3, ICAM-1, ICAM-3, EpCAM, integrin beta1, integrin beta2,
integrin alpha4/beta7, integrin alpha2, integrin alpha3, integrin
alpha4, integrin alpha5, integrin alpha6, integrin alphav,
alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1,
VLA-4, L-selectin, anti-id, E-selectin, HLA, HLA-DR, CTLA-4, T cell
receptor, B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1,
BLyS (B-lymphocyte Stimulator), complement C5, IgE, factor VII,
CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3),
Her4 (ErbB-4), Tissue Factor, VEGF, VEGFR, endothelin receptor,
VLA-4, Hapten NP-cap or NIP-cap, T cell receptor alpha/beta,
E-selectin, digoxin, placental alkaline phosphatase (PLAP) and
testicular PLAP-like alkaline phosphatase, transferrin receptor,
Carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucin MUC1,
MUC18, Heparanase I, human cardiac myosin, tumor-associated
glycoprotein-72 (TAG-72), tumor-associated antigen CA 125, Prostate
specific membrane antigen (PSMA), High molecular weight
melanoma-associated antigen (HMW-MAA), carcinoma-associated
antigen, Gcoprotein IIb/IIIa (GPIIb/IIIa), tumor-associated antigen
expressing Lewis Y related carbohydrate, human cytomegalovirus
(HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory
syncital virus RSV F, RSVF Fgp, VNRintegrin, IL-8, cytokeratin
tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB
gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, Herpes
simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB
envelope glycoprotein, and Clostridium perfringens toxin.
[0106] One skilled in the art will appreciate that the
aforementioned list of targets refers not only to specific proteins
and biomolecules, but the biochemical pathway or pathways that
comprise them. For example, reference to CTLA-4 as a target antigen
implies that the ligands and receptors that make up the T cell
co-stimulatory pathway, including CTLA-4, B7-1, B7-2, CD28, and any
other undiscovered ligands or receptors that bind these proteins,
are also targets. Thus target as used herein refers not only to a
specific biomolecule, but the set of proteins that interact with
said target and the members of the biochemical pathway to which
said target belongs. One skilled in the art will further appreciate
that any of the aforementioned target antigens, the ligands or
receptors that bind them, or other members of their corresponding
biochemical pathway, may be operably linked to the Fc variants of
the present invention in order to generate an Fc fusion. Thus for
example, an Fc fusion that targets EGFR could be constructed by
operably linking an Fc variant to EGF, TGF.alpha., or any other
ligand, discovered or undiscovered, that binds EGFR. Accordingly,
an Fc variant of the present invention could be operably linked to
EGFR in order to generate an Fc fusion that binds EGF, TGF.alpha.,
or any other ligand, discovered or undiscovered, that binds EGFR.
Thus virtually any polypeptide, whether a ligand, receptor, or some
other protein or protein domain, including but not limited to the
aforementioned targets and the proteins that compose their
corresponding biochemical pathways, may be operably linked to the
Fc variants of the present invention to develop an Fc fusion.
[0107] A number of antibodies and Fc fusions that are approved for
use, in clinical trials, or in development may benefit from the Fc
variants of the present invention. Said antibodies and Fc fusions
are herein referred to as "clinical Products and candidates". Thus
in a preferred embodiment, the Fc variants of the present invention
may find use in a range of clinical products and candidates. For
example, a number of antibodies that target CD20 may benefit from
the Fc variants of the present invention. For example the Fc
variants of the present invention may find use in an antibody that
is substantially similar to rituximab (Rituxan.RTM.,
IDEC/Genentech/Roche) (see for example U.S. Pat. No. 5,736,137), a
chimeric anti-CD20 antibody approved to treat Non-Hodgkin's
lymphoma; HuMax-CD20, an anti-CD20 currently being developed by
Genmab, an anti-CD20 antibody described in U.S. Pat. No. 5,500,362,
AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.),
and HumaLYM (Intracel). A number of antibodies that target members
of the family of epidermal growth factor receptors, including EGFR
(ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), may
benefit from the Fc variants of the present invention. For example
the Fc variants of the present invention may find use in an
antibody that is substantially similar to trastuzumab
(Herceptin.RTM., Genentech) (see for example U.S. Pat. No.
5,677,171), a humanized anti-Her2/neu antibody approved to treat
breast cancer; pertuzumab (rhuMab-2C4, Omnitarg.TM.), currently
being developed by Genentech; an anti-Her2 antibody described in
U.S. Pat. No. 4,753,894; cetuximab (Erbitux.RTM., Imclone) (U.S.
Pat. No. 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody
in clinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No.
6,235,883), currently being developed by Abgenix/Immunex/Amgen;
HuMax-EGFr (U.S. Ser. No. 10/172,317), currently being developed by
Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S.
Pat. No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys.
252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20;
Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62
(Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al.,
1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al.,
1993, Br J Cancer 1993, 67(2):247-53; Modjtahedi et al., 1996, Br J
Cancer, 73(2):228-35; Modjtahedi et al., 2003, Int J Cancer,
105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de
Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.
6,506,883; Mateo et al., 1997, Immunotechnology, 3(1):71-81);
mAb-806 (Ludwig Institue for Cancer Research, Memorial
Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA.
100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer
Institute) (PCT WO 0162931A2); and SC100 (Scancell) (PCT WO
01/88138). In another preferred embodiment, the Fc variants of the
present invention may find use in alemtuzumab (Campath.RTM.,
Millenium), a humanized monoclonal antibody currently approved for
treatment of B-cell chronic lymphocytic leukemia. The Fc variants
of the present invention may find use in a variety of antibodies or
Fc fusions that are substantially similar to other clinical
products and candidates, including but not limited to muromonab-CD3
(Orthoclone OKT3.RTM.), an anti-CD3 antibody developed by Ortho
Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin.RTM.),
an anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab
ozogamicin (Mylotarg.RTM.), an anti-CD33 (p67 protein) antibody
developed by Celltech/Wyeth, alefacept (Amevive.RTM.), an
anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro.RTM.),
developed by Centocor/Lilly, basiliximab (Simulect.RTM.), developed
by Novartis, palivizumab (Synagis.RTM.), developed by MedImmune,
infliximab (Remicade.RTM.), an anti-TNFalpha antibody developed by
Centocor, adalimumab (Humira.RTM.), an anti-TNFalpha antibody
developed by Abbott, Humicade.TM., an anti-TNFalpha antibody
developed by Celltech, etanercept (Enbrel.RTM.), an anti-TNFalpha
Fc fusion developed by Immunex/Amgen, ABX-CBL, an anti-CD147
antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody
being developed by Abgenix, ABX-MA1, an anti-MUC18 antibody being
developed by Abgenix, Pemtumomab (R1549, .sup.90Y-muHMFG1), an
anti-MUC1 In development by Antisoma, Therex (R1550), an anti-MUC1
antibody being developed by Antisoma, AngioMab (AS1405), being
developed by Antisoma, HuBC-1, being developed by Antisoma,
Thioplatin (AS1407) being developed by Antisoma, Antegren.RTM.
(natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7
antibody being developed by Biogen, VLA-1 mAb, an anti-VLA-1
integrin antibody being developed by Biogen, LTBR mAb, an
anti-lymphotoxin beta receptor (LTBR) antibody being developed by
Biogen, CAT-152, an anti-TGF.beta.2 antibody being developed by
Cambridge Antibody Technology, J695, an anti-IL-12 antibody being
developed by Cambridge Antibody Technology and Abbott, CAT-192, an
anti-TGF.beta.1 antibody being developed by Cambridge Antibody
Technology and Genzyme, CAT-213, an anti-Eotaxin1 antibody being
developed by Cambridge Antibody Technology, LymphoStat-B.TM. an
anti-Blys antibody being developed by Cambridge Antibody Technology
and Human Genome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1
antibody being developed by Cambridge Antibody Technology and Human
Genome Sciences, Inc., Avastin.TM. (bevacizumab, rhuMAb-VEGF), an
anti-VEGF antibody being developed by Genentech, an anti-HER
receptor family antibody being developed by Genentech, Anti-Tissue
Factor (ATF), an anti-Tissue Factor antibody being developed by
Genentech, Xolair.TM. (Omalizumab), an anti-IgE antibody being
developed by Genentech, Raptiva.TM. (Efalizumab), an anti-CD11a
antibody being developed by Genentech and Xoma, MLN-02 Antibody
(formerly LDP-02), being developed by Genentech and Millenium
Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by
Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab
and Amgen, HuMax-Inflam, being developed by Genmab and Medarex,
HuMax-Cancer, an anti-Heparanase I antibody being developed by
Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, being
developed by Genmab and Amgen, HuMax-TAC, being developed by
Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC
Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody
being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80
antibody being developed by IDEC Pharmaceuticals, IDEC-152, an
anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage
migration factor (MIF) antibodies being developed by IDEC
Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed
by Imclone, IMC-1C11, an anti-KDR antibody being developed by
Imclone, DC101, an anti-flk-1 antibody being developed by Imclone,
anti-VE cadherin antibodies being developed by Imclone,
CEA-Cide.TM. (labetuzumab), an anti-carcinoembryonic antigen (CEA)
antibody being developed by Immunomedics, LymphoCide.TM.
(Epratuzumab), an anti-CD22 antibody being developed by
Immunomedics, AFP-Cide, being developed by Immunomedics,
MyelomaCide, being developed by Immunomedics, LkoCide, being
developed by Immunomedics, ProstaCide, being developed by
Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by
Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex,
MDX-070 being developed by Medarex, MDX-018 being developed by
Medarex, Osidem.TM. (IDM-1), and anti-Her2 antibody being developed
by Medarex and Immuno-Designed Molecules, HuMax.TM.-CD4, an
anti-CD4 antibody being developed by Medarex and Genmab,
HuMax-IL15, an anti-IL15 antibody being developed by Medarex and
Genmab, CNTO 148, an anti-TNF.alpha. antibody being developed by
Medarex and Centocor/J&J, CNTO 1275, an anti-cytokine antibody
being developed by Centocor/J&J, MOR101 and MOR102,
anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies
being developed by MorphoSys, MOR201, an anti-fibroblast growth
factor receptor 3 (FGFR-3) antibody being developed by MorphoSys,
Nuvion.RTM. (visilizumab), an anti-CD3 antibody being developed by
Protein Design Labs, HuZAF.TM., an anti-gamma interferon antibody
being developed by Protein Design Labs, Anti-|5 |1 Integrin, being
developed by Protein Design Labs, anti-IL-12, being developed by
Protein Design Labs, ING-1, an anti-Ep-CAM antibody being developed
by Xoma, and MLN01, an anti-Beta2 integrin antibody being developed
by Xoma.
[0108] Application of the Fc variants to the aforementioned
antibody and Fc fusion clinical products and candidates is not
meant to be constrained to their precise composition. The Fc
variants of the present invention may be incorporated into the
aforementioned clinical candidates and products, or into antibodies
and Fc fusions that are substantially similar to them. The Fc
variants of the present invention may be incorporated into versions
of the aforementioned clinical candidates and products that are
humanized, affinity matured, engineered, or modified in some other
way. Furthermore, the entire polypeptide of the aforementioned
clinical products and candidates need not be used to construct a
new antibody or Fc fusion that incorporates the Fc variants of the
present invention; for example only the variable region of a
clinical product or candidate antibody, a substantially similar
variable region, or a humanized, affinity matured, engineered, or
modified version of the variable region may be used. In another
embodiment, the Fc variants of the present invention may find use
in an antibody or Fc fusion that binds to the same epitope,
antigen, ligand, or receptor as one of the aforementioned clinical
products and candidates.
[0109] The Fc variants of the present invention may find use in a
wide range of antibody and Fc fusion products. In one embodiment
the antibody or Fc fusion of the present invention is a
therapeutic, a diagnostic, or a research reagent, preferably a
therapeutic. Alternatively, the antibodies and Fc fusions of the
present invention may be used for agricultural or industrial uses.
In an alternate embodiment, the Fc variants of the present
invention compose a library that may be screened experimentally.
This library may be a list of nucleic acid or amino acid sequences,
or may be a physical composition of nucleic acids or polypeptides
that encode the library sequences. The Fc variant may find use in
an antibody composition that is monoclonal or polyclonal. The
antibodies and Fc fusions of the present invention may be agonists,
antagonists, neutralizing, inhibitory, or stimulatory. In a
preferred embodiment, the antibodies and Fc fusions of the present
invention are used to kill target cells that bear the target
antigen, for example cancer cells. In an alternate embodiment, the
antibodies and Fc fusions of the present invention are used to
block, antagonize, or agonize the target antigen, for example for
antagonizing a cytokine or cytokine receptor. In an alternately
preferred embodiment, the antibodies and Fc fusions of the present
invention are used to block, antagonize, or agonize the target
antigen and kill the target cells that bear the target antigen.
[0110] The Fc variants of the present invention may be used for
various therapeutic purposes. In a preferred embodiment, the Fc
variant proteins are administered to a patient to treat an
antibody-related disorder. A "patient" for the purposes of the
present invention includes both humans and other animals,
preferably mammals and most preferably humans. Thus the antibodies
and Fc fusions of the present invention have both human therapy and
veterinary applications. In the preferred embodiment the patient is
a mammal, and in the most preferred embodiment the patient is
human. The term "treatment" in the present invention is meant to
include therapeutic treatment, as well as prophylactic, or
suppressive measures for a disease or disorder. Thus, for example,
successful administration of an antibody or Fc fusion prior to
onset of the disease results in treatment of the disease. As
another example, successful administration of an optimized antibody
or Fc fusion after clinical manifestation of the disease to combat
the symptoms of the disease comprises treatment of the disease.
"Treatment" also encompasses administration of an optimized
antibody or Fc fusion protein after the appearance of the disease
in order to eradicate the disease. Successful administration of an
agent after onset and after clinical symptoms have developed, with
possible abatement of clinical symptoms and perhaps amelioration of
the disease, comprises treatment of the disease. Those "in need of
treatment" include mammals already having the disease or disorder,
as well as those prone to having the disease or disorder, including
those in which the disease or disorder is to be prevented. By
"antibody related disorder" or "antibody responsive disorder" or
"condition" or "disease" herein are meant a disorder that may be
ameliorated by the administration of a pharmaceutical composition
comprising an antibody or Fc fusion of the present invention.
Antibody related disorders include but are not limited to
autoimmune diseases, immunological diseases, infectious diseases,
inflammatory diseases, neurological diseases, and oncological and
neoplastic diseases including cancer. By "cancer" and "cancerous"
herein refer to or describe the physiological condition in mammals
that is typically characterized by unregulated cell growth.
Examples of cancer include but are not limited to carcinoma,
lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine
tumors, mesothelioma, schwanoma, meningioma, adenocarcinoma,
melanoma, and leukemia or lymphoid malignancies. More particular
examples of such cancers include squamous cell cancer (e.g.
epithelial squamous cell cancer), lung cancer including small-cell
lung cancer, non-small cell lung cancer, adenocarcinoma of the lung
and squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, rectal cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
or renal cancer, prostate cancer, vulval cancer, thyroid cancer,
hepatic carcinoma, anal carcinoma, penile carcinoma, testicular
cancer, esophagael cancer, tumors of the biliary tract, as well as
head and neck cancer. Furthermore, the Fc variants of the present
invention may be used to treat conditions including but not limited
to congestive heart failure (CHF), vasculitis, rosecea, acne,
eczema, myocarditis and other conditions of the myocardium,
systemic lupus erythematosus, diabetes, spondylopathies, synovial
fibroblasts, and bone marrow stroma; bone loss; Paget's disease,
osteoclastoma; multiple myeloma; breast cancer; disuse osteopenia;
malnutrition, periodontal disease, Gaucher's disease, Langerhans'
cell histiocytosis, spinal cord injury, acute septic arthritis,
osteomalacia, Cushing's syndrome, monoostotic fibrous dysplasia,
polyostotic fibrous dysplasia, periodontal reconstruction, and bone
fractures; sarcoidosis; multiple myeloma; osteolytic bone cancers,
breast cancer, lung cancer, kidney cancer and rectal cancer; bone
metastasis, bone pain management, and humoral malignant
hypercalcemia, ankylosing spondylitisa and other
spondyloarthropathies; transplantation rejection, viral infections,
hematologic neoplasisas and neoplastic-like conditions for example,
Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma,
small lymphocytic lymphoma/chronic lymphocytic leukemia, mycosis
fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large
B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia and
lymphoplasmacytic leukemia), tumors of lymphocyte precursor cells,
including B-cell acute lymphoblastic leukemia/lymphoma, and T-cell
acute lymphoblastic leukemia/lymphoma, thymoma, tumors of the
mature T and NK cells, including peripheral T-cell leukemias, adult
T-cell leukemia/T-cell lymphomas and large granular lymphocytic
leukemia, Langerhans cell histocytosis, myeloid neoplasias such as
acute myelogenous leukemias, including AML with maturation, AML
without differentiation, acute promyelocytic leukemia, acute
myelomonocytic leukemia, and acute monocytic leukemias,
myelodysplastic syndromes, and chronic myeloproliferative
disorders, including chronic myelogenous leukemia, tumors of the
central nervous system, e.g., brain tumors (glioma, neuroblastoma,
astrocytoma, medulloblastoma, ependymoma, and retinoblastoma),
solid tumors (nasopharyngeal cancer, basal cell carcinoma,
pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma,
testicular cancer, uterine, vaginal or cervical cancers, ovarian
cancer, primary liver cancer or endometrial cancer, and tumors of
the vascular system (angiosarcoma and hemagiopericytoma),
osteoporosis, hepatitis, HIV, AIDS, spondyloarthritis, rheumatoid
arthritis, inflammatory bowel diseases (IBD), sepsis and septic
shock, Crohn's Disease, psoriasis, schleraderma, graft versus host
disease (GVHD), allogenic islet graft rejection, hematologic
malignancies, such as multiple myeloma (MM), myelodysplastic
syndrome (MDS) and acute myelogenous leukemia (AML), inflammation
associated with tumors, peripheral nerve injury or demyelinating
diseases.
[0111] In one embodiment, an antibody or Fc fusion of the present
invention is administered to a patient having a disease involving
inappropriate expression of a protein. Within the scope of the
present invention this is meant to include diseases and disorders
characterized by aberrant proteins, due for example to alterations
in the amount of a protein present, the presence of a mutant
protein, or both. An overabundance may be due to any cause,
including but not limited to overexpression at the molecular level,
prolonged or accumulated appearance at the site of action, or
increased activity of a protein relative to normal. Included within
this definition are diseases and disorders characterized by a
reduction of a protein. This reduction may be due to any cause,
including but not limited to reduced expression at the molecular
level, shortened or reduced appearance at the site of action,
mutant forms of a protein, or decreased activity of a protein
relative to normal. Such an overabundance or reduction of a protein
can be measured relative to normal expression, appearance, or
activity of a protein, and said measurement may play an important
role in the development and/or clinical testing of the antibodies
and Fc fusions of the present invention.
[0112] In one embodiment, an antibody or Fc fusion of the present
invention is the only therapeutically active agent administered to
a patient. Alternatively, the antibody or Fc fusion of the present
invention is administered in combination with one or more other
therapeutic agents, including but not limited to cytotoxic agents,
chemotherapeutic agents, cytokines, growth inhibitory agents,
anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,
cardioprotectants, or other therapeutic agents. Such molecules are
suitably present in combination in amounts that are effective for
the purpose intended. The skilled medical practitioner can
determine empirically the appropriate dose or doses of other
therapeutic agents useful herein. The antibodies and Fc fusions of
the present invention may be administered concomitantly with one or
more other therapeutic regimens. For example, an antibody or Fc
fusion of the present invention may be administered to the patient
along with chemotherapy, radiation therapy, or both chemotherapy
and radiation therapy. In one embodiment, the antibody or Fc fusion
of the present invention may be administered in conjunction with
one or more antibodies or Fc fusions, which may or may not comprise
an Fc variant of the present invention.
[0113] In one embodiment, the antibodies and Fc fusions of the
present invention are administered with a chemotherapeutic agent.
By "chemotherapeutic agent" as used herein is meant a chemical
compound useful in the treatment of cancer. Examples of
chemotherapeutic agents include but are not limited to alkylating
agents such as thiotepa and cyclosphosphamide (CYTOXAN.TM.); alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; sizofuran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel
(TAXOTERE.RTM., Rhne-Poulenc Rorer, Antony, France); chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins; capecitabine; thymidylate synthase inhibitor (such as
Tomudex); cox-2 inhibitors, such as celicoxib (CELEBREX.RTM.) or
MK-0966 (VIOXX.RTM.); and pharmaceutically acceptable salts, acids
or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY
117018, onapristone, and toremifene (Fareston); and anti-androgens
such as flutamide, nilutamide, bicalutamide, leuprolide, and
goserelin; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0114] A chemotherapeutic or other cytotoxic agent may be
administered as a prodrug. By "prodrug" as used herein is meant a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, for example Wilman, 1986,
Biochemical Society Transactions, 615th Meeting Belfast,
14:375-382; and Stella et al., "Prodrugs: A Chemical Approach to
Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al.,
(ed.): 247-267, Humana Press, 1985. The prodrugs that may find use
with the present invention include but are not limited to
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs,
beta-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use with the antibodies and Fc
fusions of the present invention include but are not limited to any
of the aforementioned chemotherapeutic agents.
[0115] The antibodies and Fc fusions of the present invention may
be combined with other therapeutic regimens. For example, in one
embodiment, the patient to be treated with the antibody or Fc
fusion may also receive radiation therapy. Radiation therapy can be
administered according to protocols commonly employed in the art
and known to the skilled artisan. Such therapy includes but is not
limited to cesium, iridium, iodine, or cobalt radiation. The
radiation therapy may be whole body irradiation, or may be directed
locally to a specific site or tissue in or on the body, such as the
lung, bladder, or prostate. Typically, radiation therapy is
administered in pulses over a period of time from about 1 to 2
weeks. The radiation therapy may, however, be administered over
longer periods of time. For instance, radiation therapy may be
administered to patients having head and neck cancer for about 6 to
about 7 weeks. Optionally, the radiation therapy may be
administered as a single dose or as multiple, sequential doses. The
skilled medical practitioner can determine empirically the
appropriate dose or doses of radiation therapy useful herein. In
accordance with another embodiment of the invention, the antibody
or Fc fusion of the present invention and one or more other
anti-cancer therapies are employed to treat cancer cells ex vivo.
It is contemplated that such ex vivo treatment may be useful in
bone marrow transplantation and particularly, autologous bone
marrow transplantation. For instance, treatment of cells or
tissue(s) containing cancer cells with antibody or Fc fusion and
one or more other anti-cancer therapies, such as described above,
can be employed to deplete or substantially deplete the cancer
cells prior to transplantation in a recipient patient. It is of
course contemplated that the antibodies and Fc fusions of the
invention can be employed in combination with still other
therapeutic techniques such as surgery.
[0116] In an alternate embodiment, the antibodies and Fc fusions of
the present invention are administered with a cytokine. By
"cytokine" as used herein is meant a generic term for proteins
released by one cell population that act on another cell as
intercellular mediators. Examples of such cytokines are
lymphokines, monokines, and traditional polypeptide hormones.
Included among the cytokines are growth hormone such as human
growth hormone, N-methionyl human growth hormone, and bovine growth
hormone; parathyroid hormone; thyroxine; insulin; proinsulin;
relaxin; prorelaxin; glycoprotein hormones such as follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH), and
luteinizing hormone (LH); hepatic growth factor; fibroblast growth
factor; prolactin; placental lactogen; tumor necrosis factor-alpha
and -beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-beta; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;
insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, beta,
and -gamma; colony stimulating factors (CSFs) such as
macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and
granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1,
IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or
TNF-beta; and other polypeptide factors including LIF and kit
ligand (KL). As used herein, the term cytokine includes proteins
from natural sources or from recombinant cell culture, and
biologically active equivalents of the native sequence
cytokines.
[0117] A variety of other therapeutic agents may find use for
administration with the antibodies and Fc fusions of the present
invention. In one embodiment, the antibody or Fc fusion is
administered with an anti-angiogenic agent. By "anti-angiogenic
agent" as used herein is meant a compound that blocks, or
interferes to some degree, the development of blood vessels. The
anti-angiogenic factor may, for instance, be a small molecule or a
protein, for example an antibody, Fc fusion, or cytokine, that
binds to a growth factor or growth factor receptor involved in
promoting angiogenesis. The preferred anti-angiogenic factor herein
is an antibody that binds to Vascular Endothelial Growth Factor
(VEGF). In an alternate embodiment, the antibody or Fc fusion is
administered with a therapeutic agent that induces or enhances
adaptive immune response, for example an antibody that targets
CTLA-4. In an alternate embodiment, the antibody or Fc fusion is
administered with a tyrosine kinase inhibitor. By "tyrosine kinase
inhibitor" as used herein is meant a molecule that inhibits to some
extent tyrosine kinase activity of a tyrosine kinase. Examples of
such inhibitors include but are not limited to quinazolines, such
as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines;
pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP
60261 and CGP 62706; pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl
methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lambert);
antisense molecules (e.g. those that bind to ErbB-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering A G); pan-ErbB inhibitors such as C1-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate
(ST1571, Gleevec.RTM.; Novartis); PKI 166 (Novartis); GW2016 (Glaxo
SmithKline); C1-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen);
ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11
(Imclone); or as described in any of the following patent
publications: U.S. Pat. No. 5,804,396; PCT WO 99/09016 (American
Cyanimid); PCT WO 98/43960 (American Cyanamid); PCT WO 97/38983
(Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCT WO 99/06396
(Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc); PCT WO 96/33978
(AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980
(AstraZeneca), gefitinib (IRESSA.TM., ZD1839, AstraZeneca), and
OSI-774 (Tarceva.TM., OSI Pharmaceuticals/Genentech).
[0118] A variety of linkers may find use in the present invention
to generate Fc fusions (see definition above) or antibody- or Fc
fusion-conjugates (see definition below). By "linker", "linker
sequence", "spacer", "tethering sequence" or grammatical
equivalents thereof, herein is meant a molecule or group of
molecules (such as a monomer or polymer) that connects two
molecules and often serves to place the two molecules in a
preferred configuration. A number of strategies may be used to
covalently link molecules together. These include, but are not
limited to polypeptide linkages between N- and C-termini of
proteins or protein domains, linkage via disulfide bonds, and
linkage via chemical cross-linking reagents. In one aspect of this
embodiment, the linker is a peptide bond, generated by recombinant
techniques or peptide synthesis. Choosing a suitable linker for a
specific case where two polypeptide chains are to be connected
depends on various parameters, including but not limited to the
nature of the two polypeptide chains (e.g., whether they naturally
oligomerize), the distance between the N- and the C-termini to be
connected if known, and/or the stability of the linker towards
proteolysis and oxidation. Furthermore, the linker may contain
amino acid residues that provide flexibility. Thus, the linker
peptide may predominantly include the following amino acid
residues: Gly, Ser, Ala, or Thr. The linker peptide should have a
length that is adequate to link two molecules in such a way that
they assume the correct conformation relative to one another so
that they retain the desired activity. Suitable lengths for this
purpose include at least one and not more than 30 amino acid
residues. Preferably, the linker is from about 1 to 30 amino acids
in length, with linkers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18 19 and 20 amino acids in length being
preferred. In addition, the amino acid residues selected for
inclusion in the linker peptide should exhibit properties that do
not interfere significantly with the activity of the polypeptide.
Thus, the linker peptide on the whole should not exhibit a charge
that would be inconsistent with the activity of the polypeptide, or
interfere with internal folding, or form bonds or other
interactions with amino acid residues in one or more of the
monomers that would seriously impede the binding of receptor
monomer domains. Useful linkers include glycine-serine polymers
(including, for example, (GS)n, (GSGGS)n (SEQ ID NO:6), (GGGGS)n
(SEQ ID NO:7) and (GGGS)n (SEQ ID NO:8), where n is an integer of
at least one), glycine-alanine polymers, alanine-serine polymers,
and other flexible linkers such as the tether for the shaker
potassium channel, and a large variety of other flexible linkers,
as will be appreciated by those in the art. Glycine-serine polymers
are preferred since both of these amino acids are relatively
unstructured, and therefore may be able to serve as a neutral
tether between components. Secondly, serine is hydrophilic and
therefore able to solubilize what could be a globular glycine
chain. Third, similar chains have been shown to be effective in
joining subunits of recombinant proteins such as single chain
antibodies. Suitable linkers may also be identified by screening
databases of known three-dimensional structures for naturally
occurring motifs that can bridge the gap between two polypeptide
chains. In a preferred embodiment, the linker is not immunogenic
when administered in a human patient. Thus linkers may be chosen
such that they have low immunogenicity or are thought to have low
immunogenicity. For example, a linker may be chosen that exists
naturally in a human. In a preferred embodiment the linker has the
sequence of the hinge region of an antibody, that is the sequence
that links the antibody Fab and Fc regions; alternatively the
linker has a sequence that comprises part of the hinge region, or a
sequence that is substantially similar to the hinge region of an
antibody. Another way of obtaining a suitable linker is by
optimizing a simple linker, e.g., (Gly4Ser)n, through random
mutagenesis. Alternatively, once a suitable polypeptide linker is
defined, additional linker polypeptides can be created to select
amino acids that more optimally interact with the domains being
linked. Other types of linkers that may be used in the present
invention include artificial polypeptide linkers and inteins. In
another embodiment, disulfide bonds are designed to link the two
molecules. In another embodiment, linkers are chemical
cross-linking agents. For example, a variety of bifunctional
protein coupling agents may be used, including but not limited to
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., 1971, Science 238:1098. Chemical linkers may enable
chelation of an isotope. For example, Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody (see PCT WO 94/11026). The linker
may be cleavable, facilitating release of the cytotoxic drug in the
cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Chari et
al., 1992, Cancer Research 52: 127-131) may be used. Alternatively,
a variety of nonproteinaceous polymers, including but not limited
to polyethylene glycol (PEG), polypropylene glycol,
polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol, may find use as linkers, that is may find use
to link the Fc variants of the present invention to a fusion
partner to generate an Fc fusion, or to link the antibodies and Fc
fusions of the present invention to a conjugate.
[0119] In one embodiment, the antibody or Fc fusion of the present
invention is conjugated or operably linked to another therapeutic
compound, referred to herein as a conjugate. The conjugate may be a
cytotoxic agent, a chemotherapeutic agent, a cytokine, an
anti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, a
radioisotope, or other therapeutically active agent.
Chemotherapeutic agents, cytokines, anti-angiogenic agents,
tyrosine kinase inhibitors, and other therapeutic agents have been
described above, and all of these aforemention therapeutic agents
may find use as antibody or Fc fusion conjugates. In an alternate
embodiment, the antibody or Fc fusion is conjugated or operably
linked to a toxin, including but not limited to small molecule
toxins and enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof.
Small molecule toxins include but are not limited to calicheamicin,
maytansine (U.S. Pat. No. 5,208,020), trichothene, and CC1065. In
one embodiment of the invention, the antibody or Fc fusion is
conjugated to one or more maytansine molecules (e.g. about 1 to
about 10 maytansine molecules per antibody molecule). Maytansine
may, for example, be converted to May-SS-Me which may be reduced to
May-SH3 and reacted with modified antibody or Fc fusion (Chari et
al., 1992, Cancer Research 52: 127-131) to generate a
maytansinoid-antibody or maytansinoid-Fc fusion conjugate. Another
conjugate of interest comprises an antibody or Fc fusion conjugated
to one or more calicheamicin molecules. The calicheamicin family of
antibiotics are capable of producing double-stranded DNA breaks at
sub-picomolar concentrations. Structural analogues of calicheamicin
that may be used include but are not limited to
.gamma..sub.1.sup.1, .alpha..sub.2.sup.1, .alpha..sub.3,
N-acetyl-.gamma..sub.1.sup.1, PSAG, and .THETA..sup.1.sub.1,
(Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al.,
1998, Cancer Research 58:2925-2928) (U.S. Pat. No. 5,714,586; U.S.
Pat. No. 5,712,374; U.S. Pat. No. 5,264,586; U.S. Pat. No.
5,773,001). Dolastatin 10 analogs such as auristatin E (AE) and
monomethylauristatin E (MMAE) may find use as conjugates for the Fc
variants of the present invention (Doronina et al., 2003, Nat
Biotechnol 21(7):778-84; Francisco et al., 2003 Blood
102(4):1458-65). Useful enzymatically active toxins include but are
not limited to diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, PCT WO 93/21232. The present
invention further contemplates a conjugate or fusion formed between
an antibody or Fc fusion of the present invention and a compound
with nucleolytic activity, for example a ribonuclease or DNA
endonuclease such as a deoxyribonuclease (DNase).
[0120] In an alternate embodiment, an antibody or Fc fusion of the
present invention may be conjugated or operably linked to a
radioisotope to form a radioconjugate. A variety of radioactive
isotopes are available for the production of radioconjugate
antibodies and Fc fusions. Examples include, but are not limited
to, At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186,
Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32, and radioactive
isotopes of Lu.
[0121] In yet another embodiment, an antibody or Fc fusion of the
present invention may be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the
antibody-receptor or Fc fusion-receptor conjugate is administered
to the patient, followed by removal of unbound conjugate from the
circulation using a clearing agent and then administration of a
"ligand" (e.g. avidin) which is conjugated to a cytotoxic agent
(e.g. a radionucleotide). In an alternate embodiment, the antibody
or Fc fusion is conjugated or operably linked to an enzyme in order
to employ Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT). ADEPT may be used by conjugating or operably linking the
antibody or Fc fusion to a prodrug-activating enzyme that converts
a prodrug (e.g. a peptidyl chemotherapeutic agent, see PCT WO
81/01145) to an active anti-cancer drug. See, for example, PCT WO
88/07378 and U.S. Pat. No. 4,975,278. The enzyme component of the
immunoconjugate useful for ADEPT includes any enzyme capable of
acting on a prodrug in such a way so as to covert it into its more
active, cytotoxic form. Enzymes that are useful in the method of
this invention include but are not limited to alkaline phosphatase
useful for converting phosphate-containing prodrugs into free
drugs; arylsulfatase useful for converting sulfate-containing
prodrugs into free drugs; cytosine deaminase useful for converting
non-toxic 5-fluorocytosine into the anti-cancer drug,
5-fluorouracil; proteases, such as serratia protease, thermolysin,
subtilisin, carboxypeptidases and cathepsins (such as cathepsins B
and L), that are useful for converting peptide-containing prodrugs
into free drugs; D-alanylcarboxypeptidases, useful for converting
prodrugs that contain D-amino acid substituents;
carbohydrate-cleaving enzymes such as .beta.-galactosidase and
neuramimidase useful for converting glycosylated prodrugs into free
drugs; beta-lactamase useful for converting drugs derivatized with
.alpha.-lactams into free drugs; and penicillin amidases, such as
penicillin V amidase or penicillin G amidase, useful for converting
drugs derivatized at their amine nitrogens with phenoxyacetyl or
phenylacetyl groups, respectively, into free drugs. Alternatively,
antibodies with enzymatic activity, also known in the art as
"abzymes", can be used to convert the prodrugs of the invention
into free active drugs (see, for example, Massey, 1987, Nature 328:
457-458). Antibody-abzyme and Fc fusion-abzyme conjugates can be
prepared for delivery of the abzyme to a tumor cell population.
[0122] Other modifications of the antibodies and Fc fusions of the
present invention are contemplated herein. For example, the
antibody or Fc fusion may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol.
[0123] Pharmaceutical compositions are contemplated wherein an
antibody or Fc fusion of the present invention and one or more
therapeutically active agents are formulated. Formulations of the
antibodies and Fc fusions of the present invention are prepared for
storage by mixing said antibody or Fc fusion having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed., 1980), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, acetate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners
and other flavoring agents; fillers such as microcrystalline
cellulose, lactose, corn and other starches; binding agents;
additives; coloring agents; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). In a preferred embodiment, the
pharmaceutical composition that comprises the antibody or Fc fusion
of the present invention is in a water-soluble form, such as being
present as pharmaceutically acceptable salts, which is meant to
include both acid and base addition salts. "Pharmaceutically
acceptable acid addition salt" refers to those salts that retain
the biological effectiveness of the free bases and that are not
biologically or otherwise undesirable, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid and the like, and organic acids such as
acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic
acid, maleic acid, malonic acid, succinic acid, fumaric acid,
tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic
acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic
acid, salicylic acid and the like. "Pharmaceutically acceptable
base addition salts" include those derived from inorganic bases
such as sodium, potassium, lithium, ammonium, calcium, magnesium,
iron, zinc, copper, manganese, aluminum salts and the like.
Particularly preferred are the ammonium, potassium, sodium,
calcium, and magnesium salts. Salts derived from pharmaceutically
acceptable organic non-toxic bases include salts of primary,
secondary, and tertiary amines, substituted amines including
naturally occurring substituted amines, cyclic amines and basic ion
exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine. The
formulations to be used for in vivo administration are preferrably
sterile. This is readily accomplished by filtration through sterile
filtration membranes or other methods.
[0124] The antibodies and Fc fusions disclosed herein may also be
formulated as immunoliposomes. A liposome is a small vesicle
comprising various types of lipids, phospholipids and/or surfactant
that is useful for delivery of a therapeutic agent to a mammal.
Liposomes containing the antibody or Fc fusion are prepared by
methods known in the art, such as described in Epstein et al.,
1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, Proc
Natl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No.
4,544,545; and PCT WO 97/38731. Liposomes with enhanced circulation
time are disclosed in U.S. Pat. No. 5,013,556. The components of
the liposome are commonly arranged in a bilayer formation, similar
to the lipid arrangement of biological membranes. Particularly
useful liposomes can be generated by the reverse phase evaporation
method with a lipid composition comprising phosphatidylcholine,
cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
Liposomes are extruded through filters of defined pore size to
yield liposomes with the desired diameter. A chemotherapeutic agent
or other therapeutically active agent is optionally contained
within the liposome (Gabizon et al., 1989, J National Cancer Inst
81:1484).
[0125] The antibodies, Fc fusions, and other therapeutically active
agents may also be entrapped in microcapsules prepared by methods
including but not limited to coacervation techniques, interfacial
polymerization (for example using hydroxymethylcellulose or
gelatin-microcapsules, or poly-(methylmethacylate) microcapsules),
colloidal drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), and
macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.
Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semipermeable matrices of
solid hydrophobic polymer, which matrices are in the form of shaped
articles, e.g. films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for
example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (which are injectable microspheres composed of
lactic acid-glycolic acid copolymer and leuprolide acetate),
poly-D-(-)-3-hydroxybutyric acid, and ProLease.RTM. (commercially
available from Alkermes), which is a microsphere-based delivery
system composed of the desired bioactive molecule incorporated into
a matrix of poly-DL-lactide-co-glycolide (PLG).
[0126] The concentration of the therapeutically active antibody or
Fc fusion of the present invention in the formulation may vary from
about 0.1 to 100 weight %. In a preferred embodiment, the
concentration of the antibody or Fc fusion is in the range of 0.003
to 1.0 molar. In order to treat a patient, a therapeutically
effective dose of the antibody or Fc fusion of the present
invention may be administered. By "therapeutically effective dose"
herein is meant a dose that produces the effects for which it is
administered. The exact dose will depend on the purpose of the
treatment, and will be ascertainable by one skilled in the art
using known techniques. Dosages may range from 0.01 to 100 mg/kg of
body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body
weight, with 1 to 10 mg/kg being preferred. As is known in the art,
adjustments for antibody or Fc fusion degradation, systemic versus
localized delivery, and rate of new protease synthesis, as well as
the age, body weight, general health, sex, diet, time of
administration, drug interaction and the severity of the condition
may be necessary, and will be ascertainable with routine
experimentation by those skilled in the art.
[0127] Administration of the pharmaceutical composition comprising
an antibody or Fc fusion of the present invention, preferably in
the form of a sterile aqueous solution, may be done in a variety of
ways, including, but not limited to orally, subcutaneously,
intravenously, intranasally, intraotically, transdermally,
topically (e.g., gels, salves, lotions, creams, etc.),
intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx.RTM.
inhalable technology commercially available from Aradigm, or
Inhance.TM. pulmonary delivery system commercially available from
Inhale Therapeutics), vaginally, parenterally, rectally, or
intraocularly. In some instances, for example for the treatment of
wounds, inflammation, etc., the antibody or Fc fusion may be
directly applied as a solution or spray. As is known in the art,
the pharmaceutical composition may be formulated accordingly
depending upon the manner of introduction.
Engineering Methods
[0128] The present invention provides engineering methods that may
be used to generate Fc variants. A principal obstacle that has
hindered previous attempts at Fc engineering is that only random
attempts at modification have been possible, due in part to the
inefficiency of engineering strategies and methods, and to the
low-throughput nature of antibody production and screening. The
present invention describes engineering methods that overcome these
shortcomings. A variety of design strategies, computational
screening methods, library generation methods, and experimental
production and screening methods are contemplated. These
strategies, approaches, techniques, and methods may be applied
individually or in various combinations to engineer optimized Fc
variants.
Design Strategies
[0129] The most efficient approach to generating Fc variants that
are optimized for a desired property is to direct the engineering
efforts toward that goal. Accordingly, the present invention
teaches design strategies that may be used to engineer optimized Fc
variants. The use of a design strategy is meant to guide Fc
engineering, but is not meant to constrain an Fc variant to a
particular optimized property based on the design strategy that was
used to engineer it. At first thought this may seem
counterintuitive; however its validity is derived from the enormous
complexity of subtle interactions that determine the structure,
stability, solubility, and function of proteins and protein-protein
complexes. Although efforts can be made to predict which protein
positions, residues, interactions, etc. are important for a design
goal, often times critical ones are not predictable. Effects on
protein structure, stability, solubility, and function, whether
favorable or unfavorable, are often unforeseen. Yet there are
innumerable amino acid modifications that are detrimental or
deleterious to proteins. Thus often times the best approach to
engineering comes from generation of protein variants that are
focused generally towards a design goal but do not cause
detrimental effects. In this way, a principal objective of a design
strategy may be the generation of quality diversity. At a
simplistic level this can be thought of as stacking the odds in
one's favor. As an example, perturbation of the Fc carbohydrate or
a particular domain-domain angle, as described below, are valid
design strategies for generating optimized Fc variants, despite the
fact that how carbohydrate and domain-domain angles determine the
properties of Fc is not well understood. By reducing the number of
detrimental amino acid modifications that are screened, i.e. by
screening quality diversity, these design strategies become
practical. Thus the true value of the design strategies taught in
the present invention is their ability to direct engineering
efforts towards the generation of valuable Fc variants. The
specific value of any one resulting variant is determined after
experimentation.
[0130] One design strategy for engineering Fc variants is provided
in which interaction of Fc with some Fc ligand is altered by
engineering amino acid modifications at the interface between Fc
and said Fc ligand. Fc ligands herein may include but are not
limited to Fc.gamma.Rs, C1q, FcRn, protein A or G, and the like. By
exploring energetically favorable substitutions at Fc positions
that impact the binding interface, variants can be engineered that
sample new interface conformations, some of which may improve
binding to the Fc ligand, some of which may reduce Fc ligand
binding, and some of which may have other favorable properties.
Such new interface conformations could be the result of, for
example, direct interaction with Fc ligand residues that form the
interface, or indirect effects caused by the amino acid
modifications such as perturbation of side chain or backbone
conformations. Variable positions may be chosen as any positions
that are believed to play an important role in determining the
conformation of the interface. For example, variable positions may
be chosen as the set of residues that are within a certain
distance, for example 5 Angstroms (.ANG.), preferrably between 1
and 10 .ANG., of any residue that makes direct contact with the Fc
ligand.
[0131] An additional design strategy for generating Fc variants is
provided in which the conformation of the Fc carbohydrate at N297
is optimized. Optimization as used in this context is meant to
includes conformational and compositional changes in the N297
carbohydrate that result in a desired property, for example
increased or reduced affinity for an Fc.gamma.R. Such a strategy is
supported by the observation that the carbohydrate structure and
conformation dramatically affect Fc/Fc.gamma.R and Fc/C1q binding
(Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,
2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J Biol
Chem 276:45539-45547; Radaev et al., 2001, J Biol Chem
276:16478-16483; Shields et al., 2002, J Biol Chem 277:26733-26740;
Shinkawa et al., 2003, J Biol Chem 278:3466-3473). However the
carbohydrate makes no specific contacts with Fc.gamma.Rs. By
exploring energetically favorable substitutions at positions that
interact with carbohydrate, a quality diversity of variants can be
engineered that sample new carbohydrate conformations, some of
which may improve and some of which may reduce binding to one or
more Fc ligands. While the majority of mutations near the
Fc/carbohydrate interface appear to alter carbohydrate
conformation, some mutations have been shown to alter the
glycosylation composition (Lund et al., 1996, J Immunol
157:4963-4969; Jefferis et al., 2002, Immunol Lett 82:57-65).
[0132] Another design strategy for generating Fc variants is
provided in which the angle between the C.gamma.2 and C.gamma.3
domains is optimized Optimization as used in this context is meant
to describe conformational changes in the C.gamma.2-C.gamma.3
domain angle that result in a desired property, for example
increased or reduced affinity for an Fc.gamma.R. This angle is an
important determinant of Fc/Fc.gamma.R affinity (Radaev et al.,
2001, J Biol Chem 276:16478-16483), and a number of mutations
distal to the Fc/Fc.gamma.R interface affect binding potentially by
modulating it (Shields et al., 2001, J Biol Chem 276:6591-6604). By
exploring energetically favorable substitutions positions that
appear to play a key role in determining the C.gamma.2-C.gamma.3
angle and the flexibility of the domains relative to one another, a
quality diversity of variants can be designed that sample new
angles and levels of flexibility, some of which may be optimized
for a desired Fc property.
[0133] Another design strategy for generating Fc variants is
provided in which Fc is reengineered to eliminate the structural
and functional dependence on glycosylation. This design strategy
involves the optimization of Fc structure, stability, solubility,
and/or Fc function (for example affinity of Fc for one or more Fc
ligands) in the absence of the N297 carbohydrate. In one approach,
positions that are exposed to solvent in the absence of
glycosylation are engineered such that they are stable,
structurally consistent with Fc structure, and have no tendency to
aggregate. The C.gamma.2 is the only unpaired Ig domain in the
antibody (see FIG. 1). Thus the N297 carbohydrate covers up the
exposed hydrophobic patch that would normally be the interface for
a protein-protein interaction with another Ig domain, maintaining
the stability and structural integrity of Fc and keeping the
C.gamma.2 domains from aggregating across the central axis.
Approaches for optimizing aglycosylated Fc may involve but are not
limited to designing amino acid modifications that enhance
aglycoslated Fc stability and/or solubility by incorporating polar
and/or charged residues that face inward towards the
C.gamma.2-C.gamma.2 dimer axis, and by designing amino acid
modifications that directly enhance the aglycosylated Fc/Fc.gamma.R
interface or the interface of aglycosylated Fc with some other Fc
ligand.
[0134] An additional design strategy for engineering Fc variants is
provided in which the conformation of the C.gamma.2 domain is
optimized Optimization as used in this context is meant to describe
conformational changes in the C.gamma.2 domain angle that result in
a desired property, for example increased or reduced affinity for
an Fc.gamma.R. By exploring energetically favorable substitutions
at C.gamma.2 positions that impact the C.gamma.2 conformation, a
quality diversity of variants can be engineered that sample new
C.gamma.2 conformations, some of which may achieve the design goal.
Such new C.gamma.2 conformations could be the result of, for
example, alternate backbone conformations that are sampled by the
variant. Variable positions may be chosen as any positions that are
believed to play an important role in determining C.gamma.2
structure, stability, solubility, flexibility, function, and the
like. For example, C.gamma.2 hydrophobic core residues, that is
C.gamma.2 residues that are partially or fully sequestered from
solvent, may be reengineered. Alternatively, noncore residues may
be considered, or residues that are deemed important for
determining backbone structure, stability, or flexibility.
