MODIFIED Fc REGION OF ANTIBODY

Abstract

Polypeptides with improved stability as compared to that of a parent polypeptide were successfully obtained by modifying at least one amino acid in a loop region of the antibody Fc region. Furthermore, by combining multiple amino acid modifications in the loop region, polypeptides with maintained or enhanced Fc.gamma.R-binding activity as well as improved thermal stability, polypeptides with decreased Fc.gamma.R-binding activity as well as improved thermal stability, and polypeptides with not only improved thermal stability and adjusted Fc.gamma.R-binding activity but also decreased aggregate content, as compared to those of a parent polypeptide, were successfully obtained.

Description

TECHNICAL FIELD

[0001] The present invention provides antibody Fc regions in which the amino acid sequence of a naturally-occurring antibody Fc region has been modified, antibodies containing such an Fc region, pharmaceutical compositions containing such an antibody, and methods for producing them.

BACKGROUND ART

[0002] Antibodies are drawing attention as pharmaceuticals as they are highly stable in plasma and have few side effects. In particular, a number of IgG-type antibody pharmaceuticals are available on the market and many antibody pharmaceuticals are currently under development (Non-patent Documents 1 and 2).

[0003] Recently, active research has been conducted on enhancing antibody functions by introducing artificial amino acid modification into antibody Fc regions. Specifically, Fc regions with improved pharmacokinetics, Fc regions with enhanced ADCC activity, which is an effector function, and Fc regions with reduced ADCC activity in neutralizing antibodies have been studied (Non-Patent Documents 3-6). However, such Fc region modifications are known to cause adverse effects on physical properties of antibodies. For example, a modified Fc region with enhanced ADCC activity has been reported to show a decrease in melting temperature by approximately 20.degree. C. (Non-Patent Document 6). Furthermore, there are reports that modified Fc regions with reduced ADCC activity show a decrease in melting temperature by approximately 5.degree. C., readily undergo digestion by hydrolases, and readily degrade under acidic conditions (Non-Patent Documents 7-9). Furthermore, modified Fc regions with improved retention in blood have been reported to show reduced thermal stability and storage stability (Patent Document 1).

[0004] Thus, in most of the modified Fc regions discovered so far, the enhancement of their functions has resulted in loss of their excellent stability, which is one of the advantages of antibodies.

[0005] As an effort to improve stability, there is a report of a technique for amino acid modification in the CH2 domain to introduce cysteines (Non-Patent Document 10). It has been reported that formation of new disulfide bonds by introduction of cysteines increases the thermal stability by approximately 10.degree. C. to 20.degree. C. In this report, however, only the CH2 domain was evaluated for thermal stability; therefore, the thermal stability of the IgG form is unknown. Furthermore, the formation of additional disulfide bonds is expected to cause an increase in heterogeneity.

[0006] As described above, there has so far been no report on an Fc region that is excellent in both activity and stability.

[0007] Prior art documents related to the present invention are shown below:

PRIOR ART DOCUMENTS

Patent Documents

[0008] [Patent Document 1] WO2007/092772 [0009] [Patent Document 2] WO2010/085682

Non-patent Documents

[0009] [0010] [Non-patent Document 1] Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nature Biotechnology 23, 1073-1078 (2005) [0011] [Non-patent Document 2] Pavlou A K, Belsey M J., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 April; 59(3):389-96. [0012] [Non-patent Document 3] Hinton P R, Xiong J M, Johlfs M G, Tang M T, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 January 1; 176(1):346-56 [0013] [Non-patent Document 4] Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober R J, Ward E S., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 July; 15(7):637-40 [0014] [Non-patent Document 5] Oganesyan V, Damschroder M M, Leach W, Wu H, Dall'Acqua W F., Structural characterization of a mutated, ADCC-enhanced human Fc fragment., Mol Immunol. 2008 April; 45(7):1872-82 [0015] [Non-patent Document 6] Oganesyan V, Gao C, Shirinian L, Wu H, Dall'Acqua W F., Structural characterization of a human Fc fragment engineered for lack of effector functions., Biol Crystallogr. 2008 June; 64(Pt 6):700-4 [0016] [Non-patent Document 7] Liu H, Bulseco G G, Sun J., Effect of posttranslational modifications on the thermal stability of a recombinant monoclonal antibody., Immunol Lett. 2006 August; 106(2):144-53 [0017] [Non-patent Document 8] Gaza-Bulseco G, Liu H., Fragmentation of a recombinant monoclonal antibody at various pH., Pharm Res. 2008 August; 25(8):1881-90 [0018] [Non-patent Document 9] Raju T S, Scallon B J., Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain., Biochem Biophys Res Commun. 2006 March; 341(3):797-803 [0019] [Non-patent Document 10] Gong R, Vu B K, Feng Y, Prieto D A, Dyba M A, Walsh J D, Prabakaran P, Veenstra T D, Tarasov S G, Ishima R, Dimitrov D S., Engineered human antibody constant domains with increased stability., J Biol Chem. 2009 March; 284(21):14203-14210 [0020] [Non-patent Document 11] Remmele R L Jr, Callahan W J, Krishnan S, Zhou L, Bondarenko P V, Nichols A C, Kleemann G R, Pipes G D, Park S, Fodor S, Kras E, Brems D N., Active dimer of Epratuzumab provides insight into the complex nature of an antibody aggregate., J Pharm Sci. 2006 January; 95(1):126-45. [0021] [Non-patent Document 12] Rosenberg A S, Effects of Protein Aggregates: An Immunologic Perspective., AAPS J. 2006 August; 8(3); E501-E507

SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

[0022] The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide polypeptides whose stability has been improved by modifying amino acids in the antibody Fc region.

Means for Solving the Problems

[0023] The present inventors considered that, in the production of modified Fc regions, it would be desirable to enhance their function while maintaining antibody stability, or to recover stability that had been reduced due to functional enhancement.

[0024] The present inventors conducted dedicated studies, and as a result succeeded in obtaining polypeptides with an antibody Fc region which have been modified in at least one amino acid in a loop region of the Fc region and which thereby have improved stability as compared to that of a parent polypeptide.

[0025] Furthermore, by combining multiple amino acid modifications in the loop region, polypeptides with improved thermal stability and maintained or enhanced Fc.gamma.R-binding activity, and polypeptides with improved thermal stability and decreased Fc.gamma.R-binding activity, as compared to those of a parent polypeptide, were successfully obtained. Furthermore, polypeptides not only with improved thermal stability and adjusted Fc.gamma.R-binding activity but also with decreased aggregate content were successfully obtained.

[0026] More specifically, the present invention relates to the following:

[1] a polypeptide comprising an antibody Fc region, wherein at least one amino acid has been modified in a loop region of the Fc region, and the polypeptide has improved stability as compared to that of a parent polypeptide; [2] the polypeptide of [1], wherein the stability is assessed or determined using melting temperature (Tm) as an index; [3] the polypeptide of [1] or [2], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 236 (EU numbering), position 237 (EU numbering), position 238 (EU numbering), position 239 (EU numbering), position 247 (EU numbering), position 250 (EU numbering), position 265 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 271 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), position 327 (EU numbering), position 329 (EU numbering), position 330 (EU numbering), position 333 (EU numbering), position 335 (EU numbering), position 337 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering); [4] the polypeptide of any one of [1] to [3], which further has Fc.gamma.R-binding activity that is maintained or enhanced as compared to that of a parent polypeptide; [5] the polypeptide of any one of [1] to [3], which further has Fc.gamma.R-binding activity that is decreased as compared to that of a parent polypeptide; [6] the polypeptide of any one of [1] to [5], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering); [7] the polypeptide of any one of [1] to [4] and [6], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 266 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering); [8] the polypeptide of any one of [1] to [4], [6], and [7], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Ile, substitution of the amino acid at position 266 (EU numbering) with Ile, substitution of the amino acid at position 268 (EU numbering) with Gln, substitution of the amino acid at position 269 (EU numbering) with Asp, substitution of the amino acid at position 270 (EU numbering) with Glu, substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 300 (EU numbering) with Glu, substitution of the amino acid at position 324 (EU numbering) with His, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr; [9] the polypeptide of any one of [1] to [3], [5] and [6], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [10] the polypeptide of any one of [1] to [3], [5], [6], and [9], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with Gly, His, or Met; [11] the polypeptide of any one of [1] to [4], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 295 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering); [12] the polypeptide of any one of [1] to [4] and [11], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr; [13] the polypeptide of any one of [1] to [3] and [5], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [14] the polypeptide of any one of [1] to [3], [5], and [13], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [15] the polypeptide of any one of [1] to [3] and [5], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [16] the polypeptide of any one of [1] to [3], [5], and [15], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [17] the polypeptide of any one of [1] to [3] and [5], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [18] the polypeptide of any one of [1] to [3], [5], and [17], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys and Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [19] the polypeptide of any one of [1] to [3] and [5], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [20] the polypeptide of any one of [1] to [3], [5], and [19], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [21] the polypeptide of any one of [1] to [3] and [5], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [22] the polypeptide of any one of [1] to [3], [5], and [21], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [23] the polypeptide of any one of [1] to [4], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 247 (EU numbering), position 250 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering); [24] the polypeptide of any one of [1] to [4], and [23], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 247 (EU numbering) with Val, substitution of the amino acid at position 250 (EU numbering) with Phe, Ile, Met, Val, Trp, or Tyr, substitution of the amino acid at position 307 (EU numbering) with Ala, Gln, or Pro, substitution of the amino acid at position 309 (EU numbering) with Ala, Arg, or Pro, substitution of the amino acid at position 315 (EU numbering) with Ala, substitution of the amino acid at position 360 (EU numbering) with His, substitution of the amino acid at position 385 (EU numbering) with Asp, substitution of the amino acid at position 386 (EU numbering) with Pro, substitution of the amino acid at position 387 (EU numbering) with Glu, substitution of the amino acid at position 389 (EU numbering) with Ser, substitution of the amino acid at position 428 (EU numbering) with His, Trp, Tyr, or Phe, and substitution of the amino acid at position 433 (EU numbering) with Lys; [25] the polypeptide of any one of [1] to [3] and [5], wherein at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid position 298 (EU numbering) or position 309 (EU numbering); [26] the polypeptide of any one of [1] to [3], [5], and [25], wherein the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 298 (EU numbering) with Gly and substitution of the amino acid at position 309 (EU numbering) with Asp; [27] a method for improving stability of a polypeptide comprising an antibody Fc region as compared to that of a parent polypeptide by introducing at least one amino acid modification to a loop region of the Fc region; [28] the method of [27], wherein the stability is assessed or determined using melting temperature (Tm) as an index; [29] a method for producing a polypeptide comprising an antibody Fc region, which has at least one amino acid modification in a loop region of the Fc region, and has improved stability as compared to that of a parent polypeptide, wherein the method comprises the steps of: (a) introducing at least one amino acid modification to a polypeptide comprising an antibody Fc region at a loop region of the Fc region; (b) determining the stability of the polypeptide modified in step (a); and (c) selecting a polypeptide with improved stability as compared to that of the parent polypeptide; [30] a method for producing a polypeptide comprising an antibody Fc region, which has at least one amino acid modification in a loop region of the Fc region, and has improved stability as compared to that of a parent polypeptide, wherein the method comprises the steps of: (a) modifying a nucleic acid encoding the polypeptide so as to improve its stability as compared to that of the parent polypeptide; (b) introducing the modified nucleic acid into a host cell and culturing the cell to induce expression of the nucleic acid; and (c) collecting the polypeptide from the host cell culture; [31] the method of any one of [27] to [30], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 236 (EU numbering), position 237 (EU numbering), position 238 (EU numbering), position 239 (EU numbering), position 247 (EU numbering), position 250 (EU numbering), position 265 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 271 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), position 327 (EU numbering), position 329 (EU numbering), position 330 (EU numbering), position 333 (EU numbering), position 335 (EU numbering), position 337 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389

(EU numbering), position 428 (EU numbering), and position 433 (EU numbering); [32] the method of any one of [27] to [31], which further comprises the step of introducing a modification to maintain or enhance Fc.gamma.R-binding activity as compared to that of the parent polypeptide; [33] the method of any one of [27] to [31], which further comprises the step of introducing a modification to reduce Fc.gamma.R-binding activity as compared to that of the parent polypeptide; [34] the method of any one of [27] to [33], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering); [35] the method of any one of [27] to [32] and [34], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 266 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering); [36] the method of any one of [27] to [32], [34], and [35], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Ile, substitution of the amino acid at position 266 (EU numbering) with Ile, substitution of the amino acid at position 268 (EU numbering) with Gln, substitution of the amino acid at position 269 (EU numbering) with Asp, substitution of the amino acid at position 270 (EU numbering) with Glu, substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 300 (EU numbering) with Glu, substitution of the amino acid at position 324 (EU numbering) with His, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr; [37] the method of any one of [27] to [31], [33], and [34], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [38] the method of any one of [27] to [31], [33], [34], and [37], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with Gly, His, or Met; [39] the method of any one of [27] to [32], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 295 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering); [40] the method of any one of [27] to [32] and [39], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr; [41] the method of any one of [27] to [31] and [33], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [42] the method of any one of [27] to [31], [33], and [41], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [43] the method of any one of [27] to [31] and [33], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [44] the method of any one of [27] to [31], [33], and [43], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [45] the method of any one of [27] to [31] and [33], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [46] the method of any one of [27] to [31], [33], and [45], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [47] the method of any one of [27] to [31] and [33], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [48] the method of any one of [27] to [31], [33], and [47], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [49] the method of any one of [27] to [31] and [33], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering); [50] the method of any one of [27] to [31], [33], and [49], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly; [51] the method of any one of [27] to [32], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position(s) selected from the group consisting of position 247 (EU numbering), position 250 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering); [52] the method of any one of [27] to [32] and [51], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 247 (EU numbering) with Val, substitution of the amino acid at position 250 (EU numbering) with Phe, Ile, Met, Val, Trp, or Tyr, substitution of the amino acid at position 307 (EU numbering) with Ala, Gln, or Pro, substitution of the amino acid at position 309 (EU numbering) with Ala, Arg, or Pro, substitution of the amino acid at position 315 (EU numbering) with Ala, substitution of the amino acid at position 360 (EU numbering) with His, substitution of the amino acid at position 385 (EU numbering) with Asp, substitution of the amino acid at position 386 (EU numbering) with Pro, substitution of the amino acid at position 387 (EU numbering) with Glu, substitution of the amino acid at position 389 (EU numbering) with Ser, substitution of the amino acid at position 428 (EU numbering) with His, Trp, Tyr, or Phe, and substitution of the amino acid at position 433 (EU numbering) with Lys; [53] the method of any one of [27] to [31] and [33], wherein at least one or more amino acid mutations are introduced into the loop region of the Fc region at amino acid position 298 (EU numbering) or position 309 (EU numbering); [54] the method of any one of [27] to [31], [33], and [53], wherein the amino acid modification in the loop region of the Fc region is at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 298 with Gly and substitution of the amino acid at position 309 with Asp; [55] the method of any one of [27] to [54], wherein the modification is modification in a polypeptide comprising an Fc region of human IgG; [56] a nucleic acid encoding a polypeptide comprising an antibody Fc region, which has at least one amino acid modification in a loop region of the Fc region, and has improved stability as compared to that of a parent polypeptide; [57] a vector comprising the nucleic acid of [56]; [58] a host cell transformed with the vector of [57]; [59] a pharmaceutical composition comprising the polypeptide of any one of [1] to [26], or a polypeptide produced by the method of any one of [27] to [55]; [60] an agent for treating or preventing an immune-inflammatory disease or cancer, which comprises the pharmaceutical composition of [59]; [61] the agent of [60], wherein the immune-inflammatory disease is rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune bullous dermatosis, autoimmune adrenocortical inflammation, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Basedow's disease, juvenile diabetes, Addison's disease, myasthenia gravis, lenticular uveitis, systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, asthma, myodegeneration, cachexia, systemic scleroderma, localized scleroderma, Sjogren's syndrome, Behcet's disease, Reiter's syndrome, type I and type II diabetes, bone resorption disease, graft versus host reaction, ischemia reperfusion injury, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, malgias due to staining, aplastic anemia, hemolytic anemia, sudden thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome, Hashimoto's disease, pemphigus, IgA nephropathy, pollinosis, antiphospholipid antibody syndrome, polymyositis, Wegener's sarcoma, arteritis nodosa, mixed connective tissue disease, or fibromyalgia; [62] the agent of [60], wherein the cancer is pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatoma, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, cholangioma, bladder cancer, ureteral cancer, thyroid cancer, adrenal carcinoma, renal cancer, other glandular tissue cancers, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, or other solid organ tumors; [63] a method for treating or preventing an immune-inflammatory disease or cancer, comprising administering to a subject the polypeptide of any one of [1] to [26], or a polypeptide produced by the method of any one of [27] to [55]; [64] the polypeptide of any one of [1] to [26] or a polypeptide produced by the method of any one of [27] to [55], for use in treatment or prevention of an immune-inflammatory disease or cancer; [65] use of the polypeptide of any one of [1] to [26] or a polypeptide produced by the method of any one of [27] to [55] in the preparation of an agent for treating or preventing an immune-inflammatory disease or cancer; and [66] a method for producing an agent for treating or preventing an immune-inflammatory disease or cancer, comprising the step of using the polypeptide of any one of [1] to [26] or a polypeptide produced by the method of any one of [27] to [55].

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 shows the melting temperatures (Tm) of the produced antibodies.

[0028] FIG. 2-1 shows the chromatograms for aggregate content determination of TS20 to TS27 in which multiple modifications were combined.

[0029] FIG. 2-2 shows the chromatograms for aggregate content determination of TS28 to TS39 in which multiple modifications have been combined.

[0030] FIG. 2-3 shows the chromatograms for aggregate content determination of TS40 to TS43 in which multiple modifications have been combined.

[0031] FIG. 3 shows the relationship between the amino acid residues constituting the Fc region of IgG1, IgG2, IgG3, and IgG4 and Kabat EU numbering (which is herein also referred to as EU index).

[0032] FIG. 4 shows the sites of amino acid modification (L234-S239, D265-P271, Q295, Y296, S298, Y300, S324-S337; the shaded portions) in the loop region of the CH2 domain of Fc region B3 (SEQ ID NO: 16).

MODE FOR CARRYING OUT THE INVENTION

[0033] The present invention provides polypeptides having an antibody Fc region whose stability has been improved as compared to that of a parent polypeptide by introducing amino acid substitutions into a loop region of the Fc region.

[0034] Furthermore, the present invention provides methods for improving stability of a polypeptide having an antibody Fc region as compared to that of a parent polypeptide by introducing amino acid substitutions to a loop region of the antibody Fc region. Furthermore, the present invention provides methods for producing a polypeptide having an antibody Fc region whose stability has been improved as compared to that of a parent polypeptide by introducing amino acid substitutions to a loop region of the Fc region.

