Heterodimeric Fc polypeptide

Abstract

In one non-limiting embodiment, polypeptides comprising variant Fc regions containing amino acid changes in a parent Fc region are provided, as are methods for producing such polypeptides.

Description

[Technical field]

[0001] In one non-limiting embodiment, the present disclosure relates to polypeptides comprising variant Fc regions comprising amino acid changes in a parent Fc region, and methods for producing such polypeptides. [Background Technology]

[0002] Antibodies have attracted attention as drugs because of their high stability in the blood and fewer side effects (NPL 1 and NPL 2). Most of the antibody drugs currently on the market are human IgG1 subclass antibodies. Many studies have been conducted on the effector functions of IgG class antibodies, namely antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) and complement-dependent cytotoxicity (hereinafter referred to as CDC). It is reported that among human IgG class, IgG1 subclass antibodies have the highest ADCC activity and CDC activity (NPL 3). In addition, antibody-dependent cell-mediated phagocytosis (ADCP), which is the phagocytosis of target cells mediated by IgG class antibodies, has also been shown to be one of the antibody effector functions (NPL 4 and NPL 5).

[0003] In order for IgG antibodies to exert ADCC, CDC, and ADCP, the antibody Fc region needs to bind to antibody receptors (hereinafter referred to as FcγR) present on the surface of effector cells (such as killer cells, natural killer cells, and activated macrophages) and bind to various complement components. In humans, the FcγR protein family is reported to have isoforms FcγRIa, FcγRIIa, FcγRIIb, FbγRIIIa, and FcγRIIIb, and the allotypes of each are also reported (NPL 6).

[0004] Enhancing cytotoxic effector functions such as ADCC, ADCP and CDC is attracting attention as a promising means to enhance the anti-tumor effect of antibodies. The importance of FcγR-mediated effector functions for the anti-tumor effect of antibodies using mouse models has been reported (NLP 7 and NLP 8). In addition, correlation was observed between human clinical results and FcγRIIIa high-affinity polymorphic allotypes (V158) and low-affinity polymorphic allotypes (F158) (NPL 9). Similarly, it was also shown that clinical effects vary with FcγRIIa allotypes (H131 and R131) (NLP 10). These reports indicate that antibodies with Fc regions optimized for binding to specific FcγRs will mediate more effective effector functions, thereby exerting effective anti-tumor effects.

[0005] The balance of antibody binding activity to the activating receptors composed of FcγRIa, FcγRIIa, FcγRIIIa and FcγRIIIb and to the inhibitory receptors composed of FcγRIIb is an important factor in optimizing antibody effector functions. By using an Fc region with enhanced binding activity to activating receptors and reduced binding activity to inhibitory receptors, it is possible to confer optimal effector functions on antibodies (NPL 11). For the binding between the Fc region and FcγR, several amino acid residues within the antibody hinge region and CH2 domains, and the sugar chain (which is bound to the CH2 domain) attached to the Asn at position 297 according to EU numbering have been shown to be important (NPL 12, NPL 13 and NPL 14). For this binding site, Fc region variants with various FcγR binding properties have been studied to date, and Fc region variants with higher binding activity to activating FcγR have been obtained (PTL 1 and PTL 2). For example, Lazar et al. replaced Ser at position 239, Ala at position 330, and Ile at position 332 according to EU numbering in human IgG1 with Asp, Leu, and Glu, respectively, thereby successfully increasing its binding to human FcγRIIIa (V158) by up to about 370 times (NPL 15 and PTL 2). Shinkawa et al. successfully increased the binding to FcγRIIIa by up to about 100 times by deleting the fucose in the sugar chain attached to Asn at position 297 according to EU numbering (NPL 16). These methods introduce the same changes or the same sugar chain modifications into the two H chain Fc regions of the antibody at the same time. At the same time, it is reported that although the antibody Fc is a homodimer, it binds to FcγR at a 1:1 ratio and recognizes FcγR asymmetrically through the lower hinge and CH2 domain (NPL 17). Given the fact that the Fc region interacts asymmetrically with FcγRs, it is possible to more finely optimize IgG-FcγR interactions by introducing different modifications into each H chain. Based on this idea, methods for optimizing antibody-FcγR interactions by differently modifying the Fc region of each H chain to make the Fc asymmetrically modified have also been reported (PTL 3, PTL 4, PTL 5, and PTL 6). In fact, asymmetric modification of the Fc region resulted in variants with higher ADCC activity than afucosylated antibodies, which are existing ADCC-enhanced antibodies (PTL 5 and PTL 6).

[0006] In addition to ADCC activity, ADCP activity is also an important effector function of antibodies, and it is reported that it contributes to anti-tumor effects (NPL 18). ADCP activity can be enhanced by inhibiting the "don't eat me" signal, as represented by CD47 (NPL 18), and can also be enhanced by enhancing FcγRIIa binding ability (NPL 19). However, FcγRIIa (activating FcγR) and inhibitory FcγRIIb have very high homology in the amino acid sequence of the extracellular region, so it is difficult to selectively enhance FcγRIIa binding ability (NPL 20). Therefore, when FcγRIIa binding ability is enhanced, the binding activity to inhibitory receptor FcγRIIb is also enhanced, thereby weakening effector function. In fact, variants with significantly improved FcγRIIa binding ability also have FcγRIIb binding ability stronger than natural IgG1 (PTL 5 and PTL 6). Therefore, in order to exhibit high ADCC / ADCP activity, it is preferable to enhance the binding to FcγRIIIa and / or FcγRIIa as much as possible without enhancing the binding to FcγRIIb; however, such variants have not been reported yet.

[0007] [Citation List]

[0008] [Patent Document]

[0009] [PTL 1] WO 2000 / 042072

[0010] [PTL 2] WO 2006 / 019447

[0011] [PTL 3] WO 2012 / 058768

[0012] [PTL 4] WO 2012 / 125850

[0013] [PTL 5] WO 2013 / 002362

[0014] [PTL 6] WO 2014 / 104165

[0015] [Non-patent literature]

[0016] [NPL 1]Nature Biotechnology,23,1073-1078(2005)

[0017] [NPL 2]Eur.J.Pharm.Biopharm,59(3),389-96(2005)

[0018] [NPL 3]Chemical Immunology,65,88(1997)

[0019] [NPL 4]Cancer Res.,68,8049-8057(2008)

[0020] [NPL 5]Blood,113,3735-3743(2009)

[0021] [NPL 6]Immunol.Lett.82,57-65(2002)

[0022] [NPL 7]Pro.Nat.Acad.Sci.95:652-656(1998)

[0023] [NPL 8]Nature Medicine,6:443-446(2000)

[0024] [NPL 9]Blood,99:754-758(2002)

[0025] [NPL 10]Ann.Oncol.,22:1302-1307(2011)

[0026] [NPL 11]Science,310,1510-1512(2005)

[0027] [NPL 12]Chemical Immunology,65,88(1997)

[0028] [NPL 13]Eur.J.Immunol.23,1098(1993)

[0029] [NPL 14]Immunology,86,319(1995)

[0030] [NPL 15]Pro.Nat.Acad.Sci.103,4005-4010(2006)

[0031] [NPL 16]J.Biol.Chem.,278,3466-3473(2003)

[0032] [NPL 17]J.Biol.Chem.,276:16469-16477,(2001)

[0033] [NPL 18]JCI Insight,4:e131882,(2019)

[0034] [NPL 19]Mol.Cancer Ther.,7:2517-2527,(2008)

[0035] [NPL 20]Protein Eng.Des.Sel.,26,589-598,(2013) [Summary of the invention]

[0036] [Technical Issues]

[0037] In view of these circumstances, the present invention has been made. In one aspect, the present invention aims to provide polypeptides having improved Fc region functions (e.g., FcγR binding ability, ADCC activity, and ADCP activity) compared to conventional Fc region-containing polypeptides, as well as methods for producing such polypeptides.

[0038] [Solution to the problem]

[0039] In one non-limiting embodiment, the present disclosure provides the following:

[0040] [1] A polypeptide comprising a variant Fc region comprising an amino acid change in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid change in:

[0041] (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and

[0042] (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0043] [2] The polypeptide of [1], wherein the variant Fc region further comprises an amino acid alteration at position 326 according to EU numbering in the first polypeptide of the parent Fc region.

[0044] [3] The polypeptide of [1] or [2], wherein the variant Fc region further comprises an amino acid alteration at position 236 according to EU numbering in the second polypeptide of the parent Fc region.

[0045] [4] A polypeptide comprising a variant Fc region comprising an amino acid change in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid change in:

[0046] (i) positions 234, 235, 236, 239, 268, 270, 298 and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0047] (ii) positions 236, 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0048] [5] The polypeptide of any one of [1] to [4], wherein the variant Fc region further comprises an amino acid alteration at position 332 according to EU numbering in the first polypeptide of the parent Fc region.

[0049] [6] The polypeptide of any one of [1] to [5], wherein the variant Fc region further comprises an amino acid alteration at position 330 according to EU numbering in the first polypeptide of the parent Fc region.

[0050] [7] The polypeptide of any one of [1] to [6], wherein the variant Fc region further comprises an amino acid alteration at position 332 according to EU numbering in the second polypeptide of the parent Fc region.

[0051] [8] The polypeptide of any one of [1] to [7], wherein the variant Fc region further comprises an amino acid alteration at position 330 according to EU numbering in the second polypeptide of the parent Fc region.

[0052] [9] A polypeptide comprising a variant Fc region comprising an amino acid change in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid change in:

[0053] (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0054] (ii) positions 236, 270, 298, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0055]

[10] The polypeptide of any one of [1] to [9], wherein the variant Fc region further comprises amino acid alterations at positions 250 and 307 according to EU numbering in the first polypeptide of the parent Fc region.

[0056]

[11] The polypeptide of any one of [1] to

[10] , wherein the variant Fc region further comprises amino acid alterations at positions 250 and 307 according to EU numbering in the second polypeptide of the parent Fc region.

[0057]

[12] A polypeptide comprising a variant Fc region comprising an amino acid change in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid change at:

[0058] (i) positions 234, 235, 236, 239, 250, 268, 270, 298 and 307 according to EU numbering in the first polypeptide of the parent Fc region; and

[0059] (ii) positions 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0060]

[13] A polypeptide comprising a variant Fc region comprising an amino acid change in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid change in:

[0061] (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0062] (ii) positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0063]

[14] A polypeptide comprising a variant Fc region comprising an amino acid change in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid change at:

[0064] (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0065] (ii) positions 236, 250, 270, 298, 307, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0066]

[15] The polypeptide of any one of [1] to

[14] , comprising at least one amino acid change selected from the group consisting of:

[0067] (i) Tyr or Phe at position 234, Gln or Tyr at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met at position 330, and Glu at position 332 in the first polypeptide of the parent Fc region, according to EU numbering; and

[0068] (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, and Glu at position 334 in the second polypeptide of the parent Fc region, according to EU numbering.

[0069]

[16] The polypeptide of any one of [1] to

[15] , wherein the variant Fc region further comprises any of the following amino acid changes (a) to (f):

[0070] (a) Lys at position 356 according to EU numbering in the first polypeptide of the parent Fc region and Glu at position 439 according to EU numbering in the second polypeptide of the parent Fc region;

[0071] (b) Glu at position 439 according to EU numbering in the first polypeptide of the parent Fc region and Lys at position 356 according to EU numbering in the second polypeptide of the parent Fc region;

[0072] (c) Trp at position 366 according to EU numbering in the first polypeptide of the parent Fc region and Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the second polypeptide of the parent Fc region;

[0073] (d) Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the first polypeptide of the parent Fc region, and Trp at position 366 according to EU numbering in the second polypeptide of the parent Fc region;

[0074] (e) Cys at position 349 and Trp at position 366 according to EU numbering in the first polypeptide of the parent Fc region, and Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the second polypeptide of the parent Fc region;

[0075] (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the first polypeptide of the parent Fc region, and Cys at position 349 and Trp at position 366 according to EU numbering in the second polypeptide of the parent Fc region.

[0076]

[17] The polypeptide of any one of [1] to

[16] , wherein the variant Fc region further comprises any of the following amino acid changes (a) to (d) in the first polypeptide and / or the second polypeptide of the parent Fc region:

[0077] (a) Ala at position 434 according to EU numbering;

[0078] (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering;

[0079] (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering; and

[0080] (d) According to EU numbering, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440.