[0135] An additional design strategy for Fc optimization is
provided in which binding to an Fc.gamma.R, complement, or some
other Fc ligand is altered by modifications that modulate the
electrostatic interaction between Fc and said Fc ligand. Such
modifications may be thought of as optimization of the global
electrostatic character of Fc, and include replacement of neutral
amino acids with a charged amino acid, replacement of a charged
amino acid with a neutral amino acid, or replacement of a charged
amino acid with an amino acid of opposite charge (i.e. charge
reversal). Such modifications may be used to effect changes in
binding affinity between an Fc and one or more Fc ligands, for
example Fc.gamma.Rs. In a preferred embodiment, positions at which
electrostatic substitutions might affect binding are selected using
one of a variety of well known methods for calculation of
electrostatic potentials. In the simplest embodiment, Coulomb's law
is used to generate electrostatic potentials as a function of the
position in the protein. Additional embodiments include the use of
Debye-Huckel scaling to account for ionic strength effects, and
more sophisticated embodiments such as Poisson-Boltzmann
calculations. Such electrostatic calculations may highlight
positions and suggest specific amino acid modifications to achieve
the design goal. In some cases, these substitutions may be
anticipated to variably affect binding to different Fc ligands, for
example to enhance binding to activating Fc.gamma.Rs while
decreasing binding affinity to inhibitory Fc.gamma.Rs.
Computational Screening
[0136] A principal obstacle to obtaining valuable Fc variants is
the difficulty in predicting what amino acid modifications, out of
the enormous number of possibilities, will achieve the desired
goals. Indeed one of the principle reasons that previous attempts
at Fc engineering have failed to produce Fc variants of significant
clinical value is that approaches to Fc engineering have thus far
involved hit-or-miss approaches. The present invention provides
computational screening methods that enable quantitative and
systematic engineering of Fc variants. These methods typically use
atomic level scoring functions, side chain rotamer sampling, and
advanced optimization methods to accurately capture the
relationships between protein sequence, structure, and function.
Computational screening enables exploration of the entire sequence
space of possibilities at target positions by filtering the
enormous diversity which results. Variant libraries that are
screened computationally are effectively enriched for stable,
properly folded, and functional sequences, allowing active
optimization of Fc for a desired goal. Because of the overlapping
sequence constraints on protein structure, stability, solubility,
and function, a large number of the candidates in a library occupy
"wasted" sequence space. For example, a large fraction of sequence
space encodes unfolded, misfolded, incompletely folded, partially
folded, or aggregated proteins. This is particularly relevant for
Fc engineering because Ig domains are small beta sheet structures,
the engineering of which has proven extremely demanding (Quinn et
al., 1994, Proc Natl Acad Sci USA 91:8747-8751; Richardson et al.,
2002, Proc Natl Acad Sci USA 99:2754-2759). Even seemingly harmless
substitutions on the surface of a beta sheet can cause severe
packing conflicts, dramatically disrupting folding equilibrium
(Smith et al., 1995, Science 270:980-982); incidentally, alanine is
one of the worst beta sheet formers (Minor et al., 1994, Nature
371:264-267). The determinants of beta sheet stability and
specificity are a delicate balance between an extremely large
number of subtle interactions. Computational screening enables the
generation of libraries that are composed primarily of productive
sequence space, and as a result increases the chances of
identifying proteins that are optimized for the design goal. In
effect, computational screening yields an increased hit-rate,
thereby decreasing the number of variants that must be screened
experimentally. An additional obstacle to Fc engineering is the
need for active design of correlated or coupled mutations. For
example, the greatest Fc/Fc.gamma.R affinity enhancement observed
thus far is S298A/E333A/K334A, obtained by combining three better
binders obtained separately in an alanine scan (Shields et al.,
2001, J Biol Chem 276:6591-6604). Computational screening is
capable of generating such a three-fold variant in one experiment
instead of three separate ones, and furthermore is able to test the
functionality of all 20 amino acids at those positions instead of
just alanine. Computational screening deals with such complexity by
reducing the combinatorial problem to an experimentally tractable
size.
[0137] Computational screening, viewed broadly, has four steps: 1)
selection and preparation of the protein template structure or
structures, 2) selection of variable positions, amino acids to be
considered at those positions, and/or selection of rotamers to
model considered amino acids, 3) energy calculation, and 4)
combinatorial optimization. In more detail, the process of
computational screening can be described as follows. A
three-dimensional structure of a protein is used as the starting
point. The positions to be optimized are identified, which may be
the entire protein sequence or subset(s) thereof. Amino acids that
will be considered at each position are selected. In a preferred
embodiment, each considered amino acid may be represented by a
discrete set of allowed conformations, called rotamers. Interaction
energies are calculated between each considered amino acid and each
other considered amino acid, and the rest of the protein, including
the protein backbone and invariable residues. In a preferred
embodiment, interaction energies are calculated between each
considered amino acid side chain rotamer and each other considered
amino acid side chain rotamer and the rest of the protein,
including the protein backbone and invariable residues. One or more
combinatorial search algorithms are then used to identify the
lowest energy sequence and/or low energy sequences.
[0138] In a preferred embodiment, the computational screening
method used is substantially similar to Protein Design
Automation.RTM. (PDA.RTM.) technology, as is described in U.S. Pat.
No. 6,188,965; U.S. Pat. No. 6,269,312; U.S. Pat. No. 6,403,312;
U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser. No.
10/218,102; PCT WO 98/07254; PCT WO 01/40091; and PCT WO 02/25588.
In another preferred embodiment, a computational screening method
substantially similar to Sequence Prediction Algorithm.TM.
(SPA.TM.) technology is used, as is described in (Raha et al.,
2000, Protein Sci 9:1106-1119), U.S. Ser. No. 09/877,695, and U.S.
Ser. No. 10/071,859. In another preferred embodiment, the
computational screening methods described in U.S. Ser. No.
10/339,788, filed on Mar. 3, 2003, entitled "ANTIBODY
OPTIMIZATION", are used. In some embodiments, combinations of
different computational screening methods are used, including
combinations of PDA.RTM. technology and SPA.TM. technology, as well
as combinations of these computational methods in combination with
other design tools. Similarly, these computational methods can be
used simultaneously or sequentially, in any order.
[0139] A template structure is used as input into the computational
screening calculations. By "template structure" herein is meant the
structural coordinates of part or all of a protein to be optimized.
The template structure may be any protein for which a three
dimensional structure (that is, three dimensional coordinates for a
set of the protein's atoms) is known or may be calculated,
estimated, modeled, generated, or determined. The three dimensional
structures of proteins may be determined using methods including
but not limited to X-ray crystallographic techniques, nuclear
magnetic resonance (NMR) techniques, de novo modeling, and homology
modeling. If optimization is desired for a protein for which the
structure has not been solved experimentally, a suitable structural
model may be generated that may serve as the template for
computational screening calculations. Methods for generating
homology models of proteins are known in the art, and these methods
find use in the present invention. See for example, Luo, et al.
2002, Protein Sci 11: 1218-1226, Lehmann & Wyss, 2001, Curr
Opin Biotechnol 12(4):371-5; Lehmann et al., 2000, Biochim Biophys
Acta 1543(2):408-415; Rath & Davidson, 2000, Protein Sci,
9(12):2457-69; Lehmann et al., 2000, Protein Eng 13(1):49-57;
Desjarlais & Berg, 1993, Proc Nat/Acad Sci USA 90(6):2256-60;
Desjarlais & Berg, 1992, Proteins 12(2):101-4; Henikoff &
Henikoff, 2000, Adv Protein Chem 54:73-97; Henikoff & Henikoff,
1994, J Mol Biol 243(4):574-8; Morea et al., 2000, Methods
20:267-269. Protein/protein complexes may also be obtained using
docking methods. Suitable protein structures that may serve as
template structures include, but are not limited to, all of those
found in the Protein Data Base compiled and serviced by the
Research Collaboratory for Structural Bioinformatics (RCSB,
formerly the Brookhaven National Lab).
[0140] The template structure may be of a protein that occurs
naturally or is engineered. The template structure may be of a
protein that is substantially encoded by a protein from any
organism, with human, mouse, rat, rabbit, and monkey preferred. The
template structure may comprise any of a number of protein
structural forms. In a preferred embodiment the template structure
comprises an Fc region or a domain or fragment of Fc. In an
alternately preferred embodiment the template structure comprises
Fc or a domain or fragment of Fc bound to one or more Fc ligands,
with an Fc/Fc.gamma.R complex being preferred. The Fc in the
template structure may be glycosylated or unglycosylated. The
template structure may comprise more than one protein chain. The
template structure may additionally contain nonprotein components,
including but not limited to small molecules, substrates,
cofactors, metals, water molecules, prosthetic groups, polymers and
carbohydrates. In a preferred embodiment, the template structure is
a plurality or set of template proteins, for example an ensemble of
structures such as those obtained from NMR. Alternatively, the set
of template structures is generated from a set of related proteins
or structures, or artificially created ensembles. The composition
and source of the template structure depends on the engineering
goal. For example, for enhancement of human Fc/Fc.gamma.R affinity,
a human Fc/Fc.gamma.R complex structure or derivative thereof may
be used as the template structure. Alternatively, the uncomplexed
Fc structure may be used as the template structure. If the goal is
to enhance affinity of a human Fc for a mouse Fc.gamma.R, the
template structure may be a structure or model of a human Fc bound
to a mouse Fc.gamma.R.
[0141] The template structure may be modified or altered prior to
design calculations. A variety of methods for template structure
preparation are described in U.S. Pat. No. 6,188,965; U.S. Pat. No.
6,269,312; U.S. Pat. No. 6,403,312; U.S. Ser. No. 09/782,004; U.S.
Ser. No. 09/927,790; U.S. Ser. No. 09/877,695; U.S. Ser. No.
10/071,859, U.S. Ser. No. 10/218,102; PCT WO 98/07254; PCT WO
01/40091; and PCT WO 02/25588. For example, in a preferred
embodiment, explicit hydrogens may be added if not included within
the structure. In an alternate embodiment, energy minimization of
the structure is run to relax strain, including strain due to van
der Waals clashes, unfavorable bond angles, and unfavorable bond
lengths. Alternatively, the template structure is altered using
other methods, such as manually, including directed or random
perturbations. It is also possible to modify the template structure
during later steps of computational screening, including during the
energy calculation and combinatorial optimization steps. In an
alternate embodiment, the template structure is not modified before
or during computational screening calculations.
[0142] Once a template structure has been obtained, variable
positions are chosen. By "variable position" herein is meant a
position at which the amino acid identity is allowed to be altered
in a computational screening calculation. As is known in the art,
allowing amino acid modifications to be considered only at certain
variable positions reduces the complexity of a calculation and
enables computational screening to be more directly tailored for
the design goal. One or more residues may be variable positions in
computational screening calculations. Positions that are chosen as
variable positions may be those that contribute to or are
hypothesized to contribute to the protein property to be optimized,
for example Fc affinity for an Fc.gamma.R, Fc stability, Fc
solubility, and so forth. Residues at variable positions may
contribute favorably or unfavorably to a specific protein property.
For example, a residue at an Fc/Fc.gamma.R interface may be
involved in mediating binding, and thus this position may be varied
in design calculations aimed at improving Fc/Fc.gamma.R affinity.
As another example, a residue that has an exposed hydrophobic side
chain may be responsible for causing unfavorable aggregation, and
thus this position may be varied in design calculations aimed at
improving solubility. Variable positions may be those positions
that are directly involved in interactions that are determinants of
a particular protein property. For example, the Fc.gamma.R binding
site of Fc may be defined to include all residues that contact that
particular F.gamma.cR. By "contact" herein is meant some chemical
interaction between at least one atom of an Fc residue with at
least one atom of the bound Fc.gamma.R, with chemical interaction
including, but not limited to van der Waals interactions, hydrogen
bond interactions, electrostatic interactions, and hydrophobic
interactions. In an alternative embodiment, variable positions may
include those positions that are indirectly involved in a protein
property, i.e. such positions may be proximal to residues that are
known to or hypothesized to contribute to an Fc property. For
example, the Fc.gamma.R binding site of an Fc may be defined to
include all Fc residues within a certain distance, for example 4-10
.ANG., of any Fc residue that is in van der Waals contact with the
Fc.gamma.R. Thus variable positions in this case may be chosen not
only as residues that directly contact the Fc.gamma.R, but also
those that contact residues that contact the Fc.gamma.R and thus
influence binding indirectly. The specific positions chosen are
dependent on the design strategy being employed.
[0143] One or more positions in the template structure that are not
variable may be floated. By "floated position" herein is meant a
position at which the amino acid conformation but not the amino
acid identity is allowed to vary in a computational screening
calculation. In one embodiment, the floated position may have the
parent amino acid identity. For example, floated positions may be
positions that are within a small distance, for example 5 .ANG., of
a variable position residue. In an alternate embodiment, a floated
position may have a non-parent amino acid identity. Such an
embodiment may find use in the present invention, for example, when
the goal is to evaluate the energetic or structural outcome of a
specific mutation.
[0144] Positions that are not variable or floated are fixed. By
"fixed position" herein is meant a position at which the amino acid
identity and the conformation are held constant in a computational
screening calculation. Positions that may be fixed include residues
that are not known to be or hypothesized to be involved in the
property to be optimized. In this case the assumption is that there
is little or nothing to be gained by varying these positions.
Positions that are fixed may also include positions whose residues
are known or hypothesized to be important for maintaining proper
folding, structure, stability, solubility, and/or biological
function. For example, positions may be fixed for residues that
interact with a particular Fc ligand or residues that encode a
glycosylation site in order to ensure that binding to the Fc ligand
and proper glycosylation respectively are not perturbed. Likewise,
if stability is being optimized, it may be beneficial to fix
positions that directly or indirectly interact with an Fc ligand,
for example an Fc.gamma.R, so that binding is not perturbed. Fixed
positions may also include structurally important residues such as
cysteines participating in disulfide bridges, residues critical for
determining backbone conformation such as proline or glycine,
critical hydrogen bonding residues, and residues that form
favorable packing interactions.
[0145] The next step in computational screening is to select a set
of possible amino acid identities that will be considered at each
particular variable position. This set of possible amino acids is
herein referred to as "considered amino acids" at a variable
position. "Amino acids" as used herein refers to the set of natural
20 amino acids and any nonnatural or synthetic analogues. In one
embodiment, all 20 natural amino acids are considered.
Alternatively, a subset of amino acids, or even only one amino acid
is considered at a given variable position. As will be appreciated
by those skilled in the art, there is a computational benefit to
considering only certain amino acid identities at variable
positions, as it decreases the combinatorial complexity of the
search. Furthermore, considering only certain amino acids at
variable positions may be used to tailor calculations toward
specific design strategies. For example, for solubility
optimization of aglycosylated Fc, it may be beneficial to allow
only polar amino acids to be considered at nonpolar Fc residues
that are exposed to solvent in the absence of carbohydrate.
Nonnatural amino acids, including synthetic amino acids and
analogues of natural amino acids, may also be considered amino
acids. For example see Chin et al., 2003, Science, 301(5635):964-7;
and Chin et al., 2003, Chem Biol. 10(6):511-9.
[0146] A wide variety of methods may be used, alone or in
combination, to select which amino acids will be considered at each
position. For example, the set of considered amino acids at a given
variable position may be chosen based on the degree of exposure to
solvent. Hydrophobic or nonpolar amino acids typically reside in
the interior or core of a protein, which are inaccessible or nearly
inaccessible to solvent. Thus at variable core positions it may be
beneficial to consider only or mostly nonpolar amino acids such as
alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,
tryptophan, and methionine. Hydrophilic or polar amino acids
typically reside on the exterior or surface of proteins, which have
a significant degree of solvent accessibility. Thus at variable
surface positions it may be beneficial to consider only or mostly
polar amino acids such as alanine, serine, threonine, aspartic
acid, asparagine, glutamine, glutamic acid, arginine, lysine and
histidine. Some positions are partly exposed and partly buried, and
are not clearly protein core or surface positions, in a sense
serving as boundary residues between core and surface residues.
Thus at such variable boundary positions it may be beneficial to
consider both nonpolar and polar amino acids such as alanine,
serine, threonine, aspartic acid, asparagine, glutamine, glutamic
acid, arginine, lysine histidine, valine, isoleucine, leucine,
phenylalanine, tyrosine, tryptophan, and methionine. Determination
of the degree of solvent exposure at variable positions may be by
subjective evaluation or visual inspection of the template
structure by one skilled in the art of protein structural biology,
or by using a variety of algorithms that are known in the art.
Selection of amino acid types to be considered at variable
positions may be aided or determined wholly by computational
methods, such as calculation of solvent accessible surface area, or
using algorithms that assess the orientation of the
C.alpha.-C.beta. vectors relative to a solvent accessible surface,
as outlined in U.S. Pat. Nos. 6,188,965; 6,269,312; U.S. Pat. No.
6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S.
Ser. No. 10/218,102; PCT WO 98/07254; PCT WO 01/40091; and PCT WO
02/25588. In one embodiment, each variable position may be
classified explicitly as a core, surface, or boundary position or a
classification substantially similar to core, surface, or
boundary.
[0147] In an alternate embodiment, selection of the set of amino
acids allowed at variable positions may be hypothesis-driven.
Hypotheses for which amino acid types should be considered at
variable positions may be derived by a subjective evaluation or
visual inspection of the template structure by one skilled in the
art of protein structural biology. For example, if it is suspected
that a hydrogen bonding interaction may be favorable at a variable
position, polar residues that have the capacity to form hydrogen
bonds may be considered, even if the position is in the core.
Likewise, if it is suspected that a hydrophobic packing interaction
may be favorable at a variable position, nonpolar residues that
have the capacity to form favorable packing interactions may be
considered, even if the position is on the surface. Other examples
of hypothesis-driven approaches may involve issues of backbone
flexibility or protein fold. As is known in the art, certain
residues, for example proline, glycine, and cysteine, play
important roles in protein structure and stability. Glycine enables
greater backbone flexibility than all other amino acids, proline
constrains the backbone more than all other amino acids, and
cysteines may form disulfide bonds. It may therefore be beneficial
to include one or more of these amino acid types to achieve a
desired design goal. Alternatively, it may be beneficial to exclude
one or more of these amino acid types from the list of considered
amino acids.
[0148] In an alternate embodiment, subsets of amino acids may be
chosen to maximize coverage. In this case, additional amino acids
with properties similar to that in the template structure may be
considered at variable positions. For example, if the residue at a
variable position in the template structure is a large hydrophobic
residue, additional large hydrophobic amino acids may be considered
at that position. Alternatively, subsets of amino acids may be
chosen to maximize diversity. In this case, amino acids with
properties dissimilar to those in the template structure may be
considered at variable positions. For example, if the residue at a
variable position in the template is a large hydrophobic residue,
amino acids that are small, polar, etc. may be considered.
[0149] As is known in the art, some computational screening methods
require only the identity of considered amino acids to be
determined during design calculations. That is, no information is
required concerning the conformations or possible conformations of
the amino acid side chains. Other preferred methods utilize a set
of discrete side chain conformations, called rotamers, which are
considered for each amino acid. Thus, a set of rotamers may be
considered at each variable and floated position. Rotamers may be
obtained from published rotamer libraries (see for example, Lovel
et al., 2000, Proteins: Structure Function and Genetics 40:389-408;
Dunbrack & Cohen, 1997, Protein Science 6:1661-1681; DeMaeyer
et al., 1997, Folding and Design 2:53-66; Tuffery et al., 1991, J
Biomol Struct Dyn 8:1267-1289, Ponder & Richards, 1987, J Mol
Biol 193:775-791). As is known in the art, rotamer libraries may be
backbone-independent or backbone-dependent. Rotamers may also be
obtained from molecular mechanics or ab initio calculations, and
using other methods. In a preferred embodiment, a flexible rotamer
model is used (see Mendes et al., 1999, Proteins: Structure,
Function, and Genetics 37:530-543). Similarly, artificially
generated rotamers may be used, or augment the set chosen for each
amino acid and/or variable position. In one embodiment, at least
one conformation that is not low in energy is included in the list
of rotamers. In an alternate embodiment, the rotamer of the
variable position residue in the template structure is included in
the list of rotamers allowed for that variable position. In an
alternate embodiment, only the identity of each amino acid
considered at variable positions is provided, and no specific
conformational states of each amino acid are used during design
calculations. That is, use of rotamers is not essential for
computational screening.
[0150] Experimental information may be used to guide the choice of
variable positions and/or the choice of considered amino acids at
variable positions. As is known in the art, mutagenesis experiments
are often carried out to determine the role of certain residues in
protein structure and function, for example, which protein residues
play a role in determining stability, or which residues make up the
interface of a protein-protein interaction. Data obtained from such
experiments are useful in the present invention. For example,
variable positions for Fc/Fc.gamma.R affinity enhancement could
involve varying all positions at which mutation has been shown to
affect binding. Similarly, the results from such an experiment may
be used to guide the choice of allowed amino acid types at variable
positions. For example, if certain types of amino acid
substitutions are found to be favorable, similar types of those
amino acids may be considered. In one embodiment, additional amino
acids with properties similar to those that were found to be
favorable experimentally may be considered at variable positions.
For example, if experimental mutation of a variable position at an
Fc/Fc.gamma.R interface to a large hydrophobic residue was found to
be favorable, the user may choose to include additional large
hydrophobic amino acids at that position in the computational
screen. As is known in the art, display and other selection
technologies may be coupled with random mutagenesis to generate a
list or lists of amino acid substitutions that are favorable for
the selected property. Such a list or lists obtained from such
experimental work find use in the present invention. For example,
positions that are found to be invariable in such an experiment may
be excluded as variable positions in computational screening
calculations, whereas positions that are found to be more
acceptable to mutation or respond favorably to mutation may be
chosen as variable positions. Similarly, the results from such
experiments may be used to guide the choice of allowed amino acid
types at variable positions. For example, if certain types of amino
acids arise more frequently in an experimental selection, similar
types of those amino acids may be considered. In one embodiment,
additional amino acids with properties similar to those that were
found to be favorable experimentally may be considered at variable
positions. For example, if selected mutations at a variable
position that resides at an Fc/Fc.gamma.R interface are found to be
uncharged polar amino acids, the user may choose to include
additional uncharged polar amino acids, or perhaps charged polar
amino acids, at that position.
[0151] Sequence information may also be used to guide choice of
variable positions and/or the choice of amino acids considered at
variable positions. As is known in the art, some proteins share a
common structural scaffold and are homologous in sequence. This
information may be used to gain insight into particular positions
in the protein family. As is known in the art, sequence alignments
are often carried out to determine which protein residues are
conserved and which are not conserved. That is to say, by comparing
and contrasting alignments of protein sequences, the degree of
variability at a position may be observed, and the types of amino
acids that occur naturally at positions may be observed. Data
obtained from such analyses are useful in the present invention.
The benefit of using sequence information to choose variable
positions and considered amino acids at variable positions are
several fold. For choice of variable positions, the primary
advantage of using sequence information is that insight may be
gained into which positions are more tolerant and which are less
tolerant to mutation. Thus sequence information may aid in ensuring
that quality diversity, i.e. mutations that are not deleterious to
protein structure, stability, etc., is sampled computationally. The
same advantage applies to use of sequence information to select
amino acid types considered at variable positions. That is, the set
of amino acids that occur in a protein sequence alignment may be
thought of as being pre-screened by evolution to have a higher
chance than random for being compatible with a protein's structure,
stability, solubility, function, etc. Thus higher quality diversity
is sampled computationally. A second benefit of using sequence
information to select amino acid types considered at variable
positions is that certain alignments may represent sequences that
may be less immunogenic than random sequences. For example, if the
amino acids considered at a given variable position are the set of
amino acids which occur at that position in an alignment of human
protein sequences, those amino acids may be thought of as being
pre-screened by nature for generating no or low immune response if
the optimized protein is used as a human therapeutic.
[0152] The source of the sequences may vary widely, and include one
or more of the known databases, including but not limited to the
Kabat database (Johnson & Wu, 2001, Nucleic Acids Res
29:205-206; Johnson & Wu, 2000, Nucleic Acids Res 28:214-218),
the IMGT database (IMGT, the international ImMunoGeneTics
information system.RTM.; Lefranc et al., 1999, Nucleic Acids Res
27:209-212; Ruiz et al., 2000 Nucleic Acids Re. 28:219-221; Lefranc
et al., 2001, Nucleic Acids Res 29:207-209; Lefranc et al., 2003,
Nucleic Acids Res 31:307-310), and VBASE, SwissProt, GenBank and
Entrez, and EMBL Nucleotide Sequence Database. Protein sequence
information can be obtained, compiled, and/or generated from
sequence alignments of naturally occurring proteins from any
organism, including but not limited to mammals. Protein sequence
information can be obtained from a database that is compiled
privately. There are numerous sequence-based alignment programs and
methods known in the art, and all of these find use in the present
invention for generation of sequence alignments of proteins that
comprise Fc and Fc ligands.
[0153] Once alignments are made, sequence information can be used
to guide choice of variable positions. Such sequence information
can relate the variability, natural or otherwise, of a given
position. Variability herein should be distinguished from variable
position. Variability refers to the degree to which a given
position in a sequence alignment shows variation in the types of
amino acids that occur there. Variable position, to reiterate, is a
position chosen by the user to vary in amino acid identity during a
computational screening calculation. Variability may be determined
qualitatively by one skilled in the art of bioinformatics. There
are also methods known in the art to quantitatively determine
variability that may find use in the present invention. The most
preferred embodiment measures Information Entropy or Shannon
Entropy. Variable positions can be chosen based on sequence
information obtained from closely related protein sequences, or
sequences that are less closely related.
[0154] The use of sequence information to choose variable positions
finds broad use in the present invention. For example, if an
Fc/Fc.gamma.R interface position in the template structure is
tryptophan, and tryptophan is observed at that position in greater
than 90% of the sequences in an alignment, it may be beneficial to
leave that position fixed. In contrast, if another interface
position is found to have a greater level of variability, for
example if five different amino acids are observed at that position
with frequencies of approximately 20% each, that position may be
chosen as a variable position. In another embodiment, visual
inspection of aligned protein sequences may substitute for or aid
visual inspection of a protein structure. Sequence information can
also be used to guide the choice of amino acids considered at
variable positions. Such sequence information can relate to how
frequently an amino acid, amino acids, or amino acid types (for
example polar or nonpolar, charged or uncharged) occur, naturally
or otherwise, at a given position. In one embodiment, the set of
amino acids considered at a variable position may comprise the set
of amino acids that is observed at that position in the alignment.
Thus, the position-specific alignment information is used directly
to generate the list of considered amino acids at a variable
position in a computational screening calculation. Such a strategy
is well known in the art; see for example Lehmann & Wyss, 2001,
Curr Opin Biotechnol 12(4):371-5; Lehmann et al., 2000, Biochim
Biophys Acta 1543(2):408-415; Rath & Davidson, 2000, Protein
Sci, 9(12):2457-69; Lehmann et al., 2000, Protein Eng 13(1):49-57;
Desjarlais & Berg, 1993, Proc Natl Acad Sci USA 90(6):2256-60;
Desjarlais & Berg, 1992, Proteins 12(2):101-4; Henikoff &
Henikoff, 2000, Adv Protein Chem 54:73-97; Henikoff & Henikoff,
1994, J Mol Biol 243(4):574-8. In an alternate embodiment, the set
of amino acids considered at a variable position or positions may
comprise a set of amino acids that is observed most frequently in
the alignment. Thus, a certain criteria is applied to determine
whether the frequency of an amino acid or amino acid type warrants
its inclusion in the set of amino acids that are considered at a
variable position. As is known in the art, sequence alignments may
be analyzed using statistical methods to calculate the sequence
diversity at any position in the alignment and the occurrence
frequency or probability of each amino acid at a position. Such
data may then be used to determine which amino acids types to
consider. In the simplest embodiment, these occurrence frequencies
are calculated by counting the number of times an amino acid is
observed at an alignment position, then dividing by the total
number of sequences in the alignment. In other embodiments, the
contribution of each sequence, position or amino acid to the
counting procedure is weighted by a variety of possible mechanisms.
In a preferred embodiment, the contribution of each aligned
sequence to the frequency statistics is weighted according to its
diversity weighting relative to other sequences in the alignment. A
common strategy for accomplishing this is the sequence weighting
system recommended by Henikoff and Henikoff (Henikoff &
Henikoff, 2000, Adv Protein Chem 54:73-97; Henikoff & Henikoff,
1994, J Mol Biol 243:574-8. In a preferred embodiment, the
contribution of each sequence to the statistics is dependent on its
extent of similarity to the target sequence, i.e. the template
structure used, such that sequences with higher similarity to the
target sequence are weighted more highly. Examples of similarity
measures include, but are not limited to, sequence identity, BLOSUM
similarity score, PAM matrix similarity score, and BLAST score. In
an alternate embodiment, the contribution of each sequence to the
statistics is dependent on its known physical or functional
properties. These properties include, but are not limited to,
thermal and chemical stability, contribution to activity, and
solubility. For example, when optimizing aglycosylated Fc for
solubility, those sequences in an alignment that are known to be
most soluble (for example see Ewert et al., 2003, J Mol Biol
325:531-553), will contribute more heavily to the calculated
frequencies.
[0155] Regardless of what criteria are applied for choosing the set
of amino acids in a sequence alignment to be considered at variable
positions, use of sequence information to choose considered amino
acids finds broad use in the present invention. For example, to
optimize Fc solubility by replacing exposed nonpolar surface
residues, considered amino acids may be chosen as the set of amino
acids, or a subset of those amino acids which meet some criteria,
that are observed at that position in an alignment of protein
sequences. As another example, one or more amino acids may be added
or subtracted subjectively from a list of amino acids derived from
a sequence alignment in order to maximize coverage. For example,
additional amino acids with properties similar to those that are
found in a sequence alignment may be considered at variable
positions. For example, if an Fc position that is known to or
hypothesized to bind an Fc.gamma.R is observed to have uncharged
polar amino acids in a sequence alignment, the user may choose to
include additional uncharged polar amino acids in a computational
screening calculation, or perhaps charged polar amino acids, at
that position.
[0156] In one embodiment, sequence alignment information is
combined with energy calculation, as discussed below. For example,
pseudo energies can be derived from sequence information to
generate a scoring function. The use of a sequence-based scoring
function may assist in significantly reducing the complexity of a
calculation. However, as is appreciated by those skilled in the
art, the use of a sequence-based scoring function alone may be
inadequate because sequence information can often indicate
misleading correlations between mutations that may in reality be
structurally conflicting. Thus, in a preferred embodiment, a
structure-based method of energy calculation is used, either alone
or in combination with a sequence-based scoring function. That is,
preferred embodiments do not rely on sequence alignment information
alone as the analysis step.
[0157] Energy calculation refers to the process by which amino acid
modifications are scored. The energies of interaction are measured
by one or more scoring functions. A variety of scoring functions
find use in the present invention for calculating energies. Scoring
functions may include any number of potentials, herein referred to
as the energy terms of a scoring function, including but not
limited to a van der Waals potential, a hydrogen bond potential, an
atomic solvation potential or other solvation models, a secondary
structure propensity potential, an electrostatic potential, a
torsional potential, and an entropy potential. At least one energy
term is used to score each variable or floated position, although
the energy terms may differ depending on the position, considered
amino acids, and other considerations. In one embodiment, a scoring
function using one energy term is used. In the most preferred
embodiment, energies are calculated using a scoring function that
contains more than one energy term, for example describing van der
Waals, salvation, electrostatic, and hydrogen bond interactions,
and combinations thereof. In additional embodiments, additional
energy terms include but are not limited to entropic terms,
torsional energies, and knowledge-based energies.
[0158] A variety of scoring functions are described in U.S. Pat.
No. 6,188,965; U.S. Pat. No. 6,269,312; U.S. Pat. No. 6,403,312;
U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser. No.
09/877,695; U.S. Ser. No. 10/071,859, U.S. Ser. No. 10/218,102; PCT
WO 98/07254; PCT WO 01/40091; and PCT WO 02/25588. As will be
appreciated by those skilled in the art, scoring functions need not
be limited to physico-chemical energy terms. For example,
knowledge-based potentials may find use in the computational
screening methodology of the present invention. Such
knowledge-based potentials may be derived from protein sequence
and/or structure statistics including but not limited to threading
potentials, reference energies, pseudo energies, homology-based
energies, and sequence biases derived from sequence alignments. In
a preferred embodiment, a scoring function is modified to include
models for immunogenicity, such as functions derived from data on
binding of peptides to MHC (Major Htocompatability Complex), that
may be used to identify potentially immunogenic sequences (see for
example U.S. Ser. No. 09/903,378; U.S. Ser. No. 10/039,170; U.S.
Ser. No. 60/222,697; U.S. Ser. No. 10/339,788; PCT WO 01/21823; and
PCT WO 02/00165). In one embodiment, sequence alignment information
can be used to score amino acid substitutions. For example,
comparison of protein sequences, regardless of whether the source
of said proteins is human, monkey, mouse, or otherwise, may be used
to suggest or score amino acid mutations in the computational
screening methodology of the present invention. In one embodiment,
as is known in the art, one or more scoring functions may be
optimized or "trained" during the computational analysis, and then
the analysis re-run using the optimized system. Such altered
scoring functions may be obtained for example, by training a
scoring function using experimental data. As will be appreciated by
those skilled in the art, a number of force fields, which are
comprised of one or more energy terms, may serve as scoring
functions. Force fields include but are not limited to ab initio or
quantum mechanical force fields, semi-empirical force fields, and
molecular mechanics force fields. Scoring functions that are
knowledge-based or that use statistical methods may find use in the
present invention. These methods may be used to assess the match
between a sequence and a three-dimensional protein structure, and
hence may be used to score amino acid substitutions for fidelity to
the protein structure. In one embodiment, molecular dynamics
calculations may be used to computationally screen sequences by
individually calculating mutant sequence scores.
[0159] There are a variety of ways to represent amino acids in
order to enable efficient energy calculation. In a preferred
embodiment, considered amino acids are represented as rotamers, as
described previously, and the energy (or score) of interaction of
each possible rotamer at each variable and floated position with
the other variable and floated rotamers, with fixed position
residues, and with the backbone structure and any non-protein
atoms, is calculated. In a preferred embodiment, two sets of
interaction energies are calculated for each side chain rotamer at
every variable and floated position: the interaction energy between
the rotamer and the fixed atoms (the "singles" energy), and the
interaction energy between the variable and floated positions
rotamer and all other possible rotamers at every other variable and
floated position (the "doubles" energy). In an alternate
embodiment, singles and doubles energies are calculated for fixed
positions as well as for variable and floated positions. In an
alternate embodiment, considered amino acids are not represented as
rotamers.
[0160] An important component of computational screening is the
identification of one or more sequences that have a favorable
score, i.e. are low in energy. Determining a set of low energy
sequences from an extremely large number of possibilities is
nontrivial, and to solve this problem a combinatorial optimization
algorithm is employed. The need for a combinatorial optimization
algorithm is illustrated by examining the number of possibilities
that are considered in a typical computational screening
calculation. The discrete nature of rotamer sets allows a simple
calculation of the number of possible rotameric sequences for a
given design problem. A backbone of length n with m possible
rotamers per position will have m.sup.n possible rotamer sequences,
a number that grows exponentially with sequence length. For very
simple calculations, it is possible to examine each possible
sequence in order to identify the optimal sequence and/or one or
more favorable sequences. However, for a typical design problem,
the number of possible sequences (up to 10.sup.80 or more) is
sufficiently large that examination of each possible sequence is
intractable. A variety of combinatorial optimization algorithms may
then be used to identify the optimum sequence and/or one or more
favorable sequences. Combinatorial optimization algorithms may be
divided into two classes: (1) those that are guaranteed to return
the global minimum energy configuration if they converge, and (2)
those that are not guaranteed to return the global minimum energy
configuration, but which will always return a solution. Examples of
the first class of algorithms include but are not limited to
Dead-End Elimination (DEE) and Branch & Bound (B&B)
(including Branch and Terminate) (Gordon & Mayo, 1999,
Structure Fold Des 7:1089-98). Examples of the second class of
algorithms include, but are not limited to, Monte Carlo (MC),
self-consistent mean field (SCMF), Boltzmann sampling (Metropolis
et al., 1953, J Chem Phys 21:1087), simulated annealing
(Kirkpatrick et al., 1983, Science, 220:671-680), genetic algorithm
(GA), and Fast and Accurate Side-Chain Topology and Energy
Refinement (FASTER) (Desmet, et al., 2002, Proteins, 48:31-43). A
combinatorial optimization algorithm may be used alone or in
conjunction with another combinatorial optimization algorithm.
[0161] In one embodiment of the present invention, the strategy for
applying a combinatorial optimization algorithm is to find the
global minimum energy configuration. In an alternate embodiment,
the strategy is to find one or more low energy or favorable
sequences. In an alternate embodiment, the strategy is to find the
global minimum energy configuration and then find one or more low
energy or favorable sequences. For example, as outlined in U.S.
Pat. No. 6,269,312, preferred embodiments utilize a Dead End
Elimination (DEE) step and a Monte Carlo step. In other
embodiments, tabu search algorithms are used or combined with DEE
and/or Monte Carlo, among other search methods (see Modern
Heuristic Search Methods, edited by V. J. Rayward-Smith et al.,
1996, John Wiley & Sons Ltd.; U.S. Ser. No. 10/218,102; and PCT
WO 02/25588). In another preferred embodiment, a genetic algorithm
may be used; see for example U.S. Ser. No. 09/877,695 and U.S. Ser.
No. 10/071,859. As another example, as is more fully described in
U.S. Pat. No. 6,188,965; U.S. Pat. No. 6,269,312; U.S. Pat. No.
6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S.
Ser. No. 10/218,102; PCT WO 98/07254; PCT WO 01/40091; and PCT WO
02/25588, the global optimum may be reached, and then further
computational processing may occur, which generates additional
optimized sequences. In the simplest embodiment, design
calculations are not combinatorial. That is, energy calculations
are used to evaluate amino acid substitutions individually at
single variable positions. For other calculations it is preferred
to evaluate amino acid substitutions at more than one variable
position. In a preferred embodiment, all possible interaction
energies are calculated prior to combinatorial optimization. In an
alternatively preferred embodiment, energies may be calculated as
needed during combinatorial optimization.
Library Generation
[0162] The present invention provides methods for generating
libraries that may subsequently be screened experimentally to
single out optimized Fc variants. By "library" as used herein is
meant a set of one or more Fc variants. Library may refer to the
set of variants in any form. In one embodiment, the library is a
list of nucleic acid or amino acid sequences, or a list of nucleic
acid or amino acid substitutions at variable positions. For
example, the examples used to illustrate the present invention
below provide libraries as amino acid substitutions at variable
positions. In one embodiment, a library is a list of at least one
sequence that are Fc variants optimized for a desired property. For
example see, Filikov et al., 2002, Protein Sci 11: 1452-1461 and
Luo et al., 2002, Protein Sci 11:1218-1226. In an alternate
embodiment, a library may be defined as a combinatorial list,
meaning that a list of amino acid substitutions is generated for
each variable position, with the implication that each substitution
is to be combined with all other designed substitutions at all
other variable positions. In this case, expansion of the
combination of all possibilities at all variable positions results
in a large explicitly defined library. A library may refer to a
physical composition of polypeptides that comprise the Fc region or
some domain or fragment of the Fc region. Thus a library may refer
to a physical composition of antibodies or Fc fusions, either in
purified or unpurified form. A library may refer to a physical
composition of nucleic acids that encode the library sequences.
Said nucleic acids may be the genes encoding the library members,
the genes encoding the library members with any operably linked
nucleic acids, or expression vectors encoding the library members
together with any other operably linked regulatory sequences,
selectable markers, fusion constructs, and/or other elements. For
example, the library may be a set of mammalian expression vectors
that encode Fc library members, the protein products of which may
be subsequently expressed, purified, and screened experimentally.
As another example, the library may be a display library. Such a
library could, for example, comprise a set of expression vectors
that encode library members operably linked to some fusion partner
that enables phage display, ribosome display, yeast display,
bacterial surface display, and the like.
[0163] The library may be generated using the output sequence or
sequences from computational screening. As discussed above,
computationally generated libraries are significantly enriched in
stable, properly folded, and functional sequences relative to
randomly generated libraries. As a result, computational screening
increases the chances of identifying proteins that are optimized
for the design goal. The set of sequences in a library is
generally, but not always, significantly different from the parent
sequence, although in some cases the library preferably contains
the parent sequence. As is known in the art, there are a variety of
ways that a library may be derived from the output of computational
screening calculations. For example, methods of library generation
described in U.S. Pat. No. 6,403,312; U.S. Ser. No. 09/782,004;
U.S. Ser. No. 09/927,790; U.S. Ser. No. 10/218,102; PCT WO
01/40091; and PCT WO 02/25588 find use in the present invention. In
one embodiment, sequences scoring within a certain range of the
global optimum sequence may be included in the library. For
example, all sequences within 10 kcal/mol of the lowest energy
sequence could be used as the library. In an alternate embodiment,
sequences scoring within a certain range of one or more local
minima sequences may be used. In a preferred embodiment, the
library sequences are obtained from a filtered set. Such a list or
set may be generated by a variety of methods, as is known in the
art, for example using an algorithm such as Monte Carlo, B&B,
or SCMF. For example, the top 10.sup.3 or the top 10.sup.5
sequences in the filtered set may comprise the library.
Alternatively, the total number of sequences defined by the
combination of all mutations may be used as a cutoff criterion for
the library. Preferred values for the total number of recombined
sequences range from 10 to 10.sup.20, particularly preferred values
range from 100 to 10.sup.9. Alternatively, a cutoff may be enforced
when a predetermined number of mutations per position is reached.
In some embodiments, sequences that do not make the cutoff are
included in the library. This may be desirable in some situations,
for instance to evaluate the approach to library generation, to
provide controls or comparisons, or to sample additional sequence
space. For example, the parent sequence may be included in the
library, even if it does not make the cutoff.
[0164] Clustering algorithms may be useful for classifying
sequences derived by computational screening methods into
representative groups. For example, the methods of clustering and
their application described in U.S. Ser. No. 10/218,102 and PCT WO
02/25588, find use in the present invention. Representative groups
may be defined, for example, by similarity. Measures of similarity
include, but are not limited to sequence similarity and energetic
similarity. Thus the output sequences from computational screening
may be clustered around local minima, referred to herein as
clustered sets of sequences. For example, sets of sequences that
are close in sequence space may be distinguished from other sets.
In one embodiment, coverage within one or a subset of clustered
sets may be maximized by including in the library some, most, or
all of the sequences that make up one or more clustered sets of
sequences. For example, it may be advantageous to maximize coverage
within the one, two, or three lowest energy clustered sets by
including the majority of sequences within these sets in the
library. In an alternate embodiment, diversity across clustered
sets of sequences may be sampled by including within a library only
a subset of sequences within each clustered set. For example, all
or most of the clustered sets could be broadly sampled by including
the lowest energy sequence from each clustered set in the
library.
[0165] Sequence information may be used to guide or filter
computationally screening results for generation of a library. As
discussed, by comparing and contrasting alignments of protein
sequences, the degree of variability at a position and the types of
amino acids which occur naturally at that position may be observed.
Data obtained from such analyses are useful in the present
invention. The benefits of using sequence information have been
discussed, and those benefits apply equally to use of sequence
information to guide library generation. The set of amino acids
that occur in a sequence alignment may be thought of as being
pre-screened by evolution to have a higher chance than random at
being compatible with a protein's structure, stability, solubility,
function, and immunogenicity. The variety of sequence sources, as
well as the methods for generating sequence alignments that have
been discussed, find use in the application of sequence information
to guiding library generation. Likewise, as discussed above,
various criteria may be applied to determine the importance or
weight of certain residues in an alignment. These methods also find
use in the application of sequence information to guide library
generation. Using sequence information to guide library generation
from the results of computational screening finds broad use in the
present invention. In one embodiment, sequence information is used
to filter sequences from computational screening output. That is to
say, some substitutions are subtracted from the computational
output to generate the library. For example the resulting output of
a computational screening calculation or calculations may be
filtered so that the library includes only those amino acids, or a
subset of those amino acids that meet some criteria, for example
that are observed at that position in an alignment of sequences. In
an alternate embodiment, sequence information is used to add
sequences to the computational screening output. That is to say,
sequence information is used to guide the choice of additional
amino acids that are added to the computational output to generate
the library. For example, the output set of amino acids for a given
position from a computational screening calculation may be
augmented to include one or more amino acids that are observed at
that position in an alignment of protein sequences. In an alternate
embodiment, based on sequence alignment information, one or more
amino acids may be added to or subtracted from the computational
screening sequence output in order to maximize coverage or
diversity. For example, additional amino acids with properties
similar to those that are found in a sequence alignment may be
added to the library. For example, if a position is observed to
have uncharged polar amino acids in a sequence alignment,
additional uncharged polar amino acids may be included in the
library at that position.
[0166] Libraries may be processed further to generate subsequent
libraries. In this way, the output from a computational screening
calculation or calculations may be thought of as a primary library.
This primary library may be combined with other primary libraries
from other calculations or other libraries, processed using
subsequent calculations, sequence information, or other analyses,
or processed experimentally to generate a subsequent library,
herein referred to as a secondary library. As will be appreciated
from this description, the use of sequence information to guide or
filter libraries, discussed above, is itself one method of
generating secondary libraries from primary libraries. Generation
of secondary libraries gives the user greater control of the
parameters within a library. This enables more efficient
experimental screening, and may allow feedback from experimental
results to be interpreted more easily, providing a more efficient
design/experimentation cycle.
[0167] There are a wide variety of methods to generate secondary
libraries from primary libraries. For example, U.S. Ser. No.
10/218,102 and PCT WO 02/25588, describes methods for secondary
library generation that find use in the present invention.
Typically some selection step occurs in which a primary library is
processed in some way. For example, in one embodiment a selection
step occurs wherein some set of primary sequences are chosen to
form the secondary library. In an alternate embodiment, a selection
step is a computational step, again generally including a selection
step, wherein some subset of the primary library is chosen and then
subjected to further computational analysis, including both further
computational screening as well as techniques such as "in silico"
shuffling or recombination (see, for example U.S. Pat. No.
5,830,721; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,605,793; and
U.S. Pat. No. 5,837,458, error-prone PCR, for example using
modified nucleotides; known mutagenesis techniques including the
use of multi-cassettes; and DNA shuffling (Crameri et al., 1998,
Nature 391:288-291; Coco et al., 2001, Nat Biotechnol 19:354-9;
Coco et al., 2002, Nat Biotechnol, 20:1246-50), heterogeneous DNA
samples (U.S. Pat. No. 5,939,250); ITCHY (Ostermeier et al., 1999,
Nat Biotechnol 17:1205-1209); StEP (Zhao et al., 1998, Nat
Biotechnol 16:258-261), GSSM (U.S. Pat. No. 6,171,820 and U.S. Pat.
No. 5,965,408); in vivo homologous recombination, ligase assisted
gene assembly, end-complementary PCR, profusion (Roberts &
Szostak, 1997, Proc Natl Acad Sci USA 94:12297-12302);
yeast/bacteria surface display (Lu et al., 1995, Biotechnology
13:366-372); Seed & Aruffo, 1987, Proc Natl Acad Sci USA
84(10):3365-3369; Boder & Wittrup, 1997, Nat Biotechnol
15:553-557). In an alternate embodiment, a selection step occurs
that is an experimental step, for example any of the library
screening steps below, wherein some subset of the primary library
is chosen and then recombined experimentally, for example using one
of the directed evolution methods discussed below, to form a
secondary library. In a preferred embodiment, the primary library
is generated and processed as outlined in U.S. Pat. No.
6,403,312.
[0168] Generation of secondary and subsequent libraries finds broad
use in the present invention. In one embodiment, different primary
libraries may be combined to generate a secondary or subsequent
library. In another embodiment, secondary libraries may be
generated by sampling sequence diversity at highly mutatable or
highly conserved positions. The primary library may be analyzed to
determine which amino acid positions in the template protein have
high mutational frequency, and which positions have low mutational
frequency. For example, positions in a protein that show a great
deal of mutational diversity in computational screening may be
fixed in a subsequent round of design calculations. A filtered set
of the same size as the first would now show diversity at positions
that were largely conserved in the first library. Alternatively,
the secondary library may be generated by varying the amino acids
at the positions that have high numbers of mutations, while keeping
constant the positions that do not have mutations above a certain
frequency.