[0035] In the present invention, polypeptides generally refer to peptides or proteins of about ten or more amino acids in length. In addition, they are typically polypeptides of biological origin, but are not particularly limited. For example, they may be polypeptides composed of artificially designed sequences. They may also be any of naturally-occurring polypeptides, synthetic polypeptides, recombinant polypeptides, and such. Moreover, polypeptides may be antibodies. Preferred examples of polypeptides of the present invention include human IgG. When an antibody used is a human IgG, the type of its constant region is not limited, and human IgG isotypes (subclasses) such as IgG1, IgG2, IgG3, or IgG4 may be used.

[0036] Herein, "parent polypeptide" refers to a polypeptide which serves as a basis or reference in the production of polypeptides having an antibody Fc region of the present invention. More specifically, it can be a polypeptide having an antibody Fc region which has not yet been modified in at least one of the amino acids of the Fc region. The parent polypeptide in the present invention may be, for example, a polypeptide having an Fc region of a naturally-occurring IgG, or may be a polypeptide having an Fc region of IgG in which a modification other than the amino acid modifications of the present invention has been made to a naturally-occurring IgG.

[0037] Naturally-occurring IgGs refer to polypeptides that include an amino acid sequence identical to those of IgGs found in nature, and belong to a class of antibodies virtually encoded by immunoglobulin gamma genes. For example, naturally-occurring human IgG refers to naturally-occurring human IgG1, naturally-occurring human IgG2, naturally-occurring human IgG3, naturally-occurring human IgG4, and such. Naturally-occurring IgGs also include mutants and such that naturally occur from them.

[0038] The Fc region of a naturally-occurring IgG refers to an Fc region containing an amino acid sequence identical to that of the Fc region derived from an IgG found in nature. The Fc regions of naturally-occurring IgGs are shown in FIG. 3 (SEQ ID NOs: 11 to 14), and refer to, for example, an Fc region derived from naturally-occurring human IgG1, an Fc region derived from naturally-occurring human IgG2, an Fc region derived from naturally-occurring human IgG3, an Fc region derived from naturally-occurring human IgG4, and such. The Fc regions of naturally-occurring IgGs also include mutants and such that naturally occur from them.

[0039] In the present invention, amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991). Herein, the sites of amino acid modifications in the antibody Fc region are shown according to EU numbering based on Kabat's amino acid positions.

[0040] In the present invention, portions linking .alpha. helices and .beta. sheets are called loops, and there are no rules on their length or structure. In the present invention, a portion linking two .beta. sheets in the CH2 domain is referred to as a loop, a loop region, a loop portion, or a loop structure. Specific amino acid positions of the loop region subjected to modification in the present invention are selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 236 (EU numbering), position 237 (EU numbering), position 238 (EU numbering), position 239 (EU numbering), position 247 (EU numbering), position 250 (EU numbering), position 265 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 271 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), position 327 (EU numbering), position 329 (EU numbering), position 330 (EU numbering), position 333 (EU numbering), position 335 (EU numbering), position 337 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering).

[0041] "Fc region" refers to a region containing a fragment composed of a hinge portion or a part thereof, and CH2 and CH3 domains of an antibody molecule. An Fc region of IgG class means, for example, from cysteine at position 226 to the C terminus or from proline at position 230 to the C terminus according to Kabat's EU numbering (herein also referred to as EU index) (see FIG. 3), but is not limited thereto.

[0042] An Fc region may be obtained preferably by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies or such using a protease such as pepsin and then re-eluting a fraction adsorbed onto protein A column. The protease is not particularly limited as long as it can digest a full-length antibody so that Fab and F(ab')2 will be produced in a restrictive manner by appropriately setting the enzyme reaction conditions such as pH, and examples include pepsin and papain.

[0043] The polypeptides of the present invention include, for example, polypeptides having an Fc region with improved stability as compared to a parent polypeptide. Preferred embodiments of the polypeptides having an Fc region with improved stability as compared to a parent polypeptide include, for example, polypeptides in which at least one amino acid has been modified in a loop region of the Fc region.

[0044] The present invention provides antibody Fc regions containing an Fc region in which at least one or more amino acids have been modified in a loop region of the Fc region of an antibody (for example, human IgG (IgG1, IgG2, IgG3, and IgG4)) at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 236 (EU numbering), position 237 (EU numbering), position 238 (EU numbering), position 239 (EU numbering), position 247 (EU numbering), position 250 (EU numbering), position 265 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 271 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), position 327 (EU numbering), position 329 (EU numbering), position 330 (EU numbering), position 333 (EU numbering), position 335 (EU numbering), position 337 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering) of the IgG. For example, a polypeptide with improved stability as compared to a parent polypeptide can be provided by introducing the above-mentioned modifications to human IgG.

[0045] The present invention also provides antibody Fc regions containing an Fc region in which at least one or more amino acid mutations have been introduced into a loop region of the Fc region of an antibody (for example, human IgG (IgG1, IgG2, IgG3, and IgG4)) at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering) of the IgG.

[0046] Herein, "stability" means, for example, thermodynamic stability of a polypeptide, but is not limited thereto. Thermodynamic stability of a polypeptide can be assessed or determined, for example, by using the melting temperature (Tm) of a CH2 domain as an index. Thus, polypeptides of the present invention are preferably assessed or judged using melting temperature (Tm) as an index. Tm can be measured by CD (circular dichroism), DSC (differential scanning calorimetry), and DSF (differential scanning fluorimetry).

[0047] The above-mentioned methods used for evaluating thermal stability can evaluate the thermal stability of the CH2, CH3, and Fab domains individually, when a sample in the form of IgG is measured. When CH2 and CH3 of the Fc region are compared, CH2 has lower thermal stability; therefore, improving the thermal stability of CH2 may lead to improvement of thermal stability of the Fc region.

[0048] Furthermore, an IgG keeps a highly-controlled conformation, and the conformation and physical stability of the respective domains affect each other. That is, a modification introduced into a certain domain may have an effect on a different domain, resulting in changes in the conformation and physical stability of the entire IgG. Therefore, when evaluating effects of introduced modifications, a sample is desirably evaluated in the form of IgG. For the above-mentioned reasons, evaluation of thermal stability of the CH2 domain in the present specification has been carried out using modified antibodies produced in the form of IgG.

[0049] When CD is used, Tm is determined by observing the mean residue molar ellipticity (.theta.) changing with rising temperature. Devices for measuring CD include, for example, a circular dichroism dispersion meter (JASCO Corporation). When CD spectra are monitored at a suitable wavelength (for example, 208 nm or 222 nm) while increasing the temperature, .theta. increases at a certain temperature, and becomes constant at temperatures thereafter. The temperature corresponding to the midpoint between the .theta. at low temperatures and the .theta. at high temperatures is determined as Tm. For the measurement, for example, protein solutions prepared using citric acid, Tris, phosphate solution, and such may be used, and such solutions can be used at a concentration of several hundred .mu.g/mL.

[0050] When DSC is used, Tm is determined by observing the amount of heat changing with rising temperature. Measurement devices for DSC include MicroCal VP-DSC and Micro Cal Capillary DSC (both from GE Healthcare). When a protein solution and a buffer are enclosed in measurement cells, and temperature differences between the cells are measured while raising the temperature, the reaction becomes endothermic at a certain temperature. This temperature is determined as Tm. For the measurement, for example, protein solutions prepared using citrate buffer, TBS, PBS, histidine buffer, and such may be used, and such solutions can be used at a concentration of several ten .mu.g/mL to several hundred .mu.g/mL.

[0051] When DSF is used, Tm is determined by observing exposure of hydrophobic residues due to rising temperature, using a fluorescent reagent (for example, SYPRO Orange) that specifically binds to hydrophobic residues. A protein solution and a fluorescence reagent are mixed at an appropriate ratio. When fluorescence intensity is measured while raising the temperature using an RT-PCR instrument, an increase in the fluorescence intensity is observed at a certain temperature. This temperature is determined as Tm. Measurement devices for DSF include Rotor-Gene Q (QIAGEN), and CFX96 real-time PCR analysis system (Bio-Rad). For the measurements, for example, protein solutions prepared using PBS, histidine buffer, and such may be used, and such solutions can be used at a concentration of several ten .mu.g/mL to several hundred .mu.g/mL.

[0052] Herein, "the stability of a polypeptide is improved" means that, for example, as compared to the Tm of the CH2 domain in the Fc region of a parent polypeptide used as a control determined according to the above-mentioned methods, the Tm of the CH2 domain in the Fc region of a test polypeptide is improved by 0.1 degrees or more, preferably 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, 10 degrees or more, or 20 degrees or more.

[0053] Furthermore, the polypeptides of the present invention may also be polypeptides having an Fc region with not only improved stability as compared to that of a parent polypeptide but also maintained or enhanced binding activity to an Fc.gamma. receptor (which may also be herein referred to as Fc.gamma.R) as compared to that of the parent polypeptide. In the present invention, examples of the polypeptides having an Fc region with not only improved stability compared to that of a parent polypeptide but also maintained or enhanced Fc.gamma.R-binding activity as compared to that of the parent polypeptide include polypeptides having the amino acid positions described in any of TS1-TS8, TS20-TS27, TS44-TS50, TS52-TS55, or TS57-TS67 as shown below in the Examples.

(TS1-TS8)

[0054] Examples of the polypeptide with maintained or enhanced Fc.gamma.R-binding activity as compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced into the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 266 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering).

[0055] Preferred polypeptides in this embodiment include polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 234 (EU numbering), position 266 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering).

[0056] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Ile, substitution of the amino acid at position 266 (EU numbering) with Ile, substitution of the amino acid at position 268 (EU numbering) with Gln, substitution of the amino acid at position 269 (EU numbering) with Asp, substitution of the amino acid at position 270 (EU numbering) with Glu, substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 300 (EU numbering) with Glu, substitution of the amino acid at position 324 (EU numbering) with His, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr.

[0057] Even more preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 234 (EU numbering) with Ile, substitution of the amino acid at position 266 (EU numbering) with Ile, substitution of the amino acid at position 268 (EU numbering) with Gln, substitution of the amino acid at position 269 (EU numbering) with Asp, substitution of the amino acid at position 270 (EU numbering) with Glu, substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 300 (EU numbering) with Glu, substitution of the amino acid at position 324 (EU numbering) with His, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr.

(TS20-TS27)

[0058] Examples of the polypeptide with maintained or enhanced Fc.gamma.R-binding activity compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 295 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering).

[0059] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 295 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering).

[0060] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr.

[0061] Even more preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr.

(TS44-TS50, TS52-TS55, TS57-TS67) Examples of the polypeptide with maintained or enhanced Fc.gamma.R-binding activity compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 247 (EU numbering), position 250 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering).

[0062] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 247 (EU numbering), position 250 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering).

[0063] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 247 (EU numbering) with Val, substitution of the amino acid at position 250 (EU numbering) with Phe, Ile, Met, Val, Trp, or Tyr, substitution of the amino acid at position 307 (EU numbering) with Ala, Gln, or Pro, substitution of the amino acid at position 309 (EU numbering) with Ala, Arg, or Pro, substitution of the amino acid at position 315 (EU numbering) with Ala, substitution of the amino acid at position 360 (EU numbering) with His, substitution of the amino acid at position 385 (EU numbering) with Asp, substitution of the amino acid at position 386 (EU numbering) with Pro, substitution of the amino acid at position 387 (EU numbering) with Glu, substitution of the amino acid at position 389 (EU numbering) with Ser, substitution of the amino acid at position 428 (EU numbering) with His, Trp, Tyr, or Phe, and substitution of the amino acid at position 433 (EU numbering) with Lys.

[0064] Even more preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 247 (EU numbering) with Val, substitution of the amino acid at position 250 (EU numbering) with Phe, Ile, Met, Val, Trp, or Tyr, substitution of the amino acid at position 307 (EU numbering) with Ala, Gln, or Pro, substitution of the amino acid at position 309 (EU numbering) with Ala, Arg, or Pro, substitution of the amino acid at position 315 (EU numbering) with Ala, substitution of the amino acid at position 360 (EU numbering) with His, substitution of the amino acid at position 385 (EU numbering) with Asp, substitution of the amino acid at position 386 (EU numbering) with Pro, substitution of the amino acid at position 387 (EU numbering) with Glu, substitution of the amino acid at position 389 (EU numbering) with Ser, substitution of the amino acid at position 428 (EU numbering) with His, Trp, Tyr, or Phe, and substitution of the amino acid at position 433 (EU numbering) with Lys.

[0065] For example, when the polypeptide is an antibody, it may preferably be used as a cancer antibody, for which its effector functions are important.

[0066] Furthermore, a polypeptide of the present invention may also be a polypeptide having an Fc region with not only improved stability compared to that of its parent polypeptide but also decreased binding activity to an Fc.gamma. receptor as compared to that of the parent polypeptide. In the present invention, examples of the polypeptide having an Fc region with not only improved stability compared to that of its parent polypeptide but also decreased Fc.gamma.R-binding activity as compared to that of the parent polypeptide include polypeptides having the amino acid modification region of TS9-TS19, TS28-TS43, TS51, or TS56 as shown below in the Examples.

(TS9-TS19)

[0067] Examples of the polypeptide with decreased Fc.gamma.R-binding activity as compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0068] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0069] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with Gly, His, or Met.

[0070] Even more preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with

[0071] Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with Gly, His, or Met.

(TS28-TS34)

[0072] Examples of the polypeptide with decreased Fc.gamma.R-binding activity compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0073] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0074] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0075] Even more preferred polypeptides in this embodiment are polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

(TS28, TS29, TS36, TS37)

[0076] Examples of the polypeptide with decreased Fc.gamma.R-binding activity as compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0077] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0078] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0079] Even more preferred polypeptides in this embodiment are polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

(TS30, TS31, TS38, TS39)

[0080] Examples of the polypeptide with decreased Fc.gamma.R-binding activity compared with that of a parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0081] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0082] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0083] Even more preferred polypeptides in this embodiment are polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

(TS32, TS33, TS40, TS41)

[0084] Examples of the polypeptide with decreased Fc.gamma.R-binding activity as compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0085] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 234 (EU numbering), position 235 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0086] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0087] Even more preferred polypeptides in this embodiment are polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

(TS34, TS35, TS42, TS43)

[0088] Examples of the polypeptide with decreased Fc.gamma.R-binding activity compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid positions selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0089] Preferred polypeptides in this embodiment are polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 234 (EU numbering), position 235 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[0090] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0091] Even more preferred polypeptides in this embodiment are polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

(TS51, TS56)

[0092] Examples of the polypeptide with decreased Fc.gamma.R-binding activity compared to that of its parent polypeptide include polypeptides in which at least one or more amino acid mutations have been introduced to the loop region of the Fc region at amino acid position 298 (EU numbering) and position 309 (EU numbering).

[0093] Preferred polypeptides in this embodiment include polypeptides in which the positions of amino acid modification in the loop region of the Fc region are position 298 (EU numbering) and position 309 (EU numbering).

[0094] More preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 298 (EU numbering) with Gly and substitution of the amino acid at position 309 with Asp.

[0095] Even more preferred polypeptides in this embodiment include polypeptides in which the amino acid modifications in the loop region of the Fc region are substitution of the amino acid at position 298 (EU numbering) with Gly and substitution of the amino acid at position 309 with Asp.

[0096] For example, when the polypeptide is an antibody, it may preferably be used as a neutralizing antibody.

[0097] "Fc.gamma. receptors" refers to receptors that may bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and practically means any member of the family of proteins encoded by the Fc.gamma. receptor genes. In humans, this family includes Fc.gamma.RI (CD64) including isoforms Fc.gamma.RIa, Fc.gamma.RIb, and Fc.gamma.RIc; Fc.gamma.RI (CD32) including isoforms Fc.gamma.RIIa (including allotypes H131 (type H) and R131 (type R)), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and

[0098] 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), and any human Fc.gamma.Rs, Fc.gamma.R isoforms or allotypes yet to be discovered, but is not limited thereto. The Fc.gamma.R in the present invention includes not only human-derived Fc.gamma.Rs, but also mouse, rat, rabbit, and monkey-derived Fc.gamma.Rs but is not limited thereto, and may be derived from any organism. Mouse Fc.gamma.Rs include Fc.gamma.RI (CD64), Fc.gamma.RI (CD32), Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2 (CD16-2), and any mouse Fc.gamma.Rs, or Fc.gamma.R isoforms or allotypes yet to be discovered, but are not limited thereto. Preferred examples of Fc.gamma. receptors in the present invention include human Fc.gamma.I (CD64), Fc.gamma.IIA (CD32), Fc.gamma.IIB (CD32), Fc.gamma.IIIA (CD16), and/or Fc.gamma.IIIB (CD16).

[0099] The polynucleotide sequence and amino acid sequence of Fc.gamma.I are set forth in SEQ ID NOs: 1 (NM.sub.--000566.3) and 2 (NP.sub.--000557.1), respectively;

the polynucleotide sequence and amino acid sequence of Fc.gamma.IIA are set forth in SEQ ID NOs: 3 (BCO20823.1) and 4 (AAH20823.1), respectively; the polynucleotide sequence and amino acid sequence of Fc.gamma.IIB are set forth in SEQ ID NOs: 5 (BC146678.1) and 6 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of Fc.gamma.IIIA are set forth in SEQ ID NOs: 7 (BCO33678.1) and 8 (AAH33678.1), respectively; and the polynucleotide sequence and amino acid sequence of Fc.gamma.IIIB are set forth in SEQ ID NOs 9 (BC128562.1) and 10 (AAI28563.1), respectively (the RefSeq Registration number is indicated inside the parentheses).

[0100] In Fc.gamma.RIIa, there are two allotypes: one where the amino acid at position 131 of Fc.gamma.RIIa is histidine (type H) and the other where this amino acid is substituted with arginine (type R) (J. Exp. Med, 172: 19-25, 1990).

[0101] In the present invention, whether or not the binding activity of a polypeptide or an Fc region of the present invention towards each type of Fc.gamma.R is decreased, or maintained or enhanced can be determined, for example, as shown in the present Examples. Specifically, it can be determined by using BIACORE, which is a device for interaction analysis based on the surface plasmon resonance (SPR) phenomenon, and observing whether there is a decrease or an increase in the dissociation constant (KD) value obtained by allowing each type of Fc.gamma.R to flow as an analyte over a sensor chip onto which a polypeptide (antibody) has been immobilized or captured with Protein A, antigen peptide or such, or whether there is an increase or a decrease in the amount of change in the sensorgram value before and after allowing each type of Fc.gamma.R to flow as an analyte over a sensor chip onto which a polypeptide (antibody) has been immobilized or captured with Protein A, antigen peptide or such.