[0081]

[18] The polypeptide of any one of [1] to

[17] , wherein the binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa is enhanced in the variant Fc region compared to the parent Fc region.

[0082]

[19] The polypeptide of

[18] , wherein the binding activity to FcγRIIa and FcγRIIIa is enhanced in the variant Fc region compared to the parent Fc region.

[0083]

[20] The polypeptide of any one of [1] to

[19] , wherein the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors in the variant Fc region is improved compared to the parent Fc region.

[0084] [20-2] The polypeptide of any one of [1] to

[19] , wherein the binding activity to an activating Fcγ receptor in the variant Fc region is selectively enhanced compared to the binding activity to an inhibitory Fcγ receptor in the variant Fc region compared to the parent Fc region.

[0085] [20-3] The polypeptide of any one of [1] to

[19] , wherein the ratio of binding activity to activating Fcγ receptors to binding activity to inhibitory Fcγ receptors (A / I ratio) in the variant Fc region is higher than that in the parent Fc region.

[0086] [20-4] The polypeptide of [20-3], wherein the ratio (A / I ratio) in the polypeptide comprising the variant Fc region is 1.1 times higher or more, 1.2 times higher or more, 1.3 times higher or more, 1.4 times higher or more, 1.5 times higher or more, 1.6 times higher or more, 1.7 times higher or more, 1.8 times higher or more, 1.9 times higher or more, 2 times higher or more, 3 times higher or more, 4 times higher or more, 5 times higher or more, 6 times higher or more, 7 times higher or more, 8 times higher or more, 9 times higher or more, 10 times higher or more, 20 times higher or more, 30 times higher or more, 40 times higher or more than the ratio in the polypeptide comprising the parent Fc region. More preferably, the dosage form is 1000 fold higher, 2000 fold higher, 3000 fold higher, 4000 fold higher, 5000 fold higher, 6000 fold higher, 7000 fold higher, 8000 fold higher, 9000 fold higher, or 10,000 fold higher or more.

[0087] [20-5] The polypeptide described in [20-3], wherein the ratio (A / I ratio) in the polypeptide comprising the variant Fc region has a value of 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 11000 or more, 12000 or more, 13000 or more, 14000 or more or 15000 or more.

[0088]

[21] The polypeptide of any one of

[20] to [20-5], wherein the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, and FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb.

[0089]

[22] The polypeptide of any one of [1] to

[21] , wherein the polypeptide comprising a variant Fc region is an antibody.

[0090]

[23] A method for producing a polypeptide comprising a variant Fc region, comprising introducing amino acid changes into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid changes are introduced at the following positions:

[0091] (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and

[0092] (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0093]

[24] A method for producing a polypeptide comprising a variant Fc region, comprising introducing amino acid changes into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the amino acid changes are introduced at the following positions:

[0094] (i) positions 234, 235, 236, 239, 268, 270, 298 and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0095] (ii) positions 236, 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0096]

[25] A method for producing a polypeptide comprising a variant Fc region, comprising introducing amino acid changes into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the amino acid changes are introduced at the following positions:

[0097] (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0098] (ii) positions 236, 270, 298, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0099]

[26] An isolated nucleic acid encoding the polypeptide of any one of [1] to

[22] .

[0100]

[27] A host cell comprising the nucleic acid described in

[26] .

[0101]

[28] A method for producing a polypeptide, comprising culturing the host cell of

[27] to produce the polypeptide.

[0102]

[29] The polypeptide according to any one of [1] to

[22] , which is used for treating tumors.

[0103]

[30] The polypeptide according to any one of [1] to

[22] , which is used for damaging cells.

[0104]

[31]

[30] The polypeptide described in claim 31, wherein the cell damage is through ADCC activity, CDC activity or ADCP activity.

[0105]

[32] A pharmaceutical composition comprising the polypeptide according to any one of [1] to

[22] and a pharmaceutically acceptable carrier.

[0106]

[33] The pharmaceutical composition described in

[32] is a pharmaceutical composition for treating tumors.

[0107]

[34] The pharmaceutical composition described in

[32] is a pharmaceutical composition for damaging cells.

[0108]

[35] The pharmaceutical composition described in

[34] , wherein the cell damage is through ADCC activity, CDC activity or ADCP activity.

[0109]

[36] A method for treating tumors, comprising administering the polypeptide described in any one of [1] to

[22] or the pharmaceutical composition described in

[32] .

[0110]

[37] A method for damaging cells, comprising administering the polypeptide described in any one of [1] to

[22] or the pharmaceutical composition described in

[32] .

[0111]

[38]

[37] The method described, wherein the cell damage is through ADCC activity, CDC activity or ADCP activity.

[0112]

[39] Use of the polypeptide described in any one of [1] to

[22] in the preparation of a medicament for treating tumors.

[0113]

[40] Use of the polypeptide described in any one of [1] to

[22] in the preparation of a medicament for damaging cells.

[0114]

[41]

[40] The use described in claim 41, wherein the cell damage is through ADCC activity, CDC activity or ADCP activity.

[0115]

[42] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing amino acid changes into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and introducing the amino acid changes into the following positions:

[0116] (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and

[0117] (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0118]

[43] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing amino acid changes into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and introducing the amino acid changes into the following positions:

[0119] (i) positions 234, 235, 236, 239, 268, 270, 298 and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0120] (ii) positions 236, 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0121]

[44] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing amino acid changes into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and introducing the amino acid changes into the following positions:

[0122] (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0123] (ii) positions 236, 270, 298, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0124]

[45] The method of any one of

[42] to

[44] , wherein the functional modification is enhancement of binding activity to FcγRIIa and FcγRIIIa.

[0125]

[46] The method of any one of

[42] to

[44] , wherein the functional modification is to improve the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors.

[0126]

[47] The method of any one of

[42] to

[44] , wherein the functional modification is to selectively enhance the binding activity to an activating Fcγ receptor compared to the binding activity to an inhibitory Fcγ receptor.

[0127]

[48] ​​The method of any one of

[42] to

[44] , wherein the functional modification is to increase the ratio of binding activity to activating Fcγ receptors to binding activity to inhibitory Fcγ receptors (A / I ratio).

[0128]

[49]

[48] The polypeptide, wherein the ratio (A / I ratio) is increased by 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, compared to the ratio in a polypeptide comprising a parent Fc region. times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, 2000 times or more, 3000 times or more, 4000 times or more, 5000 times or more, 6000 times or more, 7000 times or more, 8000 times or more, 9000 times or more, or 10000 times or more.

[0129]

[50] The method of any one of

[42] to

[49] , wherein the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, and FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb.

[0130]

[51] The method according to any one of

[42] to

[44] , wherein the functional modification is enhancement of ADCC activity, CDC activity or ADCP activity. [Brief Description of the Drawings]

[0131] Figure 1 The results of an ADCC reporter gene assay are shown, in which Hepa1-6 / hEREG cells were used as target cells and Jurkat cells expressing hFcγRIIIaV were used as effector cells. Each point represents the mean fold induction value, n=2.

[0132] Figure 2 The results of an ADCC reporter gene assay are shown, in which Hepa1-6 / hEREG cells were used as target cells and hFcγRIIaH-expressing Jurkat cells were used as effector cells. Each point represents the mean fold induction value, n=3.

[0133] Figure 3The anti-tumor effects of EGL-G1d, EGL-afucosyl, and EGL-ART6 are shown in a human FcγR transgenic mouse model transplanted with Hepa1-6 / hEREG cell lines. Antibodies were administered via the tail vein at 10 mg / kg. Each point represents the mean tumor volume of a group, n=5.

[0134] Figure 4 The hC1q binding activity of each antibody with a modified Fc is shown. Each point represents the average ELISA color development value, n=2.

[0135] Figure 5 The hC1q binding activity of each antibody with a modified Fc is also shown. Each point represents the average ELISA color development value, n=2. [Specific implementation method]

[0136] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely used using conventional methodology, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, ed. (2003); Notebook (JECellis, editor, 1998) Academic Press; Animal Cell Culture (RI Freshney), editor, 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A.Doyle, JBGriffiths, and DG Newell, editors, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, editors); Gene Transfer Vectors for Mammalian Cells (JMMiller andM.P.Calos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (J. E. Coligan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0137] I. Definition

[0138] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY, 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th ed., John Wiley & Sons (New York, NY 1992), provide general guidance to those skilled in the art on many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0139] For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural form, and vice versa. It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall prevail.

[0140] As used herein, "first polypeptide" and "second polypeptide" refer to polypeptides that constitute the Fc region of an antibody. The terms "first polypeptide" and "second polypeptide" mean that their sequences differ from each other, and preferably differ at least in the CH2 domain sequence. Furthermore, the CH3 domain sequences may also differ. The polypeptides may be, for example, polypeptides that constitute the Fc region of a naturally occurring (native) IgG, or polypeptides produced by altering a polypeptide that constitutes the Fc region of a naturally occurring (native) IgG.

[0141] "Native IgG" refers to a polypeptide belonging to the class of antibodies actually encoded by the immunoglobulin gamma gene and comprising an amino acid sequence identical to that of IgG found in nature. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes mutants spontaneously generated therefrom.

[0142] The term "polypeptide" as used herein generally refers to a peptide or protein having a length of about 10 amino acids or longer. Furthermore, these are typically polypeptides derived from organisms, but are not particularly limited. For example, they may include artificially designed sequences. Furthermore, these may include any naturally occurring polypeptide, synthetic polypeptide, recombinant polypeptide, and the like. A protein molecule as used herein refers to a molecule comprising a polypeptide.

[0143] Preferred examples of the polypeptides of the present invention include antibodies. More preferred examples include natural IgG and antibodies produced by introducing modifications of natural IgG. Examples of natural IgG particularly include natural human IgG. "Native IgG" refers to a polypeptide that belongs to the class of antibodies actually encoded by the immunoglobulin gamma gene and comprises the same amino acid sequence as the IgG found in nature. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes mutants produced spontaneously by them.

[0144] The term "antibody" is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0145] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or a heavy chain variable domain, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ).

[0146] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. Antibodies are divided into five main classes: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0147] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain and / or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antigen-binding antibody to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0148] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that comprise an Fc region as defined herein.

[0149] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) in the Fc region may or may not be present. Unless otherwise indicated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also referred to as the EU index numbering, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0150] The term "polypeptide comprising an Fc region" is not particularly limited, as long as it is a polypeptide comprising an Fc region. For example, it is an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during purification of the polypeptide (e.g., antibody) or by recombinant engineering of a nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region according to the present invention may comprise a polypeptide comprising an Fc region having G446-K447, a polypeptide comprising an Fc region having G446 and not having K447, a polypeptide comprising an Fc region from which all G446-K447 have been removed, or a mixture of the three types of polypeptides.

[0151] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, and naturally occurring variants thereof.

[0152] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., from about 1 to about 30 amino acid substitutions, preferably from about 1 to about 20 amino acid substitutions, and more preferably from about 1 to about 10 amino acid substitutions, and most preferably from about 1 to about 5 amino acid substitutions, compared to a native sequence Fc region or a parent Fc region. The variant Fc region herein preferably has at least about 80% homology to a native sequence Fc region or to a parent Fc region, preferably at least about 85% homology thereto, more preferably at least about 90% homology thereto, and most preferably at least about 95% homology thereto.

[0153] " Percent (%) amino acid sequence identity " relative to a reference polypeptide sequence is defined as the percentage of the amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after the sequences are aligned and, if necessary, introduced into spaces to obtain maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0154] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, FcR is a natural human FcR. In some embodiments, FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants of those receptors and alternative spliceable forms. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibitory receptors"), which have similar amino acid sequences, the main difference being their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in, for example, Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-341 (1995). The term "FcR" herein encompasses additional FcRs, including those to be identified in the future.

[0155] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18 (12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15 (7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[0156] "Effecter cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as from blood. In certain embodiments, effector cells can be human effector cells.

[0157] "Effector functions" refer to those biological activities attributed to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0158] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays disclosed, for example, in the definitions herein.

[0159] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigens and subsequently kill the target cells with cytotoxins. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991) summarizes FcR expression on hematopoietic cells. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337 or 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0160] "Cytotoxic activity" includes, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity as described above, complement-dependent cytotoxicity (CDC) activity as described below, and T cell-mediated cytotoxicity. CDC activity refers to the cytotoxic activity of the complement system. On the other hand, ADCC activity refers to the activity of an antibody binding to an antigen present on the cell surface of a target cell, and effector cells further binding to the antibody, thereby damaging the target cell. Whether the antibody of interest has ADCC activity and whether the antibody of interest has CDC activity can be measured by known methods (e.g., Current Protocols in Immunology, Chapter 7, Immunologic studies in humans, edited by Coligan et al. (1993)).