[0169] This discussion is not meant to constrain generation of
libraries subsequent to primary libraries to secondary libraries.
As will be appreciated, primary and secondary libraries may be
processed further to generate tertiary libraries, quaternary
libraries, and so on. In this way, library generation is an
iterative process. For example, tertiary libraries may be
constructed using a variety of additional steps applied to one or
more secondary libraries; for example, further computational
processing may occur, secondary libraries may be recombined, or
subsets of different secondary libraries may be combined. In a
preferred embodiment, a tertiary library may be generated by
combining secondary libraries. For example, primary and/or
secondary libraries that analyzed different parts of a protein may
be combined to generate a tertiary library that treats the combined
parts of the protein. In an alternate embodiment, the variants from
a primary library may be combined with the variants from another
primary library to provide a combined tertiary library at lower
computational cost than creating a very long filtered set. These
combinations may be used, for example, to analyze large proteins,
especially large multi-domain proteins, of which Fc is an example.
Thus the above description of secondary library generation applies
to generating any library subsequent to a primary library, the end
result being a final library that may screened experimentally to
obtain protein variants optimized for a design goal. These examples
are not meant to constrain generation of secondary libraries to any
particular application or theory of operation for the present
invention. Rather, these examples are meant to illustrate that
generation of secondary libraries, and subsequent libraries such as
tertiary libraries and so on, is broadly useful in computational
screening methodology for library generation.
Experimental Production and Screening
[0170] The present invention provides methods for producing and
screening libraries of Fc variants. The described methods are not
meant to constrain the present invention to any particular
application or theory of operation. Rather, the provided methods
are meant to illustrate generally that one or more Fc variants or
one or more libraries of Fc variants may be produced and screened
experimentally to obtain optimized Fc variants. Fc variants may be
produced and screened in any context, whether as an Fc region as
precisely defined herein, a domain or fragment thereof, or a larger
polypeptide that comprises Fc such as an antibody or Fc fusion.
General methods for antibody molecular biology, expression,
purification, and screening are described in Antibody Engineering,
edited by Duebel & Kontermann, Springer-Verlag, Heidelberg,
2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol
5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng
2:339-76; Antibodies: A Laboratory Manual by Harlow & Lane, New
York: Cold Spring Harbor Laboratory Press, 1988.
[0171] In one embodiment of the present invention, the library
sequences are used to create nucleic acids that encode the member
sequences, and that may then be cloned into host cells, expressed
and assayed, if desired. Thus, nucleic acids, and particularly DNA,
may be made that encode each member protein sequence. These
practices are carried out using well-known procedures. For example,
a variety of methods that may find use in the present invention are
described in Molecular Cloning--A Laboratory Manual, 3rd Ed.
(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001),
and Current Protocols in Molecular Biology (John Wiley & Sons).
As will be appreciated by those skilled in the art, the generation
of exact sequences for a library comprising a large number of
sequences is potentially expensive and time consuming. Accordingly,
there are a variety of techniques that may be used to efficiently
generate libraries of the present invention. Such methods that may
find use in the present invention are described or referenced in
U.S. Pat. No. 6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No.
09/927,790; U.S. Ser. No. 10/218,102; PCT WO 01/40091; and PCT WO
02/25588. Such methods include but are not limited to gene assembly
methods, PCR-based method and methods which use variations of PCR,
ligase chain reaction-based methods, pooled oligo methods such as
those used in synthetic shuffling, error-prone amplification
methods and methods which use oligos with random mutations,
classical site-directed mutagenesis methods, cassette mutagenesis,
and other amplification and gene synthesis methods. As is known in
the art, there are a variety of commercially available kits and
methods for gene assembly, mutagenesis, vector subcloning, and the
like, and such commercial products find use in the present
invention for generating nucleic acids that encode Fc variant
members of a library.
[0172] The Fc variants of the present invention may be produced by
culturing a host cell transformed with nucleic acid, preferably an
expression vector, containing nucleic acid encoding the Fc
variants, under the appropriate conditions to induce or cause
expression of the protein. The conditions appropriate for
expression will vary with the choice of the expression vector and
the host cell, and will be easily ascertained by one skilled in the
art through routine experimentation. A wide variety of appropriate
host cells may be used, including but not limited to mammalian
cells, bacteria, insect cells, and yeast. For example, a variety of
cell lines that may find use in the present invention are described
in the ATCC.RTM. cell line catalog, available from the American
Type Culture Collection.
[0173] In a preferred embodiment, the Fc variants are expressed in
mammalian expression systems, including systems in which the
expression constructs are introduced into the mammalian cells using
virus such as retrovirus or adenovirus. Any mammalian cells may be
used, with human, mouse, rat, hamster, and primate cells being
particularly preferred. Suitable cells also include known research
cells, including but not limited to Jurkat T cells, NIH3T3, CHO,
COS, and 293 cells. In an alternately preferred embodiment, library
proteins are expressed in bacterial cells. Bacterial expression
systems are well known in the art, and include Escherichia coli (E.
coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus
lividans. In alternate embodiments, Fc variants are produced in
insect cells or yeast cells. In an alternate embodiment, Fc
variants are expressed in vitro using cell free translation
systems. In vitro translation systems derived from both prokaryotic
(e.g. E. coli) and eukaryotic (e.g. wheat germ, rabbit
reticulocytes) cells are available and may be chosen based on the
expression levels and functional properties of the protein of
interest. For example, as appreciated by those skilled in the art,
in vitro translation is required for some display technologies, for
example ribosome display. In addition, the Fc variants may be
produced by chemical synthesis methods.
[0174] The nucleic acids that encode the Fc variants of the present
invention may be incorporated into an expression vector in order to
express the protein. A variety of expression vectors may be
utilized for protein expression. Expression vectors may comprise
self-replicating extra-chromosomal vectors or vectors which
integrate into a host genome. Expression vectors are constructed to
be compatible with the host cell type. Thus expression vectors
which find use in the present invention include but are not limited
to those which enable protein expression in mammalian cells,
bacteria, insect cells, yeast, and in in vitro systems. As is known
in the art, a variety of expression vectors are available,
commercially or otherwise, that may find use in the present
invention for expressing Fc variant proteins.
[0175] Expression vectors typically comprise a protein operably
linked with control or regulatory sequences, selectable markers,
any fusion partners, and/or additional elements. By "operably
linked" herein is meant that the nucleic acid is placed into a
functional relationship with another nucleic acid sequence.
Generally, these expression vectors include transcriptional and
translational regulatory nucleic acid operably linked to the
nucleic acid encoding the Fc variant, and are typically appropriate
to the host cell used to express the protein. In general, the
transcriptional and translational regulatory sequences may include
promoter sequences, ribosomal binding sites, transcriptional start
and stop sequences, translational start and stop sequences, and
enhancer or activator sequences. As is also known in the art,
expression vectors typically contain a selection gene or marker to
allow the selection of transformed host cells containing the
expression vector. Selection genes are well known in the art and
will vary with the host cell used.
[0176] Fc variants may be operably linked to a fusion partner to
enable targeting of the expressed protein, purification, screening,
display, and the like. Fusion partners may be linked to the Fc
variant sequence via a linker sequences. The linker sequence will
generally comprise a small number of amino acids, typically less
than ten, although longer linkers may also be used. Typically,
linker sequences are selected to be flexible and resistant to
degradation. As will be appreciated by those skilled in the art,
any of a wide variety of sequences may be used as linkers. For
example, a common linker sequence comprises the amino acid sequence
GGGGS (SEQ ID NO:7). A fusion partner may be a targeting or signal
sequence that directs Fc variant protein and any associated fusion
partners to a desired cellular location or to the extracellular
media. As is known in the art, certain signaling sequences may
target a protein to be either secreted into the growth media, or
into the periplasmic space, located between the inner and outer
membrane of the cell. A fusion partner may also be a sequence that
encodes a peptide or protein that enables purification and/or
screening. Such fusion partners include but are not limited to
polyhistidine tags (His-tags) (for example H.sub.6 and H.sub.10 or
other tags for use with Immobilized Metal Affinity Chromatography
(IMAC) systems (e.g. Ni.sup.+2 affinity columns)), GST fusions, MBP
fusions, Strep-tag, the BSP biotinylation target sequence of the
bacterial enzyme BirA, and epitope tags which are targeted by
antibodies (for example c-myc tags, flag-tags, and the like). As
will be appreciated by those skilled in the art, such tags may be
useful for purification, for screening, or both. For example, an Fc
variant may be purified using a His-tag by immobilizing it to a
Ni.sup.+2 affinity column, and then after purification the same
His-tag may be used to immobilize the antibody to a Ni.sup.+2
coated plate to perform an ELISA or other binding assay (as
described below). A fusion partner may enable the use of a
selection method to screen Fc variants (see below). Fusion partners
that enable a variety of selection methods are well-known in the
art, and all of these find use in the present invention. For
example, by fusing the members of an Fc variant library to the gene
III protein, phage display can be employed (Kay et al., Phage
display of peptides and proteins: a laboratory manual, Academic
Press, San Diego, Calif., 1996; Lowman et al., 1991, Biochemistry
30:10832-10838; Smith, 1985, Science 228:1315-1317). Fusion
partners may enable Fc variants to be labeled. Alternatively, a
fusion partner may bind to a specific sequence on the expression
vector, enabling the fusion partner and associated Fc variant to be
linked covalently or noncovalently with the nucleic acid that
encodes them. For example, U.S. Ser. No. 09/642,574; U.S. Ser. No.
10/080,376; U.S. Ser. No. 09/792,630; U.S. Ser. No. 10/023,208;
U.S. Ser. No. 09/792,626; U.S. Ser. No. 10/082,671; U.S. Ser. No.
09/953,351; U.S. Ser. No. 10/097,100; U.S. Ser. No. 60/366,658; PCT
WO 00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT
WO 02/08023; PCT WO 01/28702; and PCT WO 02/07466 describe such a
fusion partner and technique that may find use in the present
invention.
[0177] The methods of introducing exogenous nucleic acid into host
cells are well known in the art, and will vary with the host cell
used. Techniques include but are not limited to dextran-mediated
transfection, calcium phosphate precipitation, calcium chloride
treatment, polybrene mediated transfection, protoplast fusion,
electroporation, viral or phage infection, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei. In the case of mammalian cells, transfection may
be either transient or stable.
[0178] In a preferred embodiment, Fc variant proteins are purified
or isolated after expression. Proteins may be isolated or purified
in a variety of ways known to those skilled in the art. Standard
purification methods include chromatographic techniques, including
ion exchange, hydrophobic interaction, affinity, sizing or gel
filtration, and reversed-phase, carried out at atmospheric pressure
or at high pressure using systems such as FPLC and HPLC.
Purification methods also include electrophoretic, immunological,
precipitation, dialysis, and chromatofocusing techniques.
Ultrafiltration and diafiltration techniques, in conjunction with
protein concentration, are also useful. As is well known in the
art, a variety of natural proteins bind Fc and antibodies, and
these proteins can find use in the present invention for
purification of Fc variants. For example, the bacterial proteins A
and G bind to the Fc region. Likewise, the bacterial protein L
binds to the Fab region of some antibodies, as of course does the
antibody's target antigen. Purification can often be enabled by a
particular fusion partner. For example, Fc variant proteins may be
purified using glutathione resin if a GST fusion is employed,
Ni.sup.+2 affinity chromatography if a His-tag is employed, or
immobilized anti-flag antibody if a flag-tag is used. For general
guidance in suitable purification techniques, see Protein
Purification: Principles and Practice, 3.sup.rd Ed., Scopes,
Springer-Verlag, NY, 1994. The degree of purification necessary
will vary depending on the screen or use of the Fc variants. In
some instances no purification is necessary. For example in one
embodiment, if the Fc variants are secreted, screening may take
place directly from the media. As is well known in the art, some
methods of selection do not involve purification of proteins. Thus,
for example, if a library of Fc variants is made into a phage
display library, protein purification may not be performed.
[0179] Fc variants may be screened using a variety of methods,
including but not limited to those that use in vitro assays, in
vivo and cell-based assays, and selection technologies. Automation
and high-throughput screening technologies may be utilized in the
screening procedures. Screening may employ the use of a fusion
partner or label. The use of fusion partners has been discussed
above. By "labeled" herein is meant that the Fc variants of the
invention have one or more elements, isotopes, or chemical
compounds attached to enable the detection in a screen. In general,
labels fall into three classes: a) immune labels, which may be an
epitope incorporated as a fusion partner that is recognized by an
antibody, b) isotopic labels, which may be radioactive or heavy
isotopes, and c) small molecule labels, which may include
fluorescent and colorimetric dyes, or molecules such as biotin that
enable other labeling methods. Labels may be incorporated into the
compound at any position and may be incorporated in vitro or in
vivo during protein expression.
[0180] In a preferred embodiment, the functional and/or biophysical
properties of Fc variants are screened in an in vitro assay. In
vitro assays may allow a broad dynamic range for screening
properties of interest. Properties of Fc variants that may be
screened include but are not limited to stability, solubility, and
affinity for Fc ligands, for example Fc.gamma.Rs. Multiple
properties may be screened simultaneously or individually. Proteins
may be purified or unpurified, depending on the requirements of the
assay. In one embodiment, the screen is a qualitative or
quantitative binding assay for binding of Fc variants to a protein
or nonprotein molecule that is known or thought to bind the Fc
variant. In a preferred embodiment, the screen is a binding assay
for measuring binding to the antibody's or Fc fusions' target
antigen. In an alternately preferred embodiment, the screen is an
assay for binding of Fc variants to an Fc ligand, including but are
not limited to the family of Fc.gamma.Rs, the neonatal receptor
FcRn, the complement protein C1q, and the bacterial proteins A and
G. Said Fc ligands may be from any organism, with humans, mice,
rats, rabbits, and monkeys preferred. Binding assays can be carried
out using a variety of methods known in the art, including but not
limited to FRET (Fluorescence Resonance Energy Transfer) and BRET
(Bioluminescence Resonance Energy Transfer)-based assays,
AlphaScreen.TM. (Amplified Luminescent Proximity Homogeneous
Assay), Scintillation Proximity Assay, ELISA (Enzyme-Linked
Immunosorbent Assay), SPR (Surface Plasmon Resonance, also known as
BIACORE.RTM.), isothermal titration calorimetry, differential
scanning calorimetry, gel electrophoresis, and chromatography
including gel filtration. These and other methods may take
advantage of some fusion partner or label of the Fc variant. Assays
may employ a variety of detection methods including but not limited
to chromogenic, fluorescent, luminescent, or isotopic labels.
[0181] The biophysical properties of Fc variant proteins, for
example stability and solubility, may be screened using a variety
of methods known in the art. Protein stability may be determined by
measuring the thermodynamic equilibrium between folded and unfolded
states. For example, Fc variant proteins of the present invention
may be unfolded using chemical denaturant, heat, or pH, and this
transition may be monitored using methods including but not limited
to circular dichroism spectroscopy, fluorescence spectroscopy,
absorbance spectroscopy, NMR spectroscopy, calorimetry, and
proteolysis. As will be appreciated by those skilled in the art,
the kinetic parameters of the folding and unfolding transitions may
also be monitored using these and other techniques. The solubility
and overall structural integrity of an Fc variant protein may be
quantitatively or qualitatively determined using a wide range of
methods that are known in the art. Methods which may find use in
the present invention for characterizing the biophysical properties
of Fc variant proteins include gel electrophoresis, chromatography
such as size exclusion chromatography and reversed-phase high
performance liquid chromatography, mass spectrometry, ultraviolet
absorbance spectroscopy, fluorescence spectroscopy, circular
dichroism spectroscopy, isothermal titration calorimetry,
differential scanning calorimetry, analytical ultra-centrifugation,
dynamic light scattering, proteolysis, and cross-linking, turbidity
measurement, filter retardation assays, immunological assays,
fluorescent dye binding assays, protein-staining assays,
microscopy, and detection of aggregates via ELISA or other binding
assay. Structural analysis employing X-ray crystallographic
techniques and NMR spectroscopy may also find use. In one
embodiment, stability and/or solubility may be measured by
determining the amount of protein solution after some defined
period of time. In this assay, the protein may or may not be
exposed to some extreme condition, for example elevated
temperature, low pH, or the presence of denaturant. Because
function typically requires a stable, soluble, and/or
well-folded/structured protein, the aforementioned functional and
binding assays also provide ways to perform such a measurement. For
example, a solution comprising an Fc variant could be assayed for
its ability to bind target antigen, then exposed to elevated
temperature for one or more defined periods of time, then assayed
for antigen binding again. Because unfolded and aggregated protein
is not expected to be capable of binding antigen, the amount of
activity remaining provides a measure of the Fc variant's stability
and solubility.
[0182] In a preferred embodiment, the library is screened using one
or more cell-based or in vivo assays. For such assays, Fc variant
proteins, purified or unpurified, are typically added exogenously
such that cells are exposed to individual variants or pools of
variants belonging to a library. These assays are typically, but
not always, based on the function of an antibody or Fc fusion that
comprises the Fc variant; that is, the ability of the antibody or
Fc fusion to bind a target antigen and mediate some biochemical
event, for example effector function, ligand/receptor binding
inhibition, apoptosis, and the like. Such assays often involve
monitoring the response of cells to antibody or Fc fusion, for
example cell survival, cell death, change in cellular morphology,
or transcriptional activation such as cellular expression of a
natural gene or reporter gene. For example, such assays may measure
the ability of Fc variants to elicit ADCC, ADCP, or CDC. For some
assays additional cells or components, that is in addition to the
target cells, may need to be added, for example example serum
complement, or effector cells such as peripheral blood monocytes
(PBMCs), NK cells, macrophages, and the like. Such additional cells
may be from any organism, preferably humans, mice, rat, rabbit, and
monkey. Antibodies and Fc fusions may cause apoptosis of certain
cell lines expressing the antibody's target antigen, or they may
mediate attack on target cells by immune cells which have been
added to the assay. Methods for monitoring cell death or viability
are known in the art, and include the use of dyes, immunochemical,
cytochemical, and radioactive reagents. For example, caspase
staining assays may enable apoptosis to be measured, and uptake or
release of radioactive substrates or fluorescent dyes such as
alamar blue may enable cell growth or activation to be monitored.
In a preferred embodiment, the DELFIA.RTM. EuTDA-based cytotoxicity
assay (Perkin Elmer, MA) is used. Alternatively, dead or damaged
target cells may be monitored by measuring the release of one or
more natural intracellular proteins, for example lactate
dehydrogenase. Transcriptional activation may also serve as a
method for assaying function in cell-based assays. In this case,
response may be monitored by assaying for natural genes or proteins
which may be upregulated, for example the release of certain
interleukins may be measured, or alternatively readout may be via a
reporter construct. Cell-based assays may also involve the measure
of morphological changes of cells as a response to the presence of
an Fc variant. Cell types for such assays may be prokaryotic or
eukaryotic, and a variety of cell lines that are known in the art
may be employed.
[0183] Alternatively, cell-based screens are performed using cells
that have been transformed or transfected with nucleic acids
encoding the Fc variants. That is, Fc variant proteins are not
added exogenously to the cells. For example, in one embodiment, the
cell-based screen utilizes cell surface display. A fusion partner
can be employed that enables display of Fc variants on the surface
of cells (Witrrup, 2001, Curr Opin Biotechnol, 12:395-399). Cell
surface display methods that may find use in the present invention
include but are not limited to display on bacteria (Georgiou et
al., 1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993, Trends
Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol 18:645-648;
Jun et al., 1998, Nat Biotechnol 16:576-80), yeast (Boder &
Wittrup, 2000, Methods Enzymol 328:430-44; Boder & Wittrup,
1997, Nat Biotechnol 15:553-557), and mammalian cells (Whitehorn et
al., 1995, Bio/technology 13:1215-1219). In an alternate
embodiment, Fc variant proteins are not displayed on the surface of
cells, but rather are screened intracellularly or in some other
cellular compartment. For example, periplasmic expression and
cytometric screening (Chen et al., 2001, Nat Biotechnol 19:
537-542), the protein fragment complementation assay (Johnsson
& Varshavsky, 1994, Proc Natl Acad Sci USA 91:10340-10344;
Pelletier et al., 1998, Proc Natl Acad Sci USA 95:12141-12146), and
the yeast two hybrid screen (Fields & Song, 1989, Nature
340:245-246) may find use in the present invention. Alternatively,
if a polypeptide that comprises the Fc variants, for example an
antibody or Fc fusion, imparts some selectable growth advantage to
a cell, this property may be used to screen or select for Fc
variants.
[0184] As is known in the art, a subset of screening methods are
those that select for favorable members of a library. Said methods
are herein referred to as "selection methods", and these methods
find use in the present invention for screening Fc variant
libraries. When libraries are screened using a selection method,
only those members of a library that are favorable, that is which
meet some selection criteria, are propagated, isolated, and/or
observed. As will be appreciated, because only the most fit
variants are observed, such methods enable the screening of
libraries that are larger than those screenable by methods that
assay the fitness of library members individually. Selection is
enabled by any method, technique, or fusion partner that links,
covalently or noncovalently, the phenotype of an Fc variant with
its genotype, that is the function of an Fc variant with the
nucleic acid that encodes it. For example the use of phage display
as a selection method is enabled by the fusion of library members
to the gene III protein. In this way, selection or isolation of
variant proteins that meet some criteria, for example binding
affinity for an Fc.gamma.R, also selects for or isolates the
nucleic acid that encodes it. Once isolated, the gene or genes
encoding Fc variants may then be amplified. This process of
isolation and amplification, referred to as panning, may be
repeated, allowing favorable Fc variants in the library to be
enriched. Nucleic acid sequencing of the attached nucleic acid
ultimately allows for gene identification.
[0185] A variety of selection methods are known in the art that may
find use in the present invention for screening Fc variant
libraries. These include but are not limited to phage display
(Phage display of peptides and proteins: a laboratory manual, Kay
et al., 1996, Academic Press, San Diego, Calif., 1996; Lowman et
al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science
228:1315-1317) and its derivatives such as selective phage
infection (Malmborg et al., 1997, J Mol Biol 273:544-551),
selectively infective phage (Krebber et al., 1997, J Mol Biol
268:619-630), and delayed infectivity panning (Benhar et al., 2000,
J Mol Biol 301:893-904), cell surface display (Witrrup, 2001, Curr
Opin Biotechnol, 12:395-399) such as display on bacteria (Georgiou
et al., 1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993,
Trends Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol
18:645-648; Jun et al., 1998, Nat Biotechnol 16:576-80), yeast
(Boder & Wittrup, 2000, Methods Enzymol 328:430-44; Boder &
Wittrup, 1997, Nat Biotechnol 15:553-557), and mammalian cells
(Whitehorn et al., 1995, Bio/technology 13:1215-1219), as well as
in vitro display technologies (Amstutz et al., 2001, Curr Opin
Biotechnol 12:400-405) such as polysome display (Mattheakis et al.,
1994, Proc Nat Acad Sci USA 91:9022-9026), ribosome display (Hanes
et al., 1997, Proc Natl Acad Sci USA 94:4937-4942), mRNA display
(Roberts & Szostak, 1997, Proc Natl Acad Sci USA
94:12297-12302; Nemoto et al., 1997, FEBS Lett 414:405-408), and
ribosome-inactivation display system (Zhou et al., 2002, J Am Chem
Soc 124, 538-543)
[0186] Other selection methods that may find use in the present
invention include methods that do not rely on display, such as in
vivo methods including but not limited to periplasmic expression
and cytometric screening (Chen et al., 2001, Nat Biotechnol
19:537-542), the protein fragment complementation assay (Johnsson
& Varshavsky, 1994, Proc Natl Acad Sci USA 91:10340-10344;
Pelletier et al., 1998, Proc Natl Acad Sci USA 95:12141-12146), and
the yeast two hybrid screen (Fields & Song, 1989, Nature
340:245-246) used in selection mode (Visintin et al., 1999, Proc
Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,
selection is enabled by a fusion partner that binds to a specific
sequence on the expression vector, thus linking covalently or
noncovalently the fusion partner and associated Fc variant library
member with the nucleic acid that encodes them. For example, U.S.
Ser. No. 09/642,574; U.S. Ser. No. 10/080,376; U.S. Ser. No.
09/792,630; U.S. Ser. No. 10/023,208; U.S. Ser. No. 09/792,626;
U.S. Ser. No. 10/082,671; U.S. Ser. No. 09/953,351; U.S. Ser. No.
10/097,100; U.S. Ser. No. 60/366,658; PCT WO 00/22906; PCT WO
01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO
01/28702; and PCT WO 02/07466 describe such a fusion partner and
technique that may find use in the present invention. In an
alternative embodiment, in vivo selection can occur if expression
of a polypeptide that comprises the Fc variant, such as an antibody
or Fc fusion, imparts some growth, reproduction, or survival
advantage to the cell.
[0187] A subset of selection methods referred to as "directed
evolution" methods are those that include the mating or breading of
favorable sequences during selection, sometimes with the
incorporation of new mutations. As will be appreciated by those
skilled in the art, directed evolution methods can facilitate
identification of the most favorable sequences in a library, and
can increase the diversity of sequences that are screened. A
variety of directed evolution methods are known in the art that may
find use in the present invention for screening Fc variant
libraries, including but not limited to DNA shuffling (PCT WO
00/42561 A3; PCT WO 01/70947 A3), exon shuffling (U.S. Pat. No.
6,365,377; Kolkman & Stemmer, 2001, Nat Biotechnol 19:423-428),
family shuffling (Crameri et al., 1998, Nature 391:288-291; U.S.
Pat. No. 6,376,246), RACHITT.TM. (Coco et al., 2001, Nat Biotechnol
19:354-359; PCT WO 02/06469), STEP and random priming of in vitro
recombination (Zhao et al., 1998, Nat Biotechnol 16:258-261; Shao
et al., 1998, Nucleic Acids Res 26:681-683), exonuclease mediated
gene assembly (U.S. Pat. No. 6,352,842; U.S. Pat. No. 6,361,974),
Gene Site Saturation Mutagenesis.TM. (U.S. Pat. No. 6,358,709),
Gene Reassembly.TM. (U.S. Pat. No. 6,358,709), SCRATCHY (Lutz et
al., 2001, Proc Natl Acad Sci USA 98:11248-11253), DNA
fragmentation methods (Kikuchi et al., Gene 236:159-167),
single-stranded DNA shuffling (Kikuchi et al., 2000, Gene
243:133-137), and AMEsystem.TM. directed evolution protein
engineering technology (Applied Molecular Evolution) (U.S. Pat. No.
5,824,514; U.S. Pat. No. 5,817,483; U.S. Pat. No. 5,814,476; U.S.
Pat. No. 5,763,192; U.S. Pat. No. 5,723,323).
[0188] The biological properties of the antibodies and Fc fusions
that comprise the Fc variants of the present invention may be
characterized in cell, tissue, and whole organism experiments. As
is know in the art, drugs are often tested in animals, including
but not limited to mice, rats, rabbits, dogs, cats, pigs, and
monkeys, in order to measure a drug's efficacy for treatment
against a disease or disease model, or to measure a drug's
pharmacokinetics, toxicity, and other properties. Said animals may
be referred to as disease models. Therapeutics are often tested in
mice, including but not limited to nude mice, SCID mice, xenograft
mice, and transgenic mice (including knockins and knockouts). For
example, an antibody or Fc fusion of the present invention that is
intended as an anti-cancer therapeutic may be tested in a mouse
cancer model, for example a xenograft mouse. In this method, a
tumor or tumor cell line is grafted onto or injected into a mouse,
and subsequently the mouse is treated with the therapeutic to
determine the ability of the antibody or Fc fusion to reduce or
inhibit cancer growth. Such experimentation may provide meaningful
data for determination of the potential of said antibody or Fc
fusion to be used as a therapeutic. Any organism, preferably
mammals, may be used for testing. For example because of their
genetic similarity to humans, monkeys can be suitable therapeutic
models, and thus may be used to test the efficacy, toxicity,
pharmacokinetics, or other property of the antibodies and Fc
fusions of the present invention. Tests of the antibodies and Fc
fusions of the present invention in humans are ultimately required
for approval as drugs, and thus of course these experiments are
contemplated. Thus the antibodies and Fc fusions of the present
invention may be tested in humans to determine their therapeutic
efficacy, toxicity, pharmacokinetics, and/or other clinical
properties.
EXAMPLES
[0189] Examples are provided below to illustrate the present
invention. These examples are not meant to constrain the present
invention to any particular application or theory of operation.
[0190] For all positions discussed in the present invention,
numbering is according to the EU index as in Kabat (Kabat et al.,
1991, Sequences of Proteins of Immunological Interest, 5th Ed.,
United States Public Health Service, National Institutes of Health,
Bethesda). Those skilled in the art of antibodies will appreciate
that this convention consists of nonsequential numbering in
specific regions of an immunoglobulin sequence, enabling a
normalized reference to conserved positions in immunoglobulin
families. Accordingly, the positions of any given immunoglobulin as
defined by the EU index will not necessarily correspond to its
sequential sequence. FIG. 3 shows the sequential and EU index
numbering schemes for the antibody alemtuzumab in order to
illustrate this principal more clearly. It should also be noted
that polymorphisms have been observed at a number of Fc positions,
including but not limited to Kabat 270, 272, 312, 315, 356, and
358, and thus slight differences between the presented sequence and
sequences in the scientific literature may exist.
Example 1
Computational Screening and Design of Fc Libraries
[0191] Computational screening calculations were carried out to
design optimized Fc variants. Fc variants were computationally
screened, constructed, and experimentally investigated over several
computation/experimentation cycles. For each successive cycle,
experimental data provided feedback into the next set of
computational screening calculations and library design. All
computational screening calculations and library design are
presented in Example 1. For each set of calculations, a table is
provided that presents the results and provides relevant
information and parameters.
[0192] Several different structures of Fc bound bound to the
extracellular domain of Fc.gamma.Rs served as template structures
for the computational screening calculations. Publicly available
Fc/Fc.gamma.R complex structures included pdb accession code 1E4K
(Sondermann et al., 2000, Nature 406:267-273.), and pdb accession
codes 1IIS and 1IIX (Radaev et al., 2001, J Biol Chem
276:16469-16477). The extracellular regions of Fc.gamma.RIIIb and
Fc.gamma.RIIIa are 96% identical, and therefore the use of the
Fc/Fc.gamma.RIIIb structure is essentially equivalent to use of
Fc.gamma.RIIIa. Nonetheless, for some calculations, a more precise
Fc/Fc.gamma.RIIIa template structure was constructed by modeling a
D129G mutation in the 1IIS and 1E4K structures (referred to as
D129G 1IIS and D129G 1E4K template structures). In addition, the
structures for human Fc bound to the extracellular domains of human
Fc.gamma.RIIb, human F158 Fc.gamma.RIIIa, and mouse Fc.gamma.RIII
were modeled using standard methods, the available Fc.gamma.R
sequence information, the aforementioned Fc/Fc.gamma.R structures,
as well as structural information for unbound complexes (pdb
accession code 1H9V)(Sondermann et al., 2001, J Mol Biol
309:737-749) (pdb accession code 1FCG)(Maxwell et al., 1999, Nat
Struct Biol 6:437-442), Fc.gamma.RIIb (pdb accession code
2FCB)(Sondermann et al., 1999, Embo J 18:1095-1103), and
Fc.gamma.RIIIb (pdb accession code 1E4J)(Sondermann et al., 2000,
Nature 406:267-273.).
[0193] Variable positions and amino acids to be considered at those
positions were chosen by visual inspection of the aforementioned
Fc/Fc.gamma.R and Fc.gamma.R structures, and using solvent
accessibility information and sequence information. Sequence
information of Fcs and Fc.gamma.Rs was particularly useful for
determining variable positions at which substitutions may provide
distinguishing affinities between activating and inhibitory
receptors. Virtually all C.gamma.2 positions were screened
computationally. The Fc structure is a homodimer of two heavy
chains (labeled chains A and B in the 1IIS, 1IIX, and 1E4K
structures) that each include the hinge and C.gamma.2-C.gamma.3
domains (shown in FIG. 2). Because the Fc.gamma.R (labeled chain C
in the 1IIS, 1IIX, and 1E4K structures) binds asymmetrically to the
Fc homodimer, each chain was often considered separately in design
calculations. For some calculations, Fc and/or Fc.gamma.R residues
proximal to variable position residues were floated, that is the
amino acid conformation but not the amino acid identity was allowed
to vary in a protein design calculation to allow for conformational
adjustments. These are indicated below the table for each set of
calculations when relevant. Considered amino acids typically
belonged to either the Core, Core XM, Surface, Boundary, Boundary
XM, or All 20 classifications, unless noted otherwise. These
classifications are defined as follows: Core=alanine, valine,
isoleucine, leucine, phenylalanine, tyrosine, tryptophan, and
methionine; Core XM=alanine, valine, isoleucine, leucine,
phenylalanine, tyrosine, and tryptophan; Surface=alanine, serine,
threonine, aspartic acid, asparagine, glutamine, glutamic acid,
arginine, lysine and histidine; Boundary=alanine, serine,
threonine, aspartic acid, asparagine, glutamine, glutamic acid,
arginine, lysine, histidine, valine, isoleucine, leucine,
phenylalanine, tyrosine, tryptophan, and methionine; Boundary
XM=Boundary=alanine, serine, threonine, aspartic acid, asparagine,
glutamine, glutamic acid, arginine, lysine, histidine, valine,
isoleucine, leucine, phenylalanine, tyrosine, and tryptophan; All
20=all 20 naturally occurring amino acids.
[0194] The majority of calculations followed one of two general
types of computational screening methods. In one method, the
conformations of amino acids at variable positions were represented
as a set of backbone-independent side chain rotamers derived from
the rotamer library of Dunbrack & Cohen (Dunbrack et al., 1997,
Protein Sci 6:1661-1681). The energies of all possible combinations
of the considered amino acids at the chosen variable positions were
calculated using a force field containing terms describing van der
Waals, salvation, electrostatic, and hydrogen bond interactions,
and the optimal (ground state) sequence was determined using a Dead
End Elimination (DEE) algorithm. As will be appreciated by those in
the art, the predicted lowest energy sequence is not necessarily
the true lowest energy sequence because of errors primarily in the
scoring function, coupled with the fact that subtle conformational
differences in proteins can result in dramatic differences in
stability. However, the predicted ground state sequence is likely
to be close to the true ground state, and thus additional favorable
diversity can be explored by evaluating the energy of sequences
that are close in sequence space and energy around the predicted
ground state. To accomplish this, as well as to generate a
diversity of sequences for a library, a Monte Carlo (MC) algorithm
was used to evaluate the energies of 1000 similar sequences around
the predicted ground state. The number of sequences out of the 1000
sequence set that contain that amino acid at that variable position
is referred to as the occupancy for that substitution, and this
value may reflect how favorable that substitution is. This
computational screening method is substantially similar to Protein
Design Automation.RTM. (PDA.RTM.) technology, as described in U.S.
Pat. No. 6,188,965; U.S. Pat. No. 6,269,312; U.S. Pat. No.
6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S.
Ser. No. 10/218,102; PCT WO 98/07254; PCT WO 01/40091; and PCT WO
02/25588, and for ease of description, is referred to as PDA.RTM.
technology throughout the examples. Tables that present the results
of these calculations provide for each variable position on the
designated chain (column 1) the amino acids considered at each
variable position (column 2), the WT Fc amino acid identity at each
variable position (column 3), the amino acid identity at each
variable position in the DEE ground state sequence (column 4), and
the set of amino acids and corresponding occupancy that are
observed in the Monte Carlo output (column 5). For example in the
first row of Table 1 below, when position 328 was varied using
boundary amino acids as the set of variable residues for that
position, L occurred 330 times in the top 1000 sequence, M occurred
302 times, etc.
[0195] Other calculations utilized a genetic algorithm (GA) to
screen for low energy sequences, with energies being calculated
during each round of "evolution" for those sequences being sampled.
The conformations of amino acids at variable and floated positions
were represented as a set of side chain rotamers derived from a
backbone-independent rotamer library using a flexible rotamer model
(Mendes et al., 1999, Proteins 37:530-543). Energies were
calculated using a force field containing terms describing van der
Waals, solvation, electrostatic, and hydrogen bond interactions.
This calculation generated a list of 300 sequences which are
predicted to be low in energy. To facilitate analysis of the
results and library generation, the 300 output sequences were
clustered computationally into 10 groups of similar sequences using
a nearest neighbor single linkage hierarchical clustering algorithm
to assign sequences to related groups based on similarity scores
(Diamond, 1995, Acta Cryst D51:127-135). That is, all sequences
within a group are most similar to all other sequences within the
same group and less similar to sequences in other groups. The
lowest energy sequence from each of these ten clusters are used as
a representative of each group, and are presented as results. This
computational screening method is substantially similar to Sequence
Prediction Algorithm.TM. (SPA.TM.) technology, as described in
(Raha et al., 2000, Protein Sci 9:1106-1119); U.S. Ser. No.
09/877,695; and U.S. Ser. No. 10/071,859, and for ease of
description, is referred to as SPA.TM. technology throughout the
examples. Tables that present the results of these calculations
provide for each variable position on the designated chain (column
1) the amino acids considered at each variable position (column 2),
the WT Fc amino acid identity at each variable position (column 3),
and the amino acid identity at the variable positions for the
lowest energy sequence from each cluster group (columns 4-13).
[0196] Computational screening was applied to design energetically
favorable interactions at the Fc/Fc.gamma.R interface at groups of
variable positions that mediate or potentially mediate binding with
Fc.gamma.R. Because the binding interface involves a large number
of Fc residues on the two different chains, and because Fc.gamma.Rs
bind asymmetrically to Fc, residues were grouped in different sets
of interacting variable positions, and designed in separate sets of
calculations. In many cases these sets were chosen as groups of
residues that were deemed to be coupled, that is the energy of one
or more residues is dependent on the identity of one or more other
residues. Various template structures were used, and in many cases
calculations explored substitutions on both chains. For many of the
variable position sets, calculations were carried out using both
the PDA.RTM. and SPA.TM. technology computational screening methods
described. The results of these calculations and relevant are
presented in Tables 1-30 below. Relevant parameters and information
are presented below each table, including the computational
screening method used, the template structure used, whether or not
that structure had carbohydrate atoms, and any residues that may
have been floated. For example, Table 2 presents results from a
PDA.RTM. calculation in which residues 120, 132, and 134 on chain C
(the Fc.gamma.RIIIb receptor) were floated.
[0197] Included within the compositions of the invention are
antibodies that have any of the listed amino acid residues in the
listed positions, either alone or in any combination (note
preferred combinations are listed in the claims, the summary and
the figures). One preferred combination is the listed amino acid
residues in the listed positions in a ground state (sometimes
referred to herein as the "global solution", as distinguished from
the wild-type). In addition, combinations between SPA.TM. proteins,
both within tables and between tables, are also included. It should
be noted that residues not listed in a given table are implied to
have not been varied, and thus remain wild-type. For example, in
the SPA.TM. calculation results presented in Table 4, column 4
(representing cluster 1) indicates a protein with the six listed
amino acids at the six listed positions (e.g. column 4 is a single
protein with a wild-type sequence except for 239E, 265G, 267S,
269Y, 270T and 299S). Thus, each of these individual proteins are
included within the invention. Alternatively, residue positions and
particular amino acids at those residue positions may be combined
between columns within a table, or between tables. Furthermore, it
should be noted that although each table indicates the presence or
absence of carbohydrate, the presence or absence of said atoms in
the computational screening calculation is not meant to imply that
Fc variants designed by such calculations should be applicable to
only aglycosylated or glycosylated Fc. Thus although the
calculations in Table 1 were run without carbohydrate atoms present
in the template structure, the resulting predicted substitutions
may be favorable in a glycosylated or aglycosylated antibody or Fc
fusion. TABLE-US-00001 TABLE 1 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 328 A Boundary L L L:
330 M: 302 E: 111 K: 62 A: 45 Q: 39 D: 36 S: 30 T: 28 N: 10 R: 7
332 A Surface I R R: 247 K: 209 Q: 130 H: 95 E: 92 T: 59 D: 51 N:
51 S: 42 A: 24 328 B Boundary L L L: 321 M: 237 T: 166 K: 73 R: 72
S: 55 Q: 20 D: 17 E: 13 A: 12 V: 10 N: 4 332 B Surface I E E: 269
Q: 180 R: 145 K: 111 D: 97 T: 78 N: 65 S: 28 A: 14 H: 13 PDA .RTM.
technology, 1IIS template structure; -carbohydrate
[0198] TABLE-US-00002 TABLE 2 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Surface S K E: 349
D: 203 K: 196 A: 95 Q: 83 S: 63 N: 10 R: 1 265 A Boundary XM D D D:
616 N: 113 L: 110 E: 104 S: 25 A: 23 Q: 9 299 A Boundary XM T I I:
669 H: 196 V: 135 327 A Boundary XM A S A: 518 S: 389 N: 67 D: 26
265 B Boundary XM D Q Q: 314 R: 247 N: 118 I: 115 A: 63 E: 55 D: 34
S: 22 K: 21 V: 11 PDA .RTM. technology; 1IIS template structure;
+carbohydrate; floated 120 C, 132 C, 134 C
[0199] TABLE-US-00003 TABLE 3 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Surface S E E: 872
Q: 69 D: 39 K: 16 A: 4 265 A Boundary XM D Y Y: 693 H: 111 E: 69 D:
62 F: 29 K: 19 R: 14 W: 2 Q: 1 267 A Boundary XM S S S: 991 A: 9
269 A Core XM E F F: 938 E: 59 Y: 3 270 A Surface D E E: 267 T: 218
K: 186 D: 89 Q: 88 R: 46 S: 34 N: 29 H: 23 A: 20 299 A Boundary XM
T H H: 486 T: 245 K: 130 E: 40 S: 39 D: 27 Q: 27 A: 4 N: 2 PDA
.RTM. technology; 1IIS template structure; -carbohydrate; floated
120 C, 122 C, 132 C, 133 C, 134 C
[0200] TABLE-US-00004 TABLE 4 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A Surface S E Q Q Q E E E Q E E 265 A All
20 D G G G G G G G G G G 267 A All 20 S S S S S S S S S S S 269 A
Core E Y Y A A V Y A A A A 270 A Surface D T S A S T T T A A A 299
A All 20 T S S S S S S S S S S SPA .TM. technology; 1IIS template
structure; +carbohydrate; floated 120 C, 122 C, 132 C, 133 C, 134
C
[0201] TABLE-US-00005 TABLE 5 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 235 A Boundary XM L T T:
195 V: 131 L: 112 W: 107 K: 85 F: 66 Y: 56 E: 52 Q: 38 S: 37 I: 34
R: 29 H: 26 N: 23 D: 9 296 A Surface Y N N: 322 D: 181 R: 172 K: 76
Y: 70 Q: 59 E: 48 S: 40 H: 20 T: 11 A: 1 298 A Surface S T T: 370
R: 343 K: 193 A: 55 S: 39 235 B Boundary XM L L L: 922 I: 78 PDA
.RTM. technology; 1IIS template structure; -carbohydrate; floated
119 C, 128 C, 157 C
[0202] TABLE-US-00006 TABLE 6 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 235 A All 20 L S S P S S S S S S S 296 A
Surface Y Q Q Q E E Q E Q Q N 298 A Surface S S K K K K S S S K S
235 B All 20 L K K K L L L L L L K SPA .TM. technology; 1IIS
template structure; +carbohydrate; floated 119 C, 128 C, 157 C
[0203] TABLE-US-00007 TABLE 7 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 B Surface S E K: 402
E: 282 H: 116 T: 67 R: 47 Q: 39 D: 26 A: 11 S: 7 N: 3 265 B
Boundary XM D W Y: 341 W: 283 I: 236 V: 77 F: 36 H: 9 T: 7 E: 4 K:
4 A: 2 D: 1 327 B Boundary XM A R R: 838 K: 86 H: 35 E: 12 T: 10 Q:
7 A: 6 D: 3 N: 3 328 B Core XM L L L: 1000 329 B Core XM P P P: 801
A: 199 330 B Core XM A Y Y: 918 F: 42 L: 22 A: 18 332 B Surface I I
I: 792 E: 202 Q: 5 K: 1 PDA .RTM. technology; 1IIS template
structure; -carbohydrate; floated 88 C, 90 C, 113 C, 114 C, 116 C,
160 C, 161 C
[0204] TABLE-US-00008 TABLE 8 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 B Surface S D T E E E E E E E E 265 B All
20 D G G K G K G G K K G 327 B All 20 A K M L L N L K L L L 328 B
Core L M M M L A M L M L L 329 B Core P P P P P P P P P P P 330 B
Core A L A A A A A A A A A 332 B Surface I I Q I I Q Q E D I I SPA
.TM. technology; 1IIS template structure; +carbohydrate; floated 88
C, 90 C, 113 C, 114 C, 116 C, 160 C, 161 C
[0205] TABLE-US-00009 TABLE 9 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A Surface S Q Q Q E Q E Q E Q Q 265 A All
20 D G G G G G G G G G G 299 A All 20 T S S A S S S S S S S 327 A
All 20 A A S S S S S S S A S 265 B All 20 D N G G G G G G G G G SPA
.TM. technology; 1IIS template structure; -carbohydrate; floated
120 C, 132 C, 134 C
[0206] TABLE-US-00010 TABLE 10 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 234 A Boundary XM L K Y:
401 L: 260 F: 151 I: 82 K: 63 H: 17 Q: 11 W: 7 R: 3 T: 2 E: 2 V: 1
235 A Boundary XM L L W: 777 L: 200 K: 12 Y: 5 I: 3 F: 2 V: 1 234 B
Boundary XM L W W: 427 Y: 203 L: 143 F: 74 I: 59 E: 32 K: 23 V: 14
D: 10 T: 7 H: 4 R: 4 235 B Boundary XM L W W: 380 Y: 380 F: 135 K:
38 L: 26 E: 15 Q: 12 H: 8 R: 4 T: 2 PDA .RTM. technology; D129G
1E4K template structure; -carbohydrate; floated 113 C, 116 C, 132
C, 155 C, 157 C
[0207] TABLE-US-00011 TABLE 11 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 234 A All 20 L G G G G G G G G G G 235 A All
20 L T L L L L L L L T L 234 B All 20 L G G G G G G G G G G 235 B
All 20 L S A S A A S S S A A SPA .TM. technology; D129G 1E4K
template structure; +carbohydrate; floated 113 C, 116 C, 132 C, 155
C, 157 C
[0208] TABLE-US-00012 TABLE 12 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Boundary XM S E E:
235 S: 122 D: 94 Q: 93 A: 74 K: 70 L: 67 T: 63 N: 57 R: 51 I: 29 V:
18 W: 15 H: 12 328 A Boundary XM L L L: 688 E: 121 K: 43 Q: 41 A:
33 D: 26 S: 14 T: 14 N: 12 R: 8 332 A Boundary XM I W I: 155 W: 95
L: 82 K: 79 E: 74 Q: 69 H: 67 V: 63 R: 57 T: 57 D: 45 S: 43 N: 42
A: 35 F: 19 Y: 18 PDA .RTM. technology; D129G 1IIS template
structure; -carbohydrate; floated 120 C
[0209] TABLE-US-00013 TABLE 13 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A All 20 S L E E Q E E K K K K 328 A All
20 L L Q L Q K L L Q K L 332 A All 20 I K K L Q A K L Q A Q SPA
.TM. technology; D129G 1IIS template structure; +carbohydrate;
floated 120 C
[0210] TABLE-US-00014 TABLE 14 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 B Boundary XM S I R:
195 I: 169 L: 126 V: 91 K: 89 E: 61 H: 52 T: 50 Q: 42 N: 35 S: 34
D: 30 A: 26 328 B Boundary XM L L L: 671 T: 165 K: 40 S: 38 E: 28
R: 17 Q: 17 V: 11 A: 8 D: 5 332 B Boundary XM I I I: 387 E: 157 L:
151 V: 78 Q: 63 K: 50 R: 33 T: 29 D: 25 A: 12 N: 8 S: 6 W: 1 PDA
.RTM. technology; D129G 1IIS template structure; -carbohydrate;
floated 90 C, 160 C, 161 C
[0211] TABLE-US-00015 TABLE 15 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 B All 20 S T L L L L L L L L L 328 B All
20 L M R M D T M L Q D L 332 B All 20 I I D Q Q Q L L T Q L SPA
.TM. technology; D129G 1IIS template structure; +carbohydrate;
floated 90 C, 160 C, 161 C
[0212] TABLE-US-00016 TABLE 16 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 B Boundary XM S T T:
164 S: 159 L: 156 E: 86 W: 76 K: 71 D: 65 A: 52 R: 43 H: 38 Q: 38
N: 31 I: 14 V: 7 328 B Boundary XM L L L: 556 E: 114 T: 84 K: 80 S:
69 Q: 36 A: 31 D: 15 R: 11 N: 4 332 B Boundary XM I W I: 188 W: 177
E: 97 L: 94 T: 59 Q: 57 V: 54 K: 52 F: 51 D: 34 H: 33 S: 27 R: 26
N: 18 A: 17 Y: 16 PDA .RTM. technology; D129G 1E4K template
structure; -carbohydrate; floated 117 C
[0213] TABLE-US-00017 TABLE 17 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 B All 20 S P S P E L L L L L L 328 B All
20 L K K K K K L K K K L 332 B All 20 I S S E L L L E L L L SPA
.TM. technology; D129G 1E4K template structure; +carbohydrate;
floated 117 C
[0214] TABLE-US-00018 TABLE 18 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Boundary XM S L K:
196 L: 171 I: 146 E: 88 V: 76 R: 75 T: 50 H: 45 D: 43 Q: 39 S: 30
N: 22 A: 19 328 A Boundary XM L W L: 517 F: 230 W: 164 H: 40 K: 29
E: 11 R: 5 T: 4 332 A Boundary XM I E I: 283 L: 217 E: 178 Q: 81 V:
64 D: 47 T: 35 K: 27 W: 18 R: 12 A: 10 Y: 7 N: 7 F: 6 S: 5 H: 3 PDA
.RTM. technology; D129G 1E4K template structure; -carbohydrate;
floated 87 C, 157 C, 158 C
[0215] TABLE-US-00019 TABLE 19 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A All 20 S F Q E T P P T P P P 328 A All
20 L K R R K K M R K M R 332 A All 20 I L L I I E I E E I I SPA
.TM. technology; D129G 1E4K template structure; +carbohydrate
atoms; floated 87 C, 157 C, 158 C
[0216] TABLE-US-00020 TABLE 20 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 240 A Core + Thr V V V:
698 M: 162 T: 140 263 A Core + Thr V V V: 966 T: 34 266 A Core +
Thr V V V: 983 T: 17 325 A Boundary N N N: 943 T: 40 A: 17 328 A
Boundary L L L: 610 M: 363 K: 27 332 A Glu I E E: 1000 PDA .RTM.