[0102] Specifically, the binding activity of an Fc region towards an Fc.gamma. receptor can be measured by the Amplified Luminescent Proximity Homogeneous Assay (ALPHA) Screen, the BIACORE method which utilizes the surface plasmon resonance (SPR) phenomena, or such, in addition to ELISA or fluorescence activated cell sorting (FACS) (Proc. Natl. Acad. Sci. USA (2006) 103 (11): 4005-4010).

[0103] ALPHA Screen is performed by ALPHA technology which uses two beads, a donor and an acceptor, based on the following principles. Luminescent signals are detected only when molecules bound to donor beads physically interact with molecules bound to the acceptor beads, and the two beads are in close proximity to each other. Laser-excited photosensitizer in the donor beads converts ambient oxygen to excited-state singlet oxygen. Singlet oxygen is dispersed around the donor beads, and when it reaches the adjacent acceptor beads, chemiluminescent reaction is induced in the beads, and light is ultimately emitted. When the molecules bound to the donor beads do not interact with the molecules bound to the acceptor beads, the chemiluminescent reaction does not take place because singlet oxygen produced by the donor beads does not reach the acceptor beads.

[0104] For example, a biotinylated polypeptide complex is bound to the donor beads, and Fc.gamma. receptor tagged with glutathione S transferase (GST) is linked to the acceptor beads. In the absence of a competing polypeptide complex having a mutant Fc region, the polypeptide complex having a wild-type Fc region interacts with the Fey receptor and produces 520-620 nm signals. The polypeptide complex having an untagged mutant Fc region competes with the polypeptide complex having a wild-type Fc region for interaction with the Fey receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence observed as a result of the competition.

[0105] Biotinylation of polypeptide complexes such as antibodies using Sulfo-NHS-biotin and such is well known. The method of expressing the Fey receptor and GST in a cell carrying a fusion gene produced by fusing a polynucleotide encoding the Fey receptor in frame with a polynucleotide encoding GST in an expressible vector, and performing purification using a glutathione column is appropriately adopted as a method for tagging an Fey receptor with GST. The obtained signals are preferably analyzed, for example, by fitting them to a one-site competition model which uses a non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0106] One of the substances (the ligand) in observation of an interaction is immobilized onto a gold thin film on a sensor chip, and by shining light from the reverse side of the sensor chip so that total reflection takes place at the interface between the gold thin film and glass, a portion of reduced reflection intensity is formed in part of the reflected light (SPR signal). When the other one of the substances (the analyte) in observation of an interaction is made to flow on the sensor chip surface and the ligand binds to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the sensor chip surface changes. The position of the SPR signal shifts as a result of this change in refractive index (on the other hand, the signal position returns when this binding dissociates). The Biacore system indicates the amount of shift mentioned above, or more specifically the time variable of mass by plotting the change in mass on the sensor chip surface on the ordinate as the measurement data (sensorgram). The amount of analyte bound to the ligand trapped on the sensor chip surface is determined from the sensorgram. Kinetic parameters such as association rate constants (ka) and dissociation rate constants (kd) are determined from the curves of the sensorgram, and the dissociation constants (KD) are determined from the ratio of these constants. In the BIACORE method, a method for measuring inhibition is preferably used. An example of the method for measuring inhibition is described in Proc. Natl. Acad. Sci USA (2006) 103 (11): 4005-4010.

[0107] In the present invention, a polypeptide with decreased Fc.gamma.R-binding activity (a polypeptide whose binding activity to Fc.gamma.R is decreased) refers to a polypeptide having at least one amino acid modification in the Fc region of a parent polypeptide (also called a polypeptide variant) which, when assayed in substantially the same amount as the parent polypeptide, binds to at least one type of Fc.gamma.R with a substantially lower binding affinity than the parent polypeptide.

[0108] For example, when the amount of the parent polypeptide bound to each Fc.gamma.R, as measured by the aforementioned method, is taken as 100, the amount of the polypeptide variant bound to each Fc.gamma.R (hereinafter referred to as "binding amount ratio") is preferably decreased to 80 or less, preferably 50 or less, 40 or less, 30 or less, 20 or less, or particularly preferably 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or 0.1 or less.

[0109] In the present invention, a polypeptide with enhanced Fc.gamma.R-binding activity (a polypeptide whose binding activity to Fc.gamma.R is enhanced) refers to a polypeptide variant which, when assayed in substantially the same amount as the parent polypeptide, binds to at least one type of Fc.gamma.R with a substantially higher binding affinity than the parent polypeptide.

[0110] For example, when the amount of the parent polypeptide bound to each Fc.gamma.R, as measured by the aforementioned method, is taken as 100, the amount of the polypeptide variant bound to each Fc.gamma.R (hereinafter referred to as "binding amount ratio") is preferably increased to 120 or more, 150 or more, 200 or more, or 300 or more.

[0111] A polypeptide with unchanged (maintained) Fc.gamma.R-binding activity (a polypeptide whose binding activity to Fc.gamma.R is unchanged (maintained)) refers to a polypeptide having at least one amino acid modification in the Fc region of a parent polypeptide (also called a polypeptide variant) which, when assayed in substantially the same amount as the parent polypeptide, binds to Fc.gamma.R with a binding affinity substantially unchanged from or equivalent to that of the parent polypeptide.

[0112] For example, when the amount of the parent polypeptide bound to each Fc.gamma.R, as measured by the aforementioned method, is taken as 100, the amount of the polypeptide variant bound to each Fc.gamma.R (hereinafter referred to as "binding amount ratio") is preferably 80 or more and 120 or less.

[0113] In the present invention, polypeptides (for example, human IgG) containing the above-mentioned modifications of the present invention may be further modified in other parts of the Fc region.

[0114] For example, such modifications to other parts of the Fc region include substituting Pro at position 238 (EU numbering) with Asp and/or substituting Leu at position 328 (EU numbering) with Glu in human IgG (IgG1, IgG2, IgG3, and IgG4). Polypeptides with maintained or decreased binding activities towards Fc.gamma.RI, Fc.gamma.RIIIa, and both R and H allotypes of Fc.gamma.RIIa, as well as enhanced Fc.gamma.RIIb-binding activity in comparison with a parent polypeptide can be provided by introducing alteration of substituting Pro at position 238 (EU numbering) with Asp or substituting Leu at position 328 (EU numbering) with Glu in human IgG.

[0115] Further alterations of the Fc region can be added to human IgG containing alterations by which Pro at position 238 (EU numbering) has been substituted with Asp and/or Leu at position 328 (EU numbering) has been substituted with Glu. Here, alteration refers to any one of, or a combination of amino acid substitutions, deletions, additions, and insertions. Additional alterations can be further included in addition to these alterations. An additional alteration can be selected from any one of, or combinations of amino acid substitutions, deletions, or modifications. For example, alterations that further decrease binding activity towards Fc.gamma.RI, Fc.gamma.RIIa (type H), Fc.gamma.RIIa (type R), or Fc.gamma.RIIIa can be added.

[0116] Preferred alterations among them are those that only reduce the binding activity towards Fc.gamma.RI, Fc.gamma.RIIa (type H), Fc.gamma.RIIa (type R), or Fc.gamma.RIIIa without reducing the binding activity toward Fc.gamma.RIIb. Preferred examples of such alterations include the following amino acid substitutions:

substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Pro at position 238 (EU numbering) with Phe, substitution of Asn at position 325 (EU numbering) with Met, substitution of Ser at position 267 (EU numbering) with Ile, substitution of Leu at position 328 (EU numbering) with Asp, substitution of Ser at position 267 (EU numbering) with Val, substitution of Leu at position 328 (EU numbering) with Trp, substitution of Ser at position 267 (EU numbering) with Gln, substitution of Ser at position 267 (EU numbering) with Met, substitution of Gly at position 236 (EU numbering) with Asp, substitution of Ala at position 327 (EU numbering) with Asn, substitution of Asn at position 325 (EU numbering) with Ser, substitution of Leu at position 235 (EU numbering) with Tyr, substitution of Val at position 266 (EU numbering) with Met, substitution of Leu at position 328 (EU numbering) with Tyr, substitution of Leu at position 235 (EU numbering) with Trp, substitution of Leu at position 235 (EU numbering) with Phe, substitution of Ser at position 239 (EU numbering) with Gly, substitution of Ala at position 327 (EU numbering) with Glu, substitution of Ala at position 327 (EU numbering) with Gly, substitution of Pro at position 238 (EU numbering) with Leu, substitution of Ser at position 239 (EU numbering) with Leu, substitution of Leu at position 328 (EU numbering) with Thr, substitution of Leu at position 328 (EU numbering) with Ser, substitution of Leu at position 328 (EU numbering) with Met, substitution of Pro at position 331 (EU numbering) with Trp, substitution of Pro at position 331 (EU numbering) with Tyr, substitution of Pro at position 331 (EU numbering) with Phe, substitution of Ala at position 327 (EU numbering) with Asp, substitution of Leu at position 328 (EU numbering) with Phe, substitution of Pro at position 271 (EU numbering) with Leu, substitution of Ser at position 267 (EU numbering) with Glu, substitution of Leu at position 328 (EU numbering) with Ala, substitution of Leu at position 328 (EU numbering) with Ile, substitution of Leu at position 328 (EU numbering) with Gln, substitution of Leu at position 328 (EU numbering) with Val, substitution of Lys at position 326 (EU numbering) with Trp, substitution of Lys at position 334 (EU numbering) with Arg, substitution of His at position 268 (EU numbering) with Gly, substitution of His at position 268 (EU numbering) with Asn, substitution of Ser at position 324 (EU numbering) with Val, substitution of Val at position 266 (EU numbering) with Leu, substitution of Pro at position 271 (EU numbering) with Gly, substitution of Ile at position 332 (EU numbering) with Phe, substitution of Ser at position 324 (EU numbering) with Ile, substitution of Glu at position 333 (EU numbering) with Pro, substitution of Tyr at position 300 (EU numbering) with Asp, substitution of Ser at position 337 (EU numbering) with Asp, substitution of Tyr at position 300 (EU numbering) with Gln, substitution of Thr at position 335 (EU numbering) with Asp, substitution of Ser at position 239 (EU numbering) with Asn, substitution of Lys at position 326 (EU numbering) with Leu, substitution of Lys at position 326 (EU numbering) with Ile, substitution of Ser at position 239 (EU numbering) with Glu, substitution of Lys at position 326 (EU numbering) with Phe, substitution of Lys at position 326 (EU numbering) with Val, substitution of Lys at position 326 (EU numbering) with Tyr, substitution of Ser at position 267 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Pro, substitution of Lys at position 326 (EU numbering) with His, substitution of Lys at position 334 (EU numbering) with Ala, substitution of Lys at position 334 (EU numbering) with Trp, substitution of His at position 268 (EU numbering) with Gln, substitution of Lys at position 326 (EU numbering) with Gln, substitution of Lys at position 326 (EU numbering) with Glu, substitution of Lys at position 326 (EU numbering) with Met, substitution of Val at position 266 (EU numbering) with Ile, substitution of Lys at position 334 (EU numbering) with Glu, substitution of Tyr at position 300 (EU numbering) with Glu, substitution of Lys at position 334 (EU numbering) with Met, substitution of Lys at position 334 (EU numbering) with Val, substitution of Lys at position 334 (EU numbering) with Thr, substitution of Lys at position 334 (EU numbering) with Ser, substitution of Lys at position 334 (EU numbering) with His, substitution of Lys at position 334 (EU numbering) with Phe, substitution of Lys at position 334 (EU numbering) with Gln, substitution of Lys at position 334 (EU numbering) with Pro, substitution of Lys at position 334 (EU numbering) with Tyr, substitution of Lys at position 334 (EU numbering) with Ile, substitution of Gln at position 295 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Asn, substitution of His at position 268 (EU numbering) with Ala, substitution of Ser at position 239 (EU numbering) with Asp, and substitution of Ser at position 267 (EU numbering) with Ala.

[0117] Furthermore, preferred alterations among these alterations are those that reduce the binding activity towards Fc.gamma.RIIa (type R) without reducing the binding activity towards Fc.gamma.RIIb. Preferred examples of such alterations include the following amino acid substitutions:

substitution of Gly at position 237 (EU numbering) with Trp, substitution of Leu at position 328 (EU numbering) with Asp, substitution of Gly at position 236 (EU numbering) with Asp, substitution of Ala at position 327 (EU numbering) with Asn, substitution of Ala at position 327 (EU numbering) with Gly, substitution of Ser at position 239 (EU numbering) with Leu, substitution of Pro at position 331 (EU numbering) with Trp, substitution of Pro at position 331 (EU numbering) with Tyr, substitution of Pro at position 331 (EU numbering) with Phe, substitution of Pro at position 271 (EU numbering) with Leu, substitution of Leu at position 328 (EU numbering) with Gln, substitution of Tyr at position 300 (EU numbering) with Asp, and substitution of Ser at position 239 (EU numbering) with Asn.

[0118] Furthermore, it is also possible to add alterations enhancing the binding activity towards Fc.gamma.RIIb by adding other alterations of the Fc region to human IgG containing substitution of Pro at position 238 (EU numbering) with Asp and/or substitution of Leu at position 328 (EU numbering) with Glu. Preferred alterations among such alterations are those that enhance only the binding activity towards Fc.gamma.RIIb without enhancing the binding activity towards Fc.gamma.RI, Fc.gamma.RIIa (type H), Fc.gamma.RIIa (type R), or Fc.gamma.RIIIa. Preferred examples of such alterations include the following amino acid substitutions:

substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Pro at position 238 (EU numbering) with Phe, substitution of Asn at position 325 (EU numbering) with Met, substitution of Ser at position 267 (EU numbering) with Ile, substitution of Leu at position 328 (EU numbering) with Asp, substitution of Ser at position 267 (EU numbering) with Val, substitution of Leu at position 328 (EU numbering) with Trp, substitution of Ser at position 267 (EU numbering) with Gln, substitution of Ser at position 267 (EU numbering) with Met, substitution of Gly at position 236 (EU numbering) with Asp, substitution of Ala at position 327 (EU numbering) with Asn, substitution of Asn at position 325 (EU numbering) with Ser, substitution of Leu at position 235 (EU numbering) with Tyr, substitution of Val at position 266 (EU numbering) with Met, substitution of Leu at position 328 (EU numbering) with Tyr, substitution of Leu at position 235 (EU numbering) with Trp, substitution of Leu at position 235 (EU numbering) with Phe, substitution of Ser at position 239 (EU numbering) with Gly, substitution of Ala at position 327 (EU numbering) with Glu, substitution of Ala at position 327 (EU numbering) with Gly, substitution of Pro at position 238 (EU numbering) with Leu, substitution of Ser at position 239 (EU numbering) with Leu, substitution of Leu at position 328 (EU numbering) with Thr, substitution of Leu at position 328 (EU numbering) with Ser, substitution of Leu at position 328 (EU numbering) with Met, substitution of Pro at position 331 (EU numbering) with Trp, substitution of Pro at position 331 (EU numbering) with Tyr, substitution of Pro at position 331 (EU numbering) with Phe, substitution of Ala at position 327 (EU numbering) with Asp, substitution of Leu at position 328 (EU numbering) with Phe, substitution of Pro at position 271 (EU numbering) with Leu, substitution of Ser at position 267 (EU numbering) with Glu, substitution of Leu at position 328 (EU numbering) with Ala, substitution of Leu at position 328 (EU numbering) with Ile, substitution of Leu at position 328 (EU numbering) with Gln, substitution of Leu at position 328 (EU numbering) with Val, substitution of Lys at position 326 (EU numbering) with Trp, substitution of Lys at position 334 (EU numbering) with Arg, substitution of His at position 268 (EU numbering) with Gly, substitution of His at position 268 (EU numbering) with Asn, substitution of Ser at position 324 (EU numbering) with Val, substitution of Val at position 266 (EU numbering) with Leu, substitution of Pro at position 271 (EU numbering) with Gly, substitution of Ile at position 332 (EU numbering) with Phe, substitution of Ser at position 324 (EU numbering) with Ile, substitution of Glu at position 333 (EU numbering) with Pro, substitution of Tyr at position 300 (EU numbering) with Asp, substitution of Ser at position 337 (EU numbering) with Asp, substitution of Tyr at position 300 (EU numbering) with Gln, substitution of Thr at position 335 (EU numbering) with Asp, substitution of Ser at position 239 (EU numbering) with Asn, substitution of Lys at position 326 (EU numbering) with Leu, substitution of Lys at position 326 (EU numbering) with Ile, substitution of Ser at position 239 (EU numbering) with Glu, substitution of Lys at position 326 (EU numbering) with Phe, substitution of Lys at position 326 (EU numbering) with Val, substitution of Lys at position 326 (EU numbering) with Tyr, substitution of Ser at position 267 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Pro, substitution of Lys at position 326 (EU numbering) with His, substitution of Lys at position 334 (EU numbering) with Ala, substitution of Lys at position 334 (EU numbering) with Trp, substitution of His at position 268 (EU numbering) with Gln, substitution of Lys at position 326 (EU numbering) with Gln, substitution of Lys at position 326 (EU numbering) with Glu, substitution of Lys at position 326 (EU numbering) with Met, substitution of Val at position 266 (EU numbering) with Ile, substitution of Lys at position 334 (EU numbering) with Glu, substitution of Tyr at position 300 (EU numbering) with Glu, substitution of Lys at position 334 (EU numbering) with Met, substitution of Lys at position 334 (EU numbering) with Val, substitution of Lys at position 334 (EU numbering) with Thr, substitution of Lys at position 334 (EU numbering) with Ser, substitution of Lys at position 334 (EU numbering) with His, substitution of Lys at position 334 (EU numbering) with Phe, substitution of Lys at position 334 (EU numbering) with Gln, substitution of Lys at position 334 (EU numbering) with Pro, substitution of Lys at position 334 (EU numbering) with Tyr, substitution of Lys at position 334 (EU numbering) with Ile, substitution of Gln at position 295 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Asn, substitution of His at position 268 (EU numbering) with Ala, substitution of Ser at position 239 (EU numbering) with Asp, and substitution of Ser at position 267 (EU numbering) with Ala.

[0119] Furthermore, preferred alterations among these alterations are those that reduce the binding activity towards Fc.gamma.RIIa (type R) without reducing the binding activity towards Fc.gamma.RIIb. Preferred examples of such alterations include the following amino acid substitutions:

substitution of Gly at position 237 (EU numbering) with Trp, substitution of Leu at position 328 (EU numbering) with Asp, substitution of Gly at position 236 (EU numbering) with Asp, substitution of Ala at position 327 (EU numbering) with Asn, substitution of Ala at position 327 (EU numbering) with Gly, substitution of Ser at position 239 (EU numbering) with Leu, substitution of Pro at position 331 (EU numbering) with Trp, substitution of Pro at position 331 (EU numbering) with Tyr, substitution of Pro at position 331 (EU numbering) with Phe, substitution of Pro at position 271 (EU numbering) with Leu, substitution of Leu at position 328 (EU numbering) with Gln, substitution of Tyr at position 300 (EU numbering) with Asp, and substitution of Ser at position 239 (EU numbering) with Asn.