[0161] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death induction in which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions that lead to the formation of pores in the membrane of the target cell. Typically, the antigen-antibody complex formed on the target cell binds and activates the complement component C1q, which in turn activates the complement cascade and leads to target cell death. In addition, complement activation may also lead to the deposition of complement components on the surface of the target cell, which leads to binding to complement receptors (e.g., CR3) on leukocytes, thereby promoting ADCC.

[0162] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to the process by which all or part of a cell coated with antibodies is incorporated into phagocytic immune cells (eg, macrophages, neutrophils, and dendritic cells) bound to the Fc region of an immunoglobulin.

[0163] An "isolated" polypeptide is one that has been separated from components of its natural environment. In some embodiments, the polypeptide is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing polypeptide purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0164] An "isolated" nucleic acid is a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0165] "Isolated nucleic acid encoding a polypeptide" refers to one or more nucleic acid molecules encoding a polypeptide (e.g., an antibody Fc region or antibody heavy and light chains or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acids present in one or more locations in a host cell.

[0166] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0167] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0168] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which it would be administered.

[0169] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0170] "Pharmaceutically acceptable carrier" refers to ingredients in pharmaceutical preparations and pharmaceutical compositions other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0171] An "effective amount" of a pharmaceutical agent (eg, a pharmaceutical preparation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive effect.

[0172] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention to attempt to alter the natural course of the individual being treated, and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, polypeptides comprising a variant Fc region of the invention are used to delay the development of a disease or slow the progression of a disease.

[0173] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive herein.

[0174] The term "tumor tissue" refers to tissue that contains at least one tumor cell. Tumor tissue is generally composed of a cluster of tumor cells (parenchyma) that forms the main body of the tumor, and connective tissue and blood vessels ("stroma") that exist between these cells and support the tumor. In some cases, the distinction between the two is clear, while in other cases, they are mixed. Tumor tissue can be infiltrated by immune cells, etc. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue in a living organism. Healthy / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0175] <Polypeptide containing variant Fc region>

[0176] In one aspect, the invention provides isolated polypeptides comprising variant Fc regions. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the variant Fc region comprises at least one amino acid residue change (e.g., substitution) compared to the corresponding sequence in the Fc region of a native sequence or reference variant sequence (collectively referred to herein as the "parent" Fc region). The Fc region of the native sequence is typically constructed as a homodimer consisting of two identical polypeptide chains. The amino acid changes in the variant Fc region of the present invention can be introduced into either of the two polypeptide chains of the parent Fc region, or introduced into the two polypeptide chains.

[0177] In some aspects, the present invention provides variant Fc regions whose functions have been modified compared to the parent Fc region. In some aspects, the variant Fc region of the present invention has enhanced binding activity to Fcγ receptors compared to the parent Fc region. In certain embodiments, the variant Fc region of the present invention has enhanced binding activity to at least one Fcγ receptor compared to the parent Fc region, and the at least one Fcγ receptor is selected from the group consisting of FcγRIa, FcγRIIa, FcβRIIb and FcγRIIIa. In some embodiments, the variant Fc region of the present invention has enhanced binding activity to FcγRIIa. In some embodiments, the variant Fc region of the present invention has enhanced binding activity to FcγRIIIa. In further embodiments, the variant Fc region of the present invention has enhanced binding activity to FcγRIIa and FcγRIIIa. On the other hand, the variant Fc region of the present invention has enhanced ADCC activity, CDC activity or ADCP activity compared to the parent Fc region.

[0178] The terms "binding activity" and "binding capacity" are used interchangeably herein and refer to the strength of the sum of the non-covalent interactions between one or more binding sites (e.g., variable region or Fc region) of a molecule (e.g., an antibody or other polypeptide) and its binding partner (e.g., an antigen or Fcγ receptor). Here, "binding activity" is not strictly limited to 1:1 interactions between binding pairs (e.g., an antibody and an antigen, or an Fc region and an Fcγ receptor). For example, when binding pairs reflect monovalent 1:1 interactions, binding activity refers to inherent binding affinity ("avidity"). When binding pairs are capable of both monovalent and multivalent binding, binding activity is the sum of each binding strength. The binding activity of molecule X to its partner Y can generally be represented by a dissociation constant (KD) or "analyte binding amount per unit amount of ligand." Affinity activity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

[0179] In certain embodiments, the binding activity of the parent Fc region and the variant Fc region can be represented by a KD (dissociation constant) value. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIa] / [KD value of the variant Fc region for FcγRIIa] is, for example, 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In further embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. Thus, the KD value of the Fc region for FcγRIIa can be the KD value of the Fc region for FcγRIIa R, the KD value of the Fc region for FcγRIIa H, or the sum or average of the two. In one embodiment, the ratio of [binding activity of the parent Fc region to FcγRIIIa] / [binding activity of the variant Fc region to FcγRIIIa] is, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 x 10 3 or larger, 2x10 3 or larger, 3x10 3 or larger, or 5x10 3 or greater. In further embodiments, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both. Thus, the KD value of the Fc region for FcγRIIIa may be the KD value of the Fc region for FcγRIIIa F, the KD value of the Fc region for FcγRIIa V, or the sum or average of the two.

[0180] In one embodiment, the variant Fc region has a KD value for FcγRIIa of, for example, 1.0×10 -6 M or smaller, 5.0x10 -7 M or smaller, 3.0x10 -7 M or smaller, 2.0x10 -7 M or smaller, 1.0x10 -7 M or smaller, 5.0x10 -8 M or smaller, 3.0x10 -8 M or smaller, 2.0x10 -8 M or smaller, 1.0x10 -8 M or smaller, 5.0x10 -9 M or smaller, 3.0x10 -9 M or smaller, 2.0x10 -9 M or smaller, or 1.0x10 -9M or less. In further embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the KD value of the variant Fc region for FcγRIIIa is, for example, 1.0x10 -6 M or smaller, 5.0x10 -7 M or smaller, 3.0x10 -7 M or smaller, 2.0x10 -7 M or smaller, 1.0x10 -7 M or smaller, 5.0x10 -8 M or smaller, 3.0x10 -8 M or smaller, 2.0x10 -8 M or smaller, 1.0x10 -8 M or smaller, 5.0x10 -9 M or smaller, 3.0x10 -9 M or smaller, 2.0x10 -9 M or smaller, 1.0x10 -9 M or smaller, 5.0x10 -10 M or smaller, 3.0x10 -10 M or smaller, 2.0x10 -10 M or smaller, or 1.0x10 -10 In further embodiments, the FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0181] In another embodiment, the binding activity of the parent and variant Fc regions can be expressed using kd (dissociation rate constant) values ​​instead of KD values.

[0182] In another embodiment, the binding activity of the parent and variant Fc regions can be represented by the amount of binding of the Fc region to the Fcγ receptor per unit amount. For example, in a surface plasmon resonance assay, the amount of binding of the Fc region immobilized on the sensor chip and the amount of binding of the Fcγ receptor further bound thereto are each measured as a resonance unit (RU). The value obtained by dividing the amount of binding of the Fcγ receptor by the amount of binding of the Fc region can be defined as the amount of binding of the Fc region to the Fcγ receptor per unit amount. Specific methods for measuring and calculating such binding amounts are described in the Examples below. In some embodiments, the ratio of [the amount of binding of the variant Fc region to FcγRIIa] / [the amount of binding of the parent Fc region to FcγRIIa] is, for example, 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In some embodiments, the ratio of [the amount of binding of the variant Fc region to FcγRIIa] / [the amount of binding of the parent Fc region to FcγRIIa] is, for example, 2 or greater, 3 or greater, 5 or greater, 10 or greater, 20 or greater, 30 or greater, 50 or greater, 100 or greater, 200 or greater, 300 or greater, 500 or greater, 1 x 10 3 or larger, 2x10 3 or larger, 3x10 3 or larger, or 5x10 3 or larger.

[0183] In certain embodiments, KD values, kd values, binding magnitude values, etc. described herein are measured or calculated by surface plasmon resonance assays performed at 25°C or 37°C (eg, see Example 2 herein).

[0184] In certain aspects, the variant Fc regions of the present invention have improved selectivity between activating and inhibitory Fcγ receptors compared to the parent Fc region. In other words, the variant Fc regions of the present invention have significantly enhanced binding activity for activating Fcγ receptors compared to inhibitory Fcγ receptors compared to the parent Fc region. In certain embodiments, the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. In some embodiments, the variant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the variant Fc regions of the present invention have improved selectivity between FcγRIIIa and FcγRIIb. In some embodiments, the variant Fc regions of the invention have improved selectivity between FcγRIIa and FcγRIIb, and between FcγRIIIa and FcγRIIb.

[0185] In certain embodiments, the binding activity of the parent Fc region and the variant Fc region can be represented by a KD (dissociation constant) value. The embodiments of the binding activity to FcγRIIa and FcγRIIIa are as described above. In one embodiment, the ratio of [KD value of the parent Fc region to FcγRIIb] / [KD value of the variant Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In another embodiment, the binding activity of the parent and variant Fc regions can be represented by a kd (dissociation rate constant) value instead of a KD value.

[0186] In another embodiment, the binding activity of the parent and variant Fc regions can be represented by the amount of binding to Fcγ receptors per unit amount of the Fc region as described above. In some embodiments, the ratio of [the amount of binding of the variant Fc region to FcγRIIb] / [the amount of binding of the parent Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the amount of binding of the variant Fc region to FcγRIIb is, for example, 0.5 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.005 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0187] In certain embodiments, the improved selectivity between activating Fcγ receptors and inhibitory Fcγ receptors is an increase in the selectivity of binding activity for activating Fcγ receptors compared to binding activity for inhibitory Fcγ receptors, or in other words, an increase in the ratio of binding activity for activating Fcγ receptors to binding activity for inhibitory Fcγ receptors (A / I ratio). This ratio (A / I ratio) is an indicator of excellent effector function. Polypeptides with a high A / I ratio can be evaluated as having excellent effector function. The binding activity of the parent Fc region and the variant Fc region for Fcγ receptors can be expressed as a KD value, a kd value, or the amount of Fcγ receptor binding per unit amount of the Fc region. The A / I ratio can be expressed using the KD value, kd value, or binding amount as follows: [KD value of inhibitory Fcγ receptor] / [KD value of activating Fcγ receptor], [kd value of inhibitory Fcγ receptor] / [kd value of activating Fcγ receptor], or [amount bound to activating Fcγ receptor] / [amount bound to inhibitory Fcγ receptor].

[0188] In one embodiment, the A / I ratio of a variant Fc region of the invention is increased by 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more compared to the A / I ratio of a parent Fc region. More, 60 times more, 70 times more, 80 times more, 90 times more, 100 times more, 200 times more, 300 times more, 400 times more, 500 times more, 600 times more, 700 times more, 800 times more, 900 times more, 1000 times more, 2000 times more, 3000 times more, 4000 times more, 5000 times more, 6000 times more, 7000 times more, 8000 times more, 9000 times more, or 10000 times more. In one embodiment, the A / I ratio of a variant Fc region of the invention has a value of 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 11000 or more, 12000 or more, 13000 or more, 14000 or more or 15000 or more. In one embodiment, the A / I ratio is the ratio of binding activity to FcγRIa to binding activity to FcγRIIb, the ratio of binding activity to FcγRIIa to binding activity to FcγRIIb, the ratio of binding activity to FcγRIIIa to binding activity to FcγRIIb, or the ratio of [the sum or average of two or three of binding activity to FcγRIa, binding activity to FcγRIIa, and binding activity to FcγRIIIa] to binding activity to FcγRIIb. In certain embodiments, FcγRIIa is FcγRIIa R, FcγRIIa H, or both. Thus, binding activity to FcγRIIa is the sum or average of binding activity to FcγRIIa R, binding activity to FcγRIIa H, or both. In certain embodiments, FcγRIIIa is FcγRIIIa F, FcγRIIIa V, or both.Therefore, the binding activity to FcγRIIIa is the binding activity to FcγRIIIa F, the binding activity to FcγRIIIa V, or the sum or average of the two.