technology; D129G 1IIS template structure; -carbohydrate; floated
273 A, 275 A, 302 A, 323 A, 134 C
[0217] TABLE-US-00021 TABLE 21 Consid- ered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 240 A All 20 V V A V V V V V V V V 263 A All
20 V V V V V V V V V V V 266 A All 20 V I V I I T V V V V I 325 A
All 20 N A N N N Q T T Q N T 328 A All 20 L K K L K L K L L L L 332
A Glu I D D D D D D D D D D SPA .TM. technology; D129G 1IIS
template structure; +carbohydrate; floated 273 A, 275 A, 302 A, 323
A, 134 C
[0218] TABLE-US-00022 TABLE 22 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 240 B Core + Thr V V V:
713 T: 287 263 B Core + Thr V V V: 992 T: 8 266 B Core + Thr V V V:
976 T: 24 325 B Boundary N N N: 453 T: 296 A: 116 D: 96 S: 30 V: 9
328 B Boundary L L L: 623 M: 194 T: 100 R: 72 K: 11 332 B Glu I E
E: 1000 PDA .RTM. technology; D129G 1IIS template structure;
-carbohydrate; floated 273 B, 275 B, 302 B, 323 B, 161 C
[0219] TABLE-US-00023 TABLE 23 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 240 B All 20 V A T A T T A A T T T 263 B All
20 V V A A T T V V T A T 266 B All 20 V V V V V V V V V I V 325 B
All 20 N N K K N K K N N N N 328 B All 20 L R L L L L L L L L L 332
A Glu I D D D D D D D D D D SPA .TM. technology; D129G 1IIS
template structure; + carbohydrate; floated 273 B, 275 B, 302 B,
323 B, 161 C
[0220] TABLE-US-00024 TABLE 24 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 240 B Core + Thr V M V:
715 M: 271 T: 12 I: 2 263 B Core + Thr V V V: 992 T: 8 266 B Core +
Thr V V V: 996 T: 4 325 B Boundary N N N: 651 T: 232 D: 64 A: 53
328 B Boundary L M M: 556 L: 407 K: 37 332 B Glu I E E: 1000 PDA
.RTM. technology; D129G 1E4K template structure; -carbohydrate;
floated 273 B, 275 B, 302 B, 323 B, 131 C
[0221] TABLE-US-00025 TABLE 25 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 240 B All 20 V T A T A A A A T A A 263 B All
20 V T W T T A T T T L L 266 B All 20 V L A T T V L T T L V 325 B
All 20 N A N A A N A A A A A 328 B All 20 L L K L L L L L L L L 332
A Glu I D D D D D D D D D D SPA .TM. technology; D129G 1E4K
template structure; + carbohydrate; floated 273 B, 275 B, 302 B,
323 B, 131 C
[0222] TABLE-US-00026 TABLE 26 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 240 A Core + Thr V V V:
876 T: 109 M: 15 263 A Core + Thr V V V: 913 T: 87 266 A Core + Thr
V V V: 969 T: 31 325 A Boundary N V V: 491 N: 236 T: 187 A: 35 D:
32 S: 19 328 A Boundary L L L: 321 W: 290 M: 271 F: 49 K: 46 R: 23
332 A Glu I E E: 1000 PDA .RTM. technology; D129G 1E4K template
structure; -carbohydrate; floated 273 A, 275 A, 302 A, 323 A, 158
C
[0223] TABLE-US-00027 TABLE 27 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 240 A All 20 V A T A A T T A A A T 263 A All
20 V T T V V T V L L V T 266 A All 20 V V V V V V V V V V V 325 A
All 20 N Q N Q Q Q Q Q Q N N 328 A All 20 L K M K K K K K K K K 332
A Glu I D D D D D D D D D D SPA .TM. technology; D129G 1E4K
template structure; + carbohydrate; floated 273 A, 275 A, 302 A,
323 A, 158 C
[0224] Computational screening calculations were aimed at designing
Fc variants to optimize the conformation of the N297 carbohydrate
and the C.gamma.2 domain. By exploring energetically favorable
substitutions at positions that interact with carbohydrate,
variants can be engineered that sample new, potentially favorable
carbohydrate conformations. Fc residues F241, F243, V262, and V264
mediate the Fc/carbohydrate interaction and thus are target
positions. The results of these design calculations are presented
in Table 28. TABLE-US-00028 TABLE 28 Considered Ground Sequences
Around Position Amino Acids WT State Ground State 241 A Core F Y Y:
172 M: 162 L: 144 F: 140 W: 110 I: 97 A: 91 V: 84 243 A Core F Y Y:
211 L: 204 W: 199 F: 160 M: 141 A: 85 262 A Core V M M: 302 I: 253
V: 243 A: 202 264 A Core V F I: 159 M: 152 V: 142 L: 140 W: 136 F:
120 Y: 104 A: 47 PDA .RTM. technology, 1IIS template structure;
-carbohydrate
[0225] Computational screening calculations were aimed at designing
Fc variants to optimize the angle between the C.gamma.3 and
C.gamma.2 domains. Residues P244, P245, P247, and W313, which
reside at the C.gamma.2/C.gamma.3 interface, appear to play a key
role in determining the C.gamma.2-C.gamma.3 angle and the
flexibility of the domains relative to one another. By exploring
energetically favorable substitutions at these positions, variants
can be designed that sample new, potentially favorable angles and
levels of flexibility. The results of these design calculations are
presented in Table 29. TABLE-US-00029 TABLE 29 Considered Ground
Sequences Around Position Amino Acids WT State Ground State 244 A
Boundary P H K: 164 H: 152 R: 110 M: 100 S: 92 N: 57 A: 54 D: 50 Q:
49 T: 46 E: 37 V: 30 L: 27 W: 23 F: 9 245 A Boundary P A A: 491 S:
378 N: 131 247 A Boundary P V V: 156 T: 125 K: 101 E: 87 Q: 79 R:
78 S: 76 A: 72 D: 72 H: 60 M: 47 N: 47 313 A Boundary W W W: 359 F:
255 Y: 128 M: 114 H: 48 K: 29 T: 24 A: 11 E: 10 V: 10 S: 9 Q: 3 PDA
.RTM. technology; 1IIS template structure; -carbohydrate
[0226] In addition to the above calculations using PDA.RTM. and
SPA.TM. computational screening methods, additional calculations
using solely an electrostatic potential were used to
computationally screen Fc variants. Calculations with Coulomb's law
and Debye-Huckel scaling highlighted a number of positions in the
Fc for which amino acid substitutions would favorably affect
binding to one or more Fc.gamma.Rs, including positions for which
replacement of a neutral amino acid with a negatively charged amino
acid may enhance binding to Fc.gamma.RIIIa, and for which
replacement of a positively charged amino acid with a neutral or
negatively charged amino acid may enhance binding to
Fc.gamma.RIIIa. These results are presented in Table 30.
TABLE-US-00030 TABLE 30 Replacement of a + residue Replacement of a
neutral residue with a - residue with a - residue H268 S239 K326
Y296 K334 A327 I332 Coulomb's law and Debye-Huckel scaling; 1IIS
template structure; +carbohydrate
[0227] Computational screening calculations were carried out to
optimize aglycosylated Fc, that is to optimize Fc structure,
stability, solubility, and Fc/Fc.gamma.R affinity in the absence of
the N297 carbohydrate. Design calculations were aimed at designing
favorable substitutions in the context of the aglycosylated Fc
template structure at residue 297, residues proximal to it,
residues at the Fc/Fc.gamma.R interface, and residues at the
Fc/carbohydrate interface. Variable positions were grouped in
different sets of interacting variable positions and designed in
separate sets of calculations, and various template structures were
used. For many of the variable position sets, calculations were
carried out using both the PDA.RTM. and SPA.TM. computational
screening methods. The results of these calculations and relevant
information are presented in Tables 31-53 below. TABLE-US-00031
TABLE 31 Considered Ground Sequences Around Position Amino Acids WT
State Ground State 265 A Boundary XM D Y Y: 531 F: 226 W: 105 H: 92
K: 21 D: 16 E: 6 T: 3 297 A Boundary XM N D A: 235 S: 229 D: 166 E:
114 N: 92 Y: 57 F: 55 Q: 25 H: 10 T: 7 K: 6 L: 3 R: 1 299 A
Boundary XM T L L: 482 Y: 186 F: 131 T: 55 S: 51 K: 31 H: 22 A: 18
E: 14 Q: 10 297 B Boundary XM N I I: 299 K: 147 V: 85 R: 82 W: 71
N: 65 D: 35 E: 35 Q: 34 S: 32 L: 31 H: 30 T: 28 A: 26 PDA .RTM.
technology; 1IIS template structure; -carbohydrate; floated 122 C,
129 C, 132 C, 155 C
[0228] TABLE-US-00032 TABLE 32 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 265 A All 20 D G G G G G G G G G G 297 A All
20 N A T A E K K A A N N 299 A All 20 T S K S K F F F F F S 297 B
All 20 N K K K K K K K K K K SPA .TM. technology; 1IIS template
structure; - carbohydrate; floated 122 C, 129 C, 132 C, 155 C
[0229] TABLE-US-00033 TABLE 33 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Surface S E E: 928
Q: 65 D: 7 265 A Boundary XM D W W: 709 Y: 248 F: 43 296 A Surface
Y H H: 449 Y: 146 E: 137 D: 89 K: 64 N: 32 T: 30 R: 25 Q: 23 S: 5
297 A Surface N E E: 471 H: 189 D: 102 T: 97 K: 96 R: 22 Q: 15 S: 8
298 A Boundary XM S R R: 353 T: 275 K: 269 A: 56 S: 38 E: 5 Q: 2 H:
2 299 A Boundary XM T F Y: 398 F: 366 L: 217 H: 15 K: 4 PDA .RTM.
technology; D129G 1IIS template structure; -carbohydrate; floated
120 C, 122 C, 128 C, 132 C, 155 C
[0230] TABLE-US-00034 TABLE 34 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A All 20 S E Q Q E Q Q Q Q Q Q 265 A All
20 D G G G G G G G G G G 296 A All 20 Y D Q N N Q N N N Q N 297 A
All 20 N A A N A D D E N N E 298 A All 20 S K K K S K K K K S K 299
A All 20 T S Y F S Y F K F S K SPA .TM. technology; D129G 1IIS
template structure; - carbohydrate; floated 120 C, 122 C, 128 C,
132 C, 155 C
[0231] TABLE-US-00035 TABLE 35 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 B Surface S E E: 417
T: 122 D: 117 Q: 94 R: 84 S: 63 K: 47 H: 29 N: 19 A: 8 265 B
Boundary XM D W W: 865 Y: 79 F: 55 K: 1 296 B Surface Y Y Y: 549 H:
97 D: 80 S: 75 N: 48 E: 45 K: 32 R: 30 Q: 28 A: 16 297 B Surface N
R R: 265 H: 224 E: 157 K: 154 Q: 75 D: 47 T: 34 N: 24 S: 13 A: 7
298 B Boundary XM S V V: 966 D: 10 T: 8 A: 8 N: 4 S: 4 299 B
Boundary XM T Y Y: 667 F: 330 H: 3 PDA .RTM. technology; D129G 1E4K
template structure; -carbohydrate; floated 117 C, 119 C, 125 C, 129
C, 152 C
[0232] TABLE-US-00036 TABLE 36 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 B All 20 S S R E K S S E E E K 265 B All
20 D A D K Y A A F F K Y 296 B All 20 Y A A A A A A A A A A 297 B
All 20 N T S T T E E E S E E 298 B All 20 S G G G G G G G G G G 299
B All 20 T L F E E Y F Y F Y Y SPA .TM. technology; D129G 1E4K
template structure; - carbohydrate; floated 117 C, 119 C, 125 C,
129 C, 152 C
[0233] TABLE-US-00037 TABLE 37 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Surface S E E: 868
Q: 92 D: 38 K: 1 N: 1 265 A Boundary XM D W W: 575 Y: 343 F: 66 H:
15 K: 1 296 A Surface Y H H: 489 Y: 103 R: 98 K: 97 Q: 64 D: 63 T:
41 N: 38 E: 7 297 A Asp N D D: 1000 298 A Boundary XM S R R: 340 K:
262 T: 255 A: 59 S: 57 E: 11 Q: 10 H: 6 299 A Boundary XM T F Y:
375 F: 323 L: 260 H: 24 K: 18 PDA .RTM. technology; D129G 1IIS
template structure; -carbohydrate; floated 120 C, 122 C, 128 C, 132
C, 155 C
[0234] TABLE-US-00038 TABLE 38 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A All 20 S E Q E E E E E E Q E 265 A All
20 D G G G G G G G G G G 296 A All 20 Y E N Q E N Q Q Q Q N 297 A
Asp N D D D D D D D D D D 298 A All 20 S K S K S K K K S K K 299 A
All 20 T S K Y S F F F F F K SPA .TM. technology; D129G 1IIS
template structure; - carbohydrate; floated 120 C, 122 C, 128 C,
132 C, 155 C
[0235] TABLE-US-00039 TABLE 39 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 B Surface S E E: 318
Q: 123 T: 109 D: 108 R: 93 S: 89 K: 69 N: 40 H: 38 A: 13 265 B
Boundary XM D W W: 745 Y: 158 F: 85 K: 9 E: 1 R: 1 H: 1 296 B
Surface Y Y Y: 390 H: 127 S: 83 R: 81 K: 78 N: 65 D: 55 E: 49 Q: 44
A: 26 T: 2 297 B Asp N D D: 1000 298 B Boundary XM S V V: 890 T: 35
A: 29 D: 19 S: 16 N: 10 E: 1 299 B Boundary XM T Y Y: 627 F: 363 H:
10 PDA .RTM. technology; D129G 1E4K template structure;
-carbohydrate; floated 117 C, 119 C, 125 C, 129 C, 152 C
[0236] TABLE-US-00040 TABLE 40 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 B All 20 S K E E Q E K Q E K Q 265 B All
20 D F K K A K Y W K L F 296 B All 20 Y A A A A A A A A A A 297 B
Asp N D D D D D D D D D D 298 B All 20 S G G G G G G G G G G 299 B
All 20 T Y Y Y Y Y Y F F Y Y SPA .TM. technology; D129G 1E4K
template structure; - carbohydrate; floated 117 C, 119 C, 125 C,
129 C, 152 C
[0237] TABLE-US-00041 TABLE 41 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Boundary XM S E E:
312 L: 148 D: 102 Q: 98 K: 64 I: 61 S: 57 A: 44 T: 39 N: 29 R: 23
V: 18 W: 5 265 A Boundary XM D W W: 363 Y: 352 F: 139 H: 77 K: 39
R: 14 D: 11 E: 4 Q: 1 297 A Asp N D D: 1000 299 A Boundary XM T Y
Y: 309 F: 224 L: 212 H: 96 K: 92 E: 28 Q: 20 R: 16 T: 2 S: 1 PDA
.RTM. technology; D129G 1IIS template structure; -carbohydrate;
floated 120 C, 122 C, 132 C, 155 C
[0238] TABLE-US-00042 TABLE 42 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A All 20 S E L L L E E E Q L E 265 A All
20 D G G G G G G G G G G 297 B Asp N D D D D D D D D D D 299 A All
20 T S K K F F F K F K F SPA .TM. technology; D129G 1IIS template
structure; - carbohydrate; floated 120 C, 122 C, 132 C, 155 C
[0239] TABLE-US-00043 TABLE 43 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 B Boundary XM S L L:
194 T: 122 S: 120 E: 111 D: 79 K: 71 A: 62 Q: 57 R: 43 H: 43 N: 37
I: 24 W: 24 V: 13 265 B Boundary XM D W Y: 248 W: 233 F: 198 K: 84
D: 57 E: 55 H: 42 R: 28 Q: 20 A: 10 T: 10 N: 8 S: 7 297 B Asp N D
D: 1000 299 B Boundary XM T Y Y: 493 F: 380 H: 76 T: 31 E: 10 D: 4
A: 3 S: 3 PDA .RTM. technology; D129G 1E4K template structure;
-carbohydrate; floated 117 C, 119 C, 129 C, 152 C
[0240] TABLE-US-00044 TABLE 44 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 B All 20 S R E P L L F P P L L 265 B All
20 D D K S F S Y A M A D 297 B Asp N D D D D D D D D D D 299 B All
20 T Y Y Y Y E Y Y Y Y Y SPA .TM. technology; D129G 1E4K template
structure; - carbohydrate; floated 117 C, 119 C, 129 C, 152 C
[0241] TABLE-US-00045 TABLE 45 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 A Boundary XM S E E:
251 L: 125 D: 120 Q: 112 S: 73 K: 65 I: 61 A: 58 T: 45 N: 35 R: 28
V: 23 W: 4 265 A Boundary XM D Y Y: 216 H: 153 K: 135 D: 109 W: 104
F: 86 R: 54 T: 38 E: 29 Q: 22 A: 21 N: 17 S: 13 L: 3 297 A Asp N D
D: 1000 PDA .RTM. technology; D129G 1IIS template structure;
-carbohydrate; floated 299 A, 120 C, 122 C, 132 C, 155 C
[0242] TABLE-US-00046 TABLE 46 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 A All 20 S S L E L Q Q E Q Q E 265 A All
20 D G G G G G G G G G G 297 A Asp N D D D D D D D D D D SPA .TM.
technology; D129G 1IIS template structure; -carbohydrate; floated
299 A, 120 C, 122 C, 132 C, 155 C
[0243] TABLE-US-00047 TABLE 47 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 239 B Boundary XM S L L:
158 S: 137 T: 125 E: 115 D: 86 K: 75 A: 62 Q: 56 H: 43 R: 39 N: 35
W: 30 I: 24 V: 15 265 B Boundary XM D Y Y: 188 W: 159 F: 156 D: 122
K: 77 E: 71 H: 61 Q: 44 R: 39 A: 24 S: 22 N: 19 T: 18 297 B Asp N D
D: 1000 PDA .RTM. technology; D129G 1E4K template structure;
-carbohydrate; floated 299 B, 117 C, 119 C, 129 C, 152 C
[0244] TABLE-US-00048 TABLE 48 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 239 B All 20 S S E P P E S P L F L 265 B All
20 D A K A M K F Y D F F 297 B Asp N D D D D D D D D D D SPA .TM.
technology; D129G 1E4K template structure; -carbohydrate; floated
299 B, 117 C, 119 C, 129 C, 152 C
[0245] TABLE-US-00049 TABLE 49 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 297 A Asp N D D: 1000
299 A Boundary XM T Y T: 123 Y: 64 H: 64 K: 64 Q: 64 F: 64 R: 63 D:
63 E: 63 S: 63 L: 63 N: 62 I: 57 A: 54 V: 52 W: 17 PDA .RTM.
technology; D129G 1IIS template structure; -carbohydrate; floated
239 A, 265 A, 120 C, 122 C, 132 C, 155 C
[0246] TABLE-US-00050 TABLE 50 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 297 A Asp N D D D D D D D D D D 299 A All 20 T
K K K K F F K K K K SPA .TM. technology; D129G 1IIS template
structure; -carbohydrate; floated 239 A, 265 A, 120 C, 122 C, 132
C, 155 C
[0247] TABLE-US-00051 TABLE 51 Considered Ground Sequences Around
Position Amino Acids WT State Ground State 297 B Asp N D D: 1000
299 B Boundary XM T Y T: 123 F: 64 Y: 64 H: 64 S: 63 N: 61 Q: 61 D:
61 E: 60 K: 58 V: 57 A: 57 R: 54 I: 52 L: 51 W: 50 PDA .RTM.
technology; D129G 1E4K template structure; -carbohydrate; floated
239 B, 265 B, 117 C, 119 C, 129 C, 152 C
[0248] TABLE-US-00052 TABLE 52 Con- sidered Amino Position Acids WT
1 2 3 4 5 6 7 8 9 10 297 B Asp N D D D D D D D D D D 299 B All 20 T
Y Y Y Y Y Y Y Y Y Y SPA .TM. technology; D129G 1E4K template
structure; -carbohydrate; floated 239 B, 265 B, 117 C, 119 C, 129
C, 152 C
[0249] Computational screening calculations were carried out to
optimize aglycosylated Fc by designing favorable substitutions at
residues that are exposed to solvent in the absence of
glycosylation such that they are stable, maintain Fc structure, and
have no tendency to aggregate. The N297 carbohydrate covers up the
exposed hydrophobic patch that would normally be the interface for
a protein-protein interaction with another Ig domain, maintaining
the stability and structural integrity of Fc and keeping the
C.gamma.2 domains from aggregating across the central axis. Key
residues for design are F241, F243, V262, and V264, which reside
behind the carbohydrate on C.gamma.2, in addition to residues such
as L328, 1332, and 1336, which are exposed nonpolar residues facing
inward towards the opposed C.gamma.2 domain, that were considered
in previously presented calculations. The importance of these
C.gamma.2 residues is supported by noting that the corresponding
residues in the C.gamma.3 domain by sequence alignment either
mediate the nonpolar interaction between the two C.gamma.3 domains
or are buried in the C.gamma.3 core. The results of these design
calculations are presented in Table 53. TABLE-US-00053 TABLE 53
Considered Ground Sequences Around Position Amino Acids WT State
Ground State 241 A Surface F E E: 190 R: 172 K: 138 H: 117 T: 93 Q:
91 D: 85 S: 49 N: 49 A: 16 243 A Surface F R R: 190 H: 164 Q: 152
E: 149 K: 92 T: 71 D: 64 N: 58 S: 42 A: 18 262 A Surface V D D: 416
E: 164 N: 138 Q: 87 T: 83 R: 44 S: 32 K: 24 A: 11 H: 1 264 A
Surface V H R: 368 H: 196 K: 147 E: 108 Q: 68 T: 34 N: 33 D: 25 S:
15 A: 6 PDA .RTM. technology; 1IIS template structure;
-carbohydrate
[0250] In a final set of calculations, a SPA.TM. computational
screening method was applied to evaluate the replacement of all
chosen variable positions with all 20 amino acids. The lowest
energy rotamer conformation for all 20 amino acids was determined,
and this energy was defined as the energy of substitution for that
amino acid at that variable position. These calculations thus
provided an energy of substitution for each of the 20 amino acids
at each variable position. The calculations used various template
structures including different Fc/Fc.gamma.RIIIb complexes (1IIS,
1IIX, 1E4K), a modeled Fc/Fc.gamma.RIIb complex, and uncomplexed Fc
(1DN2), and thus were useful for a variety of design goals aimed at
both glycosylated and aglycosylated Fc, including optimization of
Fc/Fc.gamma.R affinity, C1q affinity, Fc stability, Fc solubility,
carbohydrate conformation, and hinge conformation. Furthermore,
because these calculations provide energies for both favorable and
unfavorable substitutions, they guide substitutions that may enable
differential binding to activating versus inhibitory Fc.gamma.Rs.
Various template structures were used, and calculations explored
substitutions on both chains. The results of these calculations and
relevant parameters and information are presented in Tables 54-60
below. Column 1 lists the variable positions on chain A and B of
the template structure. Column 2 lists the wild-type amino acid
identity at each variable position. The remaining 20 columns
provide the energy for each of the natural 20 amino acids (shown in
the top row). All substitutions were normalized with respect to the
lowest energy substitution, which was set to 0 energy. For example
in Table 54, for L235 on chain A, serine is the lowest energy
substitution, and L235A is 0.9 kcal/mol less stable than L235S.
Extremely high energies were set to 20 kcal/mol for energies
between 20-50 kcal/mol, and 50 kcal/mold for energies greater than
50 kcal/mol. Favorable substitutions may be considered to be the
lowest energy substitution for each position, and substitutions
that have small energy differences from the lowest energy
substitution, for example substitutions within 1-2, 1-3, 1-5, or
1-10 kcal/mol. TABLE-US-00054 TABLE 54 Pos WT A C D E F G H I K L M
235 A L 0.9 2.8 2.8 1.5 3.2 3.2 3.4 4.9 1.6 2.1 3.2 236 A G 0 1.9
5.1 6.7 10 2.3 4.3 17.2 5.7 20 4.6 237 A G 20 20 20 50 50 0 50 50
20 50 20 239 A S 0.2 4.3 2.6 0 12.8 4.5 6.9 11.3 1.7 0.1 2.1 265 A
D 9.0 8.1 6.3 7.8 5.1 0 7.3 50 8.2 9.9 7.7 267 A S 2.1 3.3 7.3 1.4
50 7.3 20 20 0.9 2.2 5.0 269 A E 0.5 2.1 1.3 0.6 1.6 3.9 2.0 1.2
1.1 1.3 2.7 270 A D 0.3 2.8 2.3 2.0 4.0 4.0 3.4 2.4 1.2 0 2.3 296 A
Y 2.7 2.0 1.4 0 50 0 50 4.6 2.1 2.4 3.3 298 A S 0.7 2.4 6.7 3.4 20
3.9 20 6.7 0 4.1 1.4 299 A T 0.6 2.8 11.5 10.1 20 6.1 20 10.7 7.1
20 4.3 234 B L 2.1 3.2 4.1 4.2 1.6 5.3 0.1 0.7 0.6 1.0 2.0 235 B L
0.6 2.3 2.5 0.7 5.4 4.8 1.4 3.6 0.1 0 2.0 236 B G 3.1 1.3 4.4 8.2
5.2 0 1.9 20 3.1 20 4.1 237 B G 20 50 50 50 50 0 50 50 50 50 50 239
B S 0.9 2.4 3.4 1.8 5.4 5.6 2.7 3.0 0.9 0 2.0 265 B D 4.5 5.1 4.6
4.6 4.9 0 3.8 9.0 2.0 2.5 4.1 327 B A 1.8 3.4 4.7 3.9 20 7.0 20 20
0.8 0 1.9 328 B L 3.7 3.6 4.0 3.7 50 8.4 6.8 50 3.8 0 2.1 329 B P
3.4 8.6 20 20 50 8.0 16.8 50 20 20 16.9 330 B A 0.5 2.0 2.6 0.5 2.4
3.8 1.4 4.2 0 2.0 2.2 332 B I 1.5 2.7 1.2 1.6 11.9 6.8 12.9 1.2 2.9
0 1.4 Pos N P Q R S T V W Y 235 A 0.9 0.3 1.3 0.7 0 1.7 4.3 6.5 3.2
236 A 3.2 12.6 5.6 6.1 0.6 6.2 12.0 6.7 20 237 A 20 50 50 50 20 20
50 50 50 239 A 1.7 7.9 1.2 2.6 0.3 5.7 11.0 20 20 265 A 6.0 50 9.0
8.5 7.8 20 50 20 5.8 267 A 4.8 0 2.2 3.1 2.9 20 20 50 50 269 A 0 50
0.6 1.1 0.3 0.8 1.0 5.6 1.2 270 A 2.1 20 2.0 2.3 1.4 1.8 4.2 5.4
6.0 296 A 1.2 50 0.2 1.5 1.3 4.6 4.4 16.3 18.2 298 A 4.1 50 1.8 1.1
0.2 2.2 6.3 17.8 20 299 A 6.8 50 6.3 12.0 0 3.0 7.1 14.8 20 234 B
1.7 50 2.8 0.3 2.3 1.7 2.6 13.0 0 235 B 1.7 16.6 0.5 1.2 0.7 0.7
5.3 6.8 5.5 236 B 2.7 50 3.7 16.0 1.2 20 20 20 11.3 237 B 50 50 50
50 50 50 50 50 50 239 B 1.6 50 1.8 1.8 1.4 1.4 5.1 20 5.3 265 B 2.1
50 4.5 5.1 4.4 5.9 9.2 11.4 5.8 327 B 1.5 20 3.0 2.6 3.2 20 20 20
20 328 B 4.1 50 3.6 8.1 4.9 3.0 12.5 50 50 329 B 20 0 20 20 1.3
17.1 16.5 50 50 330 B 0.8 20 0.1 0.6 0.9 0.3 5.1 8.0 2.7 332 B 1.7
50 1.3 4.9 1.8 1.7 3.0 20 20 SPA .TM. technology; 1IIS template
structure; +carbohydrate atoms, no floated positions
[0251] TABLE-US-00055 TABLE 55 Pos WT A C D E F G H I K L 235 A L
0.9 2.8 2.6 1.7 3.3 3.3 3.4 5.0 1.6 2.1 236 A G 0 1.7 5.2 6.0 11.3
2.3 4.4 17.2 5.8 19.0 237 A G 20 20 20 50 50 0 50 50 20 50 238 A P
8.6 8.0 10.5 13.4 6.4 0 5.0 50 12.4 11.3 239 A S 0.1 4.2 2.5 0 20
4.5 9.0 10.8 1.8 0.2 240 A V 1.3 2.4 2.3 6.3 20 7.2 20 5.1 10.8 6.2
241 A F 0.1 1.6 1.2 0.3 0.2 4.1 1.2 10 1.3 0.1 242 A L 3.0 3.4 5.5
8.3 14.4 8.5 11.1 3.3 13.9 2.2 243 A F 1.6 2.2 2.7 0.2 1.4 5.6 2.5
0 2.2 2.0 244 A P 1.2 1.8 3.8 0.8 10.2 3.8 4.6 20 0.2 2.9 245 A P
3.9 20 20 20 20 9.1 20 20 20 20 246 A K 1.3 2.7 2.0 2.0 2.9 5.7 2.9
1.4 1.4 1.5 247 A P 1.2 2.1 0.3 0.7 4.0 3.9 3.7 1.8 1.6 1.7 248 A K
0.9 2.7 1.5 0.8 3.1 4.7 3.4 3.3 2.0 1.9 249 A D 1.2 3.7 1.6 0 20
7.3 19.7 50 1.7 20 250 A T 0 1.8 3.8 5.8 50 6.0 20 4.5 6.3 6.3 251
A L 1.1 1.9 1.2 0.5 5.8 5.1 1.9 5.6 0.9 0.7 252 A M 0.3 1.2 0.6 0
3.0 3.8 3.4 3.9 1.0 0.3 253 A I 0.7 1.7 1.1 0.2 1.8 3.5 2.2 2.0 0.3
1.2 254 A S 0.7 1.7 0.4 0.7 2.2 3.6 2.0 0.3 1.2 1.9 255 A R 1.4 2.8
2.4 2.5 0.2 5.4 1.1 17.0 1.0 2.2 256 A T 0.6 1.8 1.2 1.1 2.7 3.4
2.1 1.4 0.7 1.5 257 A P 0 7.8 20 12.9 50 6.2 50 20 12.3 12.8 258 A
E 0 1.6 4.8 2.6 1.0 4.3 2.2 14.8 4.4 6.2 259 A V 3.9 4.3 5.1 8.7 20
10.3 6.8 2.3 9.6 2.8 260 A T 1.7 2.3 3.3 1.1 20 6.6 8.6 0 0.2 1.8
261 A C 0 20 20 20 20 3.9 20 20 20 20 262 A V 1.9 3.2 0 3.3 20 7.2
20 8.3 2.9 2.9 263 A V 2.2 2.7 6.0 17.4 20 8.8 20 10 7.1 7.6 264 A
V 1.9 3.3 2.8 2.2 0 6.4 2.1 0.7 2.6 0.9 265 A D 9.0 8.1 5.9 8.6 5.3
0 7.3 50 7.9 9.7 266 A V 4.9 5.3 7.1 12.1 20 11.2 20 0.4 12.2 20
267 A S 2.3 3.5 7.2 1.3 50 7.4 20 20 0.7 1.4 268 A H 1.2 1.9 2.2
1.5 3.7 5.0 4.9 0.4 0.5 3.7 269 A E 0.3 1.9 1.3 0.5 1.3 3.7 1.9 1.1
0.8 1.2 270 A D 0.2 2.6 2.1 1.9 5.2 3.9 3.1 2.1 1.2 0 271 A P 0 5.3
8.1 9.3 20 3.1 9.1 20 6.0 9.5 272 A Q 0.8 1.9 0.9 1.2 3.0 3.2 3.7
3.7 1.6 1.8 273 A V 1.2 2.9 1.8 20 20 7.1 20 6.8 20 20 274 A K 0.4
1.8 1.4 0.8 1.9 3.9 2.4 1.4 0.7 1.1 275 A F 8.0 9.5 10.3 9.5 0 13.5
5.1 10.1 6.2 6.3 276 A N 1.3 2.4 2.4 2.2 0.8 5.1 0.8 1.2 0.6 2.3
277 A W 5.5 7.4 8.4 6.4 15.4 11.2 3.2 8.2 1.9 3.9 278 A Y 1.6 2.7
3.9 1.6 1.0 7.3 3.4 17.7 1.4 7.5 279 A V 3.1 4.1 4.0 2.2 20 8.1 9.7
8.5 0 1.4 280 A D 1.8 2.6 2.7 0.2 11.5 2.9 8.8 20 3.4 3.2 281 A G
50 50 50 50 50 0 50 50 50 50 282 A V 0.9 2.1 1.6 1.1 2.9 4.2 3.5
1.4 1.5 1.8 283 A E 0.7 1.6 0.7 0.5 1.0 4.4 1.4 0.4 1.2 1.8 284 A V
0 2.2 3.1 1.2 20 5.0 20 4.0 0.7 2.6 285 A H 0.2 1.4 3.1 1.3 3.0 2.0
2.4 3.6 1.1 2.6 286 A N 0.8 2.5 1.2 1.1 2.4 4.7 2.7 2.1 0 0.7 287 A
A 0.6 2.6 5.8 3.3 10.4 5.4 9.1 11.3 0 4.4 288 A K 0.8 2.6 2.0 1.3
3.0 3.4 3.8 2.3 1.4 1.7 289 A T 0.3 1.9 4.7 1.1 3.1 3.6 2.9 10.5
0.4 2.7 290 A K 1.7 2.2 0.5 0.6 3.0 1.3 3.0 3.7 1.7 2.1 291 A P 1.6
3.1 1.8 0.5 1.9 5.5 1.8 0.1 0.5 1.5 292 A R 1.1 2.2 3.1 0.8 5.9 4.4
8.0 5.0 0 1.6 293 A E 2.2 6.5 9.0 17.9 16.3 0 13.2 50 12.8 10.3 294
A E 1.5 2.1 2.1 0.7 8.1 2.8 3.3 2.0 2.6 1.8 295 A Q 50 50 50 50 50
0 50 50 50 50 296 A Y 2.8 2.3 1.1 0.4 50 0 50 4.6 2.2 2.3 297 A N 0
6.5 8.4 5.3 20 3.4 20 13.8 2.7 20 298 A S 0.8 2.4 5.7 2.2 20 3.7 20
6.2 0.9 9.2 299 A T 1.9 3.4 6.0 3.1 1.0 7.1 2.9 3.1 0 2.7 300 A Y
2.8 2.9 2.7 4.5 20 4.0 7.5 13.1 1.2 0 301 A R 3.0 3.5 3.8 2.8 0.8
3.4 1.8 0 1.3 0.7 302 A V 2.7 4.6 6.7 3.9 2.8 8.9 1.2 6.9 2.7 2.0
303 A V 0 2.2 3.3 1.0 6.7 4.5 5.3 1.4 2.5 3.1 304 A S 0 12.1 10.8
20 20 6.2 20 20 17.2 20 305 A V 1.1 2.3 3.3 1.2 0.3 5.4 1.2 0 0.9
1.1 306 A L 4.3 6.2 7.1 5.9 2.8 10.4 3.4 13.7 3.0 0 307 A T 1.4 3.2
3.8 2.2 6.5 5.5 4.2 0.5 0.3 4.2 308 A V 1.8 5.5 6.5 8.0 50 7.9 20
4.5 20 5.5 309 A L 1.1 2.7 0.7 0.7 1.3 4.6 2.7 0.7 1.7 1.0 310 A H
2.0 2.6 0.9 4.1 50 5.6 0.2 6.8 4.0 7.1 311 A Q 0.6 2.5 1.6 1.6 2.5
4.3 1.6 1.4 0.6 0.9 312 A N 5.4 5.1 5.9 1.3 20 0 20 10 3.4 4.8 313
A W 4.6 6.4 5.5 5.6 1.1 10.8 5.0 11.0 5.8 5.2 314 A L 2.1 2.9 4.3
2.2 5.7 6.1 7.9 5.4 0.7 0 315 A D 0.3 1.4 1.5 0.1 3.3 4.2 1.9 1.8
0.8 0.5 316 A G 50 50 50 50 50 0 50 50 50 50 317 A K 0 14.0 18.4
17.9 50 5.0 50 20 8.5 12.5 318 A E 2.0 3.0 2.7 1.7 2.7 6.7 2.6 0
1.1 1.6 319 A Y 2.9 4.4 3.9 3.4 0 8.8 1.8 20 0.5 5.2 320 A K 2.3
3.1 3.0 2.7 20 7.8 20 9.4 0 0.6 321 A C 0 3.2 20 18.8 20 6.9 20 20
20 20 322 A K 2.0 2.5 3.5 2.8 2.7 6.4 2.1 0.2 0.1 1.2 323 A V 1.5
2.8 7.3 11.9 20 8.1 20 6.0 9.6 20 324 A S 2.0 2.1 0.6 0 1.9 4.9 3.9