[0120] Furthermore, for example, amino acid substitutions that improve FcRn-binding activity (J. Immunol. 2006 January 1; 176(1): 346-56; J Biol Chem. 2006 August 18; 281(33): 23514-24; Int. Immunol. 2006 December; 18(12): 1759-69; Nat Biotechnol. 2010 February; 28(2): 157-9.; WO 2006/019447; WO 2006/053301; and WO 2009/086320), and amino acid substitutions for improving antibody heterogeneity or stability (WO 2009/041613) may be introduced into the Fc region of an antibody.

[0121] In addition to the amino acid modifications of the present invention, the following modifications can be added as necessary.

[0122] In order to regulate the plasma retention property of antibodies, it is possible to combine the amino acid modifications of the present invention with amino acid modifications for altering the antibody isoelectric point value (pI value). Modifications of the constant regions include, for example, amino acid modifications at positions 250 and 428 (EU numbering) and such described in known publications (for example, J. Immunol. 2006, 176 (1):346-356; and Nat. Biotechnol. 1997 15 (7):637-640). Modifications of the variable regions include the amino acid modifications described in WO2007/114319 and WO2009/041643. Amino acids to be modified are preferably exposed on the surface of a polypeptide having an antigen-binding activity. When a polypeptide of the present invention has a heavy chain constant region, the modifications include, for example, amino acid substitution at position 196 (EU numbering) in the amino acid sequence of the heavy chain constant region. When the heavy chain constant region is that of IgG4, the plasma retention can be enhanced, for example, by substituting lysine at position 196 with glutamine and thereby reducing the pI value. Furthermore, the plasma retention can be regulated by altering the FcRn-binding ability. Amino acid modifications that alter the FcRn-binding ability include, for example, the amino acid substitutions in the antibody heavy chain constant region described in known publications (The Journal of Biological Chemistry vo1.276, No. 9 6591-6604, 2001; and Molecular Cell, Vol. 7, 867-877, 2001).

Modifications for Improving the Stability Under Acidic Conditions

[0123] When a polypeptide of the present invention has a heavy chain constant region of IgG4, the stable four-chain structure (H2L2 structure) is preferably maintained by suppressing the dissociation of IgG4 into half-molecules under acidic conditions. Thus, arginine at amino acid position 409 (EU numbering), which plays an important role in the maintenance of the four-chain structure (Immunology 2002, 105, 9-19), is preferably substituted with lysine, the residue of the IgG1 type, which maintains the stable four-chain structure even under acidic conditions. Such modifications can be used in combination with the amino acid modifications of the present invention.

Modifications for Reducing Heterogeneity

[0124] The amino acid modifications of the present invention may be combined with the methods described in WO2009/041613. Specifically, for example, when a polypeptide of the present invention has a heavy chain constant region of IgG1, it is possible to combine the modification for deleting the C-terminal two amino acids of the IgG1 heavy chain constant region, that is, glycine and lysine at positions 446 and 447 (EU numbering), with the amino acid modifications described in the present Examples so that the heterogeneity can be reduced.

Modifications for Suppressing Deamidation Reaction

[0125] The amino acid modifications of the present invention may be combined with amino acid modifications for suppressing deamidation reaction. It has been reported that deamidation reaction tends to occur particularly at a site where asparagine (N) and glycine (G) are adjacent to each other (--NG--) (Geiger et al., J. Bio. Chem. 1987; 262:785-794). When a polypeptide of the present invention has a site where asparagine and glycine are adjacent to each other, deamidation reaction can be suppressed by modifying this amino acid sequence. Specifically, for example, either or both of asparagine and glycine are substituted with other amino acids. More specifically, for example, asparagine is substituted with aspartic acid.

[0126] Preferred examples of polypeptides of the present invention include IgG antibodies. When an IgG antibody is used as the antibody, the type of constant region is not limited, and an IgG isotypes (subclasses) such as IgG1, IgG2, IgG3, and IgG4 can be used. IgG antibodies of the present invention are preferably human IgG, and more preferably human IgG1 or human IgG4. The amino acid sequences of the heavy-chain Fc regions of human IgG1 and human IgG4 are known. A plurality of allotype sequences due to genetic polymorphisms have been described in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242 for the human IgG1 Fc region, and any of the sequences may be used in the present invention.

<Substitution>

[0127] For example, for the purpose of modifying (a)-(c) listed below, amino acid residues can be substituted with other amino acid residues:

(a) polypeptide backbone structure in the sheet-structure or helical-structure region; (b) electric charge or hydrophobicity at the target site; or (c) size of the side chain.

[0128] Amino acid residues are classified into the following groups based on their general side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, and ile; (2) neutral hydrophilic: cys, ser, thr, asn, and gln; (3) acidic: asp and glu; (4) basic: his, lys, and arg; (5) residues that affect the chain orientation: gly and pro; and (6) aromatic: trp, tyr, and phe.

[0129] Substitution between amino acid residues within each of these amino acid groups is referred to as conservative substitution, and amino acid residue substitution between different groups is referred to as non-conservative substitution. Substitutions in the present invention may be conservative substitutions or non-conservative substitutions, or a combination of conservative substitutions and non-conservative substitutions.

[0130] Amino acid sequence alterations are produced by various methods known to those skilled in the art. Such methods include the site-directed mutagenesis method (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152: 271-275; Zoller, M J, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100: 468-500; Kramer, W, Drutsa, V, Jansen, H W, Kramer, B, Pflugfelder, M, and Fritz, H J (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12: 9441-9456; Kramer W, and Fritz H J (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel, T A (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 82: 488-492), the PCR mutation method, and the cassette mutation method, but are not limited thereto.

[0131] Amino acid modification of the present invention includes post-translational modification. A specific post-translational modification may be addition or deletion of a sugar chain. For example, in the IgG1 Fc region consisting of the amino acid sequence of SEQ ID NO: 11, the amino acid residue at position 297 (EU numbering) may be sugar chain-modified. The sugar-chain structure for the modification is not limited. Generally, antibodies expressed in eukaryotic cells comprise glycosylation in the Fc region. Therefore, antibodies expressed in cells such as those below are normally modified by some type of sugar chain:

[0132] antibody-producing cells of mammals

[0133] eukaryotic cells transformed with an expression vector comprising a DNA encoding an antibody

[0134] Eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are representative animal cells used in transformation with an expression vector comprising an antibody-encoding DNA. On the other hand, the Fc regions of the present invention include those without glycosylation at this site. Antibodies whose Fc region is not glycosylated can be obtained by expressing an antibody-encoding gene in prokaryotic cells such as Escherichia coli.

[0135] Specifically, for example, sialic acid may be added to the sugar chain of an Fc region (MAbs. 2010 September-October; 2(5): 519-27).

<Antibody>

[0136] Furthermore, the present invention provides antibodies with an Fc region in which any of the above-mentioned amino acid sequences is altered.

[0137] The term "antibody/antibodies" in the present invention is used in the broadest sense, and as long as the desired biological activity is shown, it encompasses any antibodies such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecific antibodies (for example, bispecific antibodies (which may be referred to as diabodies)), chimeric antibodies, and humanized antibodies.

[0138] The antibodies of the present invention are not limited in terms of the antigen type and origin, and may be any types of antibodies. The origin of the antibodies is not particularly limited, but examples include human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0139] Methods for producing the antibodies are well known to those skilled in the art, and for example, monoclonal antibodies may be produced by the hybridoma method (Kohler and Milstein, Nature 256: 495 (1975)), or the recombination method (U.S. Pat. No. 4,816,567). Alternatively, they may be isolated from a phage antibody library (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J.Mol.Biol. 222: 581-597 (1991)). In the present invention, monoclonal antibodies include humanized antibodies and chimeric antibodies.

[0140] In addition to the above methods, B cell cloning (use for identification and cloning of the coding sequence of each antibody, isolation thereof, and construction of expression vectors for producing each antibody (particularly IgG1, IgG2, IgG3, or IgG4) etc.) as described in Bernasconi et al. (Science (2002) 298, 2199-2202) or WO2008/081008 is appropriately used as a method for obtaining antibody genes.

[0141] Moreover, the antibodies of the present invention may have an altered sugar chain. Examples of antibodies whose sugar chain has been altered include, for example, antibodies with modified glycosylation (WO99/54342 and such), antibodies deficient in fucose attached to a sugar chain (WO00/61739, WO02/31140, WO2006/067847, WO2006/067913, and such), and antibodies having a sugar chain with bisecting GlcNAc (WO02/79255 and such).

[0142] A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies prepared by grafting the CDRs of a non-human animal antibody such as a mouse antibody to a human antibody and such are known. Common genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known as a method for grafting mouse antibody CDRs to human FRs. Furthermore, it is also possible to design an amino acid sequence in which mouse CDRs have been grafted on human FRs, and then synthesize a gene of this amino acid sequence. Gene synthesis services are provided by, for example, Life Technologies, GenScript, and other companies.

[0143] A vector for expressing a humanized antibody can be produced by inserting a DNA encoding an antibody variable region in which three CDRs and four FRs are ligated and a DNA encoding a human antibody Fc region into an expression vector so that these DNAs are fused in frame. After this integration vector is transfected into a host to establish recombinant cells, these cells are cultured, and the DNA encoding the humanized antibody is expressed to produce the humanized antibody in the culture of the cells (see, European Patent Publication No. EP 239,400, and International Patent Publication No. WO 1996/002576).

[0144] As necessary, an amino acid residue in an FR may be substituted so that the CDRs of a reshaped human antibody form an appropriate antigen-binding site. For example, a mutation can be introduced into the amino acid sequence of an FR by applying the PCR method used for grafting mouse CDRs to human FRs.

[0145] A desired human antibody can be obtained by DNA immunization using a transgenic animal having the complete repertoire of human antibody genes (see International Publication Nos. WO 1993/012227, WO 1992/003918, WO 1994/002602, WO 1994/025585, WO 1996/034096, and WO 1996/033735) as an animal for immunization.

[0146] Furthermore, technologies for obtaining a human antibody by panning using a human antibody library are known. For example, a human antibody V region is expressed on the surface of a phage as a single-chain antibody (scFv) by the phage display method. The scFv-expressing phage that binds to the antigen can be selected. The DNA sequence that encodes the V region of the antigen-bound human antibody can be determined by analyzing the genes of the selected phage. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be prepared by fusing the V-region sequence in-frame with the sequence of a desired human antibody C region, and then inserting this into a suitable expression vector. The expression vector is introduced into suitable expression cells such as those described above, and the human antibody can be obtained by expressing the human antibody-encoding gene. These methods are already known (see, International Publication Nos. WO 1992/001047, WO 1992/020791, WO 1993/006213, WO 1993/011236, WO 1993/019172, WO 1995/001438, and WO 1995/15388).

[0147] Herein, there is no particular limitation on the antigen, and it may be any antigens. Examples of such antigens preferably include ligands (cytokines, chemokines, and such), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes partly containing immunoglobulins.

[0148] Examples of cytokines include interleukins 1 to 18, colony stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-.alpha., IFN-.beta., IFN-.gamma., etc.), growth factors (EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors (TNF-.alpha. and TNF-.beta.), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF, and BMP.

[0149] Examples of chemokines include CC chemokines such as CCL1 to CCL28, CXC chemokines such as CXCL1 to CXCL17, C chemokines such as XCL1 and XCL2, and CX3C chemokines such as CX3CL1.

[0150] Examples of receptors include receptors belonging to receptor families such as the hematopoietic growth factor receptor family, cytokine receptor family, tyrosine kinase-type receptor family, serine/threonine kinase-type receptor family, TNF receptor family, G protein-coupled receptor family, GPI anchor-type receptor family, tyrosine phosphatase-type receptor family, adhesion factor family, and hormone receptor family. The receptors belonging to these receptor families and their characteristics have been described in many documents such as Cooke B A., King R J B., van der Molen H J. ed. New Comprehesive Biochemistry Vol. 18B "Hormones and their Actions Part II" pp. 1-46 (1988) Elsevier Science Publishers B V; Patthy (Cell (1990) 61 (1): 13-14); Ullrich et al. (Cell (1990) 61 (2): 203-212); Massague (Cell (1992) 69 (6): 1067-1070); Miyajima et al. (Annu Rev. Immunol. (1992) 10: 295-331); Taga et al. (FASEB J. (1992) 6, 3387-3396); Fantl et al. (Annu Rev. Biochem. (1993), 62: 453-481); Smith et al. (Cell (1994) 76 (6): 959-962); and Flower D R. (Biochim. Biophys. Acta (1999) 1422 (3): 207-234).

[0151] Examples of specific receptors belonging to the above-mentioned receptor families preferably include human or mouse erythropoietin (EPO) receptors (Blood (1990) 76 (1): 31-35; and Cell (1989) 57 (2): 277-285), human or mouse granulocyte-colony stimulating factor (G-CSF) receptors (Proc. Natl. Acad. Sci. USA. (1990) 87 (22): 8702-8706, mG-CSFR; Cell (1990) 61 (2): 341-350), human or mouse thrombopoietin (TPO) receptors (Proc Natl Acad Sci USA. (1992) 89 (12): 5640-5644; EMBO J. (1993) 12(7): 2645-53), human or mouse insulin receptors (Nature (1985) 313 (6005): 756-761), human or mouse Flt-3 ligand receptors (Proc. Natl. Acad. Sci. USA. (1994) 91 (2): 459-463), human or mouse platelet-derived growth factor (PDGF) receptors (Proc. Natl. Acad. Sci. USA. (1988) 85 (10): 3435-3439), human or mouse interferon (IFN)-.alpha. and .beta. receptors (Cell (1990) 60 (2): 225-234; and Cell (1994) 77 (3): 391-400), human or mouse leptin receptors, human or mouse growth hormone (GH) receptors, human or mouse interleukin (IL)-10 receptors, human or mouse insulin-like growth factor (IGF)-I receptors, human or mouse leukemia inhibitory factor (LIF) receptors, and human or mouse ciliary neurotrophic factor (CNTF) receptors.

[0152] Cancer antigens are antigens that are expressed as cells become malignant, and they are also called tumor-specific antigens. Abnormal sugar chains that appear on cell surfaces or protein molecules when cells become cancerous are also cancer antigens, and they are also called sugar-chain cancer antigens. Examples of cancer antigens preferably include GPC3 which is a receptor belonging to the GPI anchor-type receptor family mentioned above, and is also expressed in several cancers including liver cancer (Int J Cancer. (2003) 103 (4): 455-65), as well as EpCAM which is expressed in several cancers including lung cancer (Proc Natl Acad Sci USA. (1989) 86 (1): 27-31), CA19-9, CA15-3, and sialyl SSEA-1 (SLX).

[0153] MHC antigens are roughly classified into MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, and MHC class II antigens include HLA-DR, -DQ, and -DP.

[0154] Differentiation antigens may include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, and CDw130.

[0155] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE. Immunocomplexes at least contain any component of an immunoglobulin.

[0156] Variable regions that constitute the antibodies of the present invention may be variable regions that recognize any antigen. One or more amino acid residue alterations are allowed in the amino acid sequences constituting the antibody variable regions as long as their antigen-binding activities are maintained. When altering a variable region amino acid sequence, there is no particularly limitation on the site of alteration and number of amino acids altered. For example, amino acids present in CDR and/or FR can be altered appropriately. When altering amino acids in a variable region, the binding activity is preferably maintained without particular limitation; and for example, as compared to before alteration, the binding activity is 50% or more, preferably 80% or more, and more preferably 100% or more. Furthermore, the binding activity may be increased by amino acid alterations. For example, the binding activity may be 2-, 5-, 10-times higher or such than that before alteration.

[0157] When the antigen is a soluble antigen, KD (dissociation constant) can be used as a value representing the antigen-binding activity of an antibody of the present invention. When the antigen is a membrane antigen, apparent KD (apparent dissociation constant) can be used. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be determined by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plot, and flow cytometer.

[0158] As another index for comparing the antigen-binding activity of the antibodies of the present invention, for example, k.sub.d (dissociation rate constant) can be used when the antigen is a soluble antigen, whilst apparent k.sub.d (apparent dissociation rate constant) can be used when the antigen is a membrane antigen. k.sub.d (dissociation rate constant) and apparent k.sub.d (apparent dissociation rate constant) can be determined by methods known to those skilled in the art, such as Biacore (GE Healthcare) and flow cytometer.

[0159] In the antibodies of the present invention, alteration of amino acid sequence may be at least one of amino acid residue substitution, addition, deletion, and modification. There is no particular limitation on positions to be altered and the number of amino acids to be altered. Generally, 50 amino acids or less, preferably 30 amino acids or less, more preferably 10 amino acids or less (for example, 5 amino acids or less, or 3 amino acids or less) may be altered. Alternatively, for example, alteration of 20% or less amino acid residues, or specifically 10% or less amino acid residues (for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less) in the whole amino acid sequence is acceptable. In other words, antibodies containing an amino acid sequence sharing a homology (identity) of preferably 80% or higher, more preferably 90% or higher (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) with the original amino acid sequence are also included in the antibodies of the present invention.

[0160] For example, the modification of the N-terminal glutamine of a variable region into pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Thus, when the heavy-chain N terminus is glutamine, the antibodies of the present invention also include the variable regions in which the glutamine is modified to pyroglutamic acid.

[0161] Antibody variable regions of the present invention may have any sequences, and they may be antibody variable regions of any origin, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, humanized antibodies produced by humanizing these non-human antibodies, and human antibodies. "Humanized antibodies", also referred to as "reshaped human antibodies", are antibodies in which the complementarity determining regions (CDRs) of an antibody derived from a non-human mammal, for example, a mouse antibody, are transplanted into the CDRs of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). Their common genetic recombination techniques are also known (see, European Patent Application Publication No. EP 125023 and WO 96/02576). Furthermore, these antibodies may have various amino acid substitutions introduced into their variable regions to improve their antigen binding, pharmacokinetics, stability, and antigenicity. Variable regions of the antibodies of the present invention may be able to bind antigens repeatedly due to their pH dependability in antigen binding (WO 2009/125825).

[0162] There are .kappa. chain and .lamda. chain types in antibody light-chain constant regions, but any light chain constant regions may be used. Furthermore, light-chain constant regions of the present invention may be light-chain constant regions with amino acid alterations such as substitutions, deletions, additions, and/or insertions.

[0163] For the heavy chain Fc regions of an antibody of the present invention, for example, heavy chain Fc regions of human IgG antibodies may be used. Preferred heavy chain Fc regions are those of human IgG1 antibodies and human IgG4 antibodies.

[0164] Furthermore, polypeptides of the present invention may be made into Fc fusion protein molecules by linking to other proteins, physiologically active peptides, and such.

[0165] Examples of the other proteins and biologically active peptides include receptors, adhesion molecules, ligands, and enzymes, but are not limited thereto.