[0189] In some embodiments, the variant Fc regions of the invention comprise amino acid changes at the following positions:

[0190] (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and

[0191] (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0192] In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 326 according to EU numbering in the first polypeptide of the parent Fc region. In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 236 according to EU numbering in the second polypeptide of the parent Fc region. In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 332 according to EU numbering in the first polypeptide of the parent Fc region. In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 330 according to EU numbering in the first polypeptide of the parent Fc region. In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 332 according to EU numbering in the second polypeptide of the parent Fc region. In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 330 according to EU numbering in the second polypeptide of the parent Fc region. Alternatively, the amino acid alterations described in International Publications WO2013 / 002362 and WO2014 / 104165 may also be similarly used in the present invention.

[0193] In some embodiments, the variant Fc regions of the invention comprise amino acid changes at the following positions:

[0194] (i) positions 234, 235, 236, 239, 268, 270, 298 and 330 according to EU numbering in the first polypeptide of the parent Fc region; and

[0195] (ii) positions 270, 298, 326, 330 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0196] In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 236 according to EU numbering in the second polypeptide of the parent Fc region. In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 332 according to EU numbering in the first polypeptide of the parent Fc region. In certain embodiments, the variant Fc region of the present invention further comprises an amino acid alteration at position 332 according to EU numbering in the second polypeptide of the parent Fc region. In certain embodiments, alternatively, the amino acid alterations described in International Publications WO2013 / 002362 and WO2014 / 104165 can also be similarly used in the present invention.

[0197] In some aspects, the variant Fc district of the present invention has improved stability compared to the parent Fc district. In a certain embodiment, stability is thermodynamic stability. The thermodynamic stability of a polypeptide can be determined, for example, by using a Tm value as an indicator. The Tm value can be measured using techniques known to those skilled in the art, such as circular dichroism (CD), differential scanning calorimetry (DSC), and differential scanning fluorimetry (DSF). In one embodiment, in the variant Fc district of the present invention, the Tm value in the CH2 district increases by 0.1 degree or more, 0.2 degree or more, 0.3 degree or more, 0.4 degree or more, 0.5 degree or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, or 10 degrees or more compared to the parent Fc district.

[0198] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from the group consisting of positions 250 and 307 according to EU numbering in the first polypeptide and / or the second polypeptide of the parent Fc region. Alternatively, the amino acid alterations described in WO 2013 / 11858 can be similarly used in the present invention.

[0199] In certain aspects, the variant Fc region of the present invention is composed of two polypeptide chains having different sequences from each other. On the other hand, in the variant Fc region of the present invention, heterodimerization between the first polypeptide and the second polypeptide is promoted. When using recombinant methods to produce heterodimeric proteins, it is preferred that different peptide chains preferentially associate to form heterodimers, rather than identical polypeptide chains associate to form homodimers. Whether heterodimerization in the variant Fc region is promoted can be determined by, for example, separating homodimers and heterodimers from the generated variant Fc region using techniques such as chromatography and determining the ratio of each component.

[0200] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from the group consisting of positions 349, 356, 366, 368, 407, and 439 according to EU numbering in the first polypeptide and / or the second polypeptide of the parent Fc region. Alternatively, the amino acid alterations described in WO 2006 / 106905 and WO 1996 / 027011 can also be similarly used in the present invention.

[0201] In certain aspects, the variant Fc regions of the present invention have enhanced binding activity to FcRn at acidic pH. In some embodiments, acidic pH refers to pH 4.0 to 6.5. In further embodiments, the acidic pH is at least one selected from the group consisting of pH 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain embodiments, the acidic pH is pH 5.8.

[0202] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from the group consisting of positions 428, 434, 436, 438, and 440 according to EU numbering in the first polypeptide and / or the second polypeptide of the parent Fc region. Alternatively, the amino acid alterations described in WO2016 / 125495 can also be similarly used in the present invention.

[0203] In one aspect, the variant Fc region of the invention comprises at least one amino acid alteration at at least one position selected from the group consisting of positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, 407, 428, 434, 436, 438, 439, and 440, according to EU numbering.

[0204] In one embodiment, the variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 268, 270, 298, 326, and 334, according to EU numbering. In a certain embodiment, the variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 268, 270, and 298, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 270, 298, 326, and 334, according to EU numbering, in a second polypeptide of a parent Fc region. In another embodiment, the variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 268, 270, 298, and 326, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 236, 270, 298, 326, and 334, according to EU numbering, in a second polypeptide of a parent Fc region.

[0205] In one embodiment, a variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 268, 270, 298, 326, 330, and 334, according to EU numbering. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 268, 270, 298, and 330, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 270, 298, 326, 330, and 334, according to EU numbering, in a second polypeptide of a parent Fc region.

[0206] In one embodiment, a variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 334, according to EU numbering. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, and 307, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 250, 270, 298, 307, 326, and 334, according to EU numbering, in a second polypeptide of a parent Fc region. In another embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, by EU numbering, of a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 236, 250, 270, 298, 307, 326, and 334, by EU numbering, of a second polypeptide of a parent Fc region.

[0207] In one embodiment, a variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 268, 270, 298, 326, 330, 332, and 334, according to EU numbering. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 268, 270, 298, 330, and 332, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 236, 270, 298, 326, 330, 332, and 334, according to EU numbering, in a second polypeptide of a parent Fc region.

[0208] In one embodiment, a variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, and 334, according to EU numbering. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, according to EU numbering, in a second polypeptide of a parent Fc region. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, by EU numbering, of a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, by EU numbering, of a second polypeptide of a parent Fc region.

[0209] In one embodiment, a variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334, according to EU numbering. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 332, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 236, 250, 270, 298, 307, 326, 332, and 334, according to EU numbering, in a second polypeptide of a parent Fc region.

[0210] In one embodiment, a variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334, according to EU numbering. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 332, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 250, 270, 298, 307, 326, 332, and 334, according to EU numbering, in a second polypeptide of a parent Fc region.

[0211] In one embodiment, a variant Fc region of the invention comprises amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, and 334, according to EU numbering. In a certain embodiment, a variant Fc region of the invention comprises (i) amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 330, according to EU numbering, in a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 250, 270, 298, 307, 326, 330, and 334, according to EU numbering, in a second polypeptide of a parent Fc region.

[0212] In a further embodiment, the variant Fc region of the present invention comprises at least one amino acid change selected from the group consisting of: (i) in the first polypeptide of the parent Fc region, according to EU numbering, Tyr or Phe at position 234, Gln or Tyr at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, P at position 307; ro, Asp at position 326, Met at position 330, Glu at position 332; and (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, Glu at position 334 in a second polypeptide of the parent Fc region according to EU numbering.

[0213] In a further embodiment, the variant Fc region of the present invention further comprises the following amino acid changes (a) to (f):

[0214] (a) Lys at position 356 according to EU numbering in the first polypeptide of the parent Fc region and Glu at position 439 according to EU numbering in the second polypeptide of the parent Fc region;

[0215] (b) Glu at position 439 according to EU numbering in the first polypeptide of the parent Fc region and Lys at position 356 according to EU numbering in the second polypeptide of the parent Fc region;

[0216] (c) Trp at position 366 according to EU numbering in the first polypeptide of the parent Fc region and Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the second polypeptide of the parent Fc region;

[0217] (d) Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the first polypeptide of the parent Fc region, and Trp at position 366 according to EU numbering in the second polypeptide of the parent Fc region;

[0218] (e) Cys at position 349 and Trp at position 366 according to EU numbering in the first polypeptide of the parent Fc region, and Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the second polypeptide of the parent Fc region;

[0219] (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the first polypeptide of the parent Fc region, and Cys at position 349 and Trp at position 366 according to EU numbering in the second polypeptide of the parent Fc region.

[0220] In a further aspect, the variant Fc region of the present invention further comprises any of the following amino acid changes (a) to (d) in the first polypeptide and / or the second polypeptide of the parent Fc region:

[0221] (a) Ala according to EU numbering position 434;

[0222] (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering;

[0223] (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering; and

[0224] (d) According to EU numbering, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440.

[0225] In a certain embodiment, the polypeptide comprising a variant Fc region of the invention is an antibody heavy chain constant region.

[0226] In a further embodiment, the present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 7-22 and 35-50.

[0227] In a further embodiment, the present invention provides a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 35 and a polypeptide chain of SEQ ID NO: 36, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 37 and a polypeptide chain of SEQ ID NO: 38, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 39 and a polypeptide chain of SEQ ID NO: 40, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 41 and a polypeptide chain of SEQ ID NO: 42, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 43 and a polypeptide chain of SEQ ID NO: 44, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 45 and a polypeptide chain of SEQ ID NO: 46, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 47 and a polypeptide chain of SEQ ID NO: 48, and a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 49 and a polypeptide chain of SEQ ID NO: 50.

[0228] "Fcγ receptor" (abbreviated herein as Fcγ receptor, FcγR or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies, and actually refers to any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 (H-type) and R131 (R-type)), FcγRIIb (including FcγRI Ib-1 and FcγRI Ib-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as, but not limited to, any human FcγRs, FcγR isoforms, or allotypes yet to be discovered. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. In addition, a splice variant named FcγFcγRIIb3 has also been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb also includes all splice variants registered in NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992.3. Furthermore, human FcγRIIb includes each previously reported genetic polymorphism, as well as FcγRIIb (Arthritis Rheum. 48:3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005)); and Kyogoju et al., Arthritis Rheum. 46:1242-1254 (2002)), as well as each genetic polymorphism to be reported in the future.

[0229] There are two isoforms of FcγRIIa: one in which the 131st amino acid is histidine (H-type) and the other in which the 131st amino acid is substituted with arginine (R-type) (Warrmerdam, J. Exp. Med. 172:19-25 (1990)).

[0230] FcγRs include, but are not limited to, those of human, mouse, rat, rabbit, and monkey origin, and can be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FbγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform.

[0231] The amino acid sequence of human FcγRI is shown in NP_000557.1; the amino acid sequence of human FcγRIIa is shown in AAH20823.1, etc.; the amino acid sequence of human FcγRIIb is shown in AAI46679.1, etc.; the amino acid sequence of human FcγRIIIa is shown in AAH33678.1, etc.; and the amino acid sequence of human FcγRIIIb is shown in AAI28563.1.

[0232] Unlike Fcγ receptors belonging to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides, showing 22% to 29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18(12):592-598). FcRn is expressed as a heterodimer composed of a soluble β or light chain (β2 microglobulin) and a transmembrane α chain or heavy chain. Like MHC, the FcRn α chain contains three extracellular domains (α1, α2, and α3), and its short cytoplasmic domain anchors the protein to the cell surface. The α1 and α2 domains interact with the FcRn binding domain of the antibody Fc region (Raghavan et al., Immunity (1994) 1:303-315). The amino acid sequence of human FcRn is shown in NP_004098.1; and the amino acid sequence of β2 microglobulin is shown in NP_004039.1.

[0233] As used herein, "parent Fc region" refers to the Fc region before the introduction of the amino acid changes described herein. In some embodiments, the parent Fc region is the Fc region of the native sequence (or the Fc region of a natural antibody). Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) and IgM. Antibodies can be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees or baboons). Natural antibodies can include naturally occurring mutations. The multiple allotype sequences of IgG caused by genetic polymorphism are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, and any one of them can be used in the present invention. In particular, for human IgG1, the amino acid sequence of positions 356 to 358 (EU numbering) can be DEL or EEM. In addition, for human IgG1, the amino acid at position 214 (EU numbering) can be K or R. In certain embodiments, the parent Fc region is an Fc region derived from the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4. In another embodiment, the parent Fc region is an Fc region derived from the heavy chain constant region of SEQ ID NO: 1, 28, 29, or 56. In further embodiments, the parent Fc region may be an Fc region generated by adding amino acid changes other than the amino acid changes described herein to an Fc region derived from a native sequence (reference variant sequence Fc region). Native sequence Fc regions are typically organized as homodimers composed of two identical polypeptide chains.