1.5 2.8 0.7 325 A N 2.8 3.9 8.4 3.0 20 8.3 20 0 7.7 20 326 A K 1.0
2.7 3.0 1.6 3.7 4.1 3.1 3.2 1.7 2.4 327 A A 0.9 2.8 5.8 3.1 20 6.3
16.7 14.7 2.8 20 328 A L 6.0 6.3 7.0 4.1 50 8.6 20 50 5.7 0 329 A P
1.0 2.5 0.9 0.6 4.0 3.4 3.3 1.7 1.9 2.5 330 A A 0.9 2.0 1.3 0.7 3.4
3.8 3.0 2.0 1.4 2.0 331 A P 50 50 50 50 50 0 50 50 50 50 332 A I
1.9 3.7 4.6 1.7 5.0 7.0 1.9 3.8 1.8 0 333 A E 0 3.1 3.2 0.8 4.1 4.4
4.2 16.9 3.6 2.8 334 A K 1.7 2.9 2.5 0 1.0 6.1 3.3 1.0 1.5 0.5 335
A T 0.5 3.2 4.5 2.7 4.2 4.9 4.1 20 2.1 3.1 336 A I 1.2 1.6 5.0 1.5
20 6.1 16.8 0.7 3.4 7.8 337 A S 4.8 4.8 7.5 11.5 10.1 0 5.5 50 9.9
7.0 338 A K 1.0 2.7 2.3 2.2 4.6 5.9 2.4 50 0 2.1 339 A A 1.0 2.5
0.8 1.1 4.4 3.7 3.7 2.1 1.8 2.6 340 A K 1.3 2.4 2.3 2.0 1.7 4.1 2.3
1.9 0 2.3 232 B P 1.3 3.2 2.2 2.2 4.1 2.9 3.6 1.8 2.1 2.8 233 B E
0.5 2.2 1.7 0.5 2.6 3.7 2.9 4.4 1.4 1.1 234 B L 2.9 4.0 4.8 4.9 2.0
6.1 0.8 1.5 0 1.9 235 B L 0.6 2.3 2.4 0.9 5.7 4.9 1.4 3.7 0 0 236 B
G 3.6 2.5 5.1 11.8 6.8 0 2.8 20 5.0 20 237 B G 20 50 50 50 50 0 50
50 50 50 238 B P 3.5 4.7 8.5 4.2 20 9.8 20 0 5.6 9.6 239 B S 1.0
2.5 3.4 2.0 7.2 5.7 3.1 3.1 0.6 0 240 B V 0.1 2.3 7.0 11.9 20 6.5
20 8.1 12.7 20 241 B F 0 2.0 1.4 0.8 1.0 4.0 2.0 6.5 1.1 0.6 242 B
L 2.2 3.3 6.5 6.6 6.9 7.9 4.3 0 8.7 3.9 243 B F 0.8 2.6 1.9 1.7 0.8
4.9 2.0 3.6 1.2 0.8 244 B P 1.1 2.1 4.0 1.1 11.9 3.5 5.4 20 1.4 3.2
245 B P 3.2 20 20 20 20 8.6 20 20 20 20 246 B K 0.5 2.6 1.4 1.2 2.1
4.4 1.6 0.6 0.9 1.4 247 B P 0.8 2.5 0.7 1.0 3.6 3.9 2.6 6.2 1.8 2.1
248 B K 0.2 2.2 0.2 0.6 2.2 4.1 2.5 2.4 1.7 1.0 249 B D 2.8 3.3 0
4.6 10.1 8.2 6.5 50 4.6 6.2 250 B T 0 2.2 4.9 2.8 20 6.3 20 2.2 4.3
3.2 251 B L 0 2.4 1.6 1.2 5.6 3.6 2.2 7.4 1.2 0.6 252 B M 1.3 2.4
0.8 0 1.8 5.7 2.3 0.6 1.6 0.6 253 B I 1.6 3.0 2.0 1.2 3.7 4.5 3.5
2.9 0.8 2.4 254 B S 1.0 1.5 0.8 0.6 3.8 3.8 3.2 0.5 1.9 2.5 255 B R
0.9 2.0 2.0 1.7 0 5.4 1.4 20 1.0 1.6 256 B T 0.6 2.0 1.8 1.1 2.5
3.7 1.9 1.6 1.0 1.4 257 B P 2.5 20 20 20 50 9.0 50 20 20 20 258 B E
1.5 2.4 2.7 1.4 2.7 6.4 4.2 0 0.2 5.4 259 B V 2.9 4.2 6.3 5.2 20
9.3 20 0 8.1 8.9 260 B T 0 1.6 5.3 1.9 20 4.9 20 0.6 1.1 2.8 261 B
C 0 10 20 20 20 2.6 20 20 20 20 262 B V 2.1 2.4 2.7 2.4 8.1 7.2 3.8
1.8 3.5 8.6 263 B V 2.2 3.7 4.7 11.2 20 9.1 20 15.0 13.7 2.8 264 B
V 2.1 3.0 4.6 2.7 8.6 6.8 6.6 0 1.8 1.8 265 B D 4.5 5.2 4.8 4.7 5.0
0 3.8 8.5 1.8 2.6 266 B V 5.3 5.5 7.2 12.7 20 12.0 20 2.1 20 20 267
B S 2.8 4.3 6.2 3.8 0 7.4 1.0 50 1.0 0.3 268 B H 2.6 3.7 5.1 4.1
4.9 6.0 1.8 2.6 0 2.5 269 B E 0.4 2.4 1.7 0.8 2.8 3.7 2.6 1.0 1.0
1.6 270 B D 0 1.6 1.1 7.3 4.8 4.3 2.6 20 3.8 14.5 271 B P 1.1 3.3
5.6 3.4 4.1 5.5 4.2 20 1.9 3.6 272 B Q 0.9 1.9 1.0 0.6 3.0 3.9 2.9
1.5 1.7 2.2 273 B V 3.5 4.8 6.2 8.3 20 9.2 20 4.6 8.4 3.1 274 B K
0.1 1.6 0.4 0.9 1.7 3.8 1.8 1.9 0.4 0.5 275 B F 5.7 7.0 8.4 9.2 0
11.2 3.5 9.2 7.9 5.7 276 B N 0 6.2 6.9 6.4 20 4.7 12.1 20 9.3 10
277 B W 8.3 10 10.6 9.2 2.6 14.2 7.4 12.7 6.7 7.4 278 B Y 0 2.3
17.4 4.0 50 5.1 50 20 2.8 20 279 B V 3.1 3.5 4.2 2.9 20 8.5 13.9
0.4 0 2.9 280 B D 0.5 3.0 2.1 1.5 6.7 3.1 4.7 12.6 2.9 1.6 281 B G
5.6 5.8 5.5 4.8 7.9 0 7.2 6.5 5.3 5.7 282 B V 0.4 1.9 1.1 0.6 2.9
4.1 2.1 1.3 1.0 1.4 283 B E 0.6 1.9 4.3 1.7 6.7 4.2 5.2 2.9 0.5 4.4
284 B V 0.4 2.4 2.5 1.1 20 5.9 20 1.1 1.2 6.2 285 B H 1.3 2.4 2.1
1.7 2.4 3.4 1.2 1.8 0.7 2.3 286 B N 1.2 2.7 1.0 1.1 3.0 3.1 2.6 0.8
2.0 1.9 287 B A 2.5 4.4 6.1 7.5 0 8.2 3.0 10.2 5.1 16.5 288 B K 0.4
1.9 1.9 0 2.9 3.5 2.9 2.5 1.8 2.1 289 B T 0.1 1.5 3.7 1.4 2.7 3.9
2.6 1.8 0 2.2 290 B K 0.9 1.8 0.8 0.5 2.4 0.8 2.7 3.0 1.3 1.3 291 B
P 1.2 2.1 2.5 0.5 3.9 4.6 3.4 0.7 0 3.4 292 B R 0.8 2.6 3.3 1.2 4.9
3.6 6.8 3.1 2.0 2.4 293 B E 0 3.0 4.1 2.8 7.3 3.6 5.8 5.8 2.6 4.5
294 B E 2.5 3.3 3.9 2.3 8.3 6.8 4.4 5.6 3.6 2.3 295 B Q 1.1 2.2 1.9
0.6 3.8 2.8 3.1 8.0 1.4 2.2 296 B Y 1.5 2.7 1.2 1.2 4.1 4.1 3.5 1.1
1.8 2.7 297 B N 3.9 4.5 10.1 6.0 15.5 7.3 16.7 6.6 0 5.1 298 B S
1.7 2.5 3.5 2.5 2.5 3.7 2.4 3.0 0 1.8 299 B T 0 2.7 7.2 11.1 20 4.8
20 7.5 6.9 20 300 B Y 3.8 5.2 8.0 4.3 20 8.6 20 12.2 0 4.3 301 B R
1.2 1.8 2.3 1.1 20 5.8 11.3 5.2 0.3 5.0 302 B V 3.5 4.8 5.5 3.7 0.2
9.6 1.1 0.5 2.6 3.5 303 B V 0.2 0 0.1 1.0 20 5.0 13.3 5.1 1.7 10.4
304 B S 1.5 2.3 8.2 20 20 7.6 20 7.6 20 20 305 B V 0.1 1.2 3.3 1.1
20 4.6 20 3.2 1.1 11.0 306 B L 4.7 6.8 6.3 4.3 10.4 11.1 7.8 4.2
3.0 0 307 B T 1.5 3.0 2.7 1.7 4.1 5.2 3.0 1.6 1.9 3.1 308 B V 0 0.6
7.6 20 20 6.6 20 20 16.1 15.1 309 B L 1.4 3.0 2.2 1.1 3.0 6.0 3.5
20 2.4 1.7 310 B H 2.4 2.9 2.7 4.9 20 6.8 4.4 4.8 3.1 15.0 311 B Q
0 2.2 1.3 0.7 2.1 3.3 2.4 12.6 0.6 0.9 312 B N 0 1.0 0.2 0.3 6.0
5.4 2.3 12.0 2.1 2.9 313 B W 5.3 6.6 7.3 5.4 0 11.4 6.2 20 4.0 5.2
314 B L 1.7 2.2 3.1 0 6.4 5.6 1.5 2.1 0.6 0.2 315 B D 1.4 2.3 2.4
0.7 6.0 5.5 2.3 4.8 2.2 1.0 316 B G 50 50 50 50 50 0 50 50 50 50
317 B K 0.9 2.3 4.3 2.8 1.2 4.0 0.6 13.9 0 4.8 318 B E 0.7 1.2 3.1
1.0 7.0 5.1 8.2 0.4 1.0 5.7 319 B Y 6.5 7.1 8.5 8.8 0 12.5 3.9 3.1
5.2 5.4 320 B K 3.1 4.3 7.3 4.3 20 8.6 15.0 1.4 0 11.6 321 B C 0
6.5 20 20 20 6.6 20 20 20 20 322 B K 2.3 3.2 3.5 1.8 20 7.9 20 1.1
0.6 4.9 323 B V 4.0 4.6 6.9 8.1 20 10.6 20 9.0 17.1 7.9 325 B N 3.4
5.1 9.0 4.7 20 8.2 20 16.6 16.6 20 326 B K 0.3 2.1 2.0 0.9 1.0 3.5
2.0 2.9 0.9 2.9 327 B A 1.9 3.3 4.7 3.5 20 7.0 20 20 0.3 0 328 B L
3.7 3.6 3.8 4.4 50 8.4 7.0 50 3.8 0 329 B P 3.3 8.5 20 20 50 8.0
16.5 50 18.5 20 330 B A 0.5 2.0 2.8 0.5 2.4 3.9 1.2 4.0 0 2.0 331 B
P 1.7 3.8 6.4 10.1 20 4.7 11.0 10.1 7.5 20 332 B I 1.7 2.9 1.3 1.7
14.8 7.0 13.9 1.7 3.1 0 333 B E 1.9 2.5 1.9 0 8.9 5.9 8.2 1.2 3.0
6.4 334 B K 2.9 3.9 3.7 2.6 20 8.3 12.1 1.5 2.6 5.3 335 B T 0 2.1
7.2 7.0 4.2 0.4 3.3 17.3 6.5 7.7 336 B I 0.5 1.6 2.1 0.7 20 5.0 6.1
0 1.3 5.3 337 B S 1.1 2.1 4.0 2.0 3.1 3.2 2.0 50 0 1.6 338 B K 0.6
2.3 3.0 3.0 9.4 5.3 10.6 2.2 1.1 0 339 B A 1.1 2.4 1.2 0.8 4.3 3.6
3.7 2.6 1.8 2.6 340 B K 0.9 2.0 1.4 0.8 3.0 3.4 2.9 2.1 0.8 2.5 Pos
M N P Q R S T V W Y 235 A 3.3 1.0 0.3 1.4 1.8 0 1.9 3.6 6.6 3.3 236
A 4.9 3.3 8.2 5.6 6.0 0.8 5.6 11.8 6.6 20 237 A 20 20 50 50 50 20
20 50 50 50 238 A 9.7 9.3 3.2 12.4 20 8.6 50 50 20 8.4 239 A 2.1
1.8 9.1 1.3 2.5 0.3 5.7 10.7 20 19.7 240 A 5.7 2.0 1.1 9.5 13.1 2.5
0.5 0 20 20 241 A 2.1 0.4 14.7 0.5 1.1 0.1 0 8.3 3.6 0.4 242 A 2.7
5.5 0.9 7.9 17.1 3.8 2.3 0 20 17.5 243 A 3.0 2.3 10.2 0.5 1.6 1.3
0.9 1.2 5.3 1.6 244 A 2.0 2.8 2.0 0.9 1.7 0 19.3 20 7.6 12.2 245 A
20 20 0 20 20 8.0 20 50 20 20 246 A 3.1 0.2 0 1.2 1.5 1.7 1.4 1.2
5.4 3.0 247 A 3.3 0 0.5 0.9 1.5 0.7 1.1 1.3 6.9 3.7 248 A 2.6 1.2
3.6 1.5 2.3 0.7 0 2.5 5.6 2.7 249 A 2.2 1.4 20 1.5 3.4 2.5 18.3 50
20 20 250 A 0.3 3.2 50 8.7 9.3 1.8 1.3 1.9 20 50 251 A 2.4 1.4 50 0
1.4 0.5 0.8 6.9 8.9 5.8 252 A 2.2 0.3 17.4 0.1 1.1 0.1 0.2 4.6 4.2
3.3 253 A 0.8 0.8 0.3 0 1.1 0.3 0.5 2.8 2.4 1.9 254 A 2.4 0 20 0.3
1.2 0.3 0.8 0.7 3.8 1.9 255 A 1.5 1.7 50 2.1 0 2.3 50 17.2 4.0 0.5
256 A 2.4 0 0.4 0.1 0.2 0.4 0.9 1.2 5.6 2.7 257 A 14.4 20 0.1 13.1
20 2.9 16.0 20 50 50 258 A 3.2 2.9 10.4 7.4 6.0 1.0 6.2 17.6 20 1.0
259 A 6.2 4.1 50 9.2 20 5.2 2.1 0 20 20 260 A 2.8 1.8 1.1 0.8 0.9
1.7 0.4 1.9 7.1 20 261 A 20 20 50 20 20 3.6 20 20 20 20 262 A 2.2
0.6 50 3.8 5.2 3.4 3.0 1.7 20 20
263 A 16.9 5.2 50 19.8 17.7 2.8 1.4 0 20 20 264 A 2.7 2.1 2.3 2.6
2.7 2.2 1.1 0.6 3.9 0.1 265 A 7.5 5.5 50 10.2 8.6 7.9 20 50 20 5.7
266 A 8.8 7.1 50 12.2 20 6.1 3.8 0 20 20 267 A 3.9 4.7 0 2.3 3.1
3.0 20 20 50 50 268 A 2.7 1.7 0 1.4 1.7 1.1 0.2 0.9 6.1 3.7 269 A
2.5 0 50 0.6 0.8 0.2 0.6 0.7 4.0 1.0 270 A 2.2 1.9 20 1.9 1.8 1.2
1.7 4.1 5.1 7.0 271 A 5.3 7.3 5.9 5.9 5.9 1.6 4.1 15.2 20 20 272 A
3.2 0.3 50 1.1 1.6 0 1.0 3.5 4.0 3.4 273 A 20 0 2.8 20 20 2.1 1.4
1.7 20 20 274 A 2.9 0.9 20 0 0.1 0 0.4 0.7 3.3 2.3 275 A 6.0 9.1
6.1 9.1 15.1 9.6 7.2 6.1 13.5 4.3 276 A 2.5 1.8 50 1.6 2.5 1.2 0
0.3 4.2 3.6 277 A 3.6 6.6 3.5 5.5 15.4 6.9 6.1 14.1 0 20 278 A 2.1
0 50 1.9 2.2 2.6 9.9 20 15.8 1.4 279 A 3.1 3.3 20 1.9 4.6 4.3 3.4
4.2 20 20 280 A 2.8 3.8 50 0 3.7 0.6 6.8 12.7 11.9 11.4 281 A 50 50
50 50 50 50 50 50 50 50 282 A 3.6 0.4 18.9 0.5 1.0 0 0.6 0.9 4.7
3.1 283 A 1.9 0 0.4 0.6 1.5 0.4 0.3 1.2 4.1 0.9 284 A 0.8 2.6 50
0.8 0.7 0.8 0.1 1.5 20 20 285 A 3.0 0.7 2.2 0.2 0.8 0 1.1 4.7 4.9
4.0 286 A 1.8 0.6 20 1.2 0.7 0.9 1.7 2.1 5.2 2.7 287 A 1.3 3.6 50
2.6 2.3 1.0 1.9 12.5 9.1 10.4 288 A 2.5 0.3 50 0.5 1.3 0 0.4 2.0
4.5 3.6 289 A 1.6 2.1 8.2 1.2 2.0 0 0.4 12.0 3.9 3.2 290 A 3.2 0 50
0.7 2.0 0.3 1.3 3.3 5.6 3.3 291 A 1.2 1.2 0.7 0 2.9 2.2 0.9 1.3 2.6
0.9 292 A 2.1 1.1 8.4 0.2 0.4 1.0 1.3 4.7 8.3 5.7 293 A 10.3 7.2
5.5 15.1 14.5 3.5 20 50 14.5 17.1 294 A 2.8 1.0 50 1.3 1.3 0.5 0
3.4 11.2 10.2 295 A 50 50 50 50 50 50 50 50 50 50 296 A 3.1 0.9 50
0.2 1.8 1.3 4.7 4.8 18.2 20 297 A 4.8 9.3 50 4.4 4.4 1.5 1.6 15.5
20 20 298 A 1.8 3.3 50 1.7 2.1 0 2.2 7.3 15.6 20 299 A 2.6 3.6 50
2.2 2.5 1.1 2.2 5.4 3.6 1.4 300 A 2.2 2.3 50 3.3 4.0 2.6 1.1 1.1
11.0 2.4 301 A 2.6 2.5 50 2.6 2.3 2.9 1.8 0.9 9.8 1.8 302 A 2.2 4.8
50 4.7 3.2 4.3 7.7 3.8 0 8.4 303 A 2.0 3.1 1.0 2.1 2.9 0.4 0.4 2.9
10.9 6.2 304 A 11.9 16.6 50 20 16.6 2.2 14.2 17.9 20 20 305 A 2.8
1.1 3.9 1.1 1.4 1.2 0.9 0 0.8 0.8 306 A 3.5 6.0 50 5.9 9.9 6.2 5.3
11.4 9.6 10.3 307 A 3.0 2.2 0 1.9 1.3 1.4 0.9 1.2 6.2 6.5 308 A
19.4 7.6 50 7.7 15.5 0 0.7 5.9 50 50 309 A 2.8 0 1.6 0.7 1.3 1.0
0.6 0.5 5.0 2.1 310 A 4.0 0 0.2 4.9 10 2.0 2.5 6.4 50 50 311 A 2.9
0.9 1.7 0.8 0.9 0 0.3 2.2 4.6 2.0 312 A 3.3 7.1 50 2.7 3.9 4.1 3.2
11.9 20 20 313 A 7.6 5.4 50 4.8 12.9 6.0 3.8 6.6 0 2.6 314 A 1.7
2.3 50 1.6 1.6 3.0 4.7 6.3 8.0 6.0 315 A 1.8 0.6 50 0 0.7 0 0.9 2.4
6.2 3.7 316 A 50 50 50 50 50 50 50 50 50 50 317 A 12.7 20 15.9 17.2
13.5 2.8 9.2 20 50 50 318 A 1.3 1.7 20 1.4 2.6 2.2 1.3 0 6.1 9.5
319 A 0.7 3.2 50 3.1 5.6 3.4 3.6 20 20 0.2 320 A 2.7 1.3 50 2.4 1.9
3.3 3.3 7.2 20 20 321 A 10.4 20 50 19.6 20 1.5 8.7 18.3 20 20 322 A
2.7 2.7 50 2.1 0 2.3 1.6 0.9 14.5 2.8 323 A 4.9 8.5 50 13.6 20 2.8
1.6 0 20 20 324 A 1.9 0.9 50 0.8 2.9 2.7 1.9 2.1 3.8 2.5 325 A 6.2
1.6 13.4 0.5 20 3.1 0.1 1.3 20 20 326 A 3.7 1.2 0 0.6 1.4 1.0 1.9
2.6 5.6 3.6 327 A 2.5 5.3 20 1.3 4.1 0 5.2 13.7 20 20 328 A 7.1 6.0
50 3.7 8.2 6.6 50 50 20 50 329 A 3.6 0 0.3 0.7 1.5 0.1 1.1 1.1 6.2
3.6 330 A 3.4 0 20 0.6 1.2 0.2 0.6 1.9 7.0 3.4 331 A 50 50 50 50 50
50 50 50 50 50 332 A 2.5 3.9 20 0.8 2.4 2.3 2.6 4.4 20 5.9 333 A
2.8 2.5 1.6 1.3 3.2 1.3 1.4 7.7 4.0 4.8 334 A 3.5 1.5 4.4 0.1 2.7
2.2 0.9 1.3 4.9 1.8 335 A 3.0 1.2 0 2.3 2.8 1.4 1.4 7.3 5.1 4.5 336
A 2.5 3.2 20 2.8 1.4 0.7 0.6 0 20 20 337 A 7.9 5.0 50 11.4 12.7 4.5
2.3 50 19.3 10.6 338 A 1.9 1.0 50 1.5 0.9 0.7 10.3 50 5.4 4.9 339 A
3.6 0 0.8 0.6 1.6 0.6 0.9 2.4 6.8 3.8 340 A 1.8 1.0 1.9 0.9 1.3 0.5
0.8 1.7 4.9 2.4 232 B 3.9 1.1 0 1.1 1.6 0.7 1.4 3.0 6.2 4.1 233 B
3.2 0.6 2.7 0.4 1.6 0 1.2 6.9 5.5 2.6 234 B 2.7 2.6 20 3.6 1.2 3.1
2.5 3.4 13.4 0.5 235 B 1.9 1.9 17.3 0.6 1.4 0.8 0.7 5.2 7.8 5.3 236
B 4.5 3.5 50 5.5 19.9 2.6 20 20 20 14.1 237 B 50 50 50 50 50 50 50
50 50 50 238 B 4.6 8.1 1.3 5.8 20 4.9 4.4 1.3 20 20 239 B 2.0 1.9
50 1.7 1.1 1.5 1.5 5.2 20 5.2 240 B 12.0 7.6 0 11.6 20 1.2 1.9 0.8
20 20 241 B 2.3 0.2 50 0.3 1.5 0.1 0.9 5.7 4.1 1.1 242 B 4.8 5.3 0
9.1 6.8 2.9 1.1 0.5 20 8.7 243 B 2.5 0 50 1.6 2.7 0.1 1.8 3.9 4.3
1.0 244 B 3.0 2.0 1.8 1.2 1.3 0 19.6 20 9.1 11.0 245 B 20 20 0 20
20 6.0 20 50 20 20 246 B 2.5 0.2 0.3 0.2 0.3 0.1 0 2.0 4.9 2.4 247
B 2.9 0.3 0 0.8 1.5 0.3 0.7 9.5 6.6 3.4 248 B 2.2 0 1.3 0.8 1.7 0.5
0.7 2.8 4.7 2.3 249 B 4.4 0.5 50 4.7 6.3 3.5 6.1 50 20 7.2 250 B
3.0 9.2 50 3.4 4.9 1.3 2.3 3.1 20 20 251 B 2.3 0.5 50 0.6 1.8 0.4
2.5 8.7 8.2 5.9 252 B 2.5 1.0 50 1.0 1.6 1.5 1.3 0.8 5.1 1.6 253 B
2.4 1.1 1.0 0 1.5 1.2 1.4 3.4 4.4 3.6 254 B 3.1 0.3 6.2 0.5 1.7 0
0.1 1.1 5.5 3.7 255 B 1.3 0.8 50 1.4 1.1 1.5 20 20 3.7 0.8 256 B
2.2 0 1.2 0.5 0.1 0.8 1.2 1.2 5.5 2.4 257 B 20 20 0 20 20 4.8 20 20
50 50 258 B 2.4 1.1 50 1.3 2.5 2.2 1.1 1.0 19.1 3.0 259 B 5.5 5.6
50 6.2 20 4.5 2.5 0 20 20 260 B 1.4 3.9 0.2 2.3 2.6 0.4 0.1 2.7 20
20 261 B 20 20 20 20 20 2.0 16.6 20 20 20 262 B 3.4 2.7 50 3.2 4.8
2.9 1.9 0 14.7 9.1 263 B 20 5.4 50 13.0 20 3.6 2.1 0 20 20 264 B
3.7 3.6 10.1 3.0 2.2 2.6 2.2 1.0 12.7 20 265 B 4.1 1.8 50 4.5 5.3
4.5 6.0 9.2 12.2 5.6 266 B 20 5.7 50 18.3 20 5.9 4.7 0 50 50 267 B
3.2 3.2 0.5 1.5 0.8 3.3 11.6 50 6.3 50 268 B 3.8 2.6 3.4 2.1 1.8
2.5 3.8 2.7 7.8 5.5 269 B 3.0 0 12.8 0.5 0.7 0.3 0.7 0.6 5.1 2.7
270 B 3.8 1.2 5.9 6.3 2.1 0.3 1.9 5.4 16.3 5.6 271 B 3.9 3.3 7.4
2.7 0 1.5 2.2 5.2 4.8 4.4 272 B 3.5 0.6 4.9 0 1.4 0.2 0.6 1.4 3.9
3.2 273 B 3.5 7.4 50 10.6 20 2.0 0 4.8 20 20 274 B 2.4 0.3 15.6 0.1
0 0 0.2 1.6 2.2 1.9 275 B 5.1 7.0 4.1 9.7 12.3 6.9 4.5 3.3 10.3 5.0
276 B 7.4 3.8 50 6.4 9.2 2.8 20 20 20 20 277 B 6.4 10.8 6.8 9.3
11.9 9.7 8.0 14.4 0 15.9 278 B 2.1 12.6 11.0 4.4 2.0 0.8 2.5 19.8
20 4.2 279 B 2.0 3.4 20 1.4 4.0 4.2 2.4 1.2 20 20 280 B 2.9 1.6 20
1.4 3.1 0 2.7 5.5 8.1 7.3 281 B 7.1 3.4 50 4.0 5.3 3.6 3.2 6.4 10.3
7.6 282 B 2.9 0.2 50 0 0.7 0 0.4 0.7 6.1 2.8 283 B 0.3 2.5 0 1.5
1.6 1.0 1.5 3.9 7.9 6.7 284 B 0.8 2.4 50 1.5 3.3 0 1.5 1.8 20 20
285 B 2.7 0 1.6 0.9 0.8 0.5 0.4 2.0 5.8 2.4 286 B 2.9 0 50 0.4 1.6
1.1 0.5 2.9 4.9 3.0 287 B 4.5 0.3 12.3 8.1 9.1 4.1 3.3 7.1 3.4 0.8
288 B 2.5 0.9 15.4 0.6 1.1 0.2 0.9 3.8 5.9 2.7 289 B 1.6 1.5 1.8
1.1 1.7 0 0.4 2.3 3.4 2.5 290 B 2.6 0.2 50 0.7 1.5 0 0.6 2.9 5.0
2.7 291 B 1.5 1.1 0.6 0.1 1.1 1.1 0.9 1.5 3.2 2.6 292 B 2.2 2.2
16.6 1.5 1.8 0.1 0 3.2 7.6 5.2 293 B 3.2 2.2 1.3 2.2 2.5 0 1.2 7.8
7.0 6.9 294 B 3.7 4.1 0 3.3 5.0 2.1 2.9 5.0 6.7 11.9 295 B 3.4 1.0
0.4 0.4 1.1 0 3.9 6.6 6.1 3.5 296 B 3.5 0 20 0.6 1.9 1.2 1.4 1.3
6.4 4.0 297 B 4.6 7.3 20 4.4 4.2 3.6 4.1 7.9 18.0 15.0 298 B 2.3
0.4 50 1.2 1.0 0.9 2.2 3.3 5.5 2.0 299 B 7.1 4.8 50 9.8 17.9 0.3
1.3 5.8 20 20 300 B 3.2 6.5 50 4.0 3.8 4.3 3.6 9.1 20 6.4 301 B 2.0
1.6 14.1 0.6 0.4 1.8 1.1 0 17.9 20 302 B 2.5 4.7 9.6 4.1 0.6 4.3
2.0 0 20 0.2 303 B 1.9 2.0 8.6 2.0 4.7 0.6 0.5 1.3 20 20 304 B 20
6.3 50 20 20 0 2.7 3.8 20 20 305 B 1.5 1.8 50 0.6 2.0 0.6 0 0.7 20
20 306 B 3.8 5.7 13.4 4.4 14.1 5.5 4.3 6.0 20 12.1 307 B 3.4 1.7 0
1.7 1.9 1.5 1.4 2.0 4.4 4.3 308 B 20 12.4 50 20 20 1.2 3.6 4.3 20
20 309 B 3.6 0.2 0 1.6 2.3 1.8 14.3 20 5.1 3.3 310 B 3.4 0 2.3 4.6
7.0 1.8 1.6 3.8 20 20 311 B 2.3 0.6 3.2 0.4 0.8 0.2 1.6 18.8 4.6
2.0 312 B 1.6 0.9 50 1.3 5.7 0.1 5.6 3.8 8.0 7.8 313 B 4.3 8.0 50
6.5 8.9 6.6 17.2 20 2.1 0.9 314 B 1.1 1.9 50 0.8 1.0 1.7 0.9 3.1
3.7 11.3 315 B 2.9 1.8 50 1.0 2.2 0.2 0 4.5 8.5 6.8 316 B 50 50 50
50 50 50 50 50 50 50 317 B 1.6 1.3 50 4.2 0.9 0.4 13.8 10.1 20 1.7
318 B 1.7 2.3 3.8 1.0 1.6 0.4 0 1.0 3.8 7.7 319 B 8.4 7.2 50 9.0
13.7 7.2 5.8 3.9 20 1.7 320 B 3.6 6.6 50 2.9 2.4 4.0 3.3 2.0 20 20
321 B 20 20 19.7 20 20 3.1 11.2 20 20 20 322 B 3.7 2.2 50 0.9 0.3
3.3 1.6 0 20 20 323 B 8.1 10.5 50 8.7 20 5.6 4.6 0 20 20 325 B 20 0
50 6.3 20 4.6 8.8 17.8 20 20 326 B 2.8 0.1 4.4 0 1.1 0.1 3.2 2.1
5.2 0.7 327 B 1.9 1.9 20 3.0 2.3 3.3 20 20 20 20 328 B 2.6 4.0 50
4.2 8.7 4.8 2.9 12.3 50 50 329 B 14.7 20 0 20 20 1.4 17.1 16.4 50
50 330 B 2.1 0.8 20 0 0.5 0.8 0.2 4.6 8.2 2.6 331 B 5.5 5.0 0 7.6
7.4 2.6 20 10.1 17.6 20 332 B 1.7 1.7 50 1.8 5.3 2.0 1.9 3.4 20 20
333 B 3.4 2.0 3.1 1.1 2.3 1.8 1.6 1.6 8.9 9.3 334 B 3.7 4.3 50 1.9
0 3.4 1.8 1.4 9.9 20 335 B 5.2 5.5 3.5 7.0 5.7 0.2 5.5 11.5 5.2 3.1
336 B 2.1 1.8 20 0.6 3.1 1.1 0.8 0.7 19.4 20 337 B 0.9 1.9 15.8 1.1
2.2 1.4 50 50 5.5 3.9 338 B 3.2 1.5 16.2 2.7 2.7 1.1 2.8 3.5 8.1
11.0 339 B 3.5 0 2.3 0.5 1.3 0.2 0.8 2.0 6.7 3.8 340 B 2.3 0.2 1.0
0.1 1.2 0 0.5 2.0 5.5 3.2 SPA .TM. technology; 1IIS template
structure; -carbohydrate, no floated positions
[0252] TABLE-US-00056 TABLE 56 Pos WT A C D E F G H I K L M N P Q R
S T V W Y 239 A S 0.2 4.6 2.7 0 20 4.6 14.5 11.0 1.9 0.3 2.0 1.9
8.1 1.4 2.6 0.4 5.7 11.6 20 20 240 A V 1.5 2.4 2.4 6.9 20 7.4 20
5.1 9.9 5.9 5.5 2.4 1.1 12.3 13.1 2.6 0.5 0 20 20 263 A V 2.3 2.8
6.3 16.5 20 8.8 20 9.6 7.3 7.3 15.3 4.8 50 16.4 17.4 2.8 1.4 0 20
20 264 A V 1.8 3.1 2.6 1.8 0 6.3 1.9 0.6 2.4 0.8 2.7 2.1 1.6 2.3
2.7 2.3 1.1 0.5 3.5 0 266 A V 4.9 5.2 6.9 12.3 20 11.1 20 0.8 11.9
20 8.5 6.6 50 12.5 20 6.1 3.7 0 20 20 296 A Y 3.4 2.7 1.1 0 50 0.7
50 5.0 3.6 3.5 4.2 0.9 50 0.9 2.9 2.2 5.3 5.5 16.1 18.4 299 A T 0.7
3.2 9.9 10.4 20 6.2 20 10.7 6.7 20 4.1 12.9 50 5.9 11.8 0 2.5 8.2
13.3 20 325 A N 2.5 3.5 7.7 2.5 20 8.0 20 0 6.1 20 7.8 1.2 12.8 0.8
20 2.7 0 1.0 20 20 328 A L 6.1 6.3 7.1 4.2 50 8.8 20 50 4.6 0 7.2
6.1 50 4.0 8.3 6.7 50 50 20 50 330 A A 0.9 1.8 1.2 0 2.5 4.0 2.9
1.7 1.2 1.6 2.8 0 20 0.4 1.0 0.2 0.5 1.7 6.2 2.9 332 A I 1.9 3.8
4.6 1.3 5.1 7.1 1.8 3.4 0.2 0 2.6 3.8 20 0.6 2.4 2.3 2.5 4.2 20 5.6
239 B S 1.0 2.4 3.5 2.0 6.7 5.6 2.9 3.1 0.3 0 1.9 2.1 50 1.5 1.8
1.4 1.4 5.2 20 4.2 240 B V 0.3 2.4 6.9 11.7 20 6.6 20 8.3 12.3 20
14.2 7.4 0 13.4 20 1.3 1.9 0.9 20 20 263 B V 2.4 3.9 4.5 12.5 20
9.3 20 15.8 17.1 2.1 20 5.3 50 13.8 20 3.9 2.2 0 20 20 264 B V 2.2
3.2 4.8 2.7 7.4 6.9 6.0 0 1.9 1.9 3.8 3.7 9.9 3.1 2.2 2.7 2.4 0.9
14.7 18.2 266 B V 5.4 5.5 7.5 13.2 20 12.1 20 2.6 20 20 20 5.4 50
16.1 20 6.0 4.7 0 50 50 296 B Y 1.5 2.7 1.3 1.2 4.0 4.1 3.6 1.1 1.9
2.6 3.5 0 20 0.7 1.8 1.1 1.4 1.3 6.5 4.2 299 B T 0 2.2 7.5 10.2 20
4.8 20 7.7 5.8 20 10.3 5.1 50 10.2 18.4 0.3 1.1 5.4 20 20 325 B N
3.4 5.1 8.6 5.0 20 8.2 20 16.7 20 20 20 0 19.7 6.3 20 4.6 8.6 18.2
20 20 328 B L 3.6 3.5 3.8 3.9 50 8.3 7.0 50 2.9 0 1.9 3.8 50 3.4
8.4 4.7 2.9 12.5 50 50 330 B A 0.7 2.1 2.9 0.7 2.7 4.0 1.4 4.8 0
2.2 2.3 0.8 20 0.2 0.8 1.1 0.2 4.7 7.8 3.2 332 B I 1.8 2.9 1.2 1.8
13.5 7.0 9.9 1.7 3.2 0 1.7 1.9 50 1.2 5.4 2.0 2.0 3.3 20 20 SPA
.TM. technology; D129G 1IIS template structure; +carbohydrate
[0253] TABLE-US-00057 TABLE 57 Pos WT A C D E F G H I K L M N P Q R
S T V W Y 239 A S 1.2 3.5 1.7 0 20 5.8 11.0 6.6 2.9 3.9 3.9 2.7 8.5
1.3 2.7 0.6 3.5 5.4 20 20 240 A V 1.2 2.4 6.0 14.0 20 7.1 20 6.7
9.4 10.1 7.5 4.4 1.8 14.8 20 2.0 0.4 0 20 20 263 A V 0 0.4 1.0 8.7
20 6.9 4.4 11.7 4.9 16.0 19.2 0.8 50 11.7 20 1.4 0.1 1.0 20 20 264
A V 2.9 3.7 6.3 2.8 11.6 7.6 13.2 0 3.2 3.4 4.1 4.2 7.1 2.9 3.4 3.1
1.9 0.8 12.8 16.3 266 A V 4.8 5.9 6.8 9.5 50 10.3 20 3.5 12.7 12.2
12.7 4.1 50 11.9 11.9 5.2 2.9 0 50 50 296 A Y 0.8 2.0 1.5 0.1 0.2
3.4 1.5 6.6 1.7 0.6 1.8 1.2 2.6 0 1.6 0.2 2.5 5.6 3.8 0 299 A T 1.9
3.7 7.5 0 20 7.9 14.2 2.9 0.8 3.4 4.4 2.3 50 1.9 3.0 3.5 4.1 3.3 20
20 325 A N 1.0 1.4 3.1 2.8 20 7.4 20 8.5 7.7 10.4 6.1 2.8 15.4 5.4
20 0 0.1 3.8 20 20 328 A L 2.5 5.3 4.0 1.9 50 7.5 20 20 1.6 0.2 0
2.9 50 0.4 4.8 3.2 2.9 7.0 50 50 330 A A 0.9 2.1 1.8 1.2 2.4 2.7
3.1 3.1 1.4 2.1 3.5 0.5 20 0.8 1.0 0 0.5 2.9 5.2 2.9 332 A I 2.9
3.7 3.9 0.9 6.1 7.8 2.5 0 2.7 0.8 2.8 3.5 50 0.7 3.7 2.9 2.5 1.0
8.1 6.9 239 B S 1.9 3.1 3.0 1.9 1.5 6.2 2.3 14.1 1.8 1.4 2.9 1.8 0
1.9 3.2 1.9 2.3 7.7 6.6 15.8 240 B V 0.5 1.7 5.0 13.3 20 6.6 20 1.2
12.4 12.1 8.8 4.6 6.3 20 20 1.0 0.2 0 20 20 263 B V 2.9 3.2 6.4
18.2 10.1 9.2 6.9 12.8 6.0 20 10.3 5.7 50 17.5 20 3.2 2.2 0 20 20
264 B V 2.9 3.6 4.4 3.0 8.8 7.1 6.2 0 2.3 1.9 4.5 3.4 1.7 3.2 3.5
3.5 2.0 0.9 12.0 16.4 266 B V 4.4 4.6 2.6 6.6 20 10.7 20 0 4.9 1.7
8.5 5.6 50 6.0 12.4 5.3 4.6 1.5 20 50 296 B Y 0 7.1 6.7 7.2 20 0.1
18.6 50 7.0 2.7 6.6 6.8 50 7.2 9.3 2.3 50 50 20 14.1 299 B T 0 3.2
10.4 6.0 20 5.5 20 15.9 3.2 5.9 4.4 6.4 50 5.7 9.4 1.2 1.4 13.7 20
20 325 B N 1.4 2.5 5.0 0 20 7.0 20 20 1.0 2.2 1.0 0.3 1.9 1.1 20
2.6 5.1 20 20 20 328 B L 0.4 1.3 5.6 0 50 4.5 50 50 1.9 2.4 2.4 8.3
50 0.8 16.4 1.0 1.2 50 50 50 330 B A 0.6 1.4 2.5 0.9 3.1 2.5 1.2 20
0 2.4 2.1 0.3 20 0.4 0.6 0 4.0 20 13.5 3.4 332 B I 4.3 5.3 5.7 0
11.4 9.3 4.3 2.5 5.8 2.0 4.0 6.5 17.9 3.7 5.9 4.6 4.2 3.7 20 11.6
SPA .TM. technology; D129G 1IIX template structure;
+carbohydrate
[0254] TABLE-US-00058 TABLE 58 Pos WT A C D E F G H I K L M 239 A S
1.2 2.3 2.2 1.8 7.9 5.5 7.6 0.5 0.2 1.8 2.6 240 A V 0.7 2.9 6.8 4.3
20 6.5 20 0 10.7 20 3.1 263 A V 1.7 2.9 4.6 18.8 20 8.4 5.8 15.1
2.3 14.5 2.1 264 A V 2.7 3.3 3.6 1.5 13.9 6.7 5.9 0 2.3 4.9 3.7 266
A V 3.5 3.5 5.7 12.4 20 10 20 5.7 6.3 7.8 7.4 296 A Y 2.6 50 50 50
50 0 50 50 18.5 18.0 50 299 A T 0.2 0.7 6.6 1.2 20 5.6 9.6 1.6 0.8
1.5 1.8 325 A N 3.1 3.6 7.3 2.4 20 7.7 20 20 20 10 13.1 328 A L 0.6
0 1.5 5.4 50 1.6 50 50 3.1 4.2 9.6 330 A A 1.9 2.5 4.1 2.8 4.5 4.1
3.0 3.2 1.0 2.7 3.5 332 A I 2.3 3.5 2.2 0.8 20 6.8 9.6 0 3.4 0.2
2.6 239 B S 1.4 3.6 2.5 1.4 16.8 5.8 6.2 5.0 2.5 1.4 2.0 240 B V 0
2.6 12.8 18.6 20 5.7 20 12.7 10.4 20 8.5 263 B V 1.1 2.4 3.6 20 20
7.8 17.7 11.8 4.5 20 6.3 264 B V 3.3 4.0 5.0 2.9 14.2 7.5 4.8 0 2.6
3.6 4.6 266 B V 2.9 3.3 4.9 11.3 50 9.5 20 20 20 7.9 15.0 296 B Y
2.8 50 50 50 50 0 50 50 17.7 18.7 50 299 B T 0 3.8 12.6 9.2 20 5.9
20 7.3 4.8 3.2 4.3 325 B N 0.3 2.0 5.5 2.2 50 6.1 20 0 10.5 15.5
14.6 328 B L 5.4 5.7 7.3 4.4 50 9.8 20 50 2.5 0 5.1 330 B A 0.6 1.4
3.2 1.3 3.9 3.2 2.7 4.0 1.3 3.7 3.1 332 B I 1.9 3.1 2.7 1.7 5.2 6.9
3.1 0.4 1.3 0 1.9 Pos N P Q R S T V W Y 239 A 1.4 0.9 1.3 1.9 1.5
0.8 0 8.6 9.6 240 A 9.1 2.1 7.7 20 1.4 1.1 2.4 20 20 263 A 3.2 50
20 15.0 3.6 1.2 0 20 20 264 A 3.2 1.9 2.5 3.0 3.0 2.5 0.7 19.9 19.0
266 A 5.2 50 16.6 20 4.2 1.7 0 20 50 296 A 50 50 50 50 50 50 50 50
13.6 299 A 4.8 50 1.0 9.2 0 0 1.6 20 20 325 A 3.6 50 0 20 4.0 9.7
20 20 20 328 A 1.4 50 6.9 9.6 0.6 0.1 50 50 50 330 A 2.1 20 2.4 2.6
1.3 0 3.9 7.6 5.3 332 A 2.8 14.5 3.3 4.6 2.6 1.3 0.9 10.5 20 239 B
3.8 0.3 0.5 2.4 0 1.6 5.3 20 19.5 240 B 15.1 3.1 20 20 1.0 0.2 2.4
20 20 263 B 3.3 50 20 20 3.2 1.2 0 20 20 264 B 3.5 1.7 3.1 4.1 3.9
2.9 1.3 6.9 20 266 B 4.5 50 4.9 20 1.9 0 3.6 50 50 296 B 50 50 50
50 50 50 50 50 11.3 299 B 8.0 50 12.3 8.8 0.2 2.1 4.4 20 20 325 B
1.3 10 2.4 20 2.3 2.0 1.0 20 50 328 B 5.9 50 2.8 7.4 6.1 6.4 50 50
50 330 B 0.7 20 0.6 1.3 0 0.4 4.2 8.2 3.6 332 B 2.6 7.7 1.3 2.2 2.3
1.6 2.0 10.4 5.6 SPA .TM. technology; D129G 1E4K template
structure; +carbohydrate
[0255] TABLE-US-00059 TABLE 59 Pos WT A C D E F G H I K L 239 A S
1.4 2.6 3.1 1.0 20 5.7 4.8 3.4 2.0 1.2 240 A V 2.9 3.5 3.7 4.6 20
8.2 10.8 0 9.1 3.2 263 A V 3.6 4.9 6.2 8.7 20 9.9 20 3.7 4.2 0.5
264 A V 1.8 2.8 3.3 2.0 2.9 6.2 3.1 0 2.4 0.8 266 A V 4.4 5.2 4.9
7.1 20 10.6 20 1.0 12.1 4.8 296 A Y 1.2 2.9 0.7 1.4 3.1 3.9 2.7 2.4
2.3 1.9 299 A T 0 2.6 6.0 11.5 20 5.3 20 20 6.0 20 325 A N 5.2 7.0
6.6 6.9 50 11.3 20 1.3 14.3 13.5 328 A L 4.8 5.5 7.0 3.2 20 10.5 20
50 5.1 0 330 A A 0.9 1.8 1.1 0.9 3.5 4.0 3.0 2.3 1.2 1.6 332 A I
5.3 6.4 6.7 4.8 8.2 9.9 5.2 3.1 0 3.6 239 B S 0.7 2.3 2.6 2.0 5.3
5.1 3.3 1.7 0 0 240 B V 2.3 3.0 4.1 7.3 20 8.1 20 5.1 20 11.8 263 B
V 3.2 4.3 7.3 8.3 20 9.6 20 13.3 8.5 0.6 264 B V 2.1 3.2 3.7 2.7
17.8 6.6 11.5 0 2.0 0.8 266 B V 5.0 5.0 5.2 16.3 20 11.2 20 2.3 20
14.3 296 B Y 0.9 2.3 1.0 0.5 2.7 3.7 2.5 1.2 1.3 2.1 299 B T 1.1
2.2 7.6 5.4 20 6.4 12.8 1.8 3.9 17.5 325 B N 10.1 11.5 13.1 11.2 20
15.7 20 8.6 14.3 17.1 328 B L 2.9 4.1 4.8 3.5 50 8.5 1.7 9.6 1.5 0
330 B A 0.1 2.0 1.4 1.8 1.6 4.0 3.0 2.0 0.5 0.5 332 B I 3.4 4.4 3.5
3.1 6.1 8.2 4.1 0 3.3 1.3 Pos M N P Q R S T V W Y 239 A 2.6 1.6 4.8
0 2.1 1.3 2.1 3.3 13.8 19.6 240 A 5.4 3.1 4.8 5.5 17.5 4.0 1.8 1.2
20 20 263 A 6.7 6.1 50 9.5 20 5.1 3.6 0 20 20 264 A 3.0 2.4 6.1 1.4
2.8 2.4 1.9 0.8 10.2 2.2 266 A 9.1 4.6 50 7.9 12.6 5.8 3.5 0 20 20
296 A 2.2 0 1.6 1.4 3.0 0.9 1.0 3.5 6.0 2.6 299 A 4.4 3.0 50 14.1
13.2 0.9 3.8 15.1 15.0 20 325 A 13.9 0 5.0 6.0 20 6.0 4.6 3.2 20 50
328 A 8.5 5.5 50 3.5 8.2 5.5 13.4 50 20 50 330 A 2.8 0 14.5 0.9 1.1
0.1 0.4 2.0 6.4 3.2 332 A 5.2 6.8 20 3.5 4.6 5.5 4.8 4.0 11.2 7.1
239 B 2.0 0.8 15.5 0.9 0.8 0.7 0.7 3.3 8.2 6.0 240 B 10.9 3.8 2.0
17.0 20 3.6 1.3 0 20 20 263 B 20 6.0 50 8.5 20 4.6 4.0 0 20 20 264
B 3.5 3.0 7.8 2.0 1.5 2.5 1.3 1.0 13.9 20 266 B 17.3 2.5 50 11.6 20
5.4 3.9 0 20 20 296 B 3.0 0 7.0 0.4 1.1 0.3 0.8 1.8 6.0 2.4 299 B
6.9 3.9 20 4.6 10.3 0.8 0 1.9 20 20 325 B 20 0 16.1 10.6 20 11.1
10.9 10.5 20 20 328 B 1.5 3.5 50 3.3 2.0 3.3 1.9 5.2 50 50 330 B
2.6 0 20 0.7 2.0 0.3 0.6 2.1 4.4 2.4 332 B 3.3 4.0 15.7 0.8 2.1 3.9
2.7 1.1 20 6.1 SPA .TM. technology; Fc/Fc.gamma.RIIb model template
structure; -carbohydrate
[0256] TABLE-US-00060 TABLE 60 Pos WT A C D E F G H I K L 237 A G
1.0 3.0 5.9 1.8 5.0 4.7 3.1 1.4 1.4 4.2 238 A P 3.3 5.2 9.7 4.6 20
9.2 20 0.4 19.6 5.0 239 A S 0.6 1.8 3.3 1.3 1.8 4.7 0.5 2.1 0 0.8
240 A V 1.3 2.3 2.7 8.2 20 7.0 20 6.1 8.7 0 241 A F 0 2.1 2.4 0.5
1.0 4.0 2.6 7.5 1.7 0.9 242 A L 3.7 4.7 6.6 6.7 20 9.2 20 13.0 13.8
0 243 A F 0.8 2.0 1.0 0.2 0.3 4.9 1.5 4.0 1.5 0.8 244 A P 2.8 3.7
5.5 3.2 6.4 2.9 3.6 0 0.1 4.0 245 A P 2.5 20 20 20 20 7.7 20 50 20
20 246 A K 2.1 3.5 2.8 2.5 4.0 6.5 4.5 3.4 2.7 1.9 247 A P 2.4 5.0
6.6 7.8 50 7.5 20 10 9.6 50 248 A K 2.0 4.0 4.8 4.2 20 6.9 9.6 3.3
0 2.7 249 A D 3.3 4.2 3.3 0.9 4.8 9.3 4.0 50 3.1 0 250 A T 0.1 1.5
3.0 8.0 20 6.7 20 2.5 5.4 9.5 251 A L 3.1 4.6 6.3 6.1 2.3 9.5 1.5
7.2 3.1 1.3 252 A M 3.0 3.5 5.3 2.8 20 9.4 20 4.4 0.6 5.0 253 A I
0.9 1.8 3.9 3.3 4.0 5.5 6.2 1.3 0 0.9 254 A S 0 2.4 5.5 3.4 8.1 3.9
8.5 4.2 3.3 1.7 255 A R 2.2 3.8 5.4 3.9 7.1 7.3 1.6 15.8 0 0.5 256
A T 1.1 2.3 1.7 1.5 3.2 3.9 2.5 3.1 1.1 2.0 257 A P 2.1 9.1 20 16.1
50 8.5 50 20 20 20 258 A E 0 1.6 2.3 0.6 0.9 4.2 2.1 9.4 2.4 3.5
259 A V 4.0 4.3 7.0 11.4 20 10.4 20 3.2 14.0 14.9 260 A T 2.6 2.8
3.2 0 20 7.3 5.6 1.7 3.1 5.5 261 A C 0 18.6 20 20 20 3.8 20 20 20
20 262 A V 1.8 1.5 1.3 11.1 20 7.1 20 5.8 14.6 20 263 A V 4.5 5.1
6.5 14.1 20 10.8 20 8.0 6.1 3.2 264 A V 2.6 3.2 3.7 2.0 14.0 7.0
9.0 0 2.6 2.5 265 A D 1.4 2.8 2.7 1.6 1.8 2.5 2.2 18.5 0 0.1 266 A
V 4.8 5.1 5.0 14.2 20 11.5 20 0.2 20 9.8 267 A S 2.6 4.9 5.7 3.3
1.6 7.2 2.9 0.5 1.0 2.0 268 A H 1.5 1.9 2.6 2.6 4.4 4.8 2.7 1.7 0
2.8 269 A E 0.3 2.0 0.8 0.6 2.8 3.6 2.7 1.5 1.6 1.4 270 A N 0 1.4
1.9 3.8 0.7 3.9 2.2 6.8 2.4 3.5 271 A P 0.9 2.2 5.7 6.5 3.0 5.5 2.9
12.8 5.4 15.7 272 A E 0.7 1.8 0.3 0.4 3.0 3.6 2.6 1.7 1.4 2.0 273 A
V 3.1 4.0 2.1 15.5 20 8.9 20 0.5 10.6 7.1 274 A K 0.6 2.1 1.5 0.9
2.2 4.3 2.6 1.6 1.3 1.2 275 A F 7.1 8.6 10.1 8.0 0 12.2 4.2 7.7 4.7
5.5 276 A N 0.5 1.8 1.3 0 20 5.4 20 12.6 2.6 3.2 277 A W 9.3 11.1
12.3 10.7 4.1 15.0 8.6 10.2 7.3 7.8 278 A Y 0.1 1.9 6.1 0 2.7 5.5
4.5 16.4 1.3 16.3 279 A V 3.2 4.4 5.1 3.1 20 8.2 19.7 0.3 0 1.9 280
A D 3.6 3.5 0 2.7 12.5 4.0 9.9 17.6 3.8 2.8 281 A G 50 50 50 50 50
0 50 50 50 50 282 A V 0.5 1.8 1.8 0.8 2.2 4.0 2.2 0.7 0.9 1.3 283 A
E 0.9 1.2 4.9 0 7.9 4.4 4.2 2.2 1.3 4.7 284 A V 2.1 2.6 4.4 2.6
15.0 6.7 4.4 2.0 1.8 6.3 285 A H 1.0 2.3 1.9 1.3 2.8 2.8 2.6 2.0
1.6 2.2 286 A N 0.9 1.7 0.7 0.1 3.3 3.4 3.2 0 0.9 2.1 287 A A 2.4
4.3 5.1 8.4 0 8.4 3.1 11.2 6.6 17.4 288 A K 0.7 1.9 2.0 0.9 2.7 3.9
2.6 1.0 1.3 1.9 289 A T 1.0 1.7 2.2 0.5 6.6 4.7 2.3 0.5 0.3 0.5 290
A K 1.1 2.6 2.6 1.6 3.8 5.6 5.4 3.1 0.8 0.4 291 A P 1.0 2.4 1.8 1.7
4.5 3.6 3.4 2.6 1.6 2.6 292 A R 1.5 3.2 3.2 1.9 1.7 5.2 3.0 1.8 1.0
0 293 A E 0.6 2.1 1.5 0.4 8.0 4.3 5.5 3.2 1.4 0.8 294 A E 2.3 2.5
