HK40083599B - Anti-ctla-4 antibody and use thereof - Google Patents

Description

Anti-CTLA-4 antibodies and their uses

[Technical Field]

This invention relates to anti-CTLA-4 antibodies and methods of using said antibodies.

[Background of the Invention]

The immune surveillance system monitors and eliminates mutated cells in living organisms due to gene mutations, among other things. However, the persistence of an excessive immune response can also be harmful, such as the damage to normal tissues caused by autoimmunity. Therefore, the immune system employs a negative feedback mechanism (immune checkpoints), which, once activated, suppresses the immune response (see, for example, NPL 1). Immune checkpoints are believed to play a crucial role in maintaining homeostasis within the immune system. On the other hand, it has been revealed that some tumors utilize immune checkpoints for immune escape. Currently, research on the immunosuppressive function of major immune checkpoint molecules, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1) is gaining significant traction.

CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily. Its gene was cloned in 1987 from a cDNA library of a mouse cytotoxic T cell clone (see, e.g., NPL 2). T-cell immune responses are known to be suppressed by CTLA-4. In 1996, it was reported that tumor regression was observed in cancer-bearing mice by administering anti-CTLA-4 antibodies, based on the idea that promoting T-cell activation through inhibition of CTLA-4 function would lead to cancer regression (see, e.g., NPL 3). Evaluation of the efficacy of anti-CTLA-4 antibodies in humans began in 2000, and the anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the U.S. Food and Drug Administration (FDA) in 2011 as the world's first immunostimulatory therapeutic antibody. In addition to ipilimumab, many other anti-CTLA-4 monoclonal antibodies have been produced (see, e.g., PTL 1, 2, 3, and 4), and their development as drugs is ongoing. Drugs that inhibit immune checkpoints to cancel immunosuppressive mechanisms and thus enhance immune responsiveness are called immune checkpoint inhibitors.

On the other hand, some cells with immunosuppressive functions were previously known to exist within T cells. These were identified as CD25- and CD4-positive T