[0234] Furthermore, amino acid changes made for other purposes may be combined in the variant Fc regions described herein. For example, amino acid substitutions that improve FcRn binding activity (Hinton et al., J. Immunol. 176(1):346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12):1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159 (2010); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320), as well as amino acid substitutions for improving antibody heterogeneity or stability (WO 2009 / 041613) can be added. Alternatively, the polypeptide with promotion antigen clearance characteristic described in WO 2011 / 122011, WO 2012 / 132067, WO 2013 / 046704 or WO 2013 / 180201, and the polypeptide with specific binding target tissue characteristic described in WO 2013 / 180200, the polypeptide with the characteristic of repeating in conjunction with multiple antigen molecules described in WO 2009 / 125825, WO 2012 / 073992 or WO 2013 / 047752 can be combined with variant Fc district described herein. Alternatively, in order to give the binding ability with other antigens, the amino acid changes disclosed in EP1752471 and EP1772465 can be combined in the CH3 in variant Fc district described herein. Alternatively, in order to increase plasma retention, the amino acid changes (WO2012 / 016227) that reduce constant region pI can be combined in variant Fc district described herein. Alternatively, to promote cellular uptake, amino acid changes that increase the pI of the constant region (WO 2014 / 145159) can be combined in variant Fc regions as described herein. Alternatively, to promote elimination of the target molecule from plasma, amino acid changes that increase the pI of the constant region (WO 2016 / 125495) can be combined in variant Fc regions as described herein. In one embodiment, such changes may include, for example, substitutions at at least one position selected from the group consisting of positions 311, 343, 384, 399, 400, and 413 according to EU numbering. In a further embodiment, such substitutions may be substitutions of amino acids with Lys or Arg at each position.

[0235] Methods for producing heterodimeric antibodies are not limited to these, but such antibodies can be produced by the knob-in-hole technique (see, for example, Nat. Biotechnol., (16); 677-681 (1998) and U.S. Patent No. 5731168) or by engineering electrostatic steering effects (WO2006 / 106905, WO2009 / 089004A1, J. Biol. Chem., (285), 19637-19646 (2010) etc.).

[0236] For association of heterologous polypeptides comprising a variant Fc region, techniques for inhibiting unintended association of homologous polypeptides comprising a variant Fc region by introducing electrostatic repulsion into the interface of the CH2 or CH3 domains of the Fc region may be applied, as described in WO 2006 / 106905.

[0237] Examples of amino acid residues contacting at the CH2 or CH3 domain interface of the Fc region include residue 356 (EU numbering), residue 439 (EU numbering), residue 357 (EU numbering), residue 370 (EU numbering), residue 399 (EU numbering), and residue 409 (EU numbering) in the CH3 domain.

[0238] More specifically, for example, an Fc region can be produced in which one to three pairs of amino acid residues selected from the following (1) to (3) have the same charge: (1) amino acid residues at positions 356 and 439 (EU numbering) in the CH3 domain; (2) amino acid residues at positions 357 and 370 (EU numbering) in the CH3 domain; and (3) amino acid residues at positions 399 and 409 (EU numbering) in the CH3 domain.

[0239] Furthermore, a heterologous polypeptide comprising a variant Fc region can be produced in which one to three pairs of amino acid residues selected from (1) to (3) above have the same charge in the CH3 domain of the first Fc region, and the pairs of amino acid residues selected from the above-mentioned first Fc region also have the same charge in the CH3 domain of the second Fc region, provided that the charges in the first and second Fc regions are opposite.

[0240] In the above-mentioned Fc region, for example, the negatively charged amino acid residue is preferably selected from glutamic acid (E) and aspartic acid (D), and the positively charged amino acid residue is preferably selected from lysine (K), arginine (R) and histidine (H).

[0241] Other known techniques can also be used to associate heterologous polypeptides containing variant Fc regions. Specifically, this technique is performed by replacing the amino acid side chains present in one of the Fc regions with larger side chains (knob, meaning "protrusion") and replacing the amino acid side chains present in the Fc region (hole, meaning "hole") with smaller side chains, so as to place the knob within the hole. This can promote efficient association between polypeptides containing Fc regions having different amino acid sequences (WO1996 / 027011; Ridgway et al., Prot. Eng. 9:617-621 (1996); Merchant et al., Nat. Biotech. 16:677-681 (1998)).

[0242] In addition, other known techniques can also be used for heterologous association of polypeptides containing variant Fc regions. The association of polypeptides containing Fc regions can be effectively induced using chain exchange engineered domain CH3 heterodimers (Davis et al., Prot. Eng. Des & Sel., 23: 195-202 (2010)). This technology can also be used to effectively induce association between polypeptides containing Fc regions with different amino acid sequences.

[0243] Furthermore, the heterodimeric antibody production technology using the association of antibody CH1 and CL and the association of VH and VL described in WO 2011 / 028952 can also be used.

[0244] As with the methods described in WO 2008 / 119353 and WO 2011 / 131746, a technique for producing heterodimeric antibodies by preliminarily producing two types of homodimeric antibodies, incubating the antibodies under reducing conditions to dissociate them, and allowing them to associate again can also be used.

[0245] Furthermore, techniques for generating heterodimeric antibodies by adding changes to the CH2 and CH3 domains can also be used, as with the method described in WO 2012 / 058768.

[0246] When expressing two kinds of polypeptides comprising variant Fc district with different amino acid sequences simultaneously, in order to produce the polypeptide comprising heterologous variant Fc district, usually also produce the polypeptide comprising homologous variant Fc district as impurity.In this case, the polypeptide comprising heterologous variant Fc district can be effectively obtained by using known technology to separate and purify from the polypeptide comprising homologous variant Fc district.A method has been reported, which uses ion exchange chromatography, by introducing the variable region of two types of antibody heavy chains into which amino acid changes are introduced, to produce the difference of isoelectric point between homodimeric antibody and heterodimeric antibody, so as to effectively separate and purify heterodimeric antibody (WO 2007 / 114325) from homodimeric antibody.Another method using protein A chromatography purification of heterodimeric antibody has been reported, which comprises the heterodimeric antibody of two types of heavy chains by building, and the heavy chain is derived from the mouse IgG2a combined with protein A and the rat IgG2b (WO 1998 / 050431 and WO1995 / 033844) not combined with protein A.

[0247] In addition, by replacing amino acid residues at positions 435 and 436 (EU numbering) located in the protein A binding site of the antibody heavy chain with amino acids such as Tyr or His to generate different protein A binding affinities, heterodimeric antibodies can be efficiently purified using protein A chromatography.

[0248] In the present invention, amino acid changes refer to any one of substitution, deletion, addition, insertion and modification, or a combination thereof. In the present disclosure, amino acid changes can be rewritten as amino acid mutations.

[0249] The number of amino acid changes introduced into the Fc region is not limited. In certain embodiments, it can be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, 20 or less, 22 or less, 24 or less, 26 or less, 28 or less, or 30 or less.

[0250] In one aspect, the present invention provides methods for producing polypeptides comprising variant Fc regions. In other aspects, the present invention provides methods for producing polypeptides comprising variant Fc regions whose functions have been modified. In other aspects, the present invention provides methods for modifying the function of polypeptides comprising Fc regions. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, these methods comprise introducing at least one amino acid change into a parent Fc region. In certain embodiments, these methods comprise: (i) providing a polypeptide comprising the parent Fc region; and (ii) introducing at least one amino acid change into the parent Fc region. In certain embodiments, these methods may further comprise (iii) measuring the function of the polypeptide comprising the variant Fc region. A native Fc region is typically composed of two identical polypeptide chains. The amino acid changes in the parent Fc region can be introduced into either of the two polypeptide chains of the parent Fc region, or into both polypeptide chains.

[0251] In another embodiment, a method for producing a polypeptide comprising a variant Fc region comprises: (i) providing one or more nucleic acids encoding a polypeptide comprising the parent Fc region; (ii) introducing at least one mutation into the region encoding the parent Fc region of the nucleic acid; (iii) introducing the nucleic acid produced in (ii) into a host cell; and (iv) culturing the cell in (iii) such that the polypeptide comprising the variant Fc region is expressed. In certain embodiments, the above method may further comprise (v) collecting the polypeptide comprising the variant Fc region from the host cell culture in (iv).

[0252] In certain embodiments, the nucleic acid produced in (ii) can be contained in one or more vectors (eg, expression vectors).

[0253] In some embodiments, the amino acid alterations used in the production methods of the present invention are selected from any single alteration (selected from the amino acid alterations that can be included in the variant Fc region described above), combination of single alterations, or combination alterations listed in Table 1.

[0254] The Fc region can be obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc., using a protease such as pepsin, and then reeluting the fraction adsorbed on a protein A column. The protease is not particularly limited as long as it can digest the full-length antibody, thereby producing Fab and F(ab')2 in a restricted manner by appropriately setting the enzyme reaction conditions such as pH, and examples include pepsin and papain.

[0255] In addition to the above-mentioned production methods, polypeptides comprising the variant Fc region of the present invention can be produced by other methods known in the art. Polypeptides comprising the variant Fc region produced by the production methods described herein are also included in the present invention.

[0256] In one embodiment, an isolated nucleic acid encoding a polypeptide comprising a variant Fc region of the present invention is provided. This nucleic acid can encode the amino acid sequence of a first polypeptide comprising a variant Fc region and / or the amino acid sequence of a second polypeptide comprising a variant Fc region. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises the following (e.g., transformed with): (1) a vector comprising a nucleic acid encoding the amino acid sequence of a first polypeptide comprising a variant Fc region and the amino acid sequence of a second polypeptide comprising a variant Fc region, or (2) a first vector comprising a nucleic acid encoding the amino acid sequence of a first polypeptide comprising a variant Fc region, and a second vector comprising a nucleic acid encoding the amino acid sequence of a second polypeptide comprising a variant Fc region. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of preparing a polypeptide comprising a variant Fc region of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a polypeptide comprising a variant Fc region of the invention under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

[0257] For recombinant production of polypeptides comprising variant Fc regions of the present invention, nucleic acids encoding the polypeptides are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the Fc regions of antibodies).

[0258] Suitable host cells for cloning or expressing vectors encoding polypeptides comprising a variant Fc region of the present invention include prokaryotic or eukaryotic cells.

[0259] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding polypeptides comprising variant Fc regions of the invention, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibody Fc regions with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0260] Suitable host cells for expressing glycosylated antibody Fc regions are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0261] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429.

[0262] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described in Mather et al., Annals of NY Acad. Sci. 383: 44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0.

[0263] The assays described herein or various measurement methods known in the art can be used to identify or screen the variant Fc regions provided herein, or to elucidate their physical or chemical properties or biological activities.

[0264] Described herein are assays or assays known in the art for determining the binding activity of polypeptides containing variant Fc regions to one or more FcR family members. Such binding assays include, but are not limited to, surface plasmon resonance assays, amplified luminescence proximity homogeneity assay (ALPHA) screening, ELISA, and fluorescence activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103(11):4005-4010).

[0265] In one embodiment, the binding activity of the polypeptide comprising the variant Fc region to the FcR family member can be measured using surface plasmon resonance. For example, by using known methods and reagents (e.g., protein A, protein L, protein A / G, protein G, anti-λ chain antibodies, anti-κ chain antibodies, antigenic peptides and antigenic proteins), various FcRs are used as analytes to interact with the polypeptide comprising the variant Fc region fixed or captured on the sensor chip. Alternatively, FcR can be fixed or captured on the sensor chip, and the polypeptide comprising the variant Fc region can be used as an analyte. As a result of this interaction, a binding sensorgram is obtained, and by analyzing them, the dissociation constant (KD) value of this combination can be calculated. In addition, before and after interacting with FcR, the difference in the resonance unit (RU) value in the sensorgram (i.e., the binding amount of FcR) can be used as an index of the binding activity of the polypeptide comprising the variant Fc region to FcR. Furthermore, a corrected value obtained by dividing the above-mentioned FcR binding amount by the difference in RU values ​​in the sensorgrams before and after the polypeptide containing a variant Fc region is immobilized or captured on the sensor chip (i.e., the binding amount of the polypeptide containing a variant Fc region) (i.e., the corrected value is the FcR binding amount per unit amount of the polypeptide containing a variant Fc region) can be used as an indicator of binding activity.

[0266] Any of the polypeptides comprising a variant Fc region provided herein may be used in therapeutic methods.

[0267] In one aspect, a polypeptide comprising a variant Fc region for use as a drug is provided. In a further aspect, a polypeptide comprising a variant Fc region for use in treating a tumor is provided. In certain embodiments, a polypeptide comprising a variant Fc region for use in a method of treating a tumor is provided. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of treating an individual suffering from a tumor, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In a further aspect, the present invention provides a polypeptide comprising a variant Fc region for use in damaging cells. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of damaging cells in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to damage the cells. The "individual" according to any of the above embodiments is preferably a human.