0.5 0.5 5.0 5.8 3.5 4.1 1.5 2.0 295 A Q 3.1 3.2 3.8 2.8 17.6 7.8
12.8 9.7 1.3 0 296 A Y 3.3 3.5 3.6 2.3 4.7 0 3.5 50 2.6 3.6 297 A N
1.4 2.3 3.2 1.4 4.4 4.3 3.0 0.6 0.7 2.2 298 A S 6.5 7.1 6.9 6.9
11.0 0 8.6 50 6.6 7.2 299 A T 0.3 2.2 3.0 20 0.5 4.4 0.5 5.1 1.5
16.2 300 A Y 4.1 4.8 5.1 5.2 0 9.8 2.2 11.9 3.3 2.6 301 A R 1.6 2.4
1.1 0.6 20 6.5 20 8.6 0 3.5 302 A V 3.2 4.0 5.0 4.0 20 9.0 5.6 0.3
1.6 2.3 303 A V 0.9 1.0 2.0 0 20 5.9 20 3.0 4.4 12.4 304 A S 1.1
2.3 4.1 9.2 20 7.8 20 7.6 6.9 20 305 A V 1.6 2.1 1.8 4.1 20 6.3 20
1.0 3.9 2.9 306 A L 5.1 6.8 6.7 7.0 1.7 11.5 4.9 3.8 3.9 0 307 A T
1.5 3.0 2.6 1.6 1.1 5.5 3.0 0.2 1.7 1.0 308 A V 4.0 4.5 12.8 7.2 20
10.2 20 0 17.1 5.0 309 A L 1.3 2.8 1.8 1.7 3.2 5.5 3.3 0.8 1.5 1.0
310 A H 1.6 2.2 2.1 4.0 18.6 6.3 4.2 3.5 3.2 6.7 311 A Q 0.2 1.6
1.4 0.7 1.9 3.9 1.6 0.4 0.7 0.7 312 A D 0 1.6 0.8 0.9 20 5.3 11.3
8.9 2.4 2.3 313 A W 4.0 5.5 7.0 5.1 0 10 5.1 11.0 2.7 4.2 314 A L
2.8 4.3 5.8 3.7 20 7.6 7.3 4.2 1.8 0 315 A N 0 5.1 3.1 3.4 11.1 2.6
11.3 16.1 3.4 3.4 316 A G 11.4 10 11.1 8.9 16.6 0 12.7 50 8.5 8.8
317 A K 3.1 4.8 8.1 5.6 6.5 8.4 2.5 7.3 0 5.5 318 A E 1.5 2.5 2.8
1.7 20 5.8 9.6 2.0 1.9 5.9 319 A Y 7.0 7.9 9.4 9.9 0 12.8 4.6 3.2
6.0 6.6 320 A K 1.8 2.9 6.7 1.8 20 7.1 20 1.1 0 8.1 321 A C 0 3.9
20 20 20 6.1 20 20 20 20 322 A K 2.4 3.3 6.1 3.0 20 8.0 20 1.1 0.4
16.9 323 A V 3.5 4.3 8.0 9.0 20 9.9 20 4.7 10.2 20 324 A S 0.4 2.0
0.9 0 0.2 5.0 2.5 1.3 1.3 0 325 A N 4.9 5.9 6.3 6.0 20 10.4 20 1.7
14.0 11.3 326 A K 1.6 3.6 2.4 2.4 3.8 4.9 3.0 10.6 1.4 2.9 327 A A
2.7 3.8 5.5 2.8 11.6 7.1 9.5 3.2 1.2 3.7 328 A L 2.5 3.7 4.6 1.7 50
7.4 20 5.7 1.8 0 329 A P 0.8 2.2 1.2 1.2 4.0 3.3 3.3 2.9 1.6 2.5
330 A A 0.3 1.6 2.0 1.3 3.4 3.2 2.3 2.4 1.3 2.5 331 A P 1.5 3.5 8.0
10.4 6.6 6.3 5.0 9.3 8.1 12.6 332 A I 2.4 3.5 2.6 2.2 7.9 7.2 4.0
1.3 2.3 0.7 333 A E 2.0 2.7 2.6 0 8.0 6.3 7.6 3.1 3.5 5.9 334 A K
2.1 3.4 3.5 2.0 7.1 7.1 3.5 2.0 2.2 3.0 335 A T 0.5 1.0 2.1 0.3 4.6
4.6 4.1 0.6 0.2 4.0 336 A I 0.4 1.4 2.2 0.9 13.9 4.8 4.7 0 1.3 4.3
337 A S 0.3 0.8 4.0 13.5 20 3.1 20 50 9.9 9.3 338 A K 4.7 8.3 7.1
7.6 20 9.8 20 5.5 0 2.3 339 A A 1.4 2.7 3.1 2.2 2.3 5.5 3.3 0 0.5
0.2 340 A K 1.2 3.0 2.0 1.5 1.9 3.9 2.5 2.6 0.2 2.2 237 B G 0.6 2.2
1.7 0.5 5.0 4.2 2.5 0.3 0.3 4.1 238 B P 3.0 5.3 8.9 5.4 20 9.1 20
1.1 14.4 3.6 239 B S 0.7 2.0 2.9 1.1 2.2 4.8 0.5 2.9 0 0.8 240 B V
2.0 3.0 3.4 5.3 20 7.7 20 6.4 11.3 0 241 B F 0.3 2.0 2.7 0.2 1.5
4.3 3.2 5.7 1.3 1.2 242 B L 4.0 5.1 7.0 6.5 20 9.5 20 12.9 13.6 0
243 B F 0.7 1.8 1.2 0 0.2 4.9 1.5 4.8 1.4 0.8 244 B P 2.1 3.1 5.4
2.8 7.0 3.5 3.7 0.7 0 3.6 245 B P 2.1 20 20 20 20 7.3 20 50 20 20
246 B K 1.4 2.8 1.8 2.0 3.4 5.8 3.9 2.3 1.8 1.2 247 B P 2.4 5.0 8.4
7.4 20 7.5 18.5 8.7 9.4 20 248 B K 2.0 4.1 4.6 3.7 20 6.8 9.1 3.7 0
2.2 249 B D 3.2 4.2 3.1 1.2 3.2 9.1 4.0 50 2.9 0 250 B T 0.1 1.4
3.1 7.4 20 6.7 20 2.9 5.9 8.9 251 B L 4.0 5.6 6.9 7.9 3.5 10.4 1.9
4.9 5.0 2.1 252 B M 3.0 3.6 5.6 2.9 20 9.2 20 6.0 1.0 4.9 253 B I
1.5 2.3 4.6 3.5 4.6 6.1 6.8 1.7 0 1.6 254 B S 0 2.8 6.1 3.4 8.5 4.1
9.9 4.5 4.2 1.5 255 B R 1.8 3.7 5.0 3.7 3.5 6.8 0.8 14.1 0.1 0 256
B T 1.0 2.3 1.6 1.4 3.1 3.7 2.3 3.5 1.2 1.9 257 B P 2.0 10.4 20 20
50 8.4 50 20 20 17.3 258 B E 0 1.6 2.7 0.8 0.8 3.9 2.0 12.0 2.4 4.0
259 B V 4.1 4.5 7.0 9.1 20 10.5 20 1.5 12.2 14.4 260 B T 2.4 2.7
3.0 0 20 7.2 7.2 1.6 3.0 5.3 261 B C 0 18.0 20 20 20 4.0 20 20 20
20 262 B V 1.6 1.5 0.8 20 20 7.0 20 8.4 16.8 20 263 B V 4.5 5.2 5.5
18.4 20 10.9 20 10.9 4.6 3.5 264 B V 2.6 3.0 3.7 1.6 12.8 7.1 12.2
0 2.3 3.1 265 B D 1.4 2.7 2.4 1.3 2.0 2.3 2.3 50 1.0 0 266 B V 4.9
5.4 7.0 15.9 20 11.6 20 2.7 20 20 267 B S 2.4 4.6 4.3 4.3 1.3 7.1
2.8 1.3 0.9 1.7 268 B H 2.1 2.9 3.8 2.4 6.9 5.5 2.0 1.7 0 4.9 269 B
E 0.8 2.5 0.9 1.2 3.3 4.0 2.9 1.2 2.0 2.2 270 B N 0 1.3 0.9 3.9 2.9
3.8 4.8 5.7 3.5 1.2 271 B P 0.8 2.5 6.0 7.8 3.1 5.6 4.2 16.4 5.7
16.9 272 B E 0.7 1.7 0.1 0 3.0 3.7 2.8 1.0 1.9 1.8 273 B V 4.8 5.5
5.1 16.6 20 10.8 20 3.1 9.6 7.2 274 B K 0.8 2.4 1.6 1.0 2.3 4.5 2.7
1.5 1.5 1.4 275 B F 6.9 8.5 9.9 8.0 0 12.1 3.7 8.2 4.8 5.5 276 B N
0.4 1.6 1.2 0 20 5.3 20 9.1 2.7 3.1 277 B W 8.9 10.9 11.6 10.6 4.8
14.6 7.8 10.1 6.3 7.6 278 B Y 0.8 2.1 5.6 0 1.4 6.3 3.9 18.3 1.3
12.7 279 B V 3.8 4.9 5.9 3.8 20 8.8 16.0 1.0 0 2.8 280 B D 3.5 3.4
0 1.8 12.4 3.9 9.8 17.0 3.6 2.3 281 B G 50 50 50 50 50 0 50 50 50
50 282 B V 0.4 1.8 1.7 0.8 2.2 3.9 2.1 0.8 0.9 1.3 283 B E 0.9 1.2
4.9 0 7.8 4.7 4.3 1.7 1.5 4.7 284 B V 2.3 2.7 5.0 2.6 16.4 7.0 5.5
3.1 2.0 7.0 285 B H 0.6 2.0 1.8 1.0 2.4 2.5 2.3 2.0 1.4 1.9 286 B N
1.0 1.8 0.9 0.2 3.4 3.5 3.1 0 1.4 2.2 287 B A 2.5 4.3 5.5 8.5 0 8.4
3.2 13.3 6.7 17.7 288 B K 0.6 1.7 1.6 0.8 2.5 3.7 2.5 1.0 1.4 1.7
289 B T 0.9 1.5 2.1 0.5 5.4 4.6 2.3 0 0.2 0.2 290 B K 0.7 2.4 2.5
1.2 3.2 5.2 5.2 2.4 0.2 0.1 291 B P 1.0 2.5 2.0 1.7 4.5 3.6 3.4 2.5
1.6 2.6 292 B R 1.8 3.5 3.5 2.2 1.6 5.5 2.8 2.2 1.1 0 293 B E 0.7
2.2 1.6 0.6 8.6 4.5 4.7 2.8 1.5 0.9 294 B E 2.1 2.2 0.5 0.4 5.2 5.7
3.3 2.8 1.3 3.0 295 B Q 3.3 3.1 4.0 3.1 18.8 7.6 13.1 8.3 1.2 0 296
B Y 4.2 4.4 4.5 3.6 5.6 0 4.4 50 3.4 4.4 297 B N 1.3 2.1 3.1 1.4
4.0 4.2 2.7 0.3 1.3 2.1 298 B S 5.5 6.0 6.3 5.7 9.7 0 7.5 50 5.7
6.3 299 B T 1.1 3.1 3.5 15.2 0.9 5.9 0 6.3 1.4 10.9 300 B Y 3.1 3.7
4.0 3.8 2.9 8.8 2.3 10.5 2.0 1.5 301 B R 1.5 2.3 1.4 0.3 20 6.4 20
6.8 0.1 3.8 302 B V 3.4 4.0 5.8 3.9 20 9.3 7.0 0.2 1.8 3.4 303 B V
0.2 0.2 1.2 1.2 20 5.3 20 5.8 3.9 11.4 304 B S 1.0 1.9 4.1 10.8 20
7.8 20 8.3 7.4 20 305 B V 1.5 1.8 1.7 3.9 20 6.2 20 0.7 3.2 4.2 306
B L 5.2 7.1 6.7 7.3 1.5 11.6 4.9 3.7 5.1 0 307 B T 1.6 3.0 2.5 1.9
1.1 5.5 3.0 0.2 1.6 1.2 308 B V 5.1 5.8 12.7 7.4 20 11.3 20 0 19.1
6.1 309 B L 1.3 2.8 1.9 1.7 3.2 5.4 3.3 0.9 1.5 1.0 310 B H 1.7 2.4
2.5 3.8 13.1 6.4 5.5 3.6 3.6 7.5 311 B Q 0 1.6 1.1 0.5 0.9 3.7 1.6
0.4 0.6 0.6 312 B D 0 1.7 0.8 4.6 20 5.3 20 11.0 3.1 2.9 313 B W
4.4 5.9 7.2 5.7 0 10.3 5.3 9.3 3.0 4.3 314 B L 2.8 4.4 5.7 3.7 20
7.6 8.3 4.5 1.6 0 315 B N 0 7.4 3.9 5.2 12.9 2.1 12.0 14.7 4.5 3.3
316 B G 8.9 7.7 9.2 6.9 13.3 0 10.8 50 6.7 6.8 317 B K 2.6 4.3 7.6
5.9 6.7 7.4 2.3 7.5 0 4.8 318 B E 1.7 2.6 2.9 1.6 20 6.0 9.6 1.8
2.2 6.1 319 B Y 6.9 7.8 9.3 10.1 0 12.7 4.7 3.1 6.2 7.1 320 B K 1.7
2.9 6.7 1.9 20 7.0 20 0.6 0 8.9 321 B C 0 4.5 20 20 20 6.2 20 20 20
20 322 B K 2.8 3.8 6.2 3.3 20 8.4 20 1.8 0.8 16.3 323 B V 3.5 4.4
8.8 7.6 20 9.8 20 5.4 9.2 20 324 B S 0.5 2.5 1.4 0.6 0.4 5.2 2.7
3.5 0 0.2 325 B N 4.1 5.5 6.9 5.7 20 9.6 20 1.0 10.5 11.2 326 B K
0.9 2.8 1.8 1.8 2.6 4.4 2.5 4.2 1.3 2.9 327 B A 3.1 4.4 6.1 3.2
10.3 7.4 4.0 7.0 2.8 2.5 328 B L 4.7 5.4 6.6 3.3 20 9.8 20 50 4.1 0
329 B P 0.6 2.1 0.9 1.0 3.8 3.2 2.8 2.2 1.3 2.4 330 B A 0.4 1.8 1.5
1.2 3.4 3.3 2.7 2.1 1.3 1.9 331 B P 1.6 3.6 7.8 10.6 7.3 6.5 4.6
8.9 7.7 13.6 332 B I 2.0 3.0 2.6 0.9 5.8 6.9 2.6 0 2.1 0.1 333 B E
2.2 2.8 2.7 0 8.1 6.4 7.8 3.4 3.6 6.1 334 B K 2.2 3.4 4.2 2.1 10.4
7.2 4.1 1.7 1.8 3.1 335 B T 0.5 1.1 1.9 0.8 4.8 4.6 4.4 0.5 0.3 3.7
336 B I 0.7 1.5 2.5 1.0 18.4 5.0 5.4 0.1 1.9 4.6 337 B S 0.4 1.1
4.9 10.6 20 3.4 20 50 7.9 11.0 338 B K 4.5 8.2 7.4 8.0 20 9.6 20
5.3 0 2.0 339 B A 1.5 2.8 3.0 2.1 2.4 5.5 3.3 0 0.4 0.2 340 B K 1.0
2.7 1.7 1.3 1.7 3.7 2.1 2.4 0 2.1 Pos M N P Q R S T V W Y 237 A 3.7
2.5 0.5 1.3 1.5 0 1.2 1.9 7.5 5.6 238 A 8.2 7.3 0 6.1 20 1.3 3.7
2.1 20 20 239 A 2.6 0.2 8.4 0.9 0.5 0.3 1.3 2.5 4.7 1.8 240 A 9.2
2.2 0.3 7.4 20 2.0 0 0.4 20 20 241 A 2.8 0.7 10 0.7 1.4 0 0.9 6.9
3.9 0.9 242 A 7.0 5.7 3.6 9.2 20 4.8 4.7 3.4 20 20 243 A 2.2 0.3 50
0 0.5 0.5 0.5 4.0 4.3 0.6 244 A 3.7 3.8 0.3 1.1 0.9 1.4 1.5 0.9 7.7
6.9 245 A 20 20 0 20 20 3.4 19.2 50 20 20 246 A 4.1 0 1.2 2.7 2.3
2.2 2.5 2.9 6.8 3.7 247 A 6.5 9.5 0 7.0 8.4 0.1 0.6 4.6 20 50 248 A
3.0 4.1 2.4 3.1 3.0 2.1 0.8 2.0 15.2 20 249 A 3.2 3.1 50 1.9 4.1
4.4 8.1 50 6.3 5.3 250 A 5.7 2.2 50 7.3 10.7 1.6 0.8 0 20 20 251 A
5.4 4.7 50 5.2 4.4 4.8 2.1 2.4 50 0 252 A 0 4.7 50 2.9 14.2 0.9 6.7
6.0 20 20 253 A 1.8 2.3 50 2.6 2.7 1.2 1.2 2.0 5.8 4.7 254 A 5.0
2.1 50 4.4 4.6 0.2 5.1 6.0 9.0 8.9 255 A 1.5 3.2 50 2.0 0.6 2.0 1.4
14.5 5.4 20 256 A 2.7 0 2.3 0.4 0.3 0.7 1.6 2.4 6.0 3.0 257 A 20 20
0 18.6 20 4.1 20 20 50 50 258 A 1.9 1.9 14.7 2.2 2.7 0.9 4.1 10.4
20 1.0 259 A 10.6 7.7 50 12.2 20 5.1 1.8 0 20 20 260 A 3.5 2.7 4.5
1.1 3.0 2.5 2.0 2.0 14.0 20 261 A 20 20 50 20 20 4.2 20 20 20 20
262 A 20 3.6 50 20 16.5 2.1 1.9 0 20 20 263 A 10.9 5.7 50 14.4 18.7
6.0 3.6 0 20 20 264 A 3.8 2.3 10.4 1.9 2.4 2.6 1.6 0.5 15.9 18.2
265 A 2.5 1.1 50 0.5 0.8 1.6 11.8 19.0 4.9 2.1 266 A 20 5.4 50 19.9
20 6.3 4.2 0 50 20 267 A 3.7 3.7 0 2.2 1.5 1.9 1.3 2.6 5.6 2.3 268
A 1.9 0.6 0.5 0.9 1.0 0.7 1.8 1.6 7.3 4.6 269 A 2.9 0 6.8 0.5 1.5
0.1 0.3 1.3 5.3 2.7 270 A 2.7 1.6 13.3 3.2 3.0 0.9 1.2 5.1 2.2
1.0
271 A 5.6 4.2 0 8.0 4.2 1.3 2.9 7.5 4.3 2.6 272 A 2.9 0.3 11.8 0.2
1.6 0 0.9 1.2 4.4 2.8 273 A 12.4 1.2 50 20 20 1.4 0 0.7 20 20 274 A
2.9 0.9 50 0 0.6 0.6 1.1 1.4 2.8 2.2 275 A 6.8 8.3 7.0 7.1 11.9 8.1
7.3 7.4 10.3 1.8 276 A 3.0 0.2 18.7 1.3 1.8 1.1 6.3 10.8 20 20 277
A 7.9 11.4 7.6 9.5 14.6 10.2 9.6 11.3 0 20 278 A 1.5 3.3 50 0.3 2.2
0.5 0 10 8.6 1.0 279 A 3.1 3.8 20 0.5 3.6 3.8 2.3 1.6 20 20 280 A
3.9 0.5 50 3.1 3.5 3.1 8.9 13.1 13.7 13.0 281 A 50 50 50 50 50 50
19.2 50 50 50 282 A 3.0 0.4 50 0.4 0.5 0.1 0.5 0 5.4 2.5 283 A 1.3
3.1 0.5 0.5 1.3 0.6 1.6 3.0 8.3 7.2 284 A 2.6 2.7 15.1 2.4 1.6 2.6
1.3 0 11.1 16.1 285 A 2.8 0 1.8 0.9 0.9 0.3 0.5 2.3 5.1 2.3 286 A
2.3 0.2 6.6 0.4 0.9 0.7 0.7 0.8 5.4 3.4 287 A 4.4 1.3 12.9 8.3 8.6
4.1 4.5 9.9 1.4 2.0 288 A 2.2 0.4 5.0 0.5 0.6 0 0.5 1.8 6.0 2.6 289
A 1.6 1.2 0.8 0.6 0.7 0 0.2 0.9 6.2 7.0 290 A 2.2 1.5 50 1.0 1.3
0.6 0 2.9 6.3 4.5 291 A 3.5 0 0.9 0.9 1.3 0 1.0 2.4 7.6 4.1 292 A
1.6 1.9 0.9 1.3 1.6 1.5 1.6 2.5 4.3 1.9 293 A 2.2 1.2 16.3 0 2.2
0.1 0.8 3.3 10.6 8.3 294 A 2.7 0 1.6 0.9 1.3 1.9 1.6 2.4 9.0 5.4
295 A 2.3 3.3 50 1.6 3.6 3.8 7.9 10.1 20 20 296 A 4.7 1.7 50 2.1
2.5 1.8 14.0 50 7.7 4.7 297 A 2.4 1.1 50 0.9 0 0.8 1.1 1.5 7.3 4.3
298 A 8.5 5.2 50 6.2 6.3 4.4 12.0 50 12.5 10.6 299 A 3.3 0.1 50 20
3.4 0.7 0 0.4 50 1.7 300 A 3.7 4.4 50 5.4 7.9 5.7 7.2 12.7 5.8 0.4
301 A 1.7 0.5 50 0.3 0.9 2.5 3.1 9.8 20 20 302 A 2.7 3.6 10.8 2.9
3.9 3.6 2.1 0 20 20 303 A 4.6 2.7 1.6 4.8 4.9 1.3 0.7 1.0 20 20 304
A 9.3 2.8 20 11.2 9.4 0 2.5 4.1 20 20 305 A 3.0 0.6 12.6 4.5 3.8
1.8 1.0 0 20 20 306 A 6.3 6.0 15.0 7.3 15.4 5.9 4.2 5.6 20 2.5 307
A 3.0 1.8 0 1.6 2.4 1.6 1.0 0.8 9.5 1.4 308 A 20 10.2 50 13.4 20
4.4 4.4 1.7 20 20 309 A 3.2 0 0.3 1.3 0.4 1.6 1.3 0.8 5.8 3.4 310 A
3.4 1.1 0 3.7 7.5 0.7 0.5 3.3 14.8 18.9 311 A 1.9 0 0.7 0.9 1.2 0.5
0 1.2 2.6 1.8 312 A 2.5 1.1 50 3.0 3.0 0.6 5.5 9.6 16.0 20 313 A
2.7 6.6 50 4.8 6.3 5.7 4.5 9.9 1.3 0.9 314 A 2.9 4.0 50 3.2 2.5 3.9
3.6 5.7 17.5 20 315 A 4.8 1.9 50 3.3 4.6 1.7 8.9 50 13.0 12.0 316 A
9.4 9.5 50 8.8 9.2 9.2 50 50 11.9 16.6 317 A 3.5 5.0 50 3.6 1.5 3.1
5.2 6.8 20 7.4 318 A 3.4 2.2 17.5 1.6 2.7 1.1 0 3.4 20 20 319 A 7.1
8.2 20 8.9 12.7 7.7 5.8 3.8 8.7 0.9 320 A 2.2 4.2 8.4 1.7 1.4 1.5
0.6 1.0 20 20 321 A 20 20 20 20 20 2.2 8.6 18.8 20 20 322 A 2.5 4.1
50 2.8 3.0 3.1 1.6 0 20 20 323 A 5.7 8.7 50 9.3 20 4.9 2.5 0 20 20
324 A 2.4 0.4 50 1.0 2.6 0.7 1.3 1.3 8.7 0.3 325 A 20 0 13.3 6.3 20
5.1 3.7 3.4 20 20 326 A 4.2 1.1 0 1.7 2.1 1.5 4.5 9.4 5.1 3.3 327 A
4.3 3.8 20 3.2 2.8 1.8 0 3.3 18.3 12.6 328 A 10.4 4.3 50 1.7 6.0
2.9 8.3 5.9 50 50 329 A 3.5 0.1 0.3 0.6 1.4 0 1.1 2.2 6.6 3.7 330 A
3.0 0.3 20 0.5 1.1 0 0.1 2.2 6.0 3.4 331 A 6.4 6.1 0 8.0 7.9 1.4
6.4 4.6 6.5 6.8 332 A 2.9 2.8 50 0 3.7 2.8 1.8 1.3 12.6 9.4 333 A
3.0 2.9 3.6 1.1 2.9 1.7 1.2 4.8 8.0 8.4 334 A 3.0 2.5 2.8 1.5 0 2.8
1.6 1.5 4.1 7.9 335 A 2.1 1.2 2.1 1.0 0.2 0.8 0 0.9 5.1 4.8 336 A
1.6 1.3 20 0.1 2.6 1.1 0.4 0.3 12.3 13.9 337 A 5.6 5.0 12.7 6.7 7.3
0 10.5 50 20 20 338 A 5.9 5.5 5.8 6.4 4.6 5.8 6.8 6.9 20 20 339 A
2.7 1.5 1.0 0.8 0.4 1.3 1.2 0.4 6.6 2.4 340 A 2.1 1.0 1.0 0 1.1 0.6
1.2 3.1 7.8 2.2 237 B 1.7 0.5 0.3 0 0.3 0.4 0.3 1.0 7.3 5.4 238 B
11.6 7.3 0 7.8 20 1.9 4.7 2.7 20 20 239 B 2.5 0.2 9.3 0.8 0.3 0.4
1.7 2.7 4.8 2.1 240 B 10.2 2.7 1.3 9.7 20 2.8 0.6 0.7 20 20 241 B
2.4 1.5 8.5 1.2 1.9 0 1.3 5.3 4.3 1.3 242 B 8.7 5.9 3.9 10.6 19.9
5.0 5.6 4.2 20 20 243 B 2.3 0.4 50 0 0.5 0.3 0.6 4.5 4.3 0.7 244 B
3.3 3.5 0.3 1.0 1.2 1.2 1.4 0.5 7.3 7.2 245 B 20 20 0 20 20 3.0
19.4 20 20 20 246 B 3.2 0 0.3 2.1 1.5 1.5 1.7 1.9 6.0 3.1 247 B 7.9
12.4 0 6.3 9.1 0.9 0.8 3.0 20 20 248 B 2.9 3.8 2.1 2.7 2.5 2.3 1.2
2.1 20 20 249 B 3.3 3.8 50 1.6 4.1 4.4 8.4 50 6.6 4.6 250 B 6.1 2.2
50 6.7 6.1 1.3 0.6 0 20 20 251 B 3.3 5.3 50 9.3 5.4 5.9 3.3 4.5 50
0 252 B 0 4.2 50 2.4 14.0 1.5 6.9 7.7 20 20 253 B 2.2 3.0 50 3.3
2.4 1.6 1.5 2.3 6.5 5.3 254 B 3.9 3.1 50 3.4 5.1 0.3 5.6 6.6 10 8.7
255 B 1.5 2.6 50 1.6 0 1.7 1.0 13.6 4.8 20 256 B 2.6 0 2.4 0.5 0.2
0.7 1.5 2.8 5.9 2.9 257 B 20 20 0 16.5 20 4.2 20 20 50 50 258 B 1.8
2.1 16.2 2.9 3.0 0.7 4.5 11.7 20 1.6 259 B 15.5 7.5 50 8.8 20 5.3
2.0 0 20 20 260 B 3.5 2.7 4.1 0.3 1.3 2.5 2.0 1.6 15.9 20 261 B 20
20 50 20 20 3.5 20 20 20 20 262 B 20 3.5 50 14.9 19.5 1.9 1.6 0 20
20 263 B 8.9 4.7 50 15.3 19.2 5.7 3.1 0 20 20 264 B 4.0 2.5 5.0 1.5
3.1 2.4 1.6 0.4 20 20 265 B 2.4 0.9 50 0.3 0.7 1.6 11.4 18.0 5.0
2.2 266 B 19.7 6.2 50 17.2 20 5.8 4.1 0 50 50 267 B 3.6 2.7 0 1.4
1.9 2.3 1.8 3.2 5.2 2.5 268 B 2.7 1.8 3.2 0.9 1.1 1.4 1.2 2.8 8.4
6.4 269 B 3.2 0 5.3 0.8 1.6 0.8 1.0 1.8 5.6 3.1 270 B 3.8 2.9 15.5
4.0 3.5 1.0 1.4 7.0 6.6 3.1 271 B 6.0 4.0 0 7.6 5.3 1.3 2.9 6.2 5.0
3.6 272 B 3.1 0.6 17.4 0.1 1.0 0.3 1.0 0.6 3.7 3.0 273 B 9.8 4.2 50
20 20 1.5 0 2.5 20 20 274 B 3.1 0.6 50 0 0.5 0.9 1.3 1.1 2.8 2.4
275 B 6.8 8.0 7.1 7.2 13.0 8.2 7.1 7.4 10.7 2.0 276 B 2.7 0.1 19.6
1.1 1.7 0.5 5.9 8.8 20 20 277 B 7.5 10.9 6.8 9.3 14.0 10 9.5 11.0 0
20 278 B 1.7 3.0 18.1 0.3 2.4 1.2 0.3 10.3 7.5 0.3 279 B 3.6 4.5 20
1.0 4.1 4.5 2.9 2.0 20 20 280 B 4.0 0.2 50 3.1 3.6 3.0 8.8 12.1
13.9 12.7 281 B 50 50 50 50 50 50 50 50 50 50 282 B 2.9 0.2 50 0.3
0.3 0 0.3 0 5.6 2.4 283 B 2.0 3.2 0.4 0.7 0.8 0.6 1.5 2.7 8.3 7.5
284 B 2.9 3.2 50 2.6 2.2 2.7 1.4 0 13.3 20 285 B 2.9 0 1.6 0.5 0.7
0 0.2 2.2 4.8 1.9 286 B 2.4 0.2 8.3 0.4 1.0 0.7 0.8 1.0 5.4 3.4 287
B 4.6 1.1 14.1 8.2 9.1 3.5 3.2 10.7 1.1 1.4 288 B 2.0 0.3 5.7 0.6
0.4 0 0.5 1.7 5.8 2.3 289 B 1.3 1.1 0.7 0.5 0.2 0.2 0.2 0.4 6.7 5.4
290 B 1.8 1.3 50 0.6 1.2 0.7 0 2.4 5.7 4.3 291 B 3.4 0.2 0.8 0.7
1.2 0 0.8 2.6 7.6 3.9 292 B 1.8 2.4 1.3 1.6 1.5 1.8 2.0 2.6 4.5 2.0
293 B 2.4 1.1 15.9 0 2.3 0.2 0.9 2.8 9.3 8.6 294 B 2.4 0 1.3 0.9
1.3 1.5 1.3 2.2 8.4 6.2 295 B 2.4 3.4 50 1.8 3.8 3.6 7.2 8.7 20
18.4 296 B 5.5 2.5 50 2.9 3.3 2.6 11.3 50 8.5 5.4 297 B 2.3 0.9 50
1.0 0 0.8 0.8 1.3 7.0 4.1 298 B 7.8 4.5 50 5.5 5.4 3.3 9.6 50 11.9
9.6 299 B 2.3 0.9 50 15.0 1.8 1.8 1.1 2.3 20 1.6 300 B 2.8 3.2 50
4.3 5.9 4.6 3.1 10.9 5.7 0 301 B 1.7 0.7 50 0 0.6 2.4 2.7 7.1 20 20
302 B 2.9 4.8 20 2.8 5.0 3.7 2.1 0 20 20 303 B 4.9 2.3 0.9 5.3 6.4
0.4 0 0.2 20 20 304 B 12.1 2.6 16.4 14.1 12.0 0 1.7 3.6 20 20 305 B
2.9 0.7 14.7 4.2 3.3 1.7 0.9 0 20 20 306 B 6.0 6.0 12.2 6.9 14.6
6.2 4.5 5.5 20 1.9 307 B 3.0 2.1 0 1.7 2.3 1.7 1.1 0.8 9.7 1.5 308
B 20 11.7 50 10.8 20 5.6 4.5 2.5 20 20 309 B 3.2 0 0.2 1.2 0.4 1.6
1.3 0.8 5.8 3.3 310 B 3.7 1.2 0 4.1 10.5 1.2 0.6 4.6 11.6 13.6 311
B 1.8 0 1.7 0.8 1.0 0.4 0 1.4 2.3 1.0 312 B 2.7 1.5 50 6.8 4.0 0.5
7.1 9.7 20 20 313 B 2.9 7.7 50 5.1 7.1 6.0 4.8 7.2 1.6 1.4 314 B
3.0 4.0 50 3.4 4.0 3.9 3.6 5.9 17.5 20 315 B 5.6 2.5 50 5.8 5.9 2.0
11.4 18.0 13.7 11.8 316 B 7.0 7.5 50 6.8 7.7 6.9 50 50 10 13.8 317
B 3.4 4.4 50 3.2 1.3 2.2 6.6 6.4 20 7.6 318 B 3.7 2.4 13.4 1.7 3.0
1.1 0 3.4 19.3 20 319 B 6.9 8.0 50 9.5 13.1 7.4 5.5 3.6 10.4 0.8
320 B 2.0 3.9 12.6 1.8 1.2 1.6 0.7 1.1 20 20 321 B 20 20 20 20 20
2.3 9.1 19.2 20 20 322 B 3.0 4.6 50 2.7 2.7 3.5 2.0 0 20 20 323 B
6.0 9.2 50 9.8 19.4 4.8 2.7 0 20 20 324 B 2.4 1.3 11.1 1.0 2.7 0.9
1.2 3.4 2.5 1.2 325 B 17.0 0.4 11.5 5.5 16.5 3.1 0 2.1 20 50 326 B
3.5 0.1 0 1.2 1.3 0.7 2.4 3.4 6.4 2.4 327 B 4.5 3.9 20 3.5 3.7 1.4
0 2.8 10.3 12.2 328 B 15.1 6.0 50 3.7 6.2 5.5 18.4 50 20 50 329 B
3.2 0 0.4 0.3 0.9 0 0.6 1.6 6.4 3.7 330 B 3.0 0 20 0.6 1.3 0.1 0.3
1.8 6.0 3.7 331 B 6.3 5.7 0 8.3 7.7 1.7 5.3 5.2 6.5 8.0 332 B 2.5
2.6 50 0 2.4 2.3 1.3 0.9 15.3 6.6 333 B 3.3 2.6 3.6 1.2 3.0 2.4 1.4
4.9 8.4 8.8 334 B 2.9 2.6 2.8 1.5 0 2.7 1.6 1.4 5.5 10.8 335 B 2.2
0.8 1.7 1.7 0.6 0.6 0 0.8 5.2 5.1 336 B 1.9 1.5 20 0 3.0 1.5 0.5
0.4 14.2 19.6 337 B 5.1 3.6 12.8 6.2 7.0 0 4.6 50 20 20 338 B 5.7
6.0 5.8 6.8 4.9 5.4 6.4 6.7 20 20 339 B 2.8 1.4 1.5 0.9 0.7 1.5 1.1
0.5 6.6 2.6 340 B 1.9 0.6 1.0 0 1.0 0.2 1.0 2.3 7.0 1.8 SPA .TM.
technology; 1DN2 template structure; +carbohydrate
[0257] The results of the design calculations presented above in
Tables 1-60 were used to construct a series of Fc variant libraries
for experimental production and screening. Experimental libraries
were designed in successive rounds of computational and
experimental screening. Design of subsequent Fc libraries benefited
from feedback from prior libraries, and thus typically comprised
combinations of Fc variants that showed favorable properties in the
previous screen. The entire set of Fc variants that were
constructed and experimentally tested is shown in Table 61. In this
table, row 1 lists the variable positions, and the rows that follow
indicate the amino acids at those variable positions for WT and the
Fc variants. For example, variant 18 (SEQ ID NO:26) has the
following four mutations: F241E, F243Y, V262T, and V264R. The
variable position residues that compose this set of Fc variants are
illustrated structurally in FIG. 4, and are presented in the
context of the human IgG1 Fc sequence (SEQ ID NO:2)_in FIG. 5.
TABLE-US-00061 TABLE 61 Variant SEQ ID NO Substitution(s) Variant
SEQ ID NO Substitution(s) 1 SEQ ID NO:9 V264A 50 SEQ ID NO:55 Y296Q
2 SEQ ID NO:10 V264L 51 SEQ ID NO:56 S298T 3 SEQ ID NO:11 V264I 52
SEQ ID NO:57 S298N 4 SEQ ID NO:12 F241W 53 SEQ ID NO:58 T299I 5 SEQ
ID NO:13 F241L 54 SEQ ID NO:59 A327S 6 SEQ ID NO:14 F243W 55 SEQ ID
NO:60 A327N 7 SEQ ID NO:15 F243L 56 SEQ ID NO:61 S267Q/A327S 8 SEQ
ID NO:16 F241L/F243L/V262I/V264I 57 SEQ ID NO:62 S267L/A327S 9 SEQ
ID NO:17 F241W/F243W 58 SEQ ID NO:63 A327L 10 SEQ ID NO:18
F241W/F243W/V262A/V264A 59 SEQ ID NO:64 P329F 11 SEQ ID NO:19
F241L/V262I 60 SEQ ID NO:65 A330L 12 SEQ ID NO:20 F243L/264I 61 SEQ
ID NO:66 A330Y 13 SEQ ID NO:21 F243L/V262I/V264W 62 SEQ ID NO:67
I332D 14 SEQ ID NO:22 F241Y/F243Y/V262T/V264T 63 SEQ ID NO:68 N297S
15 SEQ ID NO:23 F241E/F243R/V262E/V264R 64 SEQ ID NO:69 N297D 16
SEQ ID NO:24 F241E/F243Q/V262T/V264E 65 SEQ ID NO:70 N297S/I332E 17
SEQ ID NO:25 F241R/F243Q/V262T/V264R 66 SEQ ID NO:71 N297D/I332E 18
SEQ ID NO:26 F241E/F243Y/V262T/V264R 67 SEQ ID NO:72 N297E/I332E 19
SEQ ID NO:27 L328M 68 SEQ ID NO:73 D265Y/N297D/I332E 20 SEQ ID
NO:28 L328E 69 SEQ ID NO:74 D265Y/N297D/T299L/I332E 21 SEQ ID NO:29
L328F 70 SEQ ID NO:75 D265F/N297E/I332E 22 SEQ ID NO:30 I332E 71
SEQ ID NO:76 L328I/I332E 23 SEQ ID NO:31 L328M/I332E 72 SEQ ID
NO:77 L328Q/I332E 24 SEQ ID NO:32 P244H 73 SEQ ID NO:78 I332N 25
SEQ ID NO:33 P245A 74 SEQ ID NO:79 I332Q 26 SEQ ID NO:34 P247V 75
SEQ ID NO:80 V264T 27 SEQ ID NO:35 W313F 76 SEQ ID NO:81 V264F 28
SEQ ID NO:36 P244H/P245A/P247V 77 SEQ ID NO:82 V240I 29 SEQ ID
NO:37 P247G 78 SEQ ID NO:83 V263I 30 SEQ ID NO:38 V264I/I332E 79
SEQ ID NO:84 V266I 31 SEQ ID NO:39 F241E/F243R/V262E/V264R/I332E 80
SEQ ID NO:85 T299A 32 SEQ ID NO:40 F241E/F243Q/V262T/V264E/I332E 81
SEQ ID NO:86 T299S 33 SEQ ID NO:41 F241R/F243Q/V262T/V264R/I332E 82
SEQ ID NO:87 T299V 34 SEQ ID NO:42 F241E/F243Y/V262T/V264R/I332E 83
SEQ ID NO:88 N325Q 35 SEQ ID NO:43 S298A 84 SEQ ID NO:89 N325L 36
SEQ ID NO:44 S298A/I332E 85 SEQ ID NO:90 N325I 37 SEQ ID NO:45
S298A/E333A/K334A 86 SEQ ID NO:91 S239D 41 SEQ ID NO:46 S239E/I332E
87 SEQ ID NO:92 S239N 42 SEQ ID NO:47 S239Q/I332E 88 SEQ ID NO:93
S239F 43 SEQ ID NO:48 S239E 89 SEQ ID NO:94 S239D/I332D 44 SEQ ID
NO:49 D265G 90 SEQ ID NO:95 S239D/I332E 45 SEQ ID NO:50 D265N 91
SEQ ID NO:96 S239D/I332N 46 SEQ ID NO:51 S239E/D265G 92 SEQ ID
NO:97 S239D/I332Q 47 SEQ ID NO:52 S239E/D265N 93 SEQ ID NO:98
S239E/I332D 48 SEQ ID NO:53 S239E/D265Q 94 SEQ ID NO:99 S239E/I332N
49 SEQ ID NO:54 Y296E 95 SEQ ID NO:100 S239E/I332Q 96 SEQ ID NO:101
S239N/I332D 141 SEQ ID NO:146 V264Y 97 SEQ ID NO:102 S239N/I332E
142 SEQ ID NO:147 V266A 98 SEQ ID NO:103 S239N/I332N 143 SEQ ID
NO:148 V266T 99 SEQ ID NO:104 S239N/I332Q 144 SEQ ID NO:149 V266M
100 SEQ ID NO:105 S239Q/I332D 145 SEQ ID NO:150 E269H 101 SEQ ID
NO:106 S239Q/I332N 146 SEQ ID NO:151 E269Y 102 SEQ ID NO:107
S239Q/I332Q 147 SEQ ID NO:152 E269F 103 SEQ ID NO:108 K326E 148 SEQ
ID NO:153 E269R 104 SEQ ID NO:109 Y296D 149 SEQ ID NO:154 Y2965 105
SEQ ID NO:110 Y296N 150 SEQ ID NO:155 Y296T 106 SEQ ID NO:111
N297D/I332E/F241Y/F243Y/ 151 SEQ ID NO:156 Y296L V262T/V264T 107
SEQ ID NO:112 I332E/A330Y 152 SEQ ID NO:157 Y296I 108 SEQ ID NO:113
I332E/V264I/A330Y 153 SEQ ID NO:158 S298H 109 SEQ ID NO:114
I332E/A330L 154 SEQ ID NO:159 T299H 110 SEQ ID NO:115
I332E/V264I/A330L 155 SEQ ID NO:160 A330V 111 SEQ ID NO:116 L234D
156 SEQ ID NO:161 A330I 112 SEQ ID NO:117 L234E 157 SEQ ID NO:162
A330F 113 SEQ ID NO:118 L234N 158 SEQ ID NO:163 A330R 114 SEQ ID
NO:119 L234Q 159 SEQ ID NO:164 A330H 115 SEQ ID NO:120 L234T 160
SEQ ID NO:165 N325D 116 SEQ ID NO:121 L234H 161 SEQ ID NO:166 N325E
117 SEQ ID NO:122 L234Y 162 SEQ ID NO:167 N325A 118 SEQ ID NO:123
L234I 163 SEQ ID NO:168 N325T 119 SEQ ID NO:124 L234V 164 SEQ ID
NO.169 N325V 120 SEQ ID NO:125 L234F 165 SEQ ID NO:170 N325H 121
SEQ ID NO:126 L235D 166 SEQ ID NO:171 L328D/I332E 122 SEQ ID NO:127
L235S 167 SEQ ID NO:172 L328E/I332E 123 SEQ ID NO:128 L235N 168 SEQ
ID NO:173 L328N/I332E 124 SEQ ID NO:129 L235Q 169 SEQ ID NO:174
L328Q/I332E 125 SEQ ID NO:130 L235T 170 SEQ ID NO:175 L328V/I332E
126 SEQ ID NO:131 L235H 171 SEQ ID NO:176 L328T/I332E 127 SEQ ID
NO:132 L235Y 172 SEQ ID NO:177 L328H/I332E 128 SEQ ID NO:133 L235I
173 SEQ ID NO:178 L328I/I332E 129 SEQ ID NO:134 L235V 174 SEQ ID
NO:179 L328A 130 SEQ ID NO:135 L235F 175 SEQ ID NO:180 I332T 131
SEQ ID NO:136 S239T 176 SEQ ID NO:181 I332H 132 SEQ ID NO:137 S239H
177 SEQ ID NO:182 I332Y 133 SEQ ID NO:138 S239Y 178 SEQ ID NO:183
I332A 134 SEQ ID NO:139 V240A 179 SEQ ID NO:184 V264I/I332E/S239E
135 SEQ ID NO:140 V240I 180 SEQ ID NO:185 V264I/I332E/S239Q 136 SEQ
ID NO:141 V240M 181 SEQ ID NO:186 V264I/I332E/S239E/A330Y 137 SEQ
ID NO:142 V263A 182 SEQ ID NO:187 V264I/I332E/S239E/A330Y/S298A 138
SEQ ID NO:143 V263T 183 SEQ ID NO:188 N297D/I332E/S239D 139 SEQ ID
NO:144 V263M 184 SEQ ID NO:189 N297D/I332E/S239E 140 SEQ ID NO:145
V264M 185 SEQ ID NO:190 N297D/I332E/S239D/D265 186 SEQ ID NO:191
N297D/I332E/S239D/D2651 231 SEQ ID NO:236 E269N 187 SEQ ID NO:192
N297D/I332E/S239D/D265L 232 SEQ ID NO:237 D270Q 188 SEQ ID NO:193
N297D/I332E/S239D/D265F 233 SEQ ID NO:238 D270T 189 SEQ ID NO:194
N297D/I332E/S239D/D265Y 234 SEQ ID NO:239 D270H 190 SEQ ID NO:195
N297D/I332E/S239D/D265H 235 SEQ ID NO:240 E272S 191 SEQ ID NO:196
N297D/I332E/S239D/D265T 236 SEQ ID NO:241 E272K 192 SEQ ID NO:197
N297D/I332E/V264E 237 SEQ ID NO:242 E272I 193 SEQ ID NO:198
N297D/I332E/Y296D 238 SEQ ID NO:243 E272Y 194 SEQ ID NO:199
N297D/I332E/Y296E 239 SEQ ID NO:244 V273I 195 SEQ ID NO:200
N297D/I332E/Y296N 240 SEQ ID NO:245 K274T 196 SEQ ID NO:201
N297D/I332E/Y296Q 241 SEQ ID NO:246 K274E 197 SEQ ID NO:202
N297D/I332E/Y296H 242 SEQ ID NO:247 K274R 198 SEQ ID NO:203
N297D/I332E/Y296T 243 SEQ ID NO:248 K274L 199 SEQ ID NO:204
N297D/I332E/T299V 244 SEQ ID NO:249 K274Y 200 SEQ ID NO:205
N297D/I332E/T2991 245 SEQ ID NO:250 F275W 201 SEQ ID NO:206
N297D/I332E/T299L 246 SEQ ID NO:251 N276S 202 SEQ ID NO:207
N297D/I332E/T299F 247 SEQ ID NO:252 N276E 203 SEQ ID NO:208
N297D/I332E/T299H 248 SEQ ID NO:253 N276R 204 SEQ ID NO:209
N297D/I332E/T299E 249 SEQ ID NO:254 N276L 205 SEQ ID NO:210
N297D/I332E/A330Y 250 SEQ ID NO:255 N276Y 206 SEQ ID NO:211
N297D/I332E/S298A/A330Y 251 SEQ ID NO:256 Y278T 207 SEQ ID NO:212
S239D/I332E/A330Y 252 SEQ ID NO:257 Y278E 208 SEQ ID NO:213
S239N/I332E/A330Y 253 SEQ ID NO:258 Y278K 209 SEQ ID NO:214
S239D/I332E/A330L 254 SEQ ID NO:259 Y278W
210 SEQ ID NO:215 S239N/I332E/A330L 255 SEQ ID NO:260 E283R 211 SEQ
ID NO:216 I332E/V264I/S298A 256 SEQ ID NO:261 V302I 212 SEQ ID
NO:217 I332E/S239D/S298A 257 SEQ ID NO:262 E318R 213 SEQ ID NO:218
I332E/S239N/S298A 258 SEQ ID NO:263 K320T 214 SEQ ID NO:219
S239D/I332E/V2641 259 SEQ ID NO:264 K320D 215 SEQ ID NO:220
S239D/I332E/V264I/S298A 260 SEQ ID NO:265 K320I 216 SEQ ID NO:221
S239D/I332E/V264I/A330L 261 SEQ ID NO:266 K322T 217 SEQ ID NO:222
L328N 262 SEQ ID NO:267 K322H 218 SEQ ID NO:223 L328H 263 SEQ ID
NO:268 V323I 219 SEQ ID NO:224 S239D/I332E/A330I 264 SEQ ID NO:269
S324T 220 SEQ ID NO:225 N297D/I332E/5239D/A330L 265 SEQ ID NO:270
S324D 221 SEQ ID NO:226 P230A 266 SEQ ID NO:271 S324R 222 SEQ ID
NO:227 E233D 267 SEQ ID NO:272 S324I 223 SEQ ID NO:228 P230A/E233D
268 SEQ ID NO:273 S324V 224 SEQ ID NO:229 P230A/E233D/I332E 269 SEQ
ID NO:274 S324L 225 SEQ ID NO:230 S267T 270 SEQ ID NO:275 S324Y 226
SEQ ID NO:231 S267H 271 SEQ ID NO:276 K326L 227 SEQ ID NO:232 S267D
272 SEQ ID NO:277 K326I 228 SEQ ID NO:233 S267N 273 SEQ ID NO:278
K326T 229 SEQ ID NO:234 E269T 274 SEQ ID NO:279 A327D 230 SEQ ID
NO:235 E269L 275 SEQ ID NO:280 A327T 276 SEQ ID NO:281 A330S 290
SEQ ID NO:295 T335Y 277 SEQ ID NO:282 A330W 291 SEQ ID NO:296
L234I/L235D 278 SEQ ID NO.283 A330M 292 SEQ ID NO:297 V240I/V266I
279 SEQ ID NO:284 P331V 293 SEQ ID NO:298 S239D/A330Y/I332E/L234I
280 SEQ ID NO:285 P331H 294 SEQ ID NO:299 S239D/A330Y/I332E/L235D
281 SEQ ID NO:286 E333T 295 SEQ ID NO:300 S239D/A330Y/I332E/V240I
282 SEQ ID NO:287 E333H 296 SEQ ID NO:301 S239D/A330Y/I332E/V264T
283 SEQ ID NO:288 E333I 297 SEQ ID NO:302 S239D/A330Y/I332E/V266I
284 SEQ ID NO:289 E333Y 298 SEQ ID NO:303 S239D/A330Y/I332E/K326E
285 SEQ ID NO:290 K334I 299 SEQ ID NO:304 S239D/A330Y/I332E/K326T
286 SEQ ID NO:291 K334T 300 SEQ ID NO:305 S239D/N297D/I332E/A330Y
287 SEQ ID NO:292 K334F 301 SEQ ID NO:306 S239D/N297D/I332E/A330Y/
F241S/F243H/V262T/V264T 288 SEQ ID NO:293 T335D 302 SEQ ID NO:307
S239D/N297D/I332E/L235D 289 SEQ ID NO:294 T335R 303 SEQ ID NO:308
S239D/N297D/I332E/K326E
Example 2
Experimental Production and Screening of Fc Libraries
[0258] The majority of experimentation on the Fc variants was
carried out in the context of the anti-cancer antibody alemtuzumab
(Campath.RTM., a registered trademark of Ilex Pharmaceuticals LP).