[0166] Preferred examples of Fc fusion protein molecules of the present invention include proteins with Fc region fused to a receptor protein that binds to a target, and such examples include TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat Med. 2003 January; 9(1): 47-52; BioDrugs. 2006; 20(3): 151-60). Furthermore, a protein to be fused to a polypeptide of the present invention may be any molecule as long as it binds to a target molecule, and examples include scFv molecules (WO 2005/037989), single-domain antibody molecules (WO 2004/058821; WO 2003/002609), antibody-like molecules (Current Opinion in Biotechnology 2006, 17: 653-658; Current Opinion in Biotechnology 2007, 18: 1-10; Current Opinion in Structural Biology 1997, 7: 463-469; and Protein Science 2006, 15: 14-27) such as DARPins (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229), and Adnectin (WO 2002/032925). Furthermore, antibodies and Fc fusion protein molecules may be multispecific antibodies that bind to multiple types of target molecules or epitopes.

[0167] Furthermore, the antibodies of the present invention include antibody modification products. Such antibody modification products include, for example, antibodies linked with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Such antibody modification products can be obtained by chemically modifying antibodies of the present invention. Methods for modifying antibodies are already established in this field.

[0168] The antibodies of the present invention may also be bispecific antibodies. "Bispecific antibody" refers to an antibody that has in a single molecule variable regions that recognize different epitopes. The epitopes may be present in a single molecule or in different molecules.

[0169] The polypeptides of the present invention can be prepared by the methods known to those skilled in the art. For example, the antibodies can be prepared by the methods described below, but the methods are not limited thereto

[0170] A DNA encoding an antibody heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest and DNA encoding an antibody light chain, are expressed. A DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can be prepared, for example, by obtaining a DNA encoding the Fc region of a natural heavy chain, and introducing an appropriate substitution so that a codon encoding a particular amino acid in the Fc region encodes another amino acid of interest.

[0171] Alternatively, a DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can also be prepared by designing and then chemically synthesizing a DNA encoding a protein in which one or more amino acid residues in the Fc region of the natural heavy chain are substituted with other amino acids of interest. The position and type of amino acid substitution are not particularly limited. Furthermore, alteration is not limited to substitution, and alteration may be any of deletion, addition, or insertion, or combination thereof.

[0172] Alternatively, a DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can be prepared as a combination of partial DNAs. Such combinations of partial DNAs include, for example, the combination of a DNA encoding a variable region and a DNA encoding an Fc region, and the combination of a DNA encoding an Fab region and a DNA encoding an Fc region, but are not limited thereto. Furthermore, a DNA encoding a light chain can similarly be prepared as a combination of partial DNAs.

[0173] Methods for expressing the above-described DNAs include the methods described below. For example, a heavy chain expression vector is constructed by inserting a DNA encoding a heavy chain variable region into an expression vector along with a DNA encoding a heavy chain Fc region. Likewise, a light chain expression vector is constructed by inserting a DNA encoding a light chain variable region into an expression vector along with a DNA encoding a light chain Fc region. Alternatively, these heavy and light chain genes may be inserted into a single vector.

[0174] When inserting a DNA encoding the antibody of interest into an expression vector, the DNA is inserted so that the antibody is expressed under the control of an expression-regulating region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. In such cases, an appropriate combination of host and expression vector may be used.

[0175] Examples of the vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. Alternatively, when aiming to subclone and excise cDNA, in addition to the vectors described above, pGEM-T, pDIRECT, pT7, and such can be used.

[0176] Expression vectors are particularly useful when using vectors for producing the polypeptides of the present invention. For example, when a host cell is E. coli such as JM109, DH5a, HB101, and XL1-Blue, the expression vectors must carry a promoter that allows efficient expression in E. coli, for example, lacZ promoter (Ward et al., Nature (1989) 341: 544-546; FASEB J. (1992) 6: 2422-2427; its entirety are incorporated herein by reference), araB promoter (Better et al., Science (1988) 240: 1041-1043; its entirety are incorporated herein by reference), T7 promoter, or such. Such vectors include pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen), pEGFP, or pET (in this case, the host is preferably BL21 that expresses T7 RNA polymerase) in addition to the vectors described above.

[0177] The vectors may contain signal sequences for polypeptide secretion. As a signal sequence for polypeptide secretion, a pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169: 4379; its entirety are incorporated herein by reference) may be used when a polypeptide is secreted into the E. coli periplasm. The vector can be introduced into host cells by lipofectin method, calcium phosphate method, and DEAE-Dextran method, for example.

[0178] In addition to E. coli expression vectors, the vectors for producing the polypeptides of the present invention include mammalian expression vectors (for example, pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17): p5322; its entirety are incorporated herein by reference), pEF, and pCDM8), insect cell-derived expression vectors (for example, the "Bac-to-BAC baculovirus expression system" (Gibco-BRL) and pBacPAK8), plant-derived expression vectors (for example, pMH1 and pMH2), animal virus-derived expression vectors (for example, pHSV, pMV, and pAdexLcw), retroviral expression vectors (for example, pZIPneo), yeast expression vectors (for example, "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expression vectors (for example, pPL608 and pKTHSO), for example. When aiming for expression in animal cells such as CHO, COS, and NIH3T3 cells, the vectors must have a promoter essential for expression in cells, for example, SV40 promoter (Mulligan et al., Nature (1979) 277: 108; its entirety are incorporated herein by reference), MMTV-LTR promoter, EF1.alpha. promoter (Mizushima et al., Nucleic Acids Res. (1990) 18: 5322; its entirety are incorporated herein by reference), CAG promoter (Gene. (1990) 18: 5322; its entirety are incorporated herein by reference), and CMV promoter, and more preferably they have a gene for selecting transformed cells (for example, a drug resistance gene that allows evaluation using an agent (neomycin, G418, or such)). Vectors with such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13, for example.

[0179] In addition, the following method can be used for stable gene expression and gene copy number amplification in cells: CHO cells deficient in a nucleic acid synthesis pathway are introduced with a vector that carries a DHFR gene which compensates for the deficiency (for example, pCHOI), and the vector is amplified using methotrexate (MTX). Alternatively, the following method can be used for transient gene expression: COS cells with a gene expressing SV40 T antigen on their chromosome are transformed with a vector with an SV40 replication origin (pcD and such). Replication origins derived from polyoma virus, adenovirus, bovine papilloma virus (BPV), and such can also be used. To amplify gene copy number in host cells, the expression vectors may further carry selection markers such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase (dhfr) gene.

[0180] Antibodies can be collected, for example, by culturing transformed cells, and then separating the antibodies from the inside of the transformed cells or from the culture media. Antibodies can be separated and purified using an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, lq, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0181] Furthermore, the present invention provides methods for altering a polypeptide to produce a polypeptide with improved stability as compared to the parent polypeptide. Thus, the present invention relates to methods for improving the stability of a polypeptide having an antibody Fc region as compared to the parent polypeptide by introducing at least one amino acid alteration in a loop region of the Fc region. In the present methods, melting temperature (Tm) is preferably used as an index for evaluating or judging the stability.

[0182] In addition, the present invention provides methods for producing a polypeptide with improved stability as compared to a parent polypeptide.

[0183] As an embodiment of the production methods of the present invention, for example, a method for producing a polypeptide which has an antibody Fc region, at least one of whose amino acids in a loop region of the Fc region is altered, and which has improved stability as compared to the parent antibody, is provided.

[0184] For example, such methods include methods including the following steps:

(a) adding at least one amino acid alteration to the Fc region of a polypeptide having an antibody Fc region; (b) measuring the stability of the polypeptide altered in step (a); and (c) selecting a polypeptide with improved stability as compared to the parent polypeptide.

[0185] Preferred embodiments include a method for producing a polypeptide which has antibody Fc region, at least one of whose amino acids in a loop region of the Fc region is altered, and which has improved stability as compared to the parent polypeptide, wherein the method includes the following steps:

(a) altering a nucleic acid encoding the polypeptide so that its stability is improved as compared to the parent peptide; (b) introducing the nucleic acid into host cells and culturing them to induce expression; and (c) collecting the polypeptide from the host cell culture.

[0186] Furthermore, polypeptides (antibodies) and Fc fusion protein molecules produced by this production method are also included in the present invention.

[0187] In preferred embodiments of the above-mentioned methods, for example, a nucleic acid encoding a polypeptide having an Fc region of antibody (such as human IgG) such that at least one or more amino acids are altered at amino acid position(s) of the loop region of the Fc region selected from the group consisting of: position 234 (EU numbering), position 235 (EU numbering), position 236 (EU numbering), position 237 (EU numbering), position 238 (EU numbering), position 239 (EU numbering), position 247 (EU numbering), position 250 (EU numbering), position 265 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 271 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), position 327 (EU numbering), position 329 (EU numbering), position 330 (EU numbering), position 333 (EU numbering), position 335 (EU numbering), position 337 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering).

[0188] In the above-mentioned methods or production methods (which may be simply referred to as "methods"), it is preferred to further reduce the binding activity towards Fc.gamma.R. In order to make modifications to maintain or enhance the binding activity towards Fc.gamma.R as compared to the parent polypeptide, for example, the steps of altering the amino acid alteration sites of TS1-TS8, TS20-TS27, TS44-TS50, TS52-TS55, and TS57-TS67 as shown in the Examples described below, may be included.

[0189] Alternatively, it is preferred to further maintain or enhance the binding activity towards Fc.gamma.R in the above-mentioned methods. In order to make modifications to reduce the binding activity towards Fc.gamma.R as compared to the parent polypeptide, for example, the steps of altering the amino acid alteration sites of TS9-19, TS28-43, TS51, and TS56 as shown in the Examples described below, may be included.

[0190] The alteration sites of TS1-TS67 are as mentioned above.

[0191] In the above-mentioned methods, for example, it is preferred to alter amino acids in a polypeptide that has an Fc region of antibody (such as human IgG).

[0192] Furthermore, the present invention provides nucleic acids encoding a polypeptide which has an antibody Fc region, at least one of whose amino acids in a loop region of the Fc region is altered, and which has improved stability as compared to the parent polypeptide. The nucleic acids of the present invention may be in any form such as DNA and RNA.

[0193] The present invention also provides vectors carrying the above-described nucleic acids of the present invention. The type of vector can be appropriately selected by those skilled in the art depending on the host cells to be introduced with the vector. The vectors include, for example, those described above.

[0194] Furthermore, the present invention relates to host cells transformed with the above-described vectors of the present invention. Appropriate host cells can be selected by those skilled in the art. The host cells include, for example, those described above.

<Pharmaceutical Compositions>

[0195] The present invention provides pharmaceutical compositions comprising a polypeptide or Fc fusion protein molecule of the present invention.

[0196] The pharmaceutical compositions of the present invention can be formulated, in addition to a polypeptide or Fc-fusion protein molecule of the present invention described above, with pharmaceutically acceptable carriers by known methods. For example, the compositions can be used parenterally, when the antibodies are formulated in a sterile solution or suspension for injection using water or any other pharmaceutically acceptable liquid. For example, the compositions can be formulated by appropriately combining the antibodies or Fc-fusion protein molecules with pharmaceutically acceptable carriers or media, specifically, sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binding agents, and such, by mixing them at a unit dose and form required by generally accepted pharmaceutical implementations. Specific examples of the carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hardened castor oil 60, saccharose, carboxymethyl cellulose, corn starch, inorganic salt, and such. The content of the active ingredient in such a formulation is adjusted so that an appropriate dose within the required range can be obtained.

[0197] Sterile compositions for injection can be formulated using vehicles such as distilled water for injection, according to standard protocols.

[0198] Aqueous solutions used for injection include, for example, physiological saline and isotonic solutions containing glucose or other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These can be used in conjunction with suitable solubilizers such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and non-ionic surfactants such as Polysorbate 80.TM. and HCO-50.

[0199] Oils include sesame oils and soybean oils, and can be combined with solubilizers such as benzyl benzoate or benzyl alcohol. These may also be formulated with buffers, for example, phosphate buffers or sodium acetate buffers; analgesics, for example, procaine hydrochloride; stabilizers, for example, benzyl alcohol or phenol; or antioxidants. The prepared injections are typically aliquoted into appropriate ampules.

[0200] The administration is preferably carried out parenterally, and specifically includes injection, intranasal administration, intrapulmonary administration, and percutaneous administration. For example, injections can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0201] Furthermore, the method of administration can be appropriately selected according to the age and symptoms of the patient. A single dosage of the pharmaceutical composition containing an antibody or a polynucleotide encoding an antibody can be selected, for example, from the range of 0.0001 to 1,000 mg per kg of body weight. Alternatively, the dosage may be, for example, in the range of 0.001 to 100,000 mg/patient. However, the dosage is not limited to these values. The dosage and method of administration vary depending on the patient's body weight, age, and symptoms, and can be appropriately selected by those skilled in the art.

[0202] In the present invention, pharmaceutical compositions containing a polypeptide of the present invention mentioned above are useful as an active ingredient of therapeutic agents or preventive agents for immunological inflammatory diseases, cancer, and such. Without being limited thereto, the term "immunological inflammatory diseases" includes rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune blistering diseases, autoimmune adrenocortical disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Basedow's disease, juvenile diabetes, Addison's disease, myasthenia gravis, lens-induced uveitis, systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, asthma, muscle degeneration, cachexia, systemic scleroderma, localized scleroderma, Sjogren's syndrome, Behchet's disease, Reiter's syndrome, type I and type II diabetes, bone resorption disorder, graft-versus-host reaction, ischemia-reperfusion injury, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, malgias due to staining, aplastic anemia, hemolytic anemia, idiopathic thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome, Hashimoto's thyroiditis, pemphigus, IgA nephropathy, pollinosis, antiphospholipid antibody syndrome, polymyositis, Wegener's granulomatosis, arteritis nodosa, mixed connective tissue disease, and fibromyalgia. In the present invention, "cancer" means a physiological state in mammals that is typically characterized by uncontrolled cell growth, or refers to such a physiological state. In the present invention, the type of cancer is not particularly limited, but includes the following. Carcinoma (epithelial cancer) includes pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatoma, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, cholangioma, bladder cancer, ureteral cancer, thyroid cancer, adrenal cancer, renal cancer, and other glandular tissue cancers. Sarcoma (non-epithelial cancer) includes liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, and other solid organ tumors such as melanoma and brain tumor.

[0203] As used herein, the three-letter and single-letter codes for respective amino acids are as follows:

Alanine: Ala (A)

Arginine: Arg (R)

Asparagine: Asn (N)

[0204] Aspartic acid: Asp (D)

Cysteine: Cys (C)

Glutamine: Gln (Q)

[0205] Glutamic acid: Glu (E)

Glycine: Gly (G)

Histidine: His (H)

Isoleucine: Ile (I)

Leucine: Leu (L)

Lysine: Lys (K)

Methionine: Met (M)

Phenylalanine Phe (F)

Proline: Pro (P)

Serine: Ser (S)

Threonine: Thr (T)

Tryptophan: Trp (W)

Tyrosine: Tyr (Y)

Valine: Val (V)

[0206] All prior art documents cited herein are incorporated by reference in their entirety.

EXAMPLES

[0207] Herein below, the present invention will be specifically described with reference to the Examples, but it is not to be construed as being limited thereto. In the present Examples, the sites of amino acids modified in the Fc region were numbered according to the EU numbering system (see, Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

Example 1

Improvement of Thermal Stability Through Modification of the CH2 Domain Loop Region

[0208] Generally, .beta. sheet structure is known to readily undergo structural changes and reduction in thermal stability by amino acid mutations (Biochemistry 1994; 33:5510-5517; and Nature 1994; 367:660-663). Therefore, in the present invention, the loop regions of the Fc region were selected for mutagenesis to improve the thermal stability.

[0209] The amino acids of L234-5239, D265-P271, Q295, Y296, 5298, Y300, S324-S337 in the loop region of the CH2 domain of Fc region B3 (SEQ ID NO: 16) were substituted with each of 18 amino acids not including the original amino acid and cysteine, to produce Fc mutants (FIG. 4). These are called Fc variants. Anti-GPC3 antibodies were produced using these Fc variants. Antibodies composed of an H chain which consists of variable region GpH7 (SEQ ID NO: 17) and an Fc variant as the Fc region and an L chain which consists of variable region GpL16 (SEQ ID NO: 18) and constant region k0 (SEQ ID NO: 19) (hereinafter denoted as GpH7-Fc variant/GpL16-k0) were produced according to the method of Reference Example 1.

[0210] The melting temperature (Tm) of the produced antibodies was evaluated according to the method of Reference Example 2. In the following, unless otherwise noted, "Tm" refers to the Tm of the CH2 domain when a sample in the form of IgG was subjected to measurement. The obtained Tm data are shown in FIG. 1.

[0211] The Tm of B3, which did not contain mutations (GpH7-B3/GpL16-k0: GpH7 (SEQ ID NO: 17), B3 (SEQ ID NO: 16), GpL16 (SEQ ID NO: 18), k0 (SEQ ID NO: 19)), was approximately 68.degree. C., and mutants showing a higher Tm than this value are shown in Tables 1-1 and 1-2.

TABLE-US-00001 TABLE 1-1 Mutated residue Amino acid after mutation Tm (.degree. C.) L234 G 69.5 A 68.8 V 69.7 F 68.5 P 68.9 M 68.9 I 69.1 K 70.8 R 70.3 S 69.3 T 69.2 Y 69.0 H 69.0 N 69.1 Q 69.4 W 68.9 L235 G 69.0 A 68.8 V 69.2 F 68.9 P 68.9 M 69.0 I 69.1 K 70.7 R 70.6 S 68.9 T 68.4 Y 68.8 N 68.1 G236 A 68.4 P 68.9 L 68.4 K 69.3 R 68.6 T 68.9 H 68.2 N 68.1 G237 R 70.1 P238 I 68.1 S239 M 68.4 K 73.2 R 72.2 T 68.2 H 68.2 Q 71.2 D265 G 69.0 V266 I 69.1 L 69.7 S267 P 70.4 K 68.5 H268 A 68.5 V 68.1 F 69.6 P 69.9 M 70.0 I 69.6 L 69.9 K 70.4 R 69.2 T 68.1 Y 68.5 Q 68.7 W 68.5 E269 A 68.6 V 68.2 D 68.6 K 68.3 R 68.9 S 68.7 T 68.1 H 68.6

[0212] Table 1-2 is a continuation of Table 1-1.