[0268] In some embodiments, the tumor is a solid tumor. In solid tumors, tumor cells usually proliferate to form a colony, and tumor tissue is mainly formed by these cells. In addition, tumor tissue in an organism is usually infiltrated by immune cells such as lymphocytes, which also form part of the tumor tissue. In one embodiment, the damage to the cell is caused by ADCC activity, CDC activity or ADCP activity.

[0269] In a further aspect, the present invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In one embodiment, the medicament is for use in treating a tumor. In a further embodiment, the medicament is for use in a method of treating a tumor, comprising administering an effective amount of the medicament to an individual suffering from the tumor. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In another embodiment, the medicament is for use in damaging cells. In another embodiment, the medicament is for use in a method of damaging cells in an individual, comprising administering an effective amount of the medicament to the individual to damage the cells. The "individual" according to any of the above embodiments is preferably a human.

[0270] In a further aspect, the present invention provides a method for treating a tumor. In one embodiment, the method comprises administering to an individual suffering from such a tumor an effective amount of a polypeptide comprising a variant Fc region. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments can be a human.

[0271] In a further aspect, the present disclosure provides a method of damaging cells in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to damage the cells. In one embodiment, the "individual" is a human.

[0272] On the other hand, the present invention provides pharmaceutical preparations (pharmaceutical compositions) comprising any polypeptide containing a variant Fc region provided herein. In one embodiment, the above-mentioned pharmaceutical preparations (pharmaceutical compositions) further comprise a pharmaceutically acceptable carrier. In one embodiment, the present invention provides pharmaceutical preparations (pharmaceutical compositions) for treating tumors. In one embodiment, the present invention provides pharmaceutical preparations (pharmaceutical compositions) for damaging cells. In another embodiment, the pharmaceutical preparations (pharmaceutical compositions) comprise a polypeptide containing a variant Fc region provided herein and at least one additional therapeutic agent.

[0273] Example

[0274] [Example 1] Generation of Fc region variants with enhanced FcγR-binding ability

[0275] Although Fc variants that enhance the cytotoxic effector functions ADCC and ADCP have been previously reported, variants with symmetrically engineered CH2 domains and low-fucose antibodies produced by sugar chain modification still have room for further enhancement of FcγR binding. In addition, although the variants with asymmetrically engineered CH2 domains described in WO2013002362 and WO2014104165 have significantly enhanced FcγR-binding ability compared to symmetrically engineered Fc region variants, they still have room for further improvement. Specifically, the Fc region variant Kn125 / Hl076 (referred to herein as ART1) described in WO201300262 and WO2014104165 has significantly enhanced binding to FcγRIIIa, but its FcγRIIa binding ability is only enhanced several times compared to IgG1, so it seems necessary to further enhance it to show strong ADCP activity. Kn120 / Hl068 (herein referred to as ART2) has enhanced binding to FcγRIIa and FcγRIIIa and is expected to show strong ADCC and ADCP activity. However, its binding ability to inhibitory FcγRIIb is also enhanced, so its A / I ratio (an indicator of excellent effector function) is low. Therefore, existing methods have not yet achieved the most ideal effect, that is, "antibody modification technology that can enhance binding to activating FcγRIIa and FcγRIIIa and inhibit binding to inhibitory FcγRIIb." Therefore, in the present invention, further combinations of modifications were studied, and the generation of Fc region variants with excellent characteristics that overcome the above problems was studied.

[0276] The existing Fc region variants used as a comparative reference were prepared as follows: First, the antibody heavy chain gene H240-G1d (SEQ ID NO: 1) as described in WO2014104165 was prepared, which has a heavy chain variable region for human epidermal regulin (Epiregulin) and a heavy chain constant region sequence for human IgG1. G1d is a sequence obtained by deleting C-terminal Lys and Gly from the heavy chain constant region sequence of natural human IgG1. A knob-hole modification (which is a modification that promotes heterodimerization) (Nat. Biotechnol., 1998, 16777) was introduced into the CH3 domain of H240-G1d, and a modification that enhances FcγR binding was asymmetrically introduced into the CH2 domain to produce Fc region variants, ART1 and ART2, used as comparative references. ART1 is an Fc region variant with enhanced FcγRIIIa binding described in WO2013002362 and WO2014104165, which was prepared as follows: the modifications L234Y / L235Q / G236W / S239M / H268D / D270E / S298A for enhanced FcγR binding were introduced into the CH2 domain of H240-G1d, and Y349C / T366W were introduced into the CH3 domain to generate H240-Kn125 (SEQ ID NO: 2). In addition, D270E / K326D / A330M / K334E were introduced into the CH2 domain of H240-G1d, and D356C / T366S / L368A / Y407V were introduced into the CH3 domain to generate H240-H1076 (SEQ ID NO: 3). The plasmid containing H240-Kn125, H240-H1076 and the light chain L73-k0 (SEQ ID NO: 4) gene of the anti-human epidermidulin antibody was mixed and introduced into the human embryonic kidney cell-derived Expi293 cell line (Invitrogen) by lipofection. After 4 days of cultivation, the cells were isolated using rProtein A Sepharose 5-well plate (10 μg / mL) by methods known to those skilled in the art. TM The supernatant was purified using Fast Flow (Amersham Biosciences) to obtain an Fc variant antibody against human epiregulin (H240-Kn125 / L73-k0 / / H240-H1076 / L73-k0, abbreviated antibody name: EGL-ART1). The absorbance at 280 nm of the purified antibody solution was measured using a spectrophotometer. The concentration of the purified antibody was calculated from the measured values ​​using the extinction coefficient calculated using the PACE method (Protein Science 1995; 4: 2411-2423).

[0277] Similarly, an Fc region variant EGL-ART2 (H240-Kn220 / L73-k0 / / H240-Hl068 / L73-k0) with enhanced binding to both FcγRIIa and FcγRIIIa as described in WO2013002362 and WO2014104165 was generated. In addition, different combinations of FcγR binding-enhancing modifications, G236A, S239D, A330L, and I332E, were symmetrically introduced into the CH2 domain to generate the known FcγR binding-enhanced antibodies EGL-SDALIE (H240-Kn032 / L73-k0 / / H240-Hl032 / L73-k0), EGL-GASDIE (H240-Kn037 / L73-k0 / / H240-Hl036 / L73-k0), and EGL-GASDALIE (H240-GASDALIE / L73-k0). In addition, the afucosylated antibody EGL-afucosyl was prepared as a comparative reference and reported to have enhanced binding to FcγRIIIa (Glycobiol. Vol. 17 no. 1 pp. 104-118 (2006)). In cells where the expression of the fucose transporter gene on both homologous chromosomes is artificially suppressed, the function of the fucose transporter is suppressed. These cells can be used to obtain fucose-deficient antibodies (WO2006 / 067913, etc.). Fucose-deficient antibodies can also be obtained by producing antibodies in cells forced to express β1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II (Biotechnol, Bioeng. (2006) 93 (5), 851-861). Using these methods known to those skilled in the art, EGL-afucosyl (H240-G1d / L73-k-glycomab) was prepared.

[0278] To generate Fc region variants superior to these existing variants, new Fc region variants were generated, namely ART3, ART4, ART5, ART6, ART8, ART10, ART11, and ART12, as shown in Table 1. These variants all introduced L234F, L235Q, G236W, S239M, H268D, D270E, and S298A into one heavy chain, and D270E, S298A, K326D, and 334E into the other heavy chain. In addition to these core asymmetric modifications, these variants were generated by introducing combinations of modifications that alter FcγR binding. Specifically, for the chain that introduced L234F / L235Q / G236W / S239M / H268D / D270E / S298A, the further introduction of K326D, A330M, and I332E was investigated. For the chains with D270E / S298A / K326D / K334E, further introduction of G236A, I332E, I332D, and A330M was studied. In addition to these FcγR binding-enhancing modifications, T250V and T307P are modifications used to improve antibody stability and were introduced into both chains of ART4, ART5, ART6, ART8, ART10, ART11, and ART12, as described in WO2013118858.

[0279] The correspondence between the names of the heavy chain constant regions used herein and SEQ ID NO: is as follows: G1d (SEQ ID NO:29), Kn125 (SEQ ID NO:30), H1076 (SEQ ID NO:31), k0 (SEQ ID NO:32), Kn120 (SEQ ID NO:33), H1068 (SEQ ID NO:34), Kn443 (SEQ ID NO:35), H1408 (SEQ ID NO:36), Kn456 (SEQ ID NO:37), H1446 (SEQ ID NO:38), Kn462 (SEQ ID NO:39), H1441 (SEQ ID NO:40), Kn462 (SEQ ID NO:41), H1445 (SEQ ID NO:42), Kn461 (SEQ ID NO:43), H1443 (SEQ ID NO:44), Kn494 (SEQ ID NO:45), H1514 (SEQ ID NO:46). NO:46),Kn496(SEQ ID NO:47),H1516(SEQ ID NO:48),Kn498(SEQ ID NO:49),H1518(SEQ ID NO:50),GASDALIE(SEQ ID NO:51),Kn032(SEQ ID NO:52),H1032(SEQ ID NO:53), Kn037 (SEQ ID NO:54), H1036 (SEQ ID NO:55), G4d (SEQ ID NO:56).

[0280] (Table 1) Generated Fc region variants and introduced modifications

[0281]

[0282]

[0283] [Example 2] Evaluation of FcγR Binding of Fc Region Variants

[0284] The extracellular domain of FcγR was produced by the method described in WO2014104165. The interaction between the produced antibodies and human FcγR was analyzed by the following method using Biacore8K+. For the running buffer, 50mM sodium phosphate, 150mM NaCl and 0.05% Tween 20 (pH 7.4) were used, and the measurements were performed at 25°C. The sensor chip used was an S series SA chip (GE Healthcare) on which CaptureSelect human Fab-kappa kinetic biotin conjugate (ThermoFisher Scientific) was immobilized. The antibody of interest was captured onto the chip, and each FcγR diluted with running buffer was allowed to interact with it. The chip was regenerated using 10mM glycine-HCl (pH 1.5), and the chip was reused to capture the antibody and perform the measurement. The dissociation constant KD (mol / L) of each antibody for FcγR was calculated using Biacore Insight Evaluation software. The FcγRIIb dissociation constant adopted a steady-state affinity model, and the other FcγR dissociation constants adopted a 1:1 Langmuir binding model (Table 2).

[0285] (Table 2) Binding measurements between generated variants and human FcγR

[0286]

[0287]

[0288] In the table, "Relative KD values ​​between G1d and hFcγR" are calculated by dividing the KD value of G1d for each FcγR by the KD value of each antibody for each FcγR, indicating how much each antibody enhances binding compared to G1d. "A / I ratios" are calculated by dividing the KD of each antibody for FcγRIIb by the KD for each FcγR, indicating how much the selectivity of binding to activating FcγRs is enhanced over that to inhibitory FcγRs.

[0289] For FcγRIIIaF and Fcγ, ART3, ART4, ART5, ART6, ART8, ART10, ART11 and ART12 produced in the present invention are all enhanced compared to G1d. For both FcγRIIIaF and FcγRIIIaV, these variants are also more enhanced than the existing symmetrically modified FcγR enhancing antibodies GASDALIE, SDALIE, GASDIE and defucosyl (Afucosyl) antibodies. In addition, compared with ART2 (459.6 times) (as described in WO2014104165), ART4 (2519.9 times), ART6 (986.7 times), ART8 (1966.7 times), ART10 (1289.2 times) and ART12 (577.5 times) FcγRIIIaF binding is more enhanced. Furthermore, ART4, ART8, and ART10 showed stronger binding to FcγRIIIaF even compared to ART1 (1170.2-fold), which binds to FcγRIIIaF more strongly than ART2. Similarly, for FcγRIIIaV, ART4 (462.2-fold), ART6 (321.9-fold), ART8 (694.9-fold), and ART10 (565.5-fold) showed greater binding enhancement than ART2 (214.6-fold), and even more enhancement than ART1 (322.8-fold). ART6's binding to FcγRIIIaV was comparable to that of ART1.