Alemtuzumab binds a short linear epitope within its target antigen
CD52 (Hale et al., 1990, Tissue Antigens 35:118-127; Hale, 1995,
Immunotechnology 1:175-187). Alemtuzumab has been chosen as the
primary engineering template because its efficacy is due in part to
its ability to recruit effector cells (Dyer et al., 1989, Blood
73:1431-1439; Friend et al., 1991, Transplant Proc 23:2253-2254;
Hale et al., 1998, Blood 92:4581-4590; Glennie et al., 2000,
Immunol Today 21:403-410), and because production and use of its
antigen in binding assays are relatively straightforward. In order
to evaluate the optimized Fc variants of the present invention in
the context of other antibodies, select Fc variants were evaluated
in the anti-CD20 antibody rituximab (Rituxan.RTM., a registered
trademark of IDEC Pharmaceuticals Corporation), the anti-Her2
antibody trastuzumab (Herceptin.RTM., a registered trademark of
Genentech), and the anti-EGFR antibody cetuximab (Erbitux.RTM., a
registered trademark of Imclone). The use of alemtuzumab,
rituximab, and trastuzumab for screening purposes is not meant to
constrain the present invention to any particular antibody.
[0259] The IgG1 full length light (V.sub.L-C.sub.L) and heavy
(V.sub.H-C.gamma.1-C.gamma.2-C.gamma.3) chain antibody genes for
alemtuzumab, rituximab, and trastuzumab were constructed with
convenient end restriction sites to facilitate subcloning. The
genes were ligated into the mammalian expression vector pcDNA3.1Zeo
(Invitrogen). The V.sub.H-C.gamma.1-C.gamma.2-C.gamma.3 clone in
pcDNA3.1zeo was used as a template for mutagenesis of the Fc
region. Mutations were introduced into this clone using PCR-based
mutagenesis techniques. Fc variants were sequenced to confirm the
fidelity of the sequence. Plasmids containing heavy chain gene
(V.sub.H-C.gamma.1-C.gamma.2-C.gamma.3) (wild-type or variants)
were co-transfected with plasmid containing light chain gene
(V.sub.L-C.sub.L) into 293T cells. Media were harvested 5 days
after transfection. Expression of immunoglobulin was monitored by
screening the culture supernatant of transfectomas by western using
peroxidase-conjugated goat-anti human IgG (Jackson ImmunoResearch,
catalog #109-035-088). FIG. 6 shows expression of wild-type
alemtuzumab and variants 1 through 10 in 293T cells. Antibodies
were purified from the supernatant using protein A affinity
chromatography (Pierce, Catalog #20334. FIG. 7 shows results of the
protein purification for WT alemtuzumab. Antibody Fc variants
showed similar expression and purification results to WT. Some Fc
variants were deglycosylated in order to determine their solution
and functional properties in the absence of carbohydrate. To obtain
deglycosylated antibodies, purified alemtuzumab antibodies were
incubated with peptide-N-glycosidase (PNGase F) at 37.degree. C.
for 24 h. FIG. 8 presents an SDS PAGE gel confirming
deglycosylation for several Fc variants and WT alemtuzumab.
[0260] In order to confirm the functional fidelity of alemtuzumab
produced under these conditions, the antigenic CD52 peptide, fused
to GST, was expressed in E. coli BL21 (DE3) under IPTG induction.
Both un-induced and induced samples were run on a SDS PAGE gel, and
transferred to PVDF membrane. For western analysis, either
alemtuzumab from Sotec (final concentration 2.5 ng/ul) or media of
transfected 293T cells (final alemtuzumab concentration about
0.1-0.2 ng/ul) were used as primary antibody, and
peroxidase-conjugated goat-anti human IgG was used as secondary
antibody. FIG. 9 presents these results. The ability to bind target
antigen confirms the structural and functional fidelity of the
expressed alemtuzumab. Fc variants that have the same variable
region as WT alemtuzumab are anticipated to maintain a comparable
binding affinity for antigen.
[0261] In order to screen for Fc/Fc.gamma.R binding, the
extracellular regions of human V158 Fc.gamma.RIIIa, human F158
Fc.gamma.RIIIa, human Fc.gamma.RIIb, human Fc.gamma.RIIa, and mouse
Fc.gamma.RIII, were expressed and purified. FIG. 10 presents an SDS
PAGE gel that shows the results of expression and purification of
human V158 Fc.gamma.RIIIa. The extracellular region of this
receptor was obtained by PCR from a clone obtained from the
Mammalian Gene Collection (MGC:22630). The receptor was fused with
glutathione S-Transferase (GST) to enable screening. Tagged
Fc.gamma.RIIIa was transfected in 293T cells, and media containing
secreted Fc.gamma.RIIIa were harvested 3 days later and purified.
For western analysis, membrane was probed with anti-GST
antibody.
[0262] Binding affinity to Fc.gamma.RIIIa and Fc.gamma.RIIb was
measured for all designed Fc variants using an AlphaScreen.TM.
assay (Amplified Luminescent Proximity Homogeneous Assay (ALPHA),
PerkinElmer, Wellesley, Mass.), a bead-based non-radioactive
luminescent proximity assay. Laser excitation of a donor bead
excites oxygen, which if sufficiently close to the acceptor bead
generates a cascade of chemiluminescent events, ultimately leading
to fluorescence emission at 520-620 nm. The AlphaScreen.TM. assay
was applied as a competition assay for screening Fc variants. WT
alemtuzumab antibody was biotinylated by standard methods for
attachment to streptavidin donor beads, and GST-tagged Fc.gamma.R
was bound to glutathione chelate acceptor beads. In the absence of
competing Fc variants, WT antibody and Fc.gamma.R interact and
produce a signal at 520-620 nm. Addition of untagged Fc variant
competes with the WT Fc/Fc.gamma.R interaction, reducing
fluorescence quantitatively to enable determination of relative
binding affinities. All Fc variants were screened for V158
Fc.gamma.RIIIa binding using the AlphaScreen.TM. assay. Fc variants
were screened in the context of either alemtuzumab or trastuzumab,
and select Fc variants were also screened in the context of
rituximab and cetuximab. Select Fc variants were subsequently
screened for binding to Fc.gamma.RIIb, as well as other Fc.gamma.Rs
and Fc ligands.
[0263] FIG. 11 shows AlphaScreen.TM. data for binding to human V158
Fc.gamma.RIIIa by select Fc variants. The binding data were
normalized to the maximum and minimum luminescence signal for each
particular curve, provided by the baselines at low and high
antibody concentrations respectively. The data were fit to a one
site competition model using nonlinear regression, and these fits
are represented by the curves in the figure. These fits provide the
inhibitory concentration 50% (IC50) (i.e. the concentration
required for 50% inhibition) for each antibody, illustrated by the
dotted lines in FIG. 11, thus enabling the relative binding
affinities of Fc variants to be quantitatively determined. Here, WT
alemtuzumab has an IC50 of (4.63.times.10.sup.-9).sub.x(2)=9.2 nM,
whereas S239D has an IC50 of (3.98.times.10.sup.-10).sub.x(2)=0.8
nM. Thus S239D alemtuzumab binds 9.2 nM/0.8 nM=11.64-fold more
tightly than WT alemtuzumab to human V158 Fc.gamma.RIIIa. Similar
calculations were performed for the binding of all Fc variants to
human V158 Fc.gamma.RIIIa. Select Fc variants were also screened
for binding to human Fc.gamma.RIIb, and examples of these
AlphaScreen.TM. binding data are shown in FIG. 12. Table 62
presents the fold-enhancement or fold-reduction relative to the
parent antibody for binding of Fc variants to human V158
Fc.gamma.RIIIa (column 3) and human Fc.gamma.RIIb (column 4), as
determined by the AlphaScreen.TM. assay. For these data, a fold
above 1 indicates an enhancement in binding affinity, and a fold
below 1 indicates a reduction in binding affinity relative to WT
Fc. Data for 1-206 and 217-218 were obtained in the context of
alemtuzumab, except for those indicated with an asterix (*), which
were tested in the context of trastuzumab. All data for 207-216 and
219-303 were obtained in the context of trastuzumab.
Example 3
Selectively Enhanced Binding to Fc.gamma.Rs
[0264] A number of promising Fc variants with optimized properties
were obtained from the Fc.gamma.RIIIa and Fc.gamma.RIIb screen.
Table 62 provides Fc variants that bind more tightly to
Fc.gamma.RIIa, and thus are candidates for improving the effector
function of antibodies and Fc fusions. These include a number of
variants that comprise substitutions at 239, 264, 272, 274, 330,
and 332. FIGS. 13a and 13b show AlphaScreen.TM. binding data for
some of these Fc variants. The majority of these Fc variants
provide substantially greater Fc.gamma.RIIIa binding enhancements
over S298A/E333A/K334A (SEQ ID NO:45). TABLE-US-00062 TABLE 62
Fc.gamma.IIIa- fold; Fc.gamma.RIIIa Fc.gamma.RIIb Fc.gamma.IIb-
Variant SEQ ID NO Substitution(s) Fold Fold fold 1 SEQ ID NO:9
V264A 0.53 2 SEQ ID NO:10 V264L 0.56 3 SEQ ID NO:11 V264I 1.43 4
SEQ ID NO:12 F241W 0.29 5 SEQ ID NO:13 F241L 0.26 6 SEQ ID NO:14
F243W 0.51 7 SEQ ID NO:15 F243L 0.51 8 SEQ ID NO:16
F241L/F243L/V262I/V264I 0.09 9 SEQ ID NO:17 F241W/F243W 0.07 10 SEQ
ID NO:18 F241W/F243W/V262A/V264A 0.04 11 SEQ ID NO:19 F241L/V262I
0.06 12 SEQ ID NO:20 F243L/V264I 1.23 13 SEQ ID NO:21
F243L/V262I/V264W 0.02 14 SEQ ID NO:22 F241Y/F243Y/V262T/V264T 0.05
15 SEQ ID NO:23 F241E/F243R/V262E/V264R 0.05 16 SEQ ID NO:24
F241E/F243Q/V262T/V264E 0.07 17 SEQ ID NO:25
F241R/F243Q/V262T/V264R 0.02 18 SEQ ID NO:26
F241E/F243Y/V262T/V264R 0.05 19 SEQ ID NO:27 L328M 0.21 20 SEQ ID
NO:28 L328E 0.12 21 SEQ ID NO:29 L328F 0.24 22 SEQ ID NO:30 I332E
6.72 3.93 1.71 23 SEQ ID NO:31 L328M/I332E 2.60 24 SEQ ID NO:32
P244H 0.83 25 SEQ ID NO:33 P245A 0.25 26 SEQ ID NO:34 P247V 0.53 27
SEQ ID NO:35 W313E 0.88 28 SEQ ID NO:36 P244H/P245A/P247V 0.93 29
SEQ ID NO:37 P247G 0.54 30 SEQ ID NO:38 V264I/I332E 12.49 1.57*
7.96 31 SEQ ID NO:39 F241E/F243R/V262E/V264R/I332E 0.19 32 SEQ ID
NO:40 F241E/F243Q/V262T/V264E/I332E 33 SEQ ID NO:41
F241R/F243Q/V262T/V264R/I332E 34 SEQ ID NO:42
F241E/F243Y/V262T/V264R/I332E 0.10 35 SEQ ID NO:43 S298A 2.21 36
SEQ ID NO:44 S298A/I332E 21.73 37 SEQ ID NO:45 S298A/E333A/K334A
2.56 41 SEQ ID NO:46 S239E/I332E 5.80 3.49 1.66 42 SEQ ID NO:47
S239Q/I332E 6 60 4.68 1.41 43 SEQ ID NO:48 S239E 10.16 44 SEQ ID
NO:49 D265G <0.02 45 SEQ ID NO:50 D265N <0.02 46 SEQ ID NO:51
S239E/D265G <0.02 47 SEQ ID NO:52 S239E/D265N 0.02 48 SEQ ID
NO:53 S239E/D265Q 0.05 49 SEQ ID NO:54 Y296E 0.73 1.11 0.66 50 SEQ
ID NO:55 Y296Q 0.52 0.43 1.21 51 SEQ ID NO:56 S298T 0.94 <0.02
52 SEQ ID NO:57 S298N 0.41 <0.02 53 SEQ ID NO:58 T299I <0.02
54 SEQ ID NO:59 A327S 0.23 0.39 0.59 55 SEQ ID NO:60 A327N 0.19
1.15 0.17 56 SEQ ID NO:61 S267Q/A327S 0.03 57 SEQ ID NO:62
S267L/A327S <0.02 58 SEQ ID NO:63 A327L 0.05 59 SEQ ID NO:64
P329F <0.02 60 SEQ ID NO:65 A330L 0.73 0.38 1.92 61 SEQ ID NO:66
A330Y 1.64 0.75 2.19 62 SEQ ID NO:67 I332D 17.80 3.34 5.33 63 SEQ
ID NO:68 N297S <0.02 64 SEQ ID NO:69 N297D <0.02 65 SEQ ID
NO:70 N297S/I332E <0.02 66 SEQ ID NO:71 N297D/I332E 0.08
<0.02 67 SEQ ID NO:72 N297E/I332E <0.02 68 SEQ ID NO:73
D265Y/N297D/I332E <0.02 69 SEQ ID NO:74 D265Y/N297D/T299L/I332E
<0.02 70 SEQ ID NO:75 D265F/N297E/I332E <0.02 71 SEQ ID NO:76
L3281/I332E 7.03 72 SEQ ID NO:77 L328Q/I332E 1.54 73 SEQ ID NO:78
I332N 0.39 74 SEQ ID NO:79 I332Q 0.37 75 SEQ ID NO:80 V264T 2.73 76
SEQ ID NO:81 V264F 0.16 77 SEQ ID NO:82 V240I 3.25 78 SEQ ID NO:83
V263I 0.10 79 SEQ ID NO:84 V266I 1.86 80 SEQ ID NO:85 T299A 0.03 81
SEQ ID NO:86 T299S 0.15 82 SEQ ID NO:87 T299V <0.02 83 SEQ ID
NO:88 N325Q <0.02 84 SEQ ID NO:89 N325L <0.02 85 SEQ ID NO:90
N325I <0.02 86 SEQ ID NO:9I S239D 11.64 4.47* 2.60 87 SEQ ID
NO:92 S239N <0.02 88 SEQ ID NO:93 S239F 0.22 <0.02 89 SEQ ID
NO:94 S239D/I332D 14.10 90 SEQ ID NO:95 S239D/I332E 56.10 19.71*
2.85 91 SEQ ID NO:96 S239D/I332N 7.19 92 SEQ ID NO:97 S239D/I332Q
9.28 93 SEQ ID NO:98 S239E/I332D 9.33 94 SEQ ID NO:99 S239E/I332N
11.93 95 SEQ ID NO:100 S239E/I332Q 3.80 96 SEQ ID NO:101
S239N/I332D 3.08 97 SEQ ID NO:102 S239N/I332E 14.21 98 SEQ ID
NO:103 S239N/I332N 0.43 99 SEQ ID NO:104 S239N/I332Q 0.56 100 SEQ
ID NO:105 S239Q/I332D 5.05 101 SEQ ID NO:106 S239Q/I332N 0.39 102
SEQ ID NO:107 S239Q/I332Q 0.59 103 SEQ ID NO:108 K326E 3.85 104 SEQ
ID NO:109 Y296D 0.62 105 SEQ ID NO:110 Y296N 0.29 106 SEQ ID NO:111
N297D/I332E 0.15 107 SEQ ID NO:112 A330Y/I332E 12.02 4.40 2.73 108
SEQ ID NO:113 V264I/A330Y/I332E 12.00 3.54 3.39 109 SEQ ID NO:114
A330L/I332E 10.34 2.03 5.09 110 SEQ ID NO 115 V264I/A330L/I332E
11.15 1.79 6.23 111 SEQ ID NO:116 L234D 0.21 112 SEQ ID NO:117
L234E 1.34 2.21 0.61 113 SEQ ID NO:118 L234N 0.56 1.39 0.40 114 SEQ
ID NO:119 L234Q 0.37 115 SEQ ID NO:120 L234T 0.35 116 SEQ ID NO:121
L234H 0.33 117 SEQ ID NO:122 L234Y 1.42 1.08 1.31 118 SEQ ID NO:123
L234I 1.55 1.14 1.36 119 SEQ ID NO:124 L234V 0.38 120 SEQ ID NO:125
L234F 0.30
121 SEQ ID NO:126 L235D 1.66 3.63 0.46 122 SEQ ID NO:127 L2355 1.25
123 SEQ ID NO:128 L235N 0.40 124 SEQ ID NO:129 L235Q 0.51 125 SEQ
ID NO:130 L235T 0.52 126 SEQ ID NO:131 L235H 0.41 127 SEQ ID NO:132
L235Y 1.19 10.15 0.12 128 SEQ ID NO:133 L235I 1.10 0.94 1.17 129
SEQ ID NO:134 L235V 0.48 130 SEQ ID NO:135 L235F 0.73 3.53 0.21 131
SEQ ID NO:136 S239T 1.34 132 SEQ ID NO:137 S239H 0.20 133 SEQ ID
NO:138 S239Y 0.21 134 SEQ ID NO:139 V240A 0.70 0.14 5.00 135 SEQ ID
NO:140 V240T 136 SEQ ID NO:141 V240M 2.06 1.38 1.49 137 SEQ ID
NO:142 V263A 138 SEQ ID NO:143 V263T 0.43 139 SEQ ID NO:144 V263M
0.05 140 SEQ ID NO:145 V264M 0.26 141 SEQ ID NO:146 V264Y 1.02 0.27
3.78 142 SEQ ID NO:147 V266A <0.02 143 SEQ ID NO:148 V266T 0.45
144 SEQ ID NO:149 V266M 0.62 145 SEQ ID NO:150 E269H <0.02 146
SEQ ID NO:151 E269Y 0.12 147 SEQ ID NO:152 E269F 0.16 148 SEQ ID
NO:153 E269R 0.05 149 SEQ ID NO:154 Y296S 0.12 150 SEQ ID NO:155
Y296T <0.02 151 SEQ ID NO:156 Y296L 0.22 152 SEQ ID NO:157 Y296I
0.09 153 SEQ ID NO:158 A298H 0.27 154 SEQ ID NO:159 T299H <0.02
155 SEQ ID NO:160 A330V 0.43 156 SEQ ID NO:161 A330I 1.71 0.02 85.5
157 SEQ ID NO:162 A330F 0.60 158 SEQ ID NO:163 A330R <0.02 159
SEQ ID NO:164 A330H 0.52 160 SEQ ID NO:165 N325D 0.41 161 SEQ ID
NO:166 N325E <0.02 162 SEQ ID NO:167 N325A 0.11 163 SEQ ID
NO:168 N325T 1.10 164 SEQ ID NO:169 N325V 0.48 165 SEQ ID NO:170
N325H 0.73 166 SEQ ID NO:171 L328D/I332E 1.34 167 SEQ ID NO:172
L328E/I332E 0.20 168 SEQ ID NO:173 L328N/I332E <0.02 169 SEQ ID
NO:174 L328Q/I332E 0.70 170 SEQ ID NO:175 L328V/I332E 2.06 171 SEQ
ID NO:176 L328I/I332E 1.10 172 SEQ ID NO:177 L328H/I332E <0.02
173 SEQ ID NO:178 L328I/I332E 3.49 174 SEQ ID NO:179 L328A 0.20 175
SEQ ID NO:180 I332T 0.72 176 SEQ ID NO:181 I332H 0.46 177 SEQ ID
NO:182 I332Y 0.76 178 SEQ ID NO:183 I332A 0.89 179 SEQ ID NO:184
S239E/V264I/I332E 15.46 180 SEQ ID NO:185 S239Q/V264I/I332F 2.14
181 SEQ ID NO:186 S239E/V264I/A330Y/I332E 8.53 182 SEQ ID NO:187
S239E/V264I/S298A/A330Y/I332E 183 SEQ ID NO:188 S239D/N297D/I332E
0.28 184 SEQ ID NO:189 S239E/N297D/I332E 0.06 185 SEQ ID NO:190
S239D/D265V/N297D/I332E 0.03 186 SEQ ID NO:191
S239D/D265I/N297D/I332E 0.01 187 SEQ ID NO:192
S239D/D265L/N297D/I332E <0.02 188 SEQ ID NO:193
S239D/D265F/N297D/I332E <0.02 189 SEQ ID NO:194
S239D/D265Y/N297D/I332E 0.02 190 SEQ ID NO:195
S239D/D265H/N297D/I332E 0.04 191 SEQ ID NO:196
S239D/D265T/N297D/I332E <0.02 192 SEQ ID NO:197
V264E/N297D/I332E 0.05 193 SEQ ID NO:198 Y296D/N297D/I332E 194 SEQ
ID NO:199 Y296E/N297D/I332E <0.02 195 SEQ ID NO:200
Y296N/N297D/I332E 0.04 196 SEQ ID NO:201 Y296Q/N297D/I332E <0.02
197 SEQ ID NO:202 Y296H/N297D/I332E <0.02 198 SEQ ID NO:203
Y296T/N297D/I332E <0.02 199 SEQ ID NO:204 N297D/T299V/I332E
<0.02 200 SEQ ID NO:205 N297D/T299I/I332E <0.02 201 SEQ ID
NO:206 N297D/T299L/I332E <0.02 202 SEQ ID NO:207
N297D/T299F/I332E <0.02 203 SEQ ID NO:208 N297D/T299H/I332E
<0.02 204 SEQ ID NO:209 N297D/T299E/I332E <0.02 205 SEQ ID
NO:210 N297D/A330Y/I332E 0.43 206 SEQ ID NO:211
N297D/S298A/A330Y/I332E 0.16 207 SEQ ID NO:212 S239D/A330Y/I332E
129.58 208 SEQ ID NO:213 S239N/A330Y/I332E 14.22 209 SEQ ID NO:214
S239D/A330L/I332E 138.63 7.50 18.48 210 SEQ ID NO:215
S239N/A330L/I332E 12.95 211 SEQ ID NO:216 V264I/S298A/I332E 16.50
212 SEQ ID NO:217 S239D/S298A/I332E 295.16 6.16 47.92 213 SEQ ID
NO:218 S239N/S298A/I332E 32.14 5.15 6.24 214 SEQ ID NO:219
S239D/V264I/I332E 36.58 14.39 2.54 215 SEQ ID NO:220
S239D/V264I/S298A/I332E 216 SEQ ID NO:221 S239D/V264I/A330L/I332E
217 SEQ ID NO:222 L328N 0.59 218 SEQ ID NO:223 L328H <0.02 219
SEQ ID NO:224 S239D/I332E/A330I 59.1 220 SEQ ID NO:225
N297D/I332E/S239D/A330L 221 SEQ ID NO:226 P230A 1.09 222 SEQ ID
NO:227 E233D 0.85 223 SEQ ID NO:228 P230A/E233D 0.92 224 SEQ ID
NO:229 P230A/E233D/I332E 1.87 225 SEQ ID NO:230 S267T 226 SEQ ID
NO:231 S267H 227 SEQ ID NO:232 S267D 228 SEQ ID NO:233 S267N 229
SEQ ID NO:234 E269T <0.02 230 SEQ ID NO:235 E269L <0.02 231
SEQ ID NO:236 E269N <0.02 232 SEQ ID NO:237 D270Q <0.02 233
SEQ ID NO:238 D270T <0.02 234 SEQ ID NO.239 D270H <0.02 235
SEQ ID NO:240 E272S 236 SEQ ID NO:241 E272K 237 SEQ ID NO:242 E272I
238 SEQ ID NO:243 E272Y 8.70 239 SEQ ID NO:244 V273I 0.79 240 SEQ
ID NO:245 K274T 1.41 241 SEQ ID NO:246 K274E 6.11 242 SEQ ID NO:247
K274R 1.41 243 SEQ ID NO:248 K274L 1.09 244 SEQ ID NO:249 K274Y
1.06 245 SEQ ID NO:250 F275W 1.11
246 SEQ ID NO:251 N276S 0.41 247 SEQ ID NO:252 N276E 0.87 248 SEQ
ID NO:253 N276R 0.66 249 SEQ ID NO:254 N276L 1.07 250 SEQ ID NO:255
N276Y 0.56 251 SEQ ID NO:256 Y278T 1.87 252 SEQ ID NO:257 Y278E
0.90 253 SEQ ID NO:258 Y278K 254 SEQ ID NO:259 Y278W 0.41 255 SEQ
ID NO:260 E283R 0.67 256 SEQ ID NO 261 V302I 1.01 257 SEQ ID NO:262
E318R 1.06 258 SEQ ID NO:263 K320T 259 SEQ ID NO.264 K320D 260 SEQ
ID NO:265 K320I 261 SEQ ID NO:266 K322T 262 SEQ ID NO:267 K322H 263
SEQ ID NO:268 V323I 0.83 264 SEQ ID NO:269 S324T 265 SEQ ID NO:270
S324D 1.07 266 SEQ ID NO:271 S324R 0.71 267 SEQ ID NO:272 S324I
1.15 268 SEQ ID NO:273 S324V 117 269 SEQ ID NO:274 S324L <0.02
270 SEQ ID NO:275 S324Y 0.98 271 SEQ ID NO:276 K326L 272 SEQ ID
NO:277 K326I 1.43 273 SEQ ID NO:278 K326T 1.88 274 SEQ ID NO:279
A327D <0.02 275 SEQ ID NO:280 A327T <0.02 276 SEQ ID NO:281
A330S 277 SEQ ID NO:282 A330W 278 SEQ ID NO:283 A330M 279 SEQ ID
NO:284 P331V 280 SEQ ID NO:285 P331H 281 SEQ ID NO:286 E333T 0.78
282 SEQ ID NO:287 E333H 0.75 283 SEQ ID NO:288 E333I 284 SEQ ID
NO:289 E333Y 285 SEQ ID NO:290 K334I 286 SEQ ID NO:291 K334T 287
SEQ ID NO.292 K334F 288 SEQ ID NO:293 T335D 2.79 289 SEQ ID NO:294
T335R 2.58 290 SEQ ID NO:295 T335Y 1.56 291 SEQ ID NO:296
L234I/L235D 0.07 292 SEQ ID NO:297 V240I/V266I 1.72 293 SEQ ID
NO:298 S239D/A330Y/I332E/L234I 22.39 294 SEQ ID NO:299
S239D/A330Y/I332E/L235D 7.04 295 SEQ ID NO:300
S239D/A330Y/I332E/V240I 27.97 296 SEQ ID NO:301
S239D/A330Y/I332E/V264T 17.72 297 SEQ ID NO:302
S239D/A330Y/I332E/V266I 298 SEQ ID NO:303 S239D/A330Y/I332E/K326E
64.14 299 SEQ ID NO:304 S239D/A330Y/I332E/K326T 59.03 300 SEQ ID
NO:305 S239D/N297D/I332E/A330Y <0.02 301 SEQ ID NO:306
S239D/N297D/I332E/A330Y/ <0 02 F2415/F243H/V262T/V264T 302 SEQ
ID NO:307 S239D/N297D/I332E/L235D 303 SEQ ID NO:308
S239D/N297D/I332E/K326E
[0265] Select Fc variants were screened in the context of multiple
antibodies in order to investigate the breadth of their
applicability. AlphaScreen.TM. data for binding of select Fc
variants to human V158 Fc.gamma.RIIIa in the context of
trastuzumab, rituximab, and cetuximab are shown in FIGS. 14a, 14b,
15a, and 15b. Together with the data for alemtuzumab in FIG. 13,
the results indicate consistent binding enhancements regardless of
the antibody context, and thus that the Fc variants of the present
invention are broadly applicable to antibodies and Fc fusions.
[0266] Fc variants have been obtained that show differentially
enhanced binding to Fc.gamma.RIIa over Fc.gamma.RIIb. As discussed,
optimal effector function may result from Fc variants wherein
affinity for activating Fc.gamma.Rs is greater than affinity for
the inhibitory Fc.gamma.RIIb. AlphaScreen.TM. data directly
comparing binding to Fc.gamma.RIIIa and Fc.gamma.RIIb for two Fc
variants with this specificity profile are shown in FIGS. 16a and
16b. This concept can be defined quantitatively as the
fold-enhancement or -reduction of the activating F.gamma.R (Table
62, column 3) divided by the fold-enhancement or -reduction of the
inhibitory Fc.gamma.R (Table 62, column 4), herein referred to as
the Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold ratio. This value is
provided in Column 5 in Table 62. Table 62 shows that Fc variants
provide this specificity profile, with a
Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold ratio as high as 86:1.
[0267] Some of the most promising Fc variants of the present
invention for enhancing effector function have both substantial
increases in affinity for Fc.gamma.RIIIa and favorable
Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold ratios. These include, for
example, S239D/I332E (SEQ ID NO:95)_(Fc.gamma.RIIIa-fold=56,
Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold=3), S239D/A330Y/I332E (SEQ
ID NO:212)_(Fc.gamma.RIIIa-fold=130), S239D/A330L/I332E (SEQ ID
NO:214)_(Fc.gamma.RIIIa-fold=139,
Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold=18), and S239D/S298A/I332E
(SEQ ID NO:217) (Fc.gamma.RIIIa-fold=295,
Fc.gamma.RIIIa-fold:Fc.gamma.RIIb-fold=48). FIG. 17 shows
AlphaScreen.TM. binding data for these and other Fc variants to
human V158 Fc.gamma.RIIIa
[0268] Because there are a number of Fc.gamma.Rs that contribute to
effector function, it may be worthwhile to additionally screen Fc
variants against other receptors. FIG. 18 shows AlphaScreen.TM.
data for binding of select Fc variants to human R131 Fc.gamma.RIIa.
As can be seen, those aforementioned variants with favorable
binding enhancements and specificity profiles also show enhanced
binding to this activating receptor. The use of Fc.gamma.RIIIa,
Fc.gamma.RIIb, and Fc.gamma.RIIc for screening is not meant to
constrain experimental testing to these particular Fc.gamma.Rs;
other Fc.gamma.Rs are contemplated for screening, including but not
limited to the myriad isoforms and allotypes of Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII from humans, mice, rats, monkeys,
and the like, as previously described.
[0269] Taken together, the Fc.gamma.R binding data provided in
FIGS. 11-18 and Table 62 indicate that a number of substitions at
positions 234, 235, 239, 240, 243, 264, 266, 272, 274, 278, 325,
328, 330, and 332 are promising candidates for improving the
effector function of antibodies and Fc fusions. Because
combinations of some of these substitutions have typically resulted
in additive or synergistic binding improvements, it is anticipated
that as yet unexplored combinations of the Fc variants provided in
Table 62 will also provide favorable results. Thus all combinations
of the Fc variants in Table 62 are contemplated. Likewise,
combinations of any of the Fc variants in Table 62 with other
discovered or undiscovered Fc variants may also provide favorable
properties, and these combinations are also contemplated.
Furthermore, it is anticipated from these results that other
substitutions at positions 234, 235, 239, 240, 243, 264, 266, 325,
328, 330, and 332 may also provide favorable binding enhancements
and specificities, and thus substitutions at these positions other
than those presented in Table 62 are contemplated.
Example 4
Reduced Binding to Fc.gamma.Rs
[0270] As discussed, although there is a need for greater effector
function, for some antibody therapeutics, reduced or eliminated
effector function may be desired. Several Fc variants in Table 62
substantially reduce or ablate Fc.gamma.R binding, and thus may
find use in antibodies and Fc fusions wherein effector function is
undesirable. AlphaScreen.TM. binding data for some examples of such
variants are shown in FIGS. 19a and 19b. These Fc variants, as well
as their use in combination, may find use for eliminating effector
function when desired, for example in antibodies and Fc fusions
whose mechanism of action involves blocking or antagonism but not
killing of the cells bearing target antigen.
[0271] As discussed, one goal of the current experiments was to
obtain optimized aglycosylated Fc variants. Several Fc variants
provide significant progress towards this goal. Because it is the
site of glycosylation, substitution at N297 results in an
aglycosylated Fc. Whereas all other Fc variants that comprise a
substitution at N297 completely ablate Fc.gamma.R binding,
N297D/I332E (SEQ ID NO:71) has significant binding affinity for
Fc.gamma.RIIIa, shown in Table 62 and illustrated in FIG. 20. The
exact reason for this result is uncertain in the absence of a
high-resolution structure for this variant, although the
computational screening predictions suggest that it is potentially
due to a combination of new favorable Fc/Fc.gamma.R interactions
and favorable electrostatic properties. Indeed other electrostatic
substitutions are envisioned for further optimization of
aglycosylated Fc. Table 62 shows that other aglycosylated Fc
variants such as S239D/N297D/I332E (SEQ ID NO:188) and
N297D/A330Y/I332E (SEQ ID NO:210)_provide binding enhancements that
bring affinity for Fc.gamma.RIIIa within 0.28- and 0.43-fold
respectively of glycosylated WT alemtuzumab. Combinations of these
variants with other Fc variants that enhance Fc.gamma.R binding are
contemplated, with the goal of obtaining aglycosylated Fc variants
that bind one or more Fc.gamma.Rs with affinity that is
approximately the same as or even better than glycosylated parent
Fc. An additional set of promising Fc variants provide stability
and solubility enhancements in the absence of carbohydrate. Fc
variants that comprise substitutions at positions 241, 243, 262,
and 264, positions that do not mediate F.gamma.R binding but do
determine the interface between the carbohydrate and Fc, ablate
F.gamma.R binding, presumably because they perturb the conformation
of the carbohydrate. In deglycosylated form, however, Fc variants
F241E/F243R/V262E/V264R (SEQ ID NO:23), F241E/F243Q/V262T/V264E
(SEQ ID NO:24), F241R/F243Q/V262T/V264R (SEQ ID NO:25), and
F241E/F243Y/V262T/V264R (SEQ ID NO:261 show stronger binding to
Fc.gamma.RIIIa than in glycosylated form, as shown by the
AlphaScreen.TM. data in FIG. 21. This result indicates that these
are key positions for optimization of the structure, stability,
solubility, and function of aglycosylated Fc. Together these
results suggests that protein engineering can be used to restore
the favorable functional and solution properties of antibodies and
Fc fusions in the absence of carbohydrate, and pave the way for
aglycosylated antibodies and Fc fusions with favorable solution
properties and full functionality that comprise substitutions at
these and other Fc positions
Example 6
Affinity of Fc Variants for Polymorphic Forms of Fc.gamma.RIIIa
[0272] As discussed above, an important parameter of Fc-mediated
effector function is the affinity of Fc for both V158 and F158
polymorphic forms of Fc.gamma.RIIIa. AlphaScreen.TM. data comparing
binding of select variants to the two receptor allotypes are shown
in FIG. 22a (V158 Fc.gamma.RIIIa) and FIG. 22b (F158
Fc.gamma.RIIIa). As can be seen, all variants improve binding to
both Fc.gamma.RIIIa allotypes. These data indicate that those Fc
variants of the present invention with enhanced effector function
will be broadly applicable to the entire patient population, and
that enhancement to clinical efficacy will potentially be greatest
for the low responsive patient population who need it most.
[0273] The Fc.gamma.R binding affinities of these Fc variants were
further investigated using Surface Plasmon Resonance (SPR)
(Biacore, Uppsala, Sweden). SPR is a sensitive and extremely
quantitative method that allows for the measurement of binding
affinities of protein-protein interactions, and has been used to
effectively measure Fc/Fc.gamma.R binding (Radaev et al., 2001, J
Biol Chem 276:16478-16483). SPR thus provides an excellent
complementary binding assay to the AlphaScreen.TM. assay.
His-tagged V158 Fc.gamma.RIIIa was immobilized to an SPR chip, and
WT and Fc variant alemtuzumab antibodies were flowed over the chip
at a range of concentrations. Binding constants were obtained from
fitting the data using standard curve-fitting methods. Table 63
presents dissociation constants (Kd) for binding of select Fc
variants to V158 Fc.gamma.RIIIa and F158 Fc.gamma.RIIIa obtained
using SPR, and compares these with IC50s obtained from the
AlphaScreen.TM. assay. By dividing the Kd and IC50 for each variant
by that of WT alemtuzumab, the fold-improvements over WT (Fold) are
obtained. TABLE-US-00063 TABLE 63 SPR V158 SPR F158 AlphaScreen
.TM. AlphaScreen .TM. Fc.gamma.RIIIa Fc.gamma.RIIIa V158
Fc.gamma.RIIIa F158 Fc.gamma.RIlIa Kd Kd IC50 IC50 (nM) Fold (nM)
Fold (nM) Fold (nm) Fold WT 68 730 6.4 17.2 (SEQ ID NO:2) V264I 64
1.1 550 1.3 4.5 1.4 11.5 1.5 (SEQ ID NO:11) I332E 31 2.2 72 10.1
1.0 6.4 2.5 6.9 (SEQ ID NO:30) V264I/I332E 17 4.0 52 14.0 0.5 12.8
1.1 15.6 (SEQ ID NO:38) S298A 52 1.3 285 2.6 2.9 2.2 12.0 1.4 (SEQ
ID NO:43) S298A/E333A/ 39 1.7 156 4.7 2.5 2.6 7.5 2.3 K334A (SEQ ID
NO:45)
[0274] The SPR data corroborate the improvements to Fc.gamma.RIIIa
affinity observed by AlphaScreen.TM. assay. Table 63 further
indicates the superiority of V264I/I332E (SEQ ID NO:38) and I332E
(SEQ ID NO:30) over S298A (SEQ ID NO:43) and S298A/E333A/K334A (SEQ
ID NO:45); whereas S298A/E333A/K334A (SEQ ID NO:45) improves Fc
binding to V158 and F158 Fc.gamma.RIIa by 1.7-fold and 4.7-fold
respectively, I332E (SEQ ID NO:30) shows binding enhancements of
2.2-fold and 10.1-fold respectively, and V264I/I332E (SEQ ID NO:38)
shows binding enhancements of 4.0-fold and 14-fold respectively.
Also worth noting is that the affinity of V264I/I332E (SEQ ID
NO:38) for F158 Fc.gamma.RIIIa (52 nM) is better than that of WT
for the V158 allotype (68 nM), suggesting that this Fc variant, as
well as those with even greater improvements in binding, may enable
the clinical efficacy of antibodies for the low responsive patient
population to achieve that currently possible for high responders.
The correlation between the SPR and AlphaScreen.TM. binding
measurements are shown in FIGS. 23a-23d. FIGS. 23a and 23b show the
Kd-IC50 correlations for binding to V158 Fc.gamma.RIIIa and F158
Fc.gamma.RIIIa respectively, and FIGS. 23c and 23d show the
fold-improvement correlations for binding to V158 Fc.gamma.RIIIa
and F158 Fc.gamma.RIIIa respectively. The good fits of these data
to straight lines (r.sup.2=0.9, r.sup.2=0.84, r.sup.2=0.98, and
r.sup.2=0.90) support the accuracy the AlphaScreen.TM.
measurements, and validate its use for determining the relative
Fc.gamma.R binding affinities of Fc variants.
[0275] SPR data were also acquired for binding of select
trastuzumab Fc variants to human V158 Fc.gamma.RIIIa, F158
Fc.gamma.RIIIa, and Fc.gamma.RIIb. These data are shown in Table
64. The Fc variants tested show substantial binding enhancements to
the activating receptor Fc.gamma.RIIIa, with over 100-fold tighter
binding observed for interaction of S239D/I332E/S298A (SEQ ID
NO:217)_with F158 Fc.gamma.RIIIa. Furthermore, for the best
Fc.gamma.RIIIa binders, F158 Fc.gamma.RIIIa/Fc.gamma.RIIb ratios of
3-4 are observed. TABLE-US-00064 TABLE 64 SPR V158 SPR F158 SPR
Fc.gamma.RIIIa Fc.gamma.RIIIa Fc.gamma.RIIb Kd Kd IC50 (nM) Fold
(nM) Fold (nM) Fold WT 363.5 503 769 (SEQ ID NO:2) V264I/I332E 76.9
4.7 252 2.0 756 1.0 (SEQ ID NO:38) V264I/I332E/ 113.0 3.2 88 5.7
353 2.2 A330L (SEQ ID NO:115) S239D/I332E/ 8.2 44.3 8.9 56.5 46
16.7 A330L (SEQ ID NO:214) S239D/I332E/ 8.7 41.8 4.9 102.7 32 24.0
S298A (SEQ ID NO:217) S239D/I332E/ 12.7 28.6 6.3 79.8 35 22.0
V264I/A330L (SEQ ID NO:221)
Example 7
ADCC of Fc Variants
[0276] In order to determine the effect on effector function,
cell-based ADCC assays were performed on select Fc variants. ADCC
was measured using the DELFIA.RTM. EuTDA-based cytotoxicity assay
(Perkin Elmer, MA) with purified human peripheral blood monocytes
(PBMCs) as effector cells. Target cells were loaded with BATDA at
1.times.10.sup.6 cells/ml, washed 4 times and seeded into 96-well
plate at 10,000 cells/well. The target cells were then opsonized
using Fc variant or WT antibodies at the indicated final
concentration. Human PBMCs, isolated from buffy-coat were added at
the indicated fold-excess of target cells and the plate was
incubated at 37.degree. C. for 4 hrs. The co-cultured cells were
centrifuged at 500.times.g, supernatants were transferred to a
separate plate and incubated with Eu solution, and relative
fluorescence units were measured using a Packard Fusion.TM.
.alpha.-FP HT reader (Packard Biosciences, IL). Samples were run in
triplicate to provide error estimates (n=3, +/-S.D.). PBMCs were
allotyped for the V158 or F158 Fc.gamma.RIIIa allotype using
PCR.
[0277] ADCC assays were run on Fc variant and WT alemtuzumab using
DoHH-2 lymphoma target cells. FIG. 24a is a bar graph showing the
ADCC of these proteins at 10 ng/ml antibody. Results show that
alemtuzumab Fc variants I332E (SEQ ID NO:30), V2641 (SEQ ID NO:11),
and I332E/V264I (SEQ ID NO:38) have substantially enhanced ADCC
compared to WT alemtuzumab, with the relative ADCC enhancements
proportional to their binding improvements to Fc.gamma.RIIIa as
indicated by AlphaScreen.TM. assay and SPR. The dose dependence of
ADCC on antibody concentration is shown in FIG. 24b. The binding
data were normalized to the minimum and maximum fluorescence signal
for each particular curve, provided by the baselines at low and
high antibody concentrations respectively. The data were fit to a
sigmoidal dose-response model using nonlinear regression,
represented by the curve in the figure. The fits enable
determination of the effective concentration 50% (EC50) (i.e. the
concentration required for 50% effectiveness), which provides the
relative enhancements to ADCC for each Fc variant. The EC50s for
these binding data are analogous to the IC50s obtained from the
AlphaScreen.TM. competition data, and derivation of these values is
thus analogous to that described in Example 2 and FIG. 11. In FIG.
24b, the log(EC50)s, obtained from the fits to the data, for WT
(SEQ ID NO:2), V264I/I332E (SEQ ID NO:38), and S239D/I332E (SEQ ID
NO:95) alemtuzumab are 0.99, 0.60, and 0.49 respectively, and
therefore their respective EC50s are 9.9, 4.0, and 3.0. Thus
V264I/I332E (SEQ ID NO:38) and S239E/I332E (SEQ ID NO:95) provide a
2.5-fold and 3.3-fold enhancement respectively in ADCC over WT
alemtuzumab using PBMCs expressing heterozygous V158/F158
Fc.gamma.RIIIa. These data are summarized in Table 65 below.
TABLE-US-00065 TABLE 65 Fold log EC50 Improvement (EC50) (ng/ml)
Over WT WT 0.99 9.9 (SEQ ID NO:2) V264I/I332E 0.60 4.0 2.5 (SEQ ID
NO:38) S239D/I332E 0.49 3.0 3.3 (SEQ ID NO:95)
[0278] In order to determine whether these ADCC enhancements are
broadly applicable to antibodies, select Fc variants were evaluated
in the context of trastuzumab and rituximab. ADCC assays were run
on Fc variant and WT trastuzumab using two breast carcinoma target
cell lines BT474 and Sk-Br-3. FIG. 25a shows a bar graph
illustrating ADCC at 1 ng/ml antibody. Results indicate that V2641
(SEQ ID NO:11) and V264I/I332E (SEQ ID NO:38) trastuzumab provide
substantially enhanced ADCC compared to WT trastuzumab, with the
relative ADCC enhancements proportional to their binding
improvements to Fc.gamma.RIIIa as indicated by AlphaScreen.TM.
assay and SPR. FIGS. 25b and 25c show the dose dependence of ADCC
on antibody concentration for select Fc variants. The EC50s
obtained from the fits of these data and the relative
fold-improvements in ADCC are provided in Table 66 below.
Significant ADCC improvements are observed for I332E (SEQ ID NO:30)
trastuzumab when combined with A330L (SEQ ID NO:65) and A330Y(SEQ
ID NO:66). Furthermore, S239D/A330L/I332E (SEQ ID NO:214) provides
a substantial ADCC enhancement, greater than 300-fold for PBMCs
expressing homozygous F158/F158 Fc.gamma.RIIIa, relative to WT
trastuzumab and S298A/E333A/K334A (SEQ ID NO:45), consistent with
the Fc.gamma.R binding data observed by the AlphaScreen.TM. assay
and SPR. TABLE-US-00066 TABLE 66 Fold log EC50 Improvement (EC50)
(ng/ml) Over WT Figure 25b WT 1.1 11.5 (SEQ ID NO:2) I332E 0.34 2.2
5.2 (SEQ ID NO:30) A330Y/I332E (SEQ ID NO:112) -0.04 0.9 12.8
A330L/I332E (SEQ ID NO:114) 0.04 1.1 10.5 Figure 25d WT -0.15 0.71
(SEQ ID NO:2) S298A/E333A/K334A -0.72 0.20 3.6 (SEQ ID NO:45)
S239D/A330L/I332E -2.65 0.0022 323 (SEQ ID NO:214)
[0279] ADCC assays were run on V264I/I332E (SEQ ID NO:38), WT (SEQ
ID NO:2), and S298A/DE333A/K334A (SEQ ID NO:45) rituximab using
WIL2-S lymphoma target cells. FIG. 26a presents a bar graph showing
the ADCC of these proteins at 1 ng/ml antibody. Results indicate
that V264I/I332E (SEQ ID NO:38) rituximab provides substantially
enhanced ADCC relative to WT rituximab, as well as superior ADCC to
S298A/DE333A/K334A (SEQ ID NO:45), consistent with the
Fc.gamma.RIIIa binding improvements observed by AlphaScreen.TM.
assay and SPR. FIGS. 26b and 26c show the dose dependence of ADCC
on antibody concentration for select Fc variants. The EC50s
obtained from the fits of these data and the relative
fold-improvements in ADCC are provided in Table 67 below. As can be
seen S239D/I332E/A330L (SEQ ID NO:214) rituximab provides greater
than 900-fold enhancement in EC50 over WT for PBMCs expressing
homozygous F158/F158 Fc.gamma.RIIIa. The differences in ADCC
enhancements observed for alemtuzumab, trastuzumab, and rituximab
are likely due to the use of different PBMCs, different antibodies,
and different target cell lines TABLE-US-00067 TABLE 67 Fold log
EC50 Improvement (EC50) (ng/ml) Over WT Figure 26b WT 0.23 1.7 (SEQ
ID NO:2) S298A/E333A/K334A -0.44 0.37 4.6 (SEQ ID NO:45)
V264I/I332E -0.83 0.15 11.3 (SEQ ID NO:38) Figure 26c WT 0.77 5.9
(SEQ ID NO:2) S239D/I332E/A330L -2.20 0.0063 937 (SEQ ID
NO:214)
[0280] Thus far, ADCC data has been normalized such that the lower
and upper baselines of each Fc polypeptide are set to the minimal
and maximal fluorescence signal for that specific Fc polypeptide,
typically being the fluorescence signal at the lowest and highest
antibody concentrations respectively. Although presenting the data
in this matter enables a straightforward visual comparison of the
relative EC50s of different antibodies (hence the reason for
presenting them in this way), important information regarding the
absolute level of effector function achieved by each Fc polypeptide
is lost. FIGS. 27a and 27b present cell-based ADCC data for
trastuzumab and rituximab respectively that have been normalized
according to the absolute minimal lysis for the assay, provided by
the fluorescence signal of target cells in the presence of PBMCs
alone (no antibody), and the absolute maximal lysis for the assay,
provided by the fluorescence signal of target cells in the presence
of Triton X1000. The graphs show that the antibodies differ not
only in their EC50, reflecting their relative potency, but also in
the maximal level of ADCC attainable by the antibodies at
saturating concentrations, reflecting their relative efficacy. Thus
far these two terms, potency and efficacy, have been used loosely
to refer to desired clinical properties. In the current
experimental context, however, they are denoted as specific
quantities, and therefore are here explicitly defined. By "potency"
as used in the current experimental context is meant the EC50 of an
antibody or Fc fusion. By "efficacy" as used in the current
experimental context is meant the maximal possible effector
function of an antibody or Fc fusion at saturating levels. In
addition to the substantial enhancements to potency described thus
far, FIGS. 27a and 27b show that the Fc variants of the present
invention provide greater than 100% enhancements in efficacy over
WT trastuzumab and rituximab.
[0281] A critical parameter governing the clinical efficacy of
anti-cancer antibodies is the expression level of target antigen on
the surface of tumor cells. Thus a major clinical advantage of Fc
variants that enhance ADCC may be that it enables the targeting of
tumors that express lower levels of antigen. In To test this
hypothesis, WT and Fc variant trastuzumab antibodies were tested
for their ability to mediate ADCC against different cell lines
expressing varying levels of the Her2/neu target antigen. ADCC
assays were run with various cell lines expressing amplified to low
levels of Her2/neu receptor, including Sk-Br-3 (1.times.10.sup.6
copies), SkOV3 (.about.1.times.10.sup.5),
OVCAR3(.about.1.times.10.sup.4), and MCF-7 (.about.3.times.10.sup.3
copies), using the DELFIA EuTDA Cytotoxicity kit (PerkinElmer,
Boston, Mass.). Target cells were loaded with BATDA in batch for 25
minutes, washed multiple times with medium and seeded at 10,000
cells per well in 96-well plates. Target cells were opsonized for
15 minutes with various antibodies and concentrations (final conc.
ranging from 100 ng/ml to 0.0316 ng/ml in 1/2 log steps, including
no treatment control). Human PBMCs, isolated from buffy-coat and
allotyped as homozygous F158/F158 Fc.gamma.RIIIa were then added to
opsonized cells at 25-fold excess and co-cultured at 37.degree. C.
for 4 hrs. Thereafter, plates were centrifuged, supernatants were
removed and treated with Eu3+ solution, and relative fluorescence
units (correlating to the level of cell lysis) were measured using
a Packard Fusion.TM. .alpha.-FP HT reader (PerkinElmer, Boston,
Mass.). The experiment was carried out in triplicates. FIG. 28
shows the ADCC data comparing WT and Fc variant trastuzumab against
the four different Her2/neu.sup.+ cell lines. The S239D/I332E (SEQ
ID NO:95) and S239D/I332E/A330L (SEQ ID NO:214) variants provide
substantial ADCC enhancements over WT trastuzumab at high,
moderate, and low expression levels of target antigen. This result
suggests that the Fc variants of the present invention may broaden
the therapeutic window of anti-cancer antibodies.
[0282] Natural killer (NK) cells are a subpopulation of cells
present in PBMCs that are thought to play a significant role in
ADCC. Select Fc variants were tested in a cell-based ADCC assay in
which natural killer (NK) cells rather than PBMCs were used as
effector cells. In this assay the release of endogenous lactose
dehydrogenase (LDH), rather than EuTDA, was used to monitor cell
lysis. FIG. 29 shows that the Fc variants show substantial ADCC
enhancement when NK cells are used as effector cells. Furthermore,
together with previous assays, the results indicate that the Fc
variants of the present invention show substantial ADCC
enhancements regardless of the type of effector cell or the
detection method used.
Example 8
ADCP of Fc Variants
[0283] Another important Fc.gamma.R-mediated effector function is
ADCP. Phagocytosis of target cancer cells may not only lead to the
immediate destruction of target cells, but because phagocytosis is
a potential mechanism for antigen uptake and processing by antigen
presenting cells, enhanced ADCP may also improve the capacity of
the antibody or Fc fusion to elicit an adaptive immune response.
The ability of the Fc variants of the present invention to mediate
ADCP was therefore investigated. Monocytes were isolated from
heterozygous V158/F158 Fc.gamma.RIIIa PBMCs using a Percoll
gradient. After one week in culture in the presence of 0.1 ng/ml,
differentiated macrophages were detached with EDTA/PBS- and labeled
with the lipophilic fluorophore, PKH26, according to the
manufacturer's protocol (Sigma, St Louis, Mo.). Sk-Br-3 target
cells were labeled with PKH67 (Sigma, St Louis, Mo.), seeded in a
96-well plate at 20,000 cells per well, and treated with designated
final concentrations of WT or Fc variant trastuzumab. PKH26-labeled
macrophages were then added to the opsonized, labeled Sk-Br-3 cells
at 20,000 cells per well and the cells were co-cultured for 18 hrs
before processing cells for analysis of dual label flow cytometry.
Percent phagocytosis was determined as the number of cells
co-labeled with PKH76 and PKH26 (macrophage+Sk-Br-3) over the total
number of Sk-Br-3 in the population (phagocytosed+non-phagocytosed)
after 10,000 counts. FIG. 30 shows data comparing WT and Fc variant
trastuzumab at various antibody concentrations. The results
indicate that the S239D/I332E/A330L (SEQ ID NO:214)_variant
provides a significant enhancement in ADCP over WT trastuzumab.
Example 9
Complement Binding and Activation by Fc Variants
[0284] Complement protein C1q binds to a site on Fc that is
proximal to the Fc.gamma.R binding site, and therefore it was
prudent to determine whether the Fc variants have maintained their
capacity to recruit and activate complement. The AlphaScreen.TM.
assay was used to measure binding of select Fc variants to the
complement protein C1q. The assay was carried out with biotinylated
WT alemtuzumab antibody attached to streptavidin donor beads as
described in Example 2, and using C1q coupled directly to acceptor
beads. Binding data of V2641 (SEQ ID NO:11), I332E (SEQ ID NO:30),
S239E (SEQ ID NO:48), and V264I/I332E (SEQ ID NO:38) rituximab
shown in FIG. 31a indicate that C1q binding is uncompromised.
Cell-based CDC assays were also performed on select Fc variants to
investigate whether Fc variants maintain the capacity to activate
complement. Alamar Blue was used to monitor lysis of Fc variant and
WT rituximab-opsonized WIL2-S lymphoma cells by human serum
complement (Quidel, San Diego, Calif.). The data in FIG. 31b show
that CDC is uncompromised for the Fc variants S239E (SEQ ID NO:48),
V264I (SEQ ID NO:11), and V264I/I332E (SEQ ID NO:38) rituximab. In
contrast, FIG. 31c shows that CDC of the Fc variant
S239D/I332E/A330L (SEQ ID NO:214) is completely ablated, whereas
the S239D/I332E variant (SEQ ID NO:95)_mediates CDC that is
comparable to WT rituximab. These results indicate that protein
engineering can be used to distinguish between different effector
functions. Such control will not only enable the generation of
antibodies and Fc fusions with properties tailored for a desired
clinical outcome, but also provide a unique set of reagents with
which to experimentally investigate effector function biology.
Example 10
Protein a and FcRn Binding by Fc Variants
[0285] As discussed, bacterial proteins A and G and the neonatal Fc
receptor FcRn bind to the Fc region between the C.gamma.2 and
C.gamma.3 domains. Protein A is frequently employed for antibody
purification, and FcRn plays a key role in antibody
pharmacokinetics and transport. It was therefore important to
investigate the ability of the Fc variants of the present invention
to bind protein A and FcRn. The AlphaScreen.TM. assay was used to
measure binding of select Fc variants to protein A and human FcRn
using biotinylated WT alemtuzumab antibody attached to streptavidin
donor beads as described in Example 2, and using protein A and FcRn
coupled directly to acceptor beads. The binding data are shown in
FIG. 32 for protein A and FIG. 33 for FcRn. The results indicate
that the C.gamma.2-C.gamma.3 hinge region is unaffected by the Fc
substitutions, and importantly that the capacity of the Fc variants
to bind protein A and FcRn is uncompromised.
Example 11
Capacity of Fc Variants to Bind Mouse Fc.gamma.Rs
[0286] Optimization of Fc to nonhuman Fc.gamma.Rs may be useful for
experimentally testing Fc variants in animal models. For example,
when tested in mice (for example nude mice, SCID mice, xenograft
mice, and/or transgenic mice), antibodies and Fc fusions that
comprise Fc variants that are optimized for one or more mouse
Fc.gamma.Rs may provide valuable information with regard to
clinical efficacy, mechanism of action, and the like. In order to
evaluate whether the Fc variants of the present invention may be
useful in such experiments, affinity of select Fc variants for
mouse Fc.gamma.RIII was measured using the AlphaScreen.TM. assay.
The AlphaScreen.TM. assay was carried out using biotinylated WT
alemtuzumab attached to streptavidin donor beads as described in
Example 2, and GST-tagged mouse Fc.gamma.RIII bound to glutathione
chelate acceptor beads, expressed and purified as described in
Example 2. These binding data are shown in FIGS. 34a and 34b in the
context of alemtuzumab and trastuzumab respectively. Results show
that some Fc variants that enhance binding to human Fc.gamma.RIIIa
also enhance binding to mouse Fc.gamma.RIII. The enhancement of
mouse effector function by the Fc variants was investigated by
performing the aforementioned cell-based ADCC assays using mouse
rather than human PBMC's. FIG. 35 shows that the S239D/I332E/A330L
(SEQ ID NO:214) trastuzumab variant provides substantial ADCC
enhancement over WT in the presence of mouse immune cells. This
result indicates that the Fc variants of the present invention, or
other Fc variants that are optimized for nonhuman Fc.gamma.Rs, may
find use in experiments that use animal models.
Example 12
Validation of Fc Variants Expressed in CHO Cells
[0287] Whereas the Fc variants of the present invention were
expressed in 293T cells for screening purposes, large scale
production of antibodies is typically carried out by expression in
Chinese Hamster Ovary (CHO) cell lines. In order to evaluate the
properties of CHO-expressed Fc variants, select Fc variants and WT
alemtuzumab were expressed in CHO cells and purified as described
in Example 2. FIG. 36 shows AlphaScreen.TM. data comparing binding
of CHO- and 293T-expressed Fc variant and WT alemtuzumab to human
V158 Fc.gamma.RIIIa. The results indicate that the Fc variants of
the present invention show comparable Fc.gamma.R binding
enhancements whether expressed in 293T or CHO.
Example 13
Enhancement of Fc Variants in Fucose Minus Strain
[0288] Combinations of the Fc variants of the present invention
with other Fc modifications are contemplated with the goal of
generating novel antibodies or Fc fusions with optimized
properties. It may be beneficial to combine the Fc variants of the
present invention with other Fc modifications, including
modifications that alter effector function or interaction with one
or more Fc ligands. Such combination may provide additive,
synergistic, or novel properties in antibodies or Fc fusions. For
example, a number of methods exist for engineering different
glycoforms of Fc that alter effector function. Engineered
glycoforms may be generated by a variety of methods known in the
art, many of these techniques are based on controlling the level of
fucosylated and/or bisecting oligosaccharides that are covalently
attached to the Fc region. One method for engineering Fc glycoforms
is to express the Fc polypeptide in a cell line that generates
altered glycoforms, for example Lec-13 CHO cells. In order to
investigate the properties of Fc variants combined with engineered
glycoforms, WT and V209 (S239D/I332E/A330L(SEQ ID NO:214))
trastuzumab were expressed in Lec-13 CHO cells and purified as
described above. FIG. 37a shows AlphaScreen.TM. binding data
comparing the binding to human V158 Fc.gamma.RIIIa by WT and V209
trastuzumab expressed in 293T, CHO, and Lec-13 cells. The results
show that there is substantial synergy between the engineered
glycoforms produced by this cell line and the Fc variants of the
present invention. The cell-based ADCC assay, shown in FIG. 37b,
supports this result. Together these data indicate that other Fc
modifications, particularly engineered glycoforms, may be combined
with the Fc variants of the present invention to generate
antibodies and Fc fusions with optimized effector functions
Example 14
Therapeutic Application of Fc Variants
[0289] A number of Fc variants described in the present invention
have significant potential for improving the therapeutic efficacy
of anticancer antibodies. For illustration purposes, a number of Fc
variants of the present invention have been incorporated into the
sequence of the antibody rituximab. The WT rituximab light chain
(SEQ ID NO:3) and heavy chain (SEQ ID NO:4), described in U.S. Pat.
No. 5,736,137, are provided in FIGS. 38a and 38b. The improved
anti-CD20 antibody sequences (SEQ ID NO:5)_are provided in FIG.
38c. The improved anti-CD20 antibody sequences comprise at least
non-WT amino acid selected from the group consisting of X.sub.1,
X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, and X.sub.8.
These improved anti-CD20 antibody sequences may also comprise a
substitution Z.sub.1 and/or Z.sub.2. The use of rituximab here is
solely an example, and is not meant to constrain application of the
Fc variants to this antibody or any other particular antibody or Fc
fusion.
Example 15
A Complete Structure/Function Analysis Fc/Fc Ligand Specificity
[0290] It is clear from the results of these experiments that
protein engineering is a powerful tool for mining Fc substitutions
that significantly alter its biological function and specificity.
Given the profound clinical value of antibodies and Fc fusions, the
implication is that the protein engineering methods of the present
invention can be used to tune the clinical properties of these
important biotherapeutics. Such capability, however, demands a more
complete understanding of the relationship between the structure
and function of Fc and Fc ligands. In addition, the lack of
available information on the determinants of Fc/Fc ligand
specificity means that it is not possible to actively design Fc
variants with all desired properties as target goals. Thus it is
likely that, despite the aggressive experimental effort described
in the present invention, there are therapeutically useful Fc
variants that have not been mined, and biochemical properties of Fc
variants that remain undiscovered. Equally important to obtaining
new Fc variants for biotherapeutic application is the ability to
improve the predictiveness of the design method, thereby permitting
variants to be identified even more efficiently. Towards these
goals, a more thorough characterization of Fc/Fc ligand biology was
carried out. This included: 1) an expansion of the primary screen
to include all relevant Fc ligands, and 2) an increase in the
number of Fc variants to explore a greater set of substitutions at
all relevant Fc positions. Together this broadened approach will
enable a more thorough mining of useful Fc variants, provide a
greater understanding of Fc/Fc ligand specificity and biology, and
provide a greater data set to enable a rigorous quantitative
assessment of the predictiveness of the design methods.
[0291] Expansion of the Primary Screen
[0292] In order to better characterize the structural and
functional determinants of Fc specificity, the primary screen was
expanded to include all relevant Fc ligands. Thus all Fc variants
are tested in parallel for binding to Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIb, Fc.gamma.RIIc, Fc.gamma.RIIIa (Val158 isoform),
FcRn, and C1q. The AlphaScreen.TM. assay was used as described
above. All Fc variants were screened in the context of either
alemtuzumab or trastuzumab according to Table 62. Table 68 shows an
example of the parallel screen for a set of substitutions at Fc
positions 234 and 235. In this table, light grey indicates that Fc
variant/Fc ligand affinity is 0.5-fold or less than WT, medium grey
indicates that Fc variant/Fc ligand affinity is within 0.5-2.0 of
WT, dark grey indicates that Fc variant/Fc ligand affinity is
increased by 2-fold or greater, and white indicates that the Fc
variant/Fc ligand interaction was not measured or that the data did
not allow an accurate determination of affinity. Thus Fc variants
are grouped as those that significantly decrease, those that do not
substantially alter, and those that significantly increase binding
to a given Fc ligand. Visualization of the data in this way
provides a structure/function map of Fc, enabling a straightforward
interpretation of the results for each position such that useful
and interesting variants can be efficiently identified, and such
that predictiveness of the design method can be assessed in a
practical manner.
[0293] A number of substitutions at positions 234 and 235 show
different specificities for binding to the various Fc ligands.
Although the differences in some cases are subtle, the results
indicate that it is indeed possible to engineer Fc specificity for
different Fc ligands, even at the Fc.gamma.R interface where a
number of highly homologous receptors bind to the same site. Other
Fc variants that provide more distinct affinity differences are
presented in Table 69.
[0294] These data show even more convincingly that it is possible
to tune Fc for Fc ligand specificity, often by using very subtle
mutational differences. For example, the A330Y/I332E variant (SEQ
ID NO: 112) enhances binding to all Fc.gamma.Rs, particularly
Fc.gamma.RIIIa, as well as FcRn, while maintaining binding to C1q.
However the A330L/I332E variant (SEQ ID NO:114)_shows enhanced
binding to Fc.gamma.RI and Fc.gamma.RIIa, but has WT affinity for
the Fc.gamma.RII's. In contrast, mutations at L328 provide
preferential enhancement of the Fc.gamma.RII's over Fc.gamma.RI and
Fc.gamma.RIIa. In the case of the L328E/I332E variant (SEQ ID
NO:172), affinity for all Fc.gamma.RII's is increased, whereas
L328T/I332E (SEQ ID NO:176) provides a clear enhancement
specificity profile of
Fc.gamma.RIIc>Fc.gamma.RIIb>Fc.gamma.RIIa. In contrast, L328A
significantly enhances binding to Fc.gamma.RIIa, but provides WT
affinity for all other Fc.gamma.R's including Fc.gamma.RIIb and
Fc.gamma.RIIc. It is clear from these results that very subtle
mutational differences can provide substantial differences in
specificity. Accordingly, collections of Fc variants such as these
will not only enable the generation of antibodies and Fc fusions
that have effector function tailored for the desired outcome, but
they also provide a unique set of reagents with which to
experimentally investigate and characterize effector function
biology.
[0295] Expansion of the Fc Variant Set
[0296] Because of the incomplete information concerning the
structural and functional determinants of Fc/Fc ligand interaction,
it has not been possible to actively engineer Fc for all desired
optimization goals. The distinct specificity differences observed
in Tables 68 and 69 to the various Fc.gamma.Rs were due more to the
aggressive screening approach of the present invention; these Fc
variants were not actively designed with their particular
properties as the target goals due to the lack of structural
information for binding of Fc to the different Fc.gamma.Rs, as well
as the lack of understanding of how the structure and flexibility
of the hinge impacts Fc.gamma.R binding. Indeed the decision to
explore a large number and variety of substitutions at these
positions 234 and 235 was based on the knowledge that they are near
the Fc/Fc.gamma.R binding site, that mutations at these positions
affect Fc.gamma.R binding, and that according to computational
screening calculations a large number and variety of substitutions
are permissible at these positions. Overall, the lack of structural
information on the determinants of Fc/Fc.gamma.R specificity, the
lack of high-resolution structural information for the Fc/C1q
complex, and the inability to account for indirect effects of
substitutions on Fc/Fc ligand binding, together make it a certainty
that all of the interesting and potentially useful Fc variants will
not be explored using the current engineering methods. In order to
fully mine useful Fc variants, as well as to obtain a more complete
picture of the structural and function determinants of Fc/Fc ligand
interaction, the set of Fc variants was expanded to explore a
broader set of mutations. All Fc positions at or near the binding
sites for Fc.gamma.R's and C1q, chosen by visual inspection of the
available structures and using the information provided by the
results of previous Fc variant screening, were saturated such that
all substitutions were constructed that have not been tested
previously. At Fc positions significantly distal to the Fc.gamma.R
and C1q binding sites, a subset of select substitutions were
designed based on predicted energies in previously described
computational screening calculations, and based on available data
from existing Fc variants. This new set of Fc variants, 576 total,
is presented in Table 70. TABLE-US-00068 TABLE 70 Position WT
Substitution(s) Variant 221 D KY 801-802 222 K EY 513-514 223 T EK
803-804 224 H EY 805-806 225 T EKW 807-809 227 P EKYG 705-708 228 P
EKYG 709-712 230 P EYG 609-611 231 A EKYPG 612-616 232 P EKYG
321-324 233 E NQKRSTHAVLIFMYWG 617-632 234 L KRSAMWPG 417-424 235 L
EKRAMWPG 425-432 236 C DENQKRSTHAVLIFMYWP 713-730 237 G
DENQKRSTHVLIFMYWP 731-747 238 P DENQKRSTHVLIFMYWG 748-764 239 S
QKRVLIMWPG 325-334 241 F DEY 335-337 243 F E 515 246 K DEHY 810-813
249 D QHY 814-816 255 R EY 817-818 258 E SHY 819-821 260 T DEHY
822-825 262 V EF 826-827 264 V DENQKRSHWPG 433-443 265 D
QKRSTHVLIFMYWP 444-457 267 S EQKRVLIFMYWP 338-349 268 H
DEQKRTVLIFMWPG 350-363 269 E KSVIMWPG 765-772 270 D RSLIFMYWPG
516-525 271 P DENQKRSTHAVLIFMYWG 526-543 272 E DRTHVLFMWPG 633-643
274 K DNSHVIFMWPG 644-654 275 F L 828 276 N DTHVIFMWPG 655-664 278
Y DNQRSHVLIMPG 665-676 280 D KLWPG 544-548 281 C DKYP 829-832 282 V
EKYPG 833-837 283 E KHLYPG 838-843 284 V ENTLY 844-848 285 H DEQKYW
773-778 286 N EYPG 779-782 288 K DEY 783-785 290 K DNHLW 549-553
291 P DEQTHIG 849-855 292 R DETY 786-789 293 F NRSTHVLIFMYWPG
554-567 294 E KRSTHVLIFMYWPG 568-581 295 Q DENRSTHVIFMYWPG 582-596
296 Y KRAVMG 597-602 297* N QKRTHVLIFMYWPG 856-869 298 S DEQKRIFMYW
364-373 299 T DENQKRLFMYWPG 374-386 300 Y DENQKRSTHAVMWPG 387-401
301 R DEHY 870-873 303 V DEY 874-876 304 S DNTHL 877-881 305 V ETY
882-884 317 K EQ 885-886 318 E QHLY 887-890 320 K NSHVLFYWPG
677-686 322 K DSVIFYWPG 687-695 324 S HFMWPG 603-608 325 N
KRSFMYWPG 696-704 326 K P 458 327 A EKRHVIFMYWP 459-469 328 L
DQKRSTVIYWPG 470-481 329 P DENQKRSTHVLIMYWG 482-497 330 A ENTPG
402-406 331 P DQRTLIFMYW 498-507 332 I KRSVLFMWPG 407-416 333 E
LFMP 508-511 334 K P 512 335 T NSHVLIFMWPG 790-800 336 I EKY
891-893 337 S ENH 894-896 *Substitutions at 297 were made in the
context of S239D/I332E
[0297] All references are herein expressly incorporated by
reference.
[0298] Whereas particular embodiments of the invention have been
described above for purposes of illustration, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims.
Sequence CWU 1
1
308 1 451 PRT Homo sapiens 1 Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Asn Trp Val
Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro 50 55 60 Ser
Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln 65 70
75 80 Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp
Tyr Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195
200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315
320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445 Pro Gly Lys 450 2 227 PRT Homo sapiens 2 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 3 213 PRT Homo sapiens
3 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1
5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro
Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 4
451 PRT Homo sapiens 4 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln
Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys 210 215
220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly
Lys 450 5 451 PRT Homo sapiens misc_feature (243)..(243) Xaa can be
one of the following amino acids serine, aspartic acid, glutamic
acid, asparagine, glutamine or threonine misc_feature (244)..(244)
Xaa can be one of the following amino acids valine, isoleucine or
methionine misc_feature (268)..(268) Xaa can be one of the
following amino acids valine, isoleucine, threonine or tyrosine
misc_feature (276)..(276) Xaa can be one of the following amino
acids glutamic acid or tyrosine misc_feature (278)..(278) Xaa can
be one of the following amino acids lysine or glutamic acid
misc_feature (301)..(301) Xaa can be one of the following amino
acids asparagine or aspartic acid misc_feature (302)..(302) Xaa can
be one of the following amino acids serine or alanine misc_feature
(330)..(330) Xaa can be one of the following amino acids lysine,
glutamic acid or threonine misc_feature (334)..(334) Xaa can be one
of the following amino acids alanine, tyrosine, leucine or
isoleucine misc_feature (336)..(336) Xaa can be one of the
following amino acids isoleucine, aspartic acid, glutamic acid,
asparagine or glutamine 5 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys
Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr
Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp
Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys 210
215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly 225 230 235 240 Gly Pro Xaa Xaa Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Xaa Asp Val Ser His 260 265 270 Glu Asp Pro Xaa Val Xaa Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Xaa Xaa Thr Tyr 290 295 300 Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Xaa Ala Leu Pro Xaa Pro Xaa 325 330
335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro
Gly Lys 450 6 5 PRT Artificial synthetic 6 Gly Ser Gly Gly Ser 1 5
7 5 PRT Artificial synthetic 7 Gly Gly Gly Gly Ser 1 5 8 4 PRT
Artificial synthetic 8 Gly Gly Gly Ser 1 9 227 PRT Artificial
synthetic 9 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Ala Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115
120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly
Lys 225 10 227 PRT Artificial synthetic 10 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Leu Asp Val Ser His 35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50
55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180
185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 210 215 220 Pro Gly Lys 225 11 227 PRT Artificial
synthetic
11 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
12 227 PRT Artificial synthetic 12 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Trp Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 13 227 PRT Artificial synthetic
13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Leu Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
14 227 PRT Artificial synthetic 14 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Trp Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 15 227 PRT Artificial synthetic
15 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
16 227 PRT Artificial synthetic 16 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Leu Leu
Leu Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Ile Val Ile Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 17 227 PRT Artificial synthetic
17 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Trp Leu Trp Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
18 227 PRT Artificial synthetic 18 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Trp Leu
Trp Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Ala Val Ala Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 19 227 PRT Artificial synthetic
19 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Leu Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Ile Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
20 227 PRT Artificial synthetic 20 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Leu Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 21 227 PRT Artificial synthetic
21 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Ile Val Trp
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150
155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 22 227 PRT
Artificial synthetic 22 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Tyr Leu Tyr Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Thr Val Thr Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 23 227 PRT Artificial synthetic 23 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Glu Leu Arg Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Glu Val Arg Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 24 227 PRT
Artificial synthetic 24 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Glu Leu Gln Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Thr Val Glu Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 25 227 PRT Artificial synthetic 25 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Arg Leu Gln Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Thr Val Arg Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 26 227 PRT
Artificial synthetic 26 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Glu Leu Tyr Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Thr Val Arg Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 27 227 PRT Artificial synthetic 27 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Met Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 28 227 PRT
Artificial synthetic 28 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Glu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 29 227 PRT Artificial synthetic 29 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Phe Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 30 227 PRT
Artificial synthetic 30 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 31 227 PRT Artificial synthetic 31 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Met Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 32 227 PRT
Artificial synthetic 32 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe His Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
33 227 PRT Artificial synthetic 33 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Ala Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 34 227 PRT Artificial synthetic
34 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Val Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
35 227 PRT Artificial synthetic 35 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Phe Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 36 227 PRT Artificial synthetic
36 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe His Ala Lys Val Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
37 227 PRT Artificial synthetic 37 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Gly Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 38 227 PRT Artificial synthetic
38 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
39 227 PRT Artificial synthetic 39 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Glu Leu
Arg Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Glu Val Arg Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 40 227 PRT Artificial synthetic
40 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Glu Leu Gln Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Thr Val Glu
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
41 227 PRT Artificial synthetic 41 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Arg Leu
Gln Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Thr Val Arg Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 42 227 PRT Artificial synthetic
42 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Glu Leu Tyr Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Thr Val Arg
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
43 227 PRT Artificial synthetic 43 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1
5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ala Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 44
227 PRT Artificial synthetic 44 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 45 227 PRT Artificial synthetic 45
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5
10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ala Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Ala Ala Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 46
227 PRT Artificial synthetic 46 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 47 227 PRT Artificial synthetic 47
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5
10 15 Gly Pro Gln Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 48
227 PRT Artificial synthetic 48 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 49 227 PRT Artificial synthetic 49
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5
10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Gly
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 50
227 PRT Artificial synthetic 50 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asn Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 51 227 PRT Artificial synthetic 51
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5
10 15 Gly Pro Glu Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Gly
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 52
227 PRT Artificial synthetic 52 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asn Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 53 227 PRT Artificial synthetic 53
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5
10 15 Gly Pro Glu Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Gln
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
54 227 PRT Artificial synthetic 54 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Glu Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 55 227 PRT Artificial synthetic
55 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Gln Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
56 227 PRT Artificial synthetic 56 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Thr Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 57 227 PRT Artificial synthetic
57 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Asn Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
58 227 PRT Artificial synthetic 58 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Ile Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 59 227 PRT Artificial synthetic
59 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ser Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
60 227 PRT Artificial synthetic 60 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Asn Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 61 227 PRT Artificial synthetic
61 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Gln His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ser Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
62 227 PRT Artificial synthetic 62 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Leu His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ser Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 63 227 PRT Artificial synthetic
63 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Leu Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
64 227 PRT Artificial synthetic 64 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Phe Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 65 227 PRT Artificial synthetic 65 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20
25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Leu Pro Ile 100 105 110 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150
155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 66 227 PRT
Artificial synthetic 66 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Tyr Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 67 227 PRT Artificial synthetic 67 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Asp 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 68 227 PRT
Artificial synthetic 68 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 69 227 PRT Artificial synthetic 69 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 70 227 PRT
Artificial synthetic 70 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 71 227 PRT Artificial synthetic 71 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 72 227 PRT
Artificial synthetic 72 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Glu Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 73 227 PRT Artificial synthetic 73 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Tyr Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 74 227 PRT
Artificial synthetic 74 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Tyr Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Leu Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 75 227 PRT Artificial synthetic 75 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Phe Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Glu Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 76 227 PRT Artificial synthetic
76 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Ile Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
77 227 PRT Artificial synthetic 77 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Gln
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 78 227 PRT Artificial synthetic
78 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Asn 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
79 227 PRT Artificial synthetic 79 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Gln 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 80 227 PRT Artificial synthetic
80 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Thr
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
81 227 PRT Artificial synthetic 81 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Phe Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 82 227 PRT Artificial synthetic
82 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Ile Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
83 227 PRT Artificial synthetic 83 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Ile Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 84 227 PRT Artificial synthetic
84 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Ile Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
85 227 PRT Artificial synthetic 85 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Ala Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 86 227 PRT Artificial
synthetic 86 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Ser Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 87 227 PRT Artificial synthetic 87 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Val Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 88 227 PRT Artificial
synthetic 88 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Gln Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 89 227 PRT Artificial synthetic 89 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Leu Lys
Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 90 227 PRT Artificial
synthetic 90 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Ile Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 91 227 PRT Artificial synthetic 91 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 92 227 PRT Artificial
synthetic 92 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Asn Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 93 227 PRT Artificial synthetic 93 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Phe
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 94 227 PRT Artificial
synthetic 94 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Asp 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 95 227 PRT Artificial synthetic 95 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 96 227 PRT Artificial
synthetic 96 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Asn 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 97 227 PRT Artificial synthetic
97 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Gln 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
98 227 PRT Artificial synthetic 98 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Asp 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 99 227 PRT Artificial synthetic
99 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Glu Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Asn 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
100 227 PRT Artificial synthetic 100 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Gln 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 101 227 PRT Artificial
synthetic 101 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Asn Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Asp 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 102 227 PRT Artificial synthetic 102 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Asn Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 103 227 PRT
Artificial synthetic 103 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asn Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Asn 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 104 227 PRT Artificial synthetic 104 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Asn Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Gln 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 105 227
PRT Artificial synthetic 105 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Gln Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Asp 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 106 227 PRT Artificial synthetic
106 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Gln Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Asn 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
107 227 PRT Artificial synthetic 107 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Gln Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Gln 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 108 227
PRT Artificial synthetic 108 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Glu Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 109 227 PRT Artificial synthetic
109 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Asp Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
110 227 PRT Artificial synthetic 110 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Asn Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 111 227 PRT Artificial
synthetic 111 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Tyr Leu Tyr Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Thr Val Thr Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 112 227 PRT Artificial synthetic 112 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 113 227 PRT
Artificial synthetic 113 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Ile Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Tyr Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 114 227 PRT Artificial synthetic 114 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Leu Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 115 227
PRT Artificial synthetic 115 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Ile Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Leu Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 116 227 PRT Artificial synthetic
116 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
117 227 PRT Artificial synthetic 117 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Glu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 118 227
PRT Artificial synthetic 118 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Asn Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 119 227 PRT Artificial synthetic
119 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Gln Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
120 227 PRT Artificial synthetic 120 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Thr Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 121 227 PRT Artificial
synthetic 121 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
His Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 122 227 PRT Artificial synthetic 122 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 123 227 PRT
Artificial synthetic 123 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ile Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 124 227 PRT Artificial synthetic 124 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 125 227
PRT Artificial synthetic 125 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 126 227 PRT Artificial synthetic
126 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Asp Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
127 227 PRT Artificial synthetic 127 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Ser Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 128 227 PRT Artificial
synthetic 128 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Asn Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100
105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220
Pro Gly Lys 225 129 227 PRT Artificial synthetic 129 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Gln Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 130 227 PRT
Artificial synthetic 130 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Thr Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 131 227 PRT Artificial synthetic 131 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu His Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 132 227
PRT Artificial synthetic 132 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Tyr Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 133 227 PRT Artificial synthetic
133 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ile Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
134 227 PRT Artificial synthetic 134 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Val Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 135 227 PRT Artificial
synthetic 135 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Phe Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 136 227 PRT Artificial synthetic 136 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Thr Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 137 227 PRT
Artificial synthetic 137 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro His Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 138 227 PRT Artificial synthetic 138 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Tyr Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 139 227
PRT Artificial synthetic 139 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Ala Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 140 227 PRT Artificial synthetic 140 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Thr Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 141 227
PRT Artificial synthetic 141 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Met Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 142 227 PRT Artificial synthetic
142 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Ala Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
143 227 PRT Artificial synthetic 143 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Thr Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 144 227 PRT Artificial
synthetic 144 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Met Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 145 227 PRT Artificial synthetic 145 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Met Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 146 227 PRT
Artificial synthetic 146 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Tyr Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 147 227 PRT Artificial synthetic 147 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Ala
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 148 227
PRT Artificial synthetic 148 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Thr Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 149 227 PRT Artificial synthetic
149 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Met Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 150 227 PRT Artificial synthetic
150 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 His Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
151 227 PRT Artificial synthetic 151 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Tyr
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 152 227 PRT Artificial
synthetic 152 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Phe Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 153 227 PRT Artificial synthetic 153 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Arg Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 154 227 PRT
Artificial synthetic 154 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Ser Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 155 227 PRT Artificial synthetic 155 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Thr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 156 227
PRT Artificial synthetic 156 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Leu Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 157 227 PRT Artificial synthetic
157 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Ile Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
158 227 PRT Artificial synthetic 158 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn His Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 159 227 PRT Artificial
synthetic 159 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser His Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 160 227 PRT Artificial synthetic 160 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Val Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220 Pro Gly Lys 225 161 227 PRT Artificial synthetic 161
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5
10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ile Pro Ile 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 162
227 PRT Artificial synthetic 162 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Phe Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 163 227 PRT Artificial
synthetic 163 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Arg Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 164 227 PRT Artificial synthetic 164 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro His Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 165 227 PRT
Artificial synthetic 165 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asp Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 166 227 PRT Artificial synthetic 166 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Glu Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 167 227
PRT Artificial synthetic 167 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Ala Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 168 227 PRT Artificial synthetic
168 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Thr Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
169 227 PRT Artificial synthetic 169 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Val Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 170 227 PRT Artificial
synthetic 170 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser His Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 171 227 PRT Artificial synthetic 171 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40
45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Asp Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 172 227 PRT Artificial
synthetic 172 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Glu Pro Ala Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 173 227 PRT Artificial synthetic 173 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Asn Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 174 227 PRT
Artificial synthetic 174 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Gln Pro Ala Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 175 227 PRT Artificial synthetic 175 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Val Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 176 227
PRT Artificial synthetic 176 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Thr Pro
Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 177 227 PRT Artificial synthetic
177 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala His Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
178 227 PRT Artificial synthetic 178 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Ile
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 179 227 PRT Artificial
synthetic 179 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Ala Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 180 227 PRT Artificial synthetic 180 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Thr 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 181 227 PRT
Artificial synthetic 181 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
His 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 182 227 PRT Artificial
synthetic 182 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Tyr 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 183 227 PRT Artificial synthetic 183 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ala 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 184 227 PRT
Artificial synthetic 184 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Ile Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 185 227 PRT Artificial synthetic 185 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Gln Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 186 227
PRT Artificial synthetic 186 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Ile Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 187 227 PRT Artificial synthetic
187 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Glu Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ala Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
188 227 PRT Artificial synthetic 188 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 189 227 PRT Artificial
synthetic 189 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Glu Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 190 227 PRT Artificial synthetic 190 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Val Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 191 227 PRT
Artificial synthetic 191 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Ile Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 192 227 PRT Artificial synthetic 192 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Leu Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 193
227 PRT Artificial synthetic 193 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Phe Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 194 227 PRT Artificial
synthetic 194 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Tyr Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 195 227 PRT Artificial synthetic 195 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val His Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 196 227 PRT
Artificial synthetic 196 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Thr Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 197 227 PRT Artificial synthetic 197 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Glu Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 198 227
PRT Artificial synthetic 198 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp Asp Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 199 227 PRT Artificial synthetic
199 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Glu Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
200 227 PRT Artificial synthetic 200 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Asn Asp Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 201 227 PRT Artificial
synthetic 201 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Gln Asp Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 202 227 PRT Artificial synthetic 202 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln His Asp
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 203 227 PRT
Artificial synthetic 203 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Thr
Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 204 227 PRT
Artificial synthetic 204 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Val Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 205 227 PRT Artificial synthetic 205 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asp Ser Ile Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 206 227
PRT Artificial synthetic 206 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Leu Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 207 227 PRT Artificial synthetic
207 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asp Ser Phe Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
208 227 PRT Artificial synthetic 208 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser His Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 209 227 PRT Artificial
synthetic 209 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asp Ser Glu Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 210 227 PRT Artificial synthetic 210 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 211 227 PRT
Artificial synthetic 211 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ala Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Tyr Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 212 227 PRT Artificial synthetic 212 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 213 227
PRT Artificial synthetic 213 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asn Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 214
227 PRT Artificial synthetic 214 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Leu Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 215 227 PRT Artificial
synthetic 215 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Asn Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 216 227 PRT Artificial synthetic 216 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ala Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 217 227 PRT
Artificial synthetic 217 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 218 227 PRT Artificial synthetic 218 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Asn Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ala Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 219 227
PRT Artificial synthetic 219 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Ile Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 220 227 PRT Artificial synthetic
220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ala Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
221 227 PRT Artificial synthetic 221 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Leu Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 222 227 PRT Artificial
synthetic 222 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Asn Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 223 227 PRT Artificial synthetic 223 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala His Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 224 227 PRT
Artificial synthetic 224 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ile Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
225 227 PRT Artificial synthetic 225 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Leu Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 226 227 PRT Artificial
synthetic 226 Asp Lys Thr His Thr Cys Pro Pro Cys Ala Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 227 227 PRT Artificial synthetic 227 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Asp Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 228 227 PRT
Artificial synthetic 228 Asp Lys Thr His Thr Cys Pro Pro Cys Ala
Ala Pro Asp Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 229 227 PRT Artificial synthetic 229 Asp
Lys Thr His Thr Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 230 227
PRT Artificial synthetic 230 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Thr His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 231 227 PRT Artificial synthetic
231 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val His His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
232 227 PRT Artificial synthetic 232 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Asp His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 233 227 PRT Artificial
synthetic 233 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Asn His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 234 227 PRT Artificial synthetic 234 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Thr Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 235 227 PRT
Artificial synthetic 235 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Leu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100
105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220
Pro Gly Lys 225 236 227 PRT Artificial synthetic 236 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Asn Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 237 227 PRT
Artificial synthetic 237 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Gln Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 238 227 PRT Artificial synthetic 238 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Thr Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 239 227
PRT Artificial synthetic 239 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu His Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 240 227 PRT Artificial synthetic
240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Ser Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
241 227 PRT Artificial synthetic 241 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Lys Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 242 227 PRT Artificial
synthetic 242 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Ile Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 243 227 PRT Artificial synthetic 243 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Tyr Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 244 227 PRT
Artificial synthetic 244 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Ile Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 245 227 PRT Artificial synthetic 245 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Thr Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 246 227 PRT Artificial synthetic 246 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20
25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 35 40 45 Glu Asp Pro Glu Val Glu Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150
155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 247 227 PRT
Artificial synthetic 247 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Arg Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 248 227 PRT Artificial synthetic 248 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Leu Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 249 227
PRT Artificial synthetic 249 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Tyr Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 250 227 PRT Artificial synthetic
250 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Trp Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
251 227 PRT Artificial synthetic 251 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Ser Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 252 227 PRT Artificial
synthetic 252 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Glu Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 253 227 PRT Artificial synthetic 253 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Arg Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 254 227 PRT
Artificial synthetic 254 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Leu Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 255 227 PRT Artificial synthetic 255 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Tyr Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 256 227
PRT Artificial synthetic 256 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Thr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys 225 257 227 PRT Artificial synthetic 257 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20
25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Glu Val Asp Gly
Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150
155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 258 227 PRT
Artificial synthetic 258 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Lys Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 259 227 PRT Artificial synthetic 259 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Trp Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 260 227
PRT Artificial synthetic 260 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Arg Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 261 227 PRT Artificial synthetic
261 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Ile Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
262 227 PRT Artificial synthetic 262 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Arg Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 263 227 PRT Artificial
synthetic 263 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Thr Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 264 227 PRT Artificial synthetic 264 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Asp Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 265 227 PRT
Artificial synthetic 265 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Ile Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 266 227 PRT Artificial synthetic 266 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Thr
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 267 227
PRT Artificial synthetic 267 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65
70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys His Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 268 227 PRT Artificial synthetic
268 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Ile Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
269 227 PRT Artificial synthetic 269 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 270 227 PRT Artificial
synthetic 270 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Asp Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 271 227 PRT Artificial synthetic 271 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Arg Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 272 227 PRT
Artificial synthetic 272 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ile Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 273 227 PRT Artificial synthetic 273 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Val Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 274 227
PRT Artificial synthetic 274 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Leu Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 275 227 PRT Artificial synthetic
275 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Tyr Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
276 227 PRT Artificial synthetic 276 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Leu Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 277 227 PRT Artificial
synthetic 277 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Ile Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 278 227 PRT Artificial synthetic 278 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20
25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Thr Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150
155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 279 227 PRT
Artificial synthetic 279 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Asp Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 280 227 PRT Artificial synthetic 280 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Thr Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 281 227
PRT Artificial synthetic 281 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ser Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 282 227 PRT Artificial synthetic
282 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Trp Pro Ile 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
283 227 PRT Artificial synthetic 283 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Met Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 284 227 PRT Artificial
synthetic 284 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Val Ile 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 285 227 PRT Artificial synthetic 285 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala His Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 286 227 PRT
Artificial synthetic 286 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Thr Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 287 227 PRT Artificial synthetic 287 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 His Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 288 227
PRT Artificial synthetic 288 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Ile Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 289 227 PRT
Artificial synthetic 289 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Tyr Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 290 227 PRT Artificial synthetic 290 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Ile Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 291 227
PRT Artificial synthetic 291 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110 Glu Thr Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 292 227 PRT Artificial synthetic
292 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Phe
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
293 227 PRT Artificial synthetic 293 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Asp Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 294 227 PRT Artificial
synthetic 294 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105
110 Glu Lys Arg Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 295 227 PRT Artificial synthetic 295 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Tyr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 296 227 PRT
Artificial synthetic 296 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ile Asp Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 297 227 PRT Artificial synthetic 297 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Ile Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Ile
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 298 227
PRT Artificial synthetic 298 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ile Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 299 227 PRT Artificial synthetic
299 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Asp Gly
1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu
His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150
155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 300 227 PRT
Artificial synthetic 300 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Ile Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Tyr Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 301 227 PRT Artificial synthetic 301 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Thr Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 302 227
PRT Artificial synthetic 302 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Ile Ser His 35 40 45 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 210 215 220 Pro Gly Lys 225 303 227 PRT Artificial synthetic
303 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Glu Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
304 227 PRT Artificial synthetic 304 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu
Pro Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys 225 305 227 PRT Artificial
synthetic 305 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65 70 75 80 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Tyr Pro Glu 100 105
110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro
Gly Lys 225 306 227 PRT Artificial synthetic 306 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro
Asp Val Ser Leu His Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Thr Val Thr Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp
Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Tyr Pro Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 307 227 PRT
Artificial synthetic 307 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Asp Gly 1 5 10 15 Gly Pro Asp Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Glu 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225 308 227 PRT Artificial synthetic 308 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asp Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Glu Ala Leu Pro Ala Pro Glu 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
* * * * *