TABLE-US-00002 TABLE 1-2 Mutated residue Amino acid after mutation Tm (.degree. C.) D270 A 70.0 F 71.4 M 69.2 E 69.3 T 68.6 Y 71.2 H 71.4 N 70.2 Q 68.4 W 70.4 P271 D 68.4 Q295 M 70.8 L 69.8 Y296 G 70.9 F 69 D 69.6 E 69 S 70.6 T 68.3 N 69.9 Q 70.8 S298 G 72 A 68.1 F 68.7 M 69.7 Y300 G 68.5 P 69.9 D 68.4 E 68.6 S324 F 69 I 68.4 K 68.2 R 68.8 Y 68.6 H 69.3 N 69.7 N325 G 71.4 A 69.9 V 70.1 F 68.7 M 71.2 I 70.3 L 70.4 S 71 T 70.9 H 71.3 K326 G 69.6 A 69.2 P 68.4 R 68.8 S 69.8 N 68.9 A327 F 68.6 M 69 L 69.4 K 68.4 R 69.1 N 68.2 P329 A 68.7 A330 V 68.5 M 68.2 I 68.2 K 68.2 Y 69 H 69.5 N 68.2 E333 V 68.4 Q 68.4 T335 V 68.4 S337 R 68.1

Example 2

Preparation and Evaluation of IgG1 with Novel Fc Regions that Improve Thermal Stability

[0213] Based on the data from Example 1 and structural information (Nature 2000; 406: 267-273), several mutations presumed to be particularly effective for improving thermal stability were selected. One or more of the selected mutations were introduced into the H chains of the anti-GPC3 antibody (GpH7-Gld/GpL16-k0: GpH7 (SEQ ID NO: 17), Gld (SEQ ID NO: 15), GpL16 (SEQ ID NO: 18), k0 (SEQ ID NO: 19)) and the anti-IL6R antibody (MHO-Gld/MLO-k0: GpH7 (SEQ ID NO: 17), Gld (SEQ ID NO: 15), MLO (SEQ ID NO: 21), k0 (SEQ ID NO: 19)) to produce TS1 to TS19 (SEQ ID NOs: 26-44) shown in Table 2. At the same time, to evaluate effects on improvement of thermal stability by introduction of disulfide bond(s) in IgG form, the mutations of m01 and m02 reported in a prior document (J Biol. Chem. 2009; 284: 14203-14210) were introduced to the H chain of anti-IL6R to produce the TSm01 and TSm02 variants (SEQ ID NOs: 24 and 25). Each antibody was expressed and purified by the method of Reference Example 1.

TABLE-US-00003 TABLE 2 Variant name Alteration sites SEQ ID NO: G1d WT 15 TS01 L234I 26 TS02 V266I/H268Q/E269D/D270E 27 TS03 Q295M 28 TS04 Q295M/Y300E 29 TS05 Q295L 30 TS06 K326S/A330H 31 TS07 K326S/S324H/A330H 32 TS08 K326A/A330Y 33 TS09 L234K/L235K 34 TS10 L234K/L235R 35 TS11 L234R/L235K 36 TS12 L234R/L235R 37 TS13 S267P/H268M/D270F 38 TS14 H268M/D270F 39 TS15 H268K/D270F 40 TS16 Q295M/Y296G/S298G 41 TS17 S239K/N325G 42 TS18 S239K/N325M 43 TS19 S239K/N325H 44 TSm01 L242C/K334C 24 TSm02 V240C/K334C 25

[0214] Using the prepared antibodies, Tm values were compared by the method described in Reference Example 2. The results of Tm measurements are shown in Table 3.

TABLE-US-00004 TABLE 3 Tm (.degree. C.) Constant region name anti-GPC3 anti-IL6R TS1 69.5 70.5 TS2 69.8 70.7 TS3 71.1 72.4 TS4 62.8 63.1 TS5 70.1 71.4 TS6 70.4 71.6 TS7 69.8 70.8 TS8 70.0 71.0 TS9 n.t. 73.4 TS10 71.6 73.8 TS11 71.8 73.7 TS12 71.9 73.9 TS13 73.7 78.4 TS14 72.3 74.2 TS15 72.5 75.3 TS16 73.5 80.0 TS17 74.2 78.9 TS18 73.6 77.9 TS19 73.7 78.6 B3 69.1 n.t. TSm01 n.t. n.d. TSm02 n.t. 74.5 G1 69.4 70.4

[0215] In the results, the samples that were not evaluated are indicated as "n.t." (not tested), and the samples for which Tm was difficult to detect are indicated as "n.d." (not detected).

[0216] From the variants of TS1-TS19, the variants of TS1-3 and TS5-19 showed improvement of Tm when both the H chain sequence of anti-GPC3 and that of anti-IL6R were used. On the other hand, TS4 showed a decrease in Tm for both H chain sequences. The alteration of Q295M, which was introduced into TS4, improved Tm by approximately 2.degree. C. by itself alone (TS3), but was found to result in decreased Tm when the Y300E alteration was simultaneously introduced. According to the structural information, Q295 and Y300 are located opposite to each other on the same loop. The Q295M substitution may enhance the amino acid side chain interaction with Y300. However, it is speculated that simultaneous introduction of the Q295M and Y300E substitutions eliminated the interaction between the amino acid side chains.

[0217] As shown above, it was demonstrated that a combination of mutations improving Tm further improves Tm except for mutations having bad influence on structure. As such, in addition to TS1-3 and TS5-19 disclosed herein, it is possible to produce more combinations of Tm-improving alterations based on structural information.

[0218] With regard to TSm01 and TSm02, which were evaluated at the same time, the prior document (J Biol. Chem. 2009; 284:14203-14210) demonstrated that they increased Tm about 10.degree. C. to 20.degree. C. when the CH2 domain alone was expressed and evaluated for Tm. However, in the present examination where they were evaluated for Tm in the IgG form, multiple denaturation points were detected for TSm01, and it was difficult to clearly determine the thermal denaturation of the CH2 domain. This suggests a high possibility that TSm01 is structurally heterogeneous.

[0219] Furthermore, the Tm of TSm02 increased only by approximately 4.degree. C. as compared to that of IgG1. This demonstrates that the introduction of disulfide bonds is less effective in improving Tm in the IgG form, and the mutations found by the present inventors are more effective for improving Tm.

[0220] Tm of Fab of the anti-GPC3 antibody used in this examination is 74.7.degree. C. Therefore, with regard to TS13 to TS19, in which mutations have been introduced into the H chain of anti-GPC3, it was presumed that the fluorescence transition curves of CH2 and Fab would overlap, making accurate calculation of Tm difficult. Accordingly, as it was considered inappropriate to use the H chain of anti-GPC3 to test combinations effect on Tm, subsequent examinations were carried out using the H chain of anti-IL6R.

Example 3

Assessment of Binding of Novel Fc Regions to Human Fc.gamma. Receptors

[0221] Regarding modification of antibody Fc regions, there are reports of Fc regions with enhanced or reduced effector functions such as ADCC activity (reference: current opinion, 2009, 20, 685-691). Antibodies with enhanced effector functions may be useful mainly as antibodies for cancer therapy, and antibodies with reduced effector functions may be useful as, for example, neutralizing antibodies or receptor-Fc fusions such as Enbrel and Orencis. It is important that they are used appropriately depending on their respective purposes.

[0222] The CH2 domain, which was modified in this examination, is known to be involved in interaction with several human Fc.gamma. receptors (hereinafter denoted as hFcgRs) which affect effector functions. Therefore, the binding of TS1 and TS19 prepared by using the H chain of anti-IL6R produced in Example 2 to hFcgRs was measured, according to the method described in Reference Example 3. The measurement results are summarized in Table 4, where the binding of each antibody to each hFcgR relative to the binding of G1 to each hFcgR, which was taken as 100, was calculated.

TABLE-US-00005 TABLE 4 Variant name hFcgRIa hFcgRIIa (R) hFcgRIIa (H) hFcgRIIb hFcgRIIIa (F) hFcgRIIIa (V) TS1 96.5 67.0 76.8 61.5 62.4 83.5 TS2 98.3 67.3 123.0 61.6 159.0 130.0 TS3 97.5 89.6 115.3 84.2 63.8 90.1 TS4 99.3 148.4 105.9 219.9 73.9 99.2 TS5 97.3 128.8 142.1 157.5 86.6 106.1 TS6 100.8 122.7 120.3 142.2 103.0 90.6 TS7 99.8 116.6 118.1 125.9 98.8 86.2 TS8 102.3 119.9 100.6 132.3 164.6 124.4 TS9 -1.9 1.3 0.5 1.4 1.1 0.5 TS10 0.2 1.4 0.7 3.0 2.2 1.1 TS11 -0.2 1.5 1.3 3.4 2.6 1.4 TS12 -1.0 0.6 0.9 2.8 1.6 1.1 TS13 63.0 1.2 1.2 3.4 1.8 1.4 TS14 87.1 3.4 29.4 7.1 5.5 10.1 TS15 80.8 4.5 30.6 9.1 5.9 10.1 TS16 96.8 25.3 15.9 21.7 13.8 28.9 TS17 9.0 2.7 1.0 6.0 4.4 1.9 TS18 11.5 7.9 2.3 10.9 3.9 1.8 TS19 10.7 3.5 2.3 8.1 3.8 4.2

[0223] The results showed that while the binding of TS1 to TS8 to hFcgRs was equivalent to that of G1, the binding of TS9-19 to hFcgRs was weaker than that of G1.

Example 4

Production and Evaluation of Novel Fc Regions with Combined Mutations that Maintain Binding Ability to hFcgRs

[0224] When mutations with improving stability are introduced, it is important that antibodies for cancer therapy, whose effector functions are important, retain binding ability to hFcgRs. The mutations of TS1 to TS8, which were found to maintain binding ability to hFcgRs in Example 3, were combined to produce new variants TS20 to TS27 as shown in Table 5 (SEQ ID NOs: 45 to 52). Each antibody was expressed and purified by the method described in Reference Example 1. The prepared antibodies were subjected to Tm assessment by the method of Reference Example 2, and the results are shown in Table 6.

TABLE-US-00006 TABLE 5 Variant name Alteration sites SEQ ID NO: TS20 Q295M/K326S/A330H 45 TS21 Q295M/K326A/A330Y 46 TS22 Q295L/K326S/A330H 47 TS23 Q295L/K326A/A330Y 48 TS24 Q295M/K326S/A330Y 49 TS25 Q295M/K326A/A330H 50 TS26 Q295L/K326S/A330Y 51 TS27 Q295L/K326A/A330H 52

TABLE-US-00007 TABLE 6 Variant name Tm (.degree. C.) TS20 73.4 TS21 72.8 TS22 72.3 TS23 71.7 TS24 73.4 TS25 72.8 TS26 72.2 TS27 71.7 G1 70.4

[0225] Furthermore, binding ability to hFcgRs was measured by the method described in Reference Example 3. Binding of each antibody to each hFcgR was calculated relative to the binding of G1 to each hFcgR, which was taken as 100, and the results are shown in Table 7.

TABLE-US-00008 TABLE 7 Variant name hFcgRIa hFcgRIIa (R) hFcgRIIa (H) hFcgRIIb hFcgRIIIa (F) hFcgRIIIa (V) TS20 105.8 116.5 132.1 130.9 105.0 91.2 TS21 107.2 113.1 114.0 121.2 164.2 124.3 TS22 107.7 152.7 157.2 231.9 138.2 107.4 TS23 109.0 152.9 144.7 226.2 219.6 136.7 TS24 106.9 106.9 122.3 113.5 155.1 121.4 TS25 104.8 122.9 124.7 137.0 113.5 92.8 TS26 106.8 145.3 151.5 208.4 204.4 134.0 TS27 106.3 157.8 151.1 241.5 147.6 108.7

[0226] For all variants of TS20 to TS27, combining the mutations resulted in improved Tm while maintaining the binding ability to hFcgRs. The variants which were the most effective to improve Tm were TS20 and TS24, and they increase Tm approximately 3.degree. C. as compared to G1.

Example 5

Production and Evaluation of Novel Fc Regions with Combined Mutations that Reduce Binding Ability to hFcgRs

[0227] When mutations for improving stability are introduced into neutralizing antibodies, it is preferred to have as low effector functions as possible. The mutations of TS9 to TS19, which were found out to reduce binding affinity to hFcgRs in Example 3, were combined to produce new variants TS28 to TS43 as shown in Table 8 (SEQ ID NOs: 53 to 68). Each antibody was expressed and purified by the method described in Reference Example 1. The prepared antibodies were subjected to Tm assessment by the method of Reference Example 2, and the results are shown in Table 9.

TABLE-US-00009 TABLE 8 Variant name Alteration sites SEQ ID NO: TS28 L234K/L235K/S239K/H268K/D270F/Q295M/Y296G/S298G/N325G 53 TS29 L234K/L235K/S239K/H268K/D270F/Q295M/Y296G/S298G/N325H 54 TS30 L234K/L235K/S239K/Q295M/Y296G/S298G/N325G 55 TS31 L234K/L235K/S239K/Q295M/Y296G/S298G/N325H 56 TS32 L234K/L235K/H268K/D270F/Q295M/Y296G/S298G/N325G 57 TS33 L234K/L235K/H268K/D270F/Q295M/Y296G/S298G/N325H 58 TS34 L234K/L235K/Q295M/Y296G/S298G/N325G 59 TS35 L234K/L235K/Q295M/Y296G/S298G/N325H 60 TS36 L234K/L235R/S239K/H268K/D270F/Q295M/Y296G/S298G/N325G 61 TS37 L234K/L235R/S239K/H268K/D270F/Q295M/Y296G/S298G/N325H 62 TS38 L234K/L235R/S239K/Q295M/Y296G/S298G/N325G 63 TS39 L234K/L235R/S239K/Q295M/Y296G/S298G/N325H 64 TS40 L234K/L235R/H268K/D270F/Q295M/Y296G/S298G/N325G 65 TS41 L234K/L235R/H268K/D270F/Q295M/Y296G/S298G/N325H 66 TS42 L234K/L235R/Q295M/Y296G/S298G/N325G 67 TS43 L234K/L235R/Q295M/Y296G/S298G/N325H 68

TABLE-US-00010 TABLE 9 Variant name Tm (.degree. C.) TS28 85.8 TS29 84.6 TS30 86.6 TS31 86.2 TS32 84.9 TS33 83.5 TS34 85.3 TS35 84.8 TS36 86.0 TS37 85.0 TS38 86.6 TS39 85.9 TS40 85.1 TS41 83.8 TS42 85.4 TS43 84.9 G1 70.4

[0228] Furthermore, binding ability to hFcgRs was measured by the method described in Reference Example 3. Binding of each antibody to each hFcgR was calculated relative to the binding of G1 to each hFcgR, which was taken as 100, and the results are shown in Table 10.

TABLE-US-00011 TABLE 10 Variant name hFcgRIa hFcgRIIa (R) hFcgRIIa (H) hFcgRIIb hFcgRIIIa (F) hFcgRIIIa (V) TS28 5.8 1.9 1.7 7.2 3.8 1.7 TS29 5.1 2.1 0.7 7.2 4.2 1.6 TS30 0.1 1.2 0.2 3.8 0.7 1.1 TS31 0.2 1.3 -0.5 3.6 1.9 1.2 TS32 -0.5 0.9 1.0 1.5 0.8 0.9 TS33 -0.9 1.2 0.7 4.6 0.7 1.0 TS34 -0.9 0.9 1.4 4.2 1.8 1.5 TS35 0.5 1.1 1.7 6.2 2.0 1.5 TS36 -0.4 1.0 0.7 4.1 0.7 1.1 TS37 0.6 0.9 0.7 4.5 1.4 0.6 TS38 0.3 2.3 1.7 5.8 2.9 0.9 TS39 0.0 1.5 1.0 2.5 2.4 0.8 TS40 -1.8 1.2 0.7 0.3 0.5 1.0 TS41 0.2 1.2 1.4 4.2 2.4 1.0 TS42 -0.2 1.3 1.6 6.9 2.5 1.3 TS43 -0.9 1.7 -0.1 6.2 2.7 1.1

[0229] For all the variants of TS28 to TS43, combining the mutations increased Tm by 13.degree. C. or more. Furthermore, combining the mutations greatly reduced the binding ability to FcgRs.

Example 6

Assessment of Aggregate Content of IgG1 with Novel Fc Regions Having Improved Thermal Stability

[0230] Since aggregates affect storage stability and immunogenicity, it is preferred that mutations for improving thermal stability should not increase the content of aggregate. Accordingly, TS20 to TS43, which combine multiple mutations, and TSm01, and TSm02 were assessed for aggregate content by the method of Reference Example 4. The chromatograms for the measurement are shown in FIG. 2, and the aggregate contents are shown in Table 11.

TABLE-US-00012 TABLE 11 Variant name Aggregate content (%) TS20 1.73 TS21 1.70 TS22 1.84 TS23 1.71 TS24 1.66 TS25 1.45 TS26 1.82 TS27 1.96 TS28 2.00 TS29 3.03 TS30 2.72 TS31 2.67 TS32 2.08 TS33 3.77 TS34 2.31 TS35 2.90 TS36 1.86 TS37 1.69 TS38 2.22 TS39 2.19 TS40 1.53 TS41 2.49 TS42 2.04 TS43 1.95 TSm01 7.44 TSm02 4.11 G1 1.49

[0231] TS20 to TS27, which maintain the binding ability to hFcgRs, showed comparable aggregation content to G1, demonstrating that these thermal stability-improving mutations were found not to have a large effect on aggregate content. On the other hand, for TS28 to TS43, which greatly reduced the binding ability to hFcgRs, while the amount of aggregates was slightly increased in TS29, 33, and 35, the other variants showed almost the same aggregate amount as that of G1. In contrast, the disulfide bond-introduced variants TSm01 and TSm02 showed a significant increase in the aggregate content, that is, about 5 times and 3 times that of G1, respectively.

[0232] The above-mentioned results demonstrate that the mutations discovered in this examination by the present inventors are first-ever mutations that improved the thermal stability while maintaining the physicochemical properties.

Example 7

Production and Evaluation of IgG1 with Novel Fc Regions that Improve Thermal Stability

[0233] The above-mentioned result showed that introducing mutations to the CH2 domain improves the Tm. Therefore, regions that had not been examined for introduction of mutations in Example 1 were examined for whether there were any mutations to improve the Tm, and new variants TS44 to TS67 as shown in Table 12 were produced (SEQ ID NOs: 69-92). Each antibody was expressed and purified by the method described in Reference Example 1 and the results are shown in Table 13. Furthermore, binding ability to hFcgRs was measured by the method described in Reference Example 3. Binding of each antibody to each hFcgR was calculated relative to the binding of G1 to each hFcgR, which was taken as 100, and the results are shown in Table 14.

TABLE-US-00013 TABLE 12 Variant name Alteration sites SEQ ID NO: TS44 P247V 69 TS45 T250F 70 TS46 T250I 71 TS47 T250M 72 TS48 T250V 73 TS49 T250W 74 TS50 T250Y 75 TS51 S298G 76 TS52 T307A 77 TS53 T307Q 78 TS54 T307P 79 TS55 L309A 80 TS56 L309D 81 TS57 L309R 82 TS58 L309P 83 TS59 N315A 84 TS60 K360H 85 TS61 G385D/Q386P/N389S 86 TS62 P387E 87 TS63 M428H 88 TS64 M428W 89 TS65 M428Y 90 TS66 M428F 91 TS67 H433K 92

TABLE-US-00014 TABLE 13 Variant name Tm (.degree. C.) TS44 72.0 TS45 74.1 TS46 73.9 TS47 69.3 TS48 75.5 TS49 71.5 TS50 75.1 TS51 73.5 TS52 70.8 TS53 71.2 TS54 74.1 TS55 71.4 TS56 72.5 TS57 72.6 TS58 68.3 TS59 71.8 TS60 70.2 TS61 70.1 TS62 72.0 TS63 69.3 TS64 74.7 TS65 73.3 TS66 72.8 TS67 70.3 G1 70.4

TABLE-US-00015 TABLE 14 Variant name hFcgRIa hFcgRIIa (R) hFcgRIIa (H) hFcgRIIb hFcgRIIIa (F) hFcgRIIIa (V) TS44 101.3 103.4 110.2 112.6 92.0 102.9 TS45 104.9 125.1 119.3 150.2 62.8 84.7 TS46 99.2 102.3 104.6 104.6 66.1 89.6 TS47 100.7 126.6 114.4 162.9 49.8 72.0 TS48 101.5 111.3 108.4 113.2 66.0 89.4 TS49 99.6 123.7 126.6 148.3 82.0 96.9 TS50 101.5 136.6 129.3 169.4 83.5 98.9 TS51 99.2 55.8 27.4 40.8 36.0 58.9 TS52 98.8 98.3 101.8 97.2 64.6 89.0 TS53 101.2 99.0 102.8 95.9 66.4 90.8 TS54 102.7 103.3 103.5 102.3 66.8 90.6 TS55 99.2 101.4 104.1 106.4 70.2 90.5 TS56 67.0 76.7 79.2 74.3 47.2 69.1 TS57 101.1 97.0 98.1 97.8 61.4 86.2 TS58 96.4 68.7 82.2 70.4 41.5 61.2 TS59 101.8 99.1 100.0 99.0 73.4 96.1 TS60 104.7 102.7 104.8 99.7 70.3 94.2 TS61 100.9 106.2 106.4 112.4 73.4 96.6 TS62 101.3 99.7 101.8 95.6 68.9 91.7 TS63 98.6 102.1 102.2 108.5 71.7 90.3 TS64 99.4 114.9 110.8 124.4 89.2 104.7 TS65 100.8 108.9 110.8 111.4 76.3 96.8 TS66 103.2 111.5 112.9 114.6 78.8 99.2 TS67 99.0 99.7 103.1 97.7 66.0 91.7

[0234] The variant with the highest Tm in this examination was TS48, and increased Tm approximately 5.degree. C. The other variants also showed improvement in the Tm. The binding ability of TS51 and TS56 to hFcgRs was slightly decreased, and the others maintained their hFcgRs-binding ability.

Reference Example 1

Production of Antibody Expression Vectors and Expression and Purification of Antibodies

[0235] Amino acid substitutions were introduced by methods known to those skilled in the art using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, and such, or the In fusion Advantage PCR cloning kit (TAKARA), and then expression vectors were constructed. The nucleotide sequences of the obtained expression vectors were determined by methods known to those skilled in the art. The produced plasmids were transiently introduced into human embryonic kidney cancer-derived cell line HEK293H (Invitrogen) or FreeStyle293 (Invitrogen) to express antibodies. From the resulting culture supernatant, antibodies were purified by methods known to those skilled in the art using the rProtein A Sepharose.TM. Fast Flow (GE Healthcare). The concentrations of the purified antibodies were determined by measuring absorbance at 280 nm using a spectrophotometer, and calculating from the measured values using an extinction coefficient determined by the PACE method (Protein Science 1995; 4: 2411-2423).

Reference Example 2

Assessment of Melting Temperature (Tm) of the Modified Antibodies by Differential Scanning Fluorimetry

[0236] In this examination, thermal stability was assessed by measuring the melting temperature (Tm) of antibodies by differential scanning fluorimetry using Rotor-Gene Q (QIAGEN). It has been reported that this method shows a good correlation with Tm assessment that uses differential scanning calorimetry, which is a widely known method for evaluating thermal stability of antibodies (Journal of Pharmaceutical Science 2010; 4: 1707-1720).

[0237] The 5000 times concentrated SYPRO orange (Molecular Probes) was diluted with PBS (Sigma), and then added to the antibody solutions to prepare measurement samples. A 20 .mu.L aliquot of each sample was placed into a measurement tube, and the temperature was increased from 30.degree. C. to 99.degree. C. at 0.4.degree. C. increments with an equilibration time of approximately six seconds before measurement of fluorescence intensity at 470 nm (excitation wavelength)/555 nm (fluorescence wavelength).

[0238] The data were analyzed using Rotor-Gene Q Series Software (QIAGEN) to calculate the temperature at which fluorescence transition was observed, and this temperature was defined as Tm.

Reference Example 3

Assessment of Binding Ability to hFcgRs

[0239] Interactions between antibodies and hFcgRs (hFcgRIa, hFcgRIIa(R), hFcgRIIa(H), hFcgRIIb, hFcgRIIIa(F), and hFcgRIIIa(V)) were analyzed using Biacore T100 (GE Healthcare). HBS-EP+ (GE Healthcare) was used for the running buffer, and the measurement temperature was set at 25.degree. C. Protein A (Invitrogen) was immobilized by the amino coupling method, and an antibody of interest was captured thereon. Then, hFc.gamma.R solutions diluted using the running buffer were injected to allow interaction with the captured antibody at a flow rate of 30 .mu.L/min for five minutes for hFc.gamma.RIa, and at a flow rate of 5 .mu.L/min for one minute for the other hFc.gamma.Rs. The level of binding ability to the antibody was determined, and the results were compared among the antibodies. Since the hFc.gamma.R binding level depends on the amount of captured antibodies, the FcgR-binding levels were corrected so that the capture level of each antibody would be 200 RU (resonance unit) for hFcgRIa and 1000 RU (resonance unit) for the other hFcgRs. The captured antibodies were washed by allowing 10 mM glycine-HCl (pH 1.5) to react for 30 seconds at a flow rate of 30 .mu.L/min to recycle the chip for reuse.

Reference Example 4

Evaluation of Aggregate Content

[0240] The aggregate content in the purified antibody was evaluated by SEC analysis using the Alliance system (Waters). The mobile phase was 50 mM phosphate buffer containing 300 mM sodium chloride (pH7.0, Isekyu), the analytical column was G3000SW.sub.XL (TOSOH), and measurements were taken at a wavelength of 215 nm. The data were analyzed using Empower2 (Waters). Components that eluted as higher molecular weight species than the monomer were collectively taken as aggregates, and their content was calculated.

INDUSTRIAL APPLICABILITY

[0241] In the reported antibody Fc region variants, the stability of the Fc region is reduced in most cases. In addition, it is important that an antibody Fc region has effector functions, that is, binds to Fc.gamma.Rs. By the present invention, it is possible to provide highly stable Fc region variants maintaining Fc.gamma.R-binding, or highly stable Fc region variants with reduced Fc.gamma.R-binding.

Sequence CWU 1

1

9212268DNAHomo sapiens 1aatatcttgc atgttacaga tttcactgct cccaccagct tggagacaac atgtggttct 60tgacaactct gctcctttgg gttccagttg atgggcaagt ggacaccaca aaggcagtga 120tcactttgca gcctccatgg gtcagcgtgt tccaagagga aaccgtaacc ttgcactgtg 180aggtgctcca tctgcctggg agcagctcta cacagtggtt tctcaatggc acagccactc 240agacctcgac ccccagctac agaatcacct ctgccagtgt caatgacagt ggtgaataca 300ggtgccagag aggtctctca gggcgaagtg accccataca gctggaaatc cacagaggct 360ggctactact gcaggtctcc agcagagtct tcacggaagg agaacctctg gccttgaggt 420gtcatgcgtg gaaggataag ctggtgtaca atgtgcttta ctatcgaaat ggcaaagcct 480ttaagttttt ccactggaat tctaacctca ccattctgaa aaccaacata agtcacaatg 540gcacctacca ttgctcaggc atgggaaagc atcgctacac atcagcagga atatctgtca 600ctgtgaaaga gctatttcca gctccagtgc tgaatgcatc tgtgacatcc ccactcctgg 660aggggaatct ggtcaccctg agctgtgaaa caaagttgct cttgcagagg cctggtttgc 720agctttactt ctccttctac atgggcagca agaccctgcg aggcaggaac acatcctctg 780aataccaaat actaactgct agaagagaag actctgggtt atactggtgc gaggctgcca 840cagaggatgg aaatgtcctt aagcgcagcc ctgagttgga gcttcaagtg cttggcctcc 900agttaccaac tcctgtctgg tttcatgtcc ttttctatct ggcagtggga ataatgtttt 960tagtgaacac tgttctctgg gtgacaatac gtaaagaact gaaaagaaag aaaaagtggg 1020atttagaaat ctctttggat tctggtcatg agaagaaggt aatttccagc cttcaagaag 1080acagacattt agaagaagag ctgaaatgtc aggaacaaaa agaagaacag ctgcaggaag 1140gggtgcaccg gaaggagccc cagggggcca cgtagcagcg gctcagtggg tggccatcga 1200tctggaccgt cccctgccca cttgctcccc gtgagcactg cgtacaaaca tccaaaagtt 1260caacaacacc agaactgtgt gtctcatggt atgtaactct taaagcaaat aaatgaactg 1320acttcaactg ggatacattt ggaaatgtgg tcatcaaaga tgacttgaaa tgaggcctac 1380tctaaagaat tcttgaaaaa cttacaagtc aagcctagcc tgataatcct attacatagt 1440ttgaaaaata gtattttatt tctcagaaca aggtaaaaag gtgagtgggt gcatatgtac 1500agaagattaa gacagagaaa cagacagaaa gagacacaca cacagccagg agtgggtaga 1560tttcagggag acaagaggga atagtataga caataaggaa ggaaatagta cttacaaatg 1620actcctaagg gactgtgaga ctgagagggc tcacgcctct gtgttcagga tacttagttc 1680atggcttttc tctttgactt tactaaaaga gaatgtctcc atacgcgttc taggcataca 1740agggggtaac tcatgatgag aaatggatgt gttattcttg ccctctcttt tgaggctctc 1800tcataacccc tctatttcta gagacaacaa aaatgctgcc agtcctaggc ccctgccctg 1860taggaaggca gaatgtaact gttctgtttg tttaacgatt aagtccaaat ctccaagtgc 1920ggcactgcaa agagacgctt caagtgggga gaagcggcga taccatagag tccagatctt 1980gcctccagag atttgcttta ccttcctgat tttctggtta ctaattagct tcaggatacg 2040ctgctctcat acttgggctg tagtttggag acaaaatatt ttcctgccac tgtgtaacat 2100agctgaggta aaaactgaac tatgtaaatg actctactaa aagtttaggg aaaaaaaaca 2160ggaggagtat gacacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 22682374PRTHomo sapiens 2Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300 Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310 315 320 Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335 Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350 Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys 355 360 365 Glu Pro Gln Gly Ala Thr 370 31396DNAHomo sapiens 3gggatgacta tggagaccca aatgtctcag aatgtatgtc ccagaaacct gtggctgctt 60caaccattga cagttttgct gctgctggct tctgcagaca gtcaagctgc tcccccaaag 120gctgtgctga aacttgagcc cccgtggatc aacgtgctcc aggaggactc tgtgactctg 180acatgccagg gggctcgcag ccctgagagc gactccattc agtggttcca caatgggaat 240ctcattccca cccacacgca gcccagctac aggttcaagg ccaacaacaa tgacagcggg 300gagtacacgt gccagactgg ccagaccagc ctcagcgacc ctgtgcatct gactgtgctt 360tccgaatggc tggtgctcca gacccctcac ctggagttcc aggagggaga aaccatcatg 420ctgaggtgcc acagctggaa ggacaagcct ctggtcaagg tcacattctt ccagaatgga 480aaatcccaga aattctccca tttggatccc accttctcca tcccacaagc aaaccacagt 540cacagtggtg attaccactg cacaggaaac ataggctaca cgctgttctc atccaagcct 600gtgaccatca ctgtccaagt gcccagcatg ggcagctctt caccaatggg ggtcattgtg 660gctgtggtca ttgcgactgc tgtagcagcc attgttgctg ctgtagtggc cttgatctac 720tgcaggaaaa agcggatttc agccaattcc actgatcctg tgaaggctgc ccaatttgag 780ccacctggac gtcaaatgat tgccatcaga aagagacaac ttgaagaaac caacaatgac 840tatgaaacag ctgacggcgg ctacatgact ctgaacccca gggcacctac tgacgatgat 900aaaaacatct acctgactct tcctcccaac gaccatgtca acagtaataa ctaaagagta 960acgttatgcc atgtggtcat actctcagct tgctgagtgg atgacaaaaa gaggggaatt 1020gttaaaggaa aatttaaatg gagactggaa aaatcctgag caaacaaaac cacctggccc 1080ttagaaatag ctttaacttt gcttaaacta caaacacaag caaaacttca cggggtcata 1140ctacatacaa gcataagcaa aacttaactt ggatcatttc tggtaaatgc ttatgttaga 1200aataagacaa ccccagccaa tcacaagcag cctactaaca tataattagg tgactaggga 1260ctttctaaga agatacctac ccccaaaaaa caattatgta attgaaaacc aaccgattgc 1320ctttattttg cttccacatt ttcccaataa atacttgcct gtgactaaaa aaaaaaaaaa 1380aaaaaaaaaa aaaaaa 13964316PRTHomo sapiens 4Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu 1 5 10 15 Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp 35 40 45 Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala 50 55 60 Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu 65 70 75 80 Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn 85 90 95 Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp 100 105 110 Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro 115 120 125 His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser 130 135 140 Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys 145 150 155 160 Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala 165 170 175 Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr 180 185 190 Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser 195 200 205 Met Gly Ser Ser Ser Pro Met Gly Val Ile Val Ala Val Val Ile Ala 210 215 220 Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys 225 230 235 240 Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala 245 250 255 Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln 260 265 270 Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met 275 280 285 Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu 290 295 300 Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315 51497DNAHomo sapiens 5tgtgactgct gtgctctggg cgccagctcg ctccagggag tgatgggaat cctgtcattc 60ttacctgtcc ttgccactga gagtgactgg gctgactgca agtcccccca gccttggggt 120catatgcttc tgtggacagc tgtgctattc ctggctcctg ttgctgggac acctgcagct 180cccccaaagg ctgtgctgaa actcgagccc cagtggatca acgtgctcca ggaggactct 240gtgactctga catgccgggg gactcacagc cctgagagcg actccattca gtggttccac 300aatgggaatc tcattcccac ccacacgcag cccagctaca ggttcaaggc caacaacaat 360gacagcgggg agtacacgtg ccagactggc cagaccagcc tcagcgaccc tgtgcatctg 420actgtgcttt ctgagtggct ggtgctccag acccctcacc tggagttcca ggagggagaa 480accatcgtgc tgaggtgcca cagctggaag gacaagcctc tggtcaaggt cacattcttc 540cagaatggaa aatccaagaa attttcccgt tcggatccca acttctccat cccacaagca 600aaccacagtc acagtggtga ttaccactgc acaggaaaca taggctacac gctgtactca 660tccaagcctg tgaccatcac tgtccaagct cccagctctt caccgatggg gatcattgtg 720gctgtggtca ctgggattgc tgtagcggcc attgttgctg ctgtagtggc cttgatctac 780tgcaggaaaa agcggatttc agccaatccc actaatcctg atgaggctga caaagttggg 840gctgagaaca caatcaccta ttcacttctc atgcacccgg atgctctgga agagcctgat 900gaccagaacc gtatttagtc tccattgtct tgcattggga tttgagaaga aaatcagaga 960gggaagatct ggtatttcct ggcctaaatt ccccttgggg aggacaggga gatgctgcag 1020ttccaaaaga gaaggtttct tccagagtca tctacctgag tcctgaagct ccctgtcctg 1080aaagccacag acaatatggt cccaaataac cgactgcacc ttctgtgctt cagctcttct 1140tgacatcaag gctcttccgt tccacatcca cacagccaat ccaattaatc aaaccactgt 1200tattaacaga taatagcaac ttgggaaatg cttatgttac aggttacgtg agaacaatca 1260tgtaaatcta tatgatttca gaaatgttaa aatagactaa cctctaccag cacattaaaa 1320gtgattgttt ctgggtgata aaattattga tgatttttat tttctttatt tttctataaa 1380gatcatatat tacttttata ataaaacatt ataaaaacaa aaaaaaaaaa aaaaaaaaaa 1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 14976291PRTHomo sapiens 6Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp 1 5 10 15 Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr 20 25 30 Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro 35 40 45 Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu 50 55 60 Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp 65 70 75 80 Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln 85 90 95 Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr 100 105 110 Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val 115 120 125 Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu 130 135 140 Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu 145 150 155 160 Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg 165 170 175 Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly 180 185 190 Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys 195 200 205 Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile 210 215 220 Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala 225 230 235 240 Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Pro 245 250 255 Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn Thr Ile Thr 260 265 270 Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln 275 280 285 Asn Arg Ile 290 72137DNAHomo sapiens 7cttgtccact ccagtgtggc atcatgtggc agctgctcct cccaactgct ctgctacttc 60tagtttcagc tggcatgcgg actgaagatc tcccaaaggc tgtggtgttc ctggagcctc 120aatggtacag ggtgctcgag aaggacagtg tgactctgaa gtgccaggga gcctactccc 180ctgaggacaa ttccacacag tggtttcaca atgagagcct catctcaagc caggcctcga 240gctacttcat tgacgctgcc acagttgacg acagtggaga gtacaggtgc cagacaaacc 300tctccaccct cagtgacccg gtgcagctag aagtccatat cggctggctg ttgctccagg 360cccctcggtg ggtgttcaag gaggaagacc ctattcacct gaggtgtcac agctggaaga 420acactgctct gcataaggtc acatatttac agaatggcaa aggcaggaag tattttcatc 480ataattctga cttctacatt ccaaaagcca cactcaaaga cagcggctcc tacttctgca 540gggggcttgt tgggagtaaa aatgtgtctt cagagactgt gaacatcacc atcactcaag 600gtttgtcagt gtcaaccatc tcatcattct ttccacctgg gtaccaagtc tctttctgct 660tggtgatggt actccttttt gcagtggaca caggactata tttctctgtg aagacaaaca 720ttcgaagctc aacaagagac tggaaggacc ataaatttaa atggagaaag gaccctcaag 780acaaatgacc cccatcccat gggggtaata agagcagtag cagcagcatc tctgaacatt 840tctctggatt tgcaacccca tcatcctcag gcctctctac aagcagcagg aaacatagaa 900ctcagagcca gatcccttat ccaactctcg acttttcctt ggtctccagt ggaagggaaa 960agcccatgat cttcaagcag ggaagcccca gtgagtagct gcattcctag aaattgaagt 1020ttcagagcta cacaaacact ttttctgtcc caaccgttcc ctcacagcaa agcaacaata 1080caggctaggg atggtaatcc tttaaacata caaaaattgc tcgtgttata aattacccag 1140tttagagggg aaaaaaaaac aattattcct aaataaatgg ataagtagaa ttaatggttg 1200aggcaggacc atacagagtg tgggaactgc tggggatcta gggaattcag tgggaccaat 1260gaaagcatgg ctgagaaata gcaggtagtc caggatagtc taagggaggt gttcccatct 1320gagcccagag ataagggtgt cttcctagaa cattagccgt agtggaatta acaggaaatc 1380atgagggtga cgtagaattg agtcttccag gggactctat cagaactgga ccatctccaa 1440gtatataacg atgagtcctc ttaatgctag gagtagaaaa tggtcctagg aaggggactg 1500aggattgcgg tggggggtgg ggtggaaaag aaagtacaga acaaaccctg tgtcactgtc 1560ccaagttgct aagtgaacag aactatctca gcatcagaat gagaaagcct gagaagaaag 1620aaccaaccac aagcacacag gaaggaaagc gcaggaggtg aaaatgcttt cttggccagg 1680gtagtaagaa ttagaggtta atgcagggac tgtaaaacca ccttttctgc ttcaatatct 1740aattcctgtg tagctttgtt cattgcattt attaaacaaa tgttgtataa ccaatactaa 1800atgtactact gagcttcgct gagttaagtt atgaaacttt caaatccttc atcatgtcag 1860ttccaatgag gtggggatgg agaagacaat tgttgcttat gaaagaaagc tttagctgtc 1920tctgttttgt aagctttaag cgcaacattt cttggttcca ataaagcatt ttacaagatc 1980ttgcatgcta ctcttagata gaagatggga aaaccatggt aataaaatat gaatgataaa 2040aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 21378254PRTHomo sapiens 8Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165

170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ser Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 9820DNAHomo sapiens 9cactccagtg tggcatcatg tggcagctgc tcctcccaac tgctctgcta cttctagttt 60cagctggcat gcggactgaa gatctcccaa aggctgtggt gttcctggag cctcaatggt 120acagcgtgct tgagaaggac agtgtgactc tgaagtgcca gggagcctac tcccctgagg 180acaattccac acagtggttt cacaatgaga gcctcatctc aagccaggcc tcgagctact 240tcattgacgc tgccacagtc aacgacagtg gagagtacag gtgccagaca aacctctcca 300ccctcagtga cccggtgcag ctagaagtcc atatcggctg gctgttgctc caggcccctc 360ggtgggtgtt caaggaggaa gaccctattc acctgaggtg tcacagctgg aagaacactg 420ctctgcataa ggtcacatat ttacagaatg gcaaagacag gaagtatttt catcataatt 480ctgacttcca cattccaaaa gccacactca aagatagcgg ctcctacttc tgcagggggc 540ttgttgggag taaaaatgtg tcttcagaga ctgtgaacat caccatcact caaggtttgg 600cagtgtcaac catctcatca ttctctccac ctgggtacca agtctctttc tgcttggtga 660tggtactcct ttttgcagtg gacacaggac tatatttctc tgtgaagaca aacatttgaa 720gctcaacaag agactggaag gaccataaac ttaaatggag aaaggaccct caagacaaat 780gacccccatc ccatgggagt aataagagca gtggcagcag 82010233PRTHomo sapiens 10Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile 225 230 11330PRTArtificial Sequencean artificially synthesized sequence 11Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 12326PRTArtificial Sequencean artificially synthesized sequence 12Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 13377PRTArtificial Sequencean artificially synthesized sequence 13Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 14327PRTArtificial Sequencean artificially synthesized sequence 14Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 15328PRTArtificial Sequencean artificially synthesized sequence 15Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 16328PRTArtificial Sequencean artificially synthesized sequence 16Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105

110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Glu Ser Leu Ser Leu Ser Pro 325 17115PRTArtificial Sequencean artificially synthesized sequence 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser Phe 50 55 60 Gln Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 18112PRTArtificial Sequencean artificially synthesized sequence 18Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Gln Ala Ser Glu Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 100 105 110 19107PRTArtificial Sequencean artificially synthesized sequence 19Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 20119PRTArtificial Sequencean artificially synthesized sequence 20Gln 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 Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser 115 21107PRTArtificial Sequencean artificially synthesized sequence 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 22443PRTArtificial Sequencean artificially synthesized sequence 22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser Phe 50 55 60 Gln Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 23447PRTArtificial Sequencean artificially synthesized sequence 23Gln 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 Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 24328PRTArtificial Sequencean artificially synthesized sequence 24Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Cys Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Cys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 25328PRTArtificial Sequencean artificially synthesized sequence 25Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Cys Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Cys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 26328PRTArtificial Sequencean artificially synthesized sequence 26Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ile Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 27328PRTArtificial Sequencean artificially synthesized sequence 27Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Ile Ser Gln Asp Glu Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 28328PRTArtificial Sequencean artificially synthesized sequence 28Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 29328PRTArtificial Sequencean artificially synthesized sequence 29Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Tyr Asn Ser Thr Glu Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 30328PRTArtificial Sequencean artificially synthesized sequence 30Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 31328PRTArtificial Sequencean artificially synthesized sequence 31Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ser Ala Leu Pro His Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 32328PRTArtificial Sequencean artificially synthesized sequence 32Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170

175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val His Asn 195 200 205 Ser Ala Leu Pro His Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 33328PRTArtificial Sequencean artificially synthesized sequence 33Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ala Ala Leu Pro Tyr Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 34328PRTArtificial Sequencean artificially synthesized sequence 34Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 35328PRTArtificial Sequencean artificially synthesized sequence 35Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 36328PRTArtificial Sequencean artificially synthesized sequence 36Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Arg Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 37328PRTArtificial Sequencean artificially synthesized sequence 37Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Arg Arg Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 38328PRTArtificial Sequencean artificially synthesized sequence 38Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Pro Met Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 39328PRTArtificial Sequencean artificially synthesized sequence 39Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115

120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Met Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 40328PRTArtificial Sequencean artificially synthesized sequence 40Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 41328PRTArtificial Sequencean artificially synthesized sequence 41Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 42328PRTArtificial Sequencean artificially synthesized sequence 42Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 43328PRTArtificial Sequencean artificially synthesized sequence 43Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Met 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 44328PRTArtificial Sequencean artificially synthesized sequence 44Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 45328PRTArtificial Sequencean artificially synthesized sequence 45Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ser Ala Leu Pro His Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 46328PRTArtificial Sequencean artificially synthesized sequence 46Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ala Ala Leu Pro Tyr Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 47328PRTArtificial Sequencean artificially synthesized sequence 47Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ser Ala Leu Pro His Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 48328PRTArtificial Sequencean artificially synthesized sequence 48Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ala Ala Leu Pro Tyr Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 49328PRTArtificial Sequencean artificially synthesized sequence 49Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ser Ala Leu Pro Tyr Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 50328PRTArtificial Sequencean artificially synthesized sequence 50Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ala Ala Leu Pro His Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 51328PRTArtificial Sequencean artificially synthesized sequence 51Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ser Ala Leu Pro Tyr Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 52328PRTArtificial Sequencean artificially synthesized sequence 52Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Ala Ala Leu Pro His Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 53328PRTArtificial Sequencean artificially synthesized sequence 53Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala

Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 54328PRTArtificial Sequencean artificially synthesized sequence 54Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 55328PRTArtificial Sequencean artificially synthesized sequence 55Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 56328PRTArtificial Sequencean artificially synthesized sequence 56Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 57328PRTArtificial Sequencean artificially synthesized sequence 57Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 58328PRTArtificial Sequencean artificially synthesized sequence 58Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 59328PRTArtificial Sequencean artificially synthesized sequence 59Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 60328PRTArtificial Sequencean artificially synthesized sequence 60Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 61328PRTArtificial Sequencean artificially synthesized sequence 61Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 62328PRTArtificial Sequencean artificially synthesized sequence 62Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 63328PRTArtificial Sequencean artificially synthesized sequence 63Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 64328PRTArtificial Sequencean artificially synthesized sequence 64Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 65328PRTArtificial Sequencean artificially synthesized sequence 65Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 66328PRTArtificial Sequencean artificially synthesized sequence 66Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Lys Glu Phe Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245

250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 67328PRTArtificial Sequencean artificially synthesized sequence 67Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Gly 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 68328PRTArtificial Sequencean artificially synthesized sequence 68Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Lys Arg Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Met Gly Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser His 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 69328PRTArtificial Sequencean artificially synthesized sequence 69Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Val Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 70328PRTArtificial Sequencean artificially synthesized sequence 70Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Phe Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 71328PRTArtificial Sequencean artificially synthesized sequence 71Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Ile Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 72328PRTArtificial Sequencean artificially synthesized sequence 72Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Met Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 73328PRTArtificial Sequencean artificially synthesized sequence 73Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 74328PRTArtificial Sequencean artificially synthesized sequence 74Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Trp Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 75328PRTArtificial Sequencean artificially synthesized sequence 75Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Tyr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 76328PRTArtificial Sequencean artificially synthesized sequence 76Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Gly Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 77328PRTArtificial Sequencean artificially synthesized sequence 77Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Ala Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 78328PRTArtificial Sequencean artificially synthesized sequence 78Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Gln Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 79328PRTArtificial Sequencean artificially synthesized sequence 79Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 80328PRTArtificial Sequencean artificially synthesized sequence 80Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser

His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Ala 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 81328PRTArtificial Sequencean artificially synthesized sequence 81Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 82328PRTArtificial Sequencean artificially synthesized sequence 82Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Arg 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 83328PRTArtificial Sequencean artificially synthesized sequence 83Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Pro 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 84328PRTArtificial Sequencean artificially synthesized sequence 84Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Ala Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 85328PRTArtificial Sequencean artificially synthesized sequence 85Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr His Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 86328PRTArtificial Sequencean artificially synthesized sequence 86Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Asp Pro Pro Glu Ser 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 87328PRTArtificial Sequencean artificially synthesized sequence 87Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90

95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Glu Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 88328PRTArtificial Sequencean artificially synthesized sequence 88Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val His His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 89328PRTArtificial Sequencean artificially synthesized sequence 89Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Trp His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 90328PRTArtificial Sequencean artificially synthesized sequence 90Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Tyr His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 91328PRTArtificial Sequencean artificially synthesized sequence 91Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Phe His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 92328PRTArtificial Sequencean artificially synthesized sequence 92Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu Lys Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325

Claims

1.-62. (canceled)

63. A polypeptide comprising a modified antibody Fc region, wherein the amino acid sequence of the modified antibody Fc region differs from the sequence of a naturally occurring IgG Fc region at one or more positions including at least one position in a loop region of the IgG Fc region, and wherein the thermal stability of the polypeptide is greater than the thermal stability of a control protein identical to the polypeptide except at the at least one position in the loop region.

64. The polypeptide of claim 63, wherein the thermal stability is determined by measuring the melting temperature (Tm) of the polypeptide.

65. The polypeptide of claim 63, wherein the polypeptide binds to a human Fc.gamma.R receptor with a binding activity that is the same as or greater than the binding activity of the control protein for the human Fc.gamma.R receptor.

66. The polypeptide of claim 63, wherein the polypeptide binds to a human Fc.gamma.R receptor with a binding activity that is lower than the binding activity of the control protein for the human Fc.gamma.R receptor.

67. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 235, 236, 237, 238, 239, 247, 250, 265, 266, 267, 268, 269, 270, 271, 295, 296, 298, 300, 307, 309, 315, 324, 325, 326, 327, 329, 330, 333, 335, 337, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering).

68. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 235, 239, 266, 267, 268, 269, 270, 295, 296, 298, 300, 324, 325, 326, and 330 (all positions by EU numbering).

69. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 266, 268, 269, 270, 295, 300, 324, 326, and 330 (all positions by EU numbering).

70. The polypeptide of claim 69, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Ile at position 234, Ile at position 266, Gln at position 268, Asp at position 269, Glu at position 270, Met or Leu at position 295, Glu at position 300, His at position 324, Ser or Ala at position 326, and His or Tyr at position 330.

71. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 235, 239, 267, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering).

72. The polypeptide of claim 71, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Lys or Arg at position 234, Lys or Arg at position 235, Lys at position 239, Pro at position 267, Met or Lys at position 268, Phe at position 270, Met at position 295, Gly at position 296, Gly at position 298, and Gly, His, or Met at position 325.

73. The polypeptide of claim 63, wherein the at least one position is selected from positions 295, 326, and 330 (all positions by EU numbering).

74. The polypeptide of claim 73, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Met or Leu at position 295, Ser or Ala at position 326, and His or Tyr at position 330.

75. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 235, 239, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering).

76. The polypeptide of claim 75, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Lys at position 234, Lys or Arg at position 235, Lys or Ser at position 239, Lys or His at position 268, Phe or Asp at position 270, Met at position 295, Gly at position 296, Gly at position 298, and His or Gly at position 325.

77. The polypeptide of claim 75, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Lys at position 234, Lys or Arg at position 235, Lys at position 239, Lys at position 268, Phe at position 270, Met at position 295, Gly at position 296, Gly at position 298, and His or Gly at position 325.

78. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 235, 239, 295, 296, 298, and 325 (all positions by EU numbering).

79. The polypeptide of claim 78, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Lys at position 234, Lys or Arg at position 235, Lys at position 239, Met at position 295, Gly at position 296, Gly at position 298, and His or Gly at position 325.

80. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 235, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering).

81. The polypeptide of claim 80, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Lys at position 234, Lys or Arg at position 235, Lys at position 268, Phe at position 270, Met at position 295, Gly at position 296, Gly at position 298, and His or Gly at position 325.

82. The polypeptide of claim 63, wherein the at least one position is selected from positions 234, 235, 295, 296, 298, and 325 (all positions by EU numbering).

83. The polypeptide of claim 82, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Lys at position 234, Lys or Arg at position 235, Met at position 295, Gly at position 296, Gly at position 298, and His or Gly at position 325.

84. The polypeptide of claim 63, wherein the at least one position is selected from positions 247, 250, 307, 309, 315, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering).

85. The polypeptide of claim 84, wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid: Val at position 247, Phe, Ile, Met, Val, Trp, or Tyr at position 250, Ala, Gln, or Pro at position 307, Ala, Arg, or Pro at position 309, Ala at position 315, His at position 360, Asp at position 385, Pro at position 386, Glu at position 387, Ser at position 389, His, Trp, Tyr, or Phe at position 428, and Lys at position 433.

86. The polypeptide of claim 63, wherein the at least one position is selected from positions 298 and 309 (all positions by EU numbering).

87. The polypeptide of claim 86, wherein one or both of the following positions (EU numbering) in the modified antibody Fc region are occupied by the indicated amino acid: Gly at position 298, and Asp at position 309.

88. A method of producing the polypeptide of claim 63, comprising: identifying a first nucleotide sequence encoding a first polypeptide comprising a first antibody Fc region comprising one or more loop regions, wherein the amino acid sequences of the one or more loop regions of the first antibody Fc region are identical to the amino acid sequences of the corresponding loop regions of a naturally occurring IgG Fc region; generating a DNA comprising a second nucleotide sequence encoding a second polypeptide that differs from the first polypeptide at one or more positions, including one or more positions in at least one loop region; expressing the DNA, thereby producing the second polypeptide; and collecting the second polypeptide.

89. The method of claim 88, further comprising measuring the melting temperature of the second polypeptide; determining that the melting temperature of the second polypeptide is higher than the melting temperature of the first polypeptide; and determining, based on the relative melting temperatures of the first and second polypeptides, that the second polypeptide is thermally more stable than the first polypeptide.

90. The method of claim 88, wherein the one or more positions are selected from positions 234, 235, 236, 237, 238, 239, 247, 250, 265, 266, 267, 268, 269, 270, 271, 295, 296, 298, 300, 307, 309, 315, 324, 325, 326, 327, 329, 330, 333, 335, 337, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering).

91. The method of claim 88, wherein the one or more positions are selected from positions 234, 235, 239, 266, 267, 268, 269, 270, 295, 296, 298, 300, 324, 325, 326, and 330 (all positions by EU numbering).

92. The method of claim 88, wherein the one or more positions are selected from positions 234, 266, 268, 269, 270, 295, 300, 324, 326, and 330 (all positions by EU numbering).

93. The method of claim 88, wherein the one or more positions are selected from positions 234, 235, 239, 267, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering).

94. The method of claim 88, wherein the one or more positions are selected from positions 295, 326, and 330 (all positions by EU numbering).

95. The method of claim 88, wherein the one or more positions are selected from positions 234, 235, 239, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering).

96. The method of claim 88, wherein the one or more positions are selected from positions 234, 235, 239, 295, 296, 298, and 325 (all positions by EU numbering).

97. The method of claim 88, wherein the one or more positions are selected from positions 234, 235, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering).

98. The method of claim 88, wherein the one or more positions are selected from positions 234, 235, 295, 296, 298, and 325 (all positions by EU numbering).

99. The method of claim 88, wherein the one or more positions are selected from positions 247, 250, 307, 309, 315, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering).

100. The method of claim 88, wherein the one or more positions are selected from positions 298 and 309 (by EU numbering).

101. A nucleic acid that encodes a polypeptide comprising a modified antibody Fc region, wherein the amino acid sequence of the modified antibody Fc region differs from the sequence of a naturally occurring IgG Fc region at one or more positions including at least one position in a loop region of the IgG Fc region, and wherein the thermal stability of the polypeptide is greater than the thermal stability of a control protein identical to the polypeptide except at the at least one position in the loop region.

102. A vector comprising the nucleic acid of claim 101.

103. A host cell comprising the vector of claim 102.

104. A pharmaceutical composition comprising the polypeptide of claim 63.

105. A method for treating an immune-inflammatory disease in a subject in need of such treatment, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 104.

106. The method of claim 105, wherein the immune-inflammatory disease is selected from rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune bullous dermatosis, autoimmune adrenocortical inflammation, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Basedow's disease, juvenile diabetes, Addison's disease, myasthenia gravis, lenticular uveitis, systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, asthma, myodegeneration, cachexia, systemic scleroderma, localized scleroderma, Sjogren's syndrome, Behcet's disease, Reiter's syndrome, type I and type II diabetes, bone resorption disease, graft versus host reaction, ischemia reperfusion injury, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, malgias due to staining, aplastic anemia, hemolytic anemia, sudden thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome, Hashimoto's disease, pemphigus, IgA nephropathy, pollinosis, antiphospholipid antibody syndrome, polymyositis, Wegener's sarcoma, arteritis nodosa, mixed connective tissue disease, and fibromyalgia.

107. A method for treating cancer in a subject in need of such treatment, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 104.

108. The method of claim 107, wherein the cancer is selected from pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatoma, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, cholangioma, bladder cancer, ureteral cancer, thyroid cancer, adrenal carcinoma, renal cancer, other glandular tissue cancers, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, or other solid organ tumors.