[0290] For FcγRIIaH, compared to G1d, ART3 (32.1-fold), ART4 (6.3-fold), ART5 (33.9-fold), ART6 (118.0-fold), ART8 (15.0-fold), ART10 (2.7-fold), ART11 (4.9-fold), and ART12 (3.1-fold) all enhanced. In particular, even compared to ART2 (14.7-fold), (an FcγRIIa-enhanced antibody with an asymmetrically modified CH2 domain as described in WO2014104165), ART3, ART5, ART6, and ART8 enhanced more. In addition, even compared to the existing symmetrically modified FcγRIIa-enhanced antibody GASDIE (16.4-fold), ART3, ART5, and ART6 enhanced more, and therefore are expected to show stronger ADCP activity than any existing variant. Regarding binding to FcγRIIaR, all ART3 (13.7-fold), ART4 (3.2-fold), ART5 (10.4-fold), ART6 (24.1-fold), ART8 (13.2-fold), ART10 (1.5-fold), and ART11 (1.3-fold) were enhanced compared to G1d, but the existing variants GASDIE (24.7-fold) and ART2 (49.0-fold) were more enhanced than these variants. However, it should be noted here that the selectivity for activating FcγRs is significant. In contrast to activating FcγRs, since the inhibitory receptor FcγRIIb induces intracellular signals that suppress immune responses, it is expected to inhibit signals from activating FcγRs. In fact, it has been reported that the anti-tumor effect of antibodies is enhanced in FcγRIIb knockout mice (Nature Medicine 2000, 64443-436). Furthermore, within each mouse IgG subclass, a correlation was observed between antitumor activity and the binding ratio (A / I ratio) to activating and inhibitory FcγRs (Science 20053101510-1512). Therefore, to exert stronger effector functions, an antibody with enhanced binding to activating FcγRs and reduced binding to FcγRIIb may be necessary. However, the high sequence homology between FcγRIIaR and FcγRIIb makes it difficult to confer selectivity, and it is difficult to claim that previously reported variants possess excellent selectivity.The results showed that the new variants ART10 (A / I ratio: 6.6), ART11 (A / I ratio: 11.5), ART12 (A / I ratio: 12.8), ART4 (A / I ratio: 22.5), ART8 (A / I ratio: 28.1), ART6 (A / I ratio: 42.4), ART5 (A / I ratio: 49.9) and ART3 (A / I ratio: 52.4) generated in the present invention were all superior to G1d (A / I ratio: 6.1) in terms of the A / I ratio for FcγRIIaR, and among them, ART4, ART8, ART6, ART5 and ART3 were superior to ART2 (A / I ratio: 13.0) and GASDIE (A / I ratio: 18.6). Similarly, in terms of the A / I ratio for FcγRIIaH, ART10 (A / I ratio: 17.3), ART8 (A / I ratio: 47.2), ART12 (A / I ratio: 60.3), ART11 (A / I ratio: 63.3), ART4 (A / I ratio: 65.2), ART3 (A / I ratio: 180.7), ART5 (A / I ratio: 240.1), and ART6 (A / I ratio: 307.0) were superior to G1d (A / I ratio: 8.9). Among them, ART8, ART12, ART11, ART4, ART3, ART5, and ART6 showed better A / I ratios than ART2 (A / I ratio: 5.8) and GASDIE (A / I ratio: 18.2). From the above results, it can be seen that ART4, ART8, ART3, ART5, and ART6 are antibodies with better A / I ratios than existing antibodies that enhance FcγRIIaR. Furthermore, it can be said that ART3, ART5, and ART6 are antibodies having better binding ability and A / I ratio than existing antibodies that are enhanced for FcγRIIaH.

[0291] For FcγRIIIaF, the A / I ratios of ART3 (A / I ratio 396.1), ART5 (A / I ratio 398.8), ART11 (A / I ratio 694.1), ART6 (A / I ratio 975.5), ART8 (A / I ratio 2350.3), ART10 (A / I ratio 3159.9), ART12 (A / I ratio 4309.9) and ART4 (A / I ratio 9943.2) were all better than G1d (A / I ratio 3.4). Among them, ART4 showed a better A / I ratio than ART1 (A / I ratio 4947.2), where ART1 is an FcγRIIIa-specific enhanced variant described in WO2014104165. Similarly, for FcγRIIIaV, ART3 (A / I ratio 2003.7), ART5 (A / I ratio 2064.8), ART6 (A / I ratio 2625.7), ART11 (A / I ratio 3974.6), ART8 (A / I ratio 6852.0), ART12 (A / I ratio 9721.0), ART10 (A / I ratio 11436.1), and ART4 (A / I ratio 15047.3) all showed better ratios than G1d (A / I ratio 28.1) and the existing enhanced variant Afucosyl (A / I ratio 298.1). Among them, ART4 and ART10 showed better A / I ratios than ART1 (A / I ratio 11261.3), where ART1 is an FcγRIIIa-specific enhanced variant described in WO2014104165. Based on the above results, it can be said that ART4 is an antibody that has superior binding ability to both FcγRIIIaF and FcγRIIIaV and A / I ratio compared to the existing enhanced antibody ART1.

[0292] [Example 3] Evaluation of Antibodies with Modified Fc Regions by ADCC Reporter Gene Bioassay

[0293] (3-1) Production of human epiregulin-expressing cells (Hepa1-6 / hEREG)

[0294] The mouse hepatocellular carcinoma cell line Hepa1-6 was purchased from ATCC. The human EREG (hEREG) gene was introduced into the cells by transfection, and clones expressing constitutively expressed hEREG were selected. Zeocin was used to select for the hEREG gene. Hepa1-6 / hEREG cells were maintained and passaged in D-MEM (high glucose) medium (SIGMA) supplemented with 10% FBS (SIGMA) and 400 μg / mL Zeocin.

[0295] (3-2) Evaluation by ADCC reporter gene bioassay

[0296] For the measurement of in vitro ADCC activity, hFcγRIIIaV ADCC reporter bioassay, effector cells, propagation model (Promega) was used. As target cells, 10 μL was adjusted to 5×10 5 10 cells / mL of Hepa1-6 / hEREG cells were added to each well of a 384-well plate. The culture medium used was an assay buffer (96% RPMI, 4% FBS). Next, the antibody produced in Example 1 and the negative control EGL-G4d (heavy chain SEQ ID NO: 28, light chain SEQ ID NO: 4) (which has the sequence of human IgG4) were diluted with assay buffer, and 11 serial dilutions were performed starting from a final concentration of 1 μg / mL at a common ratio of 10, and then 10 μL was added to each well. Finally, as an effector cell solution, 10 μL was added to adjust the concentration to 3×10 6 The Jurkat cells expressing hFcγRIIIaV of 1% 4% 4% 2% 6% 2% 3% 4% 6% 2% 3 ...

[0297] The fold induction value was determined by dividing the luminescence value of each well by the luminescence value of the well without antibody addition, and was used as an indicator for evaluating the ADCC of each antibody. Figure 1 The EC50 value of each sample was calculated using JMP 11.2.1 (SAS Institute Inc.) and is shown in Table 3.

[0298] (Table 3) EC50 values ​​of hFcγRIIIaV-mediated reporter gene induction activity of each modified Fc-containing antibody

[0299] Abbreviation for antibody EC50 value (μg / mL) EGL-G1d 4.63E-02 EGL-ART1 7.16E-04 EGL-ART2 1.35E-03 EGL-ART3 1.53E-03 EGL-ART4 7.89E-04 EGL-ART5 1.67E-03 EGL-ART6 1.50E-03 EGL-ART8 1.07E-03 EGL-ART10 1.07E-04 EGL-ART11 4.13E-04 EGL-ART12 3.04E-04 EGL-GASDALIE 2.08E-03 EGL-SDALIE 1.78E-03 EGL-GASDIE 3.70E-03 EGL-G4d N / A EGL-afuCosy| 5.16E-03

[0300] These results indicate that the antibodies with modified Fc produced this time exhibited stronger reporter gene induction activity on Hepa1-6 / hEREG cells compared to wild-type human IgG1 constant regions. The results in Table 3 also show that all of these variants exhibited activity at lower concentrations compared to variants with symmetrically engineered CH2 domains and low-fucose antibodies produced by sugar chain modification. Among the variants produced this time, ART3, ART4, ART5, ART6, ART8, ART10, ART11, and ART12 showed activity at the same concentration or lower than ART2. In particular, ART4, ART10, ART11, and ART12 exhibited activity even at lower concentrations compared to ART1 (which has more enhanced hFcγRIIIaV binding than ART2).

[0301] [Example 4] Evaluation of Antibodies with Modified Fc Regions by ADCP Reporter Gene Bioassay

[0302] For the measurement of in vitro ADCP activity, hFcγRIIaH ADCP reporter gene bioassay, core kit (Promega) was used. As target cells, 10 μL of cells were adjusted to 1×10 6 cells / mL of Hepa1-6 / hEREG cells were added to each well of a 384-well plate. Assay buffer (96% RPMI, 4% FBS) was used as a culture medium. Next, the antibodies produced in Example 1 were diluted with assay buffer to a final concentration of 0, 0.001, 0.01, 0.1, 1, and 10 μg / mL, respectively, and then 10 μL was added to each well. Finally, as an effector cell solution, 10 μL of Jurkat cells expressing hFcγRIIaH attached to the kit were added and mixed for a total of 30 μL. The mixture was then allowed to stand in a 5% CO2 incubator at 37°C for 6 hours. The cell density of Jurkat cells expressing hFcγRIIaH was 9.68×10 5 Cells / mL. The plate was then allowed to stand at room temperature for 15 minutes, and 30 μL of Bio-Glo reagent was added to each well. For Bio-Glo reagent, the Bio-Glo luciferase assay system (buffer and substrate) was used. Subsequently, the luminescence of each well was measured using a plate reader. The fold induction value was determined by dividing the luminescence value of each well by the luminescence value of the well without adding the antibody, and was used as an indicator for evaluating the ADCP of each antibody. The results are shown in FIG. Figure 2 The EC50 value of each sample was calculated using JMP 11.2.1 (SAS Institute Inc.) and is shown in Table 4.

[0303] (Table 4) EC50 values ​​of hFcγRIIaH-mediated reporter gene induction activity of each modified Fc-containing antibody

[0304] Abbreviation for antibody EC50 value (μg / mL) EGL-G1d N / A EGL-ART1 3.53E-02 EGL-ART2 2.18E-02 EGL-ART3 1.58E-02 EGL-ART4 4.32E-02 EGL-ART5 2.45E-02 EGL-ART6 1.92E-02 EGL-ART8 1.71E-02 EGL-ART10 4.19E-02 EGL-ART11 3.67E-02 EGL-ART12 8.50E-02 EGL-GASDALIE 3.24E-02 EGL-SDALIE N / A EGL-GASDIE 1.49E-02 EGL-G4d N / A EGL-afucosyl N / A

[0305] These results indicate that the antibodies with modified Fc produced this time exhibited stronger reporter gene induction activity on Hepa1-6 / hEREG cells compared to wild-type human IgG1 constant regions. The results in Table 4 also show that they exhibit activity at lower concentrations compared to variants with symmetrically engineered CH2 domains and low-fucose antibodies produced by sugar chain modifications. In addition, among the variants produced this time, ART2, ART3, ART5, ART6, and ART8 were active at lower concentrations compared to ART1. In particular, ART3, ART6, and ART8 exhibited activity at lower concentrations even compared to ART2 (which has stronger hFcγRIIaH binding).

[0306] [Example 5] Evaluation of the anti-tumor effect of antibodies with modified Fc regions in a syngeneic tumor cell transplantation model using human FcγR transgenic mice

[0307] (5-1) Cell lines

[0308] The Hepa1-6 / hEREG cells produced in Example 3-1 were maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (SIGMA) and 400 μg / mL Zeocin.

[0309] (5-2) Generation of Syngeneic Tumor Transplant Mouse Model

[0310] For efficacy testing, human FcγR transgenic mice were used (Proc Natl Acad Sci USA, 2012 Apr 17; 109(16): 6181-6186). 16-week-old male mice were intraperitoneally administered with 100 μL / head of anti-asialo GM1 antibody (aGM1, WAKO) to increase cell engraftment. On the second day of aGM1 administration, a 1:1 mixture of Hepa1-6 / hEREG cells and matrix gel (CORNING) was subcutaneously administered to transplant 1x10 7 When the average volume of transplanted tumors reached about 300 mm 3 Up to 500mm 3 , determine to establish the model.

[0311] The volume of the transplanted tumor was calculated using the following formula:

[0312] Tumor volume = long diameter x short diameter x short diameter / 2

[0313] (5-3) Preparation of the drug to be administered

[0314] In view of the A / I ratio results in Example 2 and the strength of the reporter gene induction activity in Examples 3 and 4, EGL-ART6 produced in the present invention is expected to have the most potent anti-tumor activity. Therefore, as reagents to be administered to the Hepa1-6 / hEREG cell transplantation model, an anti-hEREG control antibody (EGL-G1d) and an anti-hEREG antibody with enhanced FcγR binding (EGL afucosyl and EGL-ART6) were produced by the same method as in Example 1 and prepared at 1 mg / mL using His buffer (150 mM NaCl, 20 mM His-HCl buffer pH 6.0).

[0315] (5-4) Drug Administration for Antitumor Effect Measurement

[0316] Seven days after transplantation, EGL-G1d, EGL-afucosyl, and EGL-ART6 were administered at a dose of 10 mg / kg via the tail vein.

[0317] The detailed information of drug treatment in the antitumor effect measurement is shown in Table 5.

[0318] (Table 5) Measurement of anti-tumor effects in the Hepa1-6 / hEREG cell transplantation model

[0319]

[0320] (5-5) Evaluation of anti-tumor effects

[0321] The antitumor effect was evaluated by calculating the tumor volume using the formula shown in (5-2).

[0322] The TGI (tumor growth inhibition) value was calculated using the following formula:

[0323] TGI=(1-(average tumor volume of the target group at the time of measurement - average tumor volume before antibody administration) / (average tumor volume of the control group at the time of measurement - average tumor volume before antibody administration)) x 100. Therefore, the FcγR binding-enhancing antibodies EGL-afucosil and EGL-ART6 administered at 10 mg / kg showed an efficacy of TGI=80 or higher on day 19 after administration. On the other hand, the control antibody EGL-G1d showed TGI=31( Figure 3 ). This confirms that the in vivo anti-tumor effect of EGL-ART6 produced in the present invention is also enhanced compared to the expected EGL-afucosil.

[0324] [Example 6] Evaluation of C1q Binding Activity of Antibodies Having Modified Fc Regions

[0325] Although it has been reported that the CDC activity of antibodies contributes to anti-tumor effects (Nat. Immunol., 2017, 18889), it is known to cause side effects derived from CDC activity, such as infusion-related reactions (J. Immunol. 2008 1802294-2298 and Br. J. Haematol. 2001, 115807-811). Therefore, even when developing antibody drugs with enhanced ADCC activity and ADCP activity, the degree of CDC activity can be selected according to the target disease. The interaction between complement and Fc is mediated by C1q. According to reports analyzing the interaction between C1q and Fc (Science, 2018, 359, 794-797 and Molecular Immunology 2012, 51, 66-72), the FcγR and C1q interaction sites on the Fc region appear to partially overlap. These articles report that residues at EU numbering positions 329, 330, and 331 on the Fc region are important for interaction with C1q. Furthermore, residues at EU numbering positions 268, 270, and 298 also contribute to C1q binding. These positions and surrounding residues were modified in the present invention to enhance FcγR binding. Therefore, the resulting Fc region variants may also have enhanced or reduced binding to C1q, potentially enabling manipulation of CDC activity during antibody drug development. Therefore, the resulting Fc region variants were evaluated for C1q binding.

[0326] ELISA was performed on the anti-human epiregulin antibodies produced in Examples 1 and 3. In addition, the buffers shown in Table 6 were prepared as needed. The antigen used was human C1q protein (hC1q).

[0327] (Table 6) Buffer composition used in human C1q ELISA

[0328] buffer composition Blocking / Dilution Buffer: TBS, 0.1% Tween 20, 0.5% BSA, 1x Block ace powder Wash buffer PBST, pH 7.4 Stop buffer 0.5 mol / L sulfuric acid

[0329] First, a 96-well Maxisorp plate (Thermo Fisher) was coated overnight at 4°C with 50 μL of a solution containing each antibody prepared at 30, 10, 3, 1, 0.3, 0.1, and 0.03 μg / mL in PBS. Each well of the plate was washed with wash buffer to remove the antibody that was not bound to the plate, and then the wells were blocked with 200 μL of blocking / dilution buffer at room temperature for 2 hours or longer. After removing the blocking / dilution buffer from each well, hC1q (Calbiochem) prepared to a final concentration of 3 μg / mL in the blocking / dilution solution was added at 50 μL / well. The plate was then allowed to stand at room temperature for 1 hour to allow hC1q to bind to the antibody in each well. After washing with wash buffer, 50 μL of HRP-conjugated anti-hC1q antibody (AbDSerotec) diluted with blocking / dilution buffer was added to each well and the plate was incubated while standing for 1 hour. After washing with wash buffer, TMB single solution (Invitrogen) was added. The color reaction of the solution in each well was stopped by adding a stop buffer, and then the color was measured by absorbance at 450 nm and 690 nm. The buffer used was a buffer containing the composition shown in Table 6. The measurement results are shown in Table 6. Figure 4 and Figure 5 shown.

[0330] like Figure 4 and Figure 5As shown, among the variants evaluated, ART3, ART5, and ART11 had enhanced C1q binding compared to G1d. The binding abilities of afucosyl and ART8 were comparable to those of G1d. Furthermore, compared to G1d, ART1, ART2, ART4, ART6, ART10, ART12, GASDALIE, SDALIE, and GASDIE showed reduced C1q binding. Among these, the C1q binding abilities of ART1, ART2, ART4, ART6, ART12, GASDALIE, SDALIE, and GASDIE were reduced to the same level as G4d, which has a human IgG4 sequence. Since human IgG4 is considered to have minimal CDC activity (J. Immunol. Methods 2005 306 151-160), these variants whose C1q binding was reduced to the same level as G4d had almost no CDC activity, similar to IgG4. Common amino acid modifications shared by these variants with greatly reduced C1q binding include modifications at Ala330 or Ile332. These regions, especially Ala330, are very important regions for interacting with C1q. This suggests that it is the modification introduced into this site that greatly reduces binding to C1q. On the other hand, compared to G1d, ART3, ART5, and ART11, which have enhanced C1q binding, do not have modifications introduced at positions 330 or 332, indicating that the enhanced binding is due to the effects of the S298A modification or the modification at position 326, which is believed to enhance binding to C1q (Science, 2018, 359, 794-797).

[0331] Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples herein should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

[0332] [Industrial Applicability]

[0333] The present invention provides polypeptides containing Fc region variants that bind more strongly to activating FcγRIIa and FcγRIIIa and exhibit reduced binding to inhibitory FcγRIIb. The polypeptides disclosed herein exhibit high ADCC / ADCP activity and are useful for anti-tumor therapy (e.g., treatment and / or prevention of inflammatory diseases, treatment and / or prevention of various cancers, etc.). Sequence Listing <110> Chugai Pharmaceutical Co., Ltd. <120> Heterodimeric FC polypeptide <130> C1-A2023PP <150> PCT / JP2020 / 032547 <151> 2020-08-28 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-G1d <400> 1 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 2 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn125 <400> 2 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met <30]]35 40 45 [[ID=三十二]]Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Tyr Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 3 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl076 <400> 3<​​​​​​​​​​​​​​​​​​​​​​​​​​​​Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Met Pro Ile Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 4 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> L73-k0 <400> 4 Asp 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 His Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Gln Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro [[ID=3*]]65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Glu Gln Leu Arg Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 5 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn120 <400> 5 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr It should be noted that there was a small error in the original text where "145" was written as a full-width number. It has been corrected to the half-width "145" in the translation for better consistency.20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Tyr Tyr Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Asp Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 6 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl068 <400> 6 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Lys Pro Ile Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 7 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn443 <400> 7 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 8 <211> 444[[ID=z8]] <212> PRT <213> Artificial Sequence <220> <223> H240-Hl408 <400> 8 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Ile Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 9 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn456 <400> 9 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Met Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 10 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl446 <400> 10 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Met Pro Glu Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 11 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn462 <400> 11 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 12 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl441 <400> 12 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Ile Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 13 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn462 <400> 13 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 14 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl445 <400> 14 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Lys Pro Asp Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 15 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn461 <400> 15 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 16 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl443 <400> 16 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Glu Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 17 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn494 <400> 17 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 18 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl514 <400> 18 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Glu Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 19 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn496 <400> 19 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 20 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl516 <400> 20 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Ile Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 21 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn498 <400> 21 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Met Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 22 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl518 <400> 22 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Asp Ala Leu Pro Met Pro Ile Glu Glu Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 23 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240 - GASDALIE <400> 23 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 24 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn032 <400> 24 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 25 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl032 <400> 25 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 26 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Kn037 <400> 26 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 27 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> H240-Hl036<​​​​​​​​​​Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 28 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> H240 - G4d <400> 28 Gln Asp Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Arg Lys Gln Thr Lys Tyr Arg Glu Lys Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 <210> 29 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> G1d <400> 29 Ala 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 <210> 30 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn125 <400> 30 Ala 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 Tyr Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 31 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl076 <400> 31 Ala 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 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 Asp Ala Leu Pro Met Pro Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> k0 <400> 32 Arg 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 <210> 33 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn120 <400> 33 Ala 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 Tyr Tyr Trp Gly Pro Met 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 Asp 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 Ala 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 Asp Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 34 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl068 <400> 34 Ala 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 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 Asp Ala Leu Pro Lys Pro Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 35 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn443 <400> 35 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Asp 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 Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 36 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl408 <400> 36 Ala 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 [[ID=3B]]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[[ID=3B]] 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 Ala 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 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 Ala 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 Asp Ala Leu Pro Ala Pro Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 37 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn456 <400> 37 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Met Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 38 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl446 <400> 38 Ala 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 Ala 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 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 Ala 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 Asp Ala Leu Pro Met Pro Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 39 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn462 <400> 39 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Asp 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 Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 40 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl441 <400> 40 Ala 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 Ala 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 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 Ala 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 Asp Ala Leu Pro Ala Pro Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 41 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn462 <400> 41 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Asp 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 Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 42 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl445 <400> 42 Ala 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 Ala 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 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 Ala 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 Asp Ala Leu Pro Lys Pro Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 43<00​​​​​​<220> <223> Kn461 <400> 43 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Asp Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 44 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl443 <400> 44 Ala 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 Ala 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 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 Ala 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 Asp Ala Leu Pro Ala Pro Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 45 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn494 <400> 45 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 46 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl514 <400> 46 Ala 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 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 Ala 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 Asp Ala Leu Pro Ala Pro Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 47 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn496 <400> 47 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 48 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl516 <400> 48 Ala 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 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 Ala 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 Asp Ala Leu Pro Ala Pro Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 49 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn498 <400> 49 Ala 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 Phe Gln Trp Gly Pro Met 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 Asp Glu 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 Ala 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 Met Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 50 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl518 <400> 50 Ala 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 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 Ala 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 Asp Ala Leu Pro Met Pro Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 51 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> GASDALIE <400> 51 Ala 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 Ala Gly Pro Asp 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 Leu Pro Glu 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 <210> 52 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn032 <400> 52 Ala 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 Asp 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 Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 53 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl032 <400> 53 Ala 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 Asp 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 Leu Pro Glu 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 Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 54 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Kn037 <400> 54 Ala 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 Ala Gly Pro Asp 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 Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp 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 <210> 55 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Hl036 <400> 55 Ala 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 Ala Gly Pro Asp 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 Glu 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 Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val 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 <210> 56 <211> 325 <212> PRT <213> Artificial Sequence <220> <223> G4d <400> 56 Ala 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 Pro 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 325

Claims

1. A variant Fc region, wherein a set of amino acid changes are introduced into the CH2 domain of a parent Fc region, wherein the parent Fc region is the Fc region of native human IgG1 and is composed of two polypeptide chains, and wherein the set of amino acid changes in the CH2 domain is selected from the group consisting of (a) to (d) below: (a) Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, and Met at position 330 in the first polypeptide of the parent Fc region, according to EU numbering; and in the second polypeptide of the parent Fc region, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met at position 330, and Glu at position 334, according to EU numbering; (b) Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Met at position 330, and Glu at position 332 in the first polypeptide of the parent Fc region, according to EU numbering; and in the second polypeptide of the parent Fc region, according to EU numbering: Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met at position 330, Glu at position 332, and Glu at position 334; (c) Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, and Pro at position 307 in the first polypeptide of the parent Fc region, according to EU numbering; and Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, and Glu at position 334, according to EU numbering in the second polypeptide of the parent Fc region; and (d) Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, and Glu at position 332 in the first polypeptide of the parent Fc region, according to EU numbering; and According to EU numbering, in the second polypeptide of the parent Fc region, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Glu at position 332, and Glu at position 334.

2. The variant Fc region of claim 1, wherein the variant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to the parent Fc region.

3. The variant Fc region of claim 1, wherein the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors in the variant Fc region is improved compared to the parent Fc region.

4. An antibody comprising the variant Fc region of any one of claims 1-3.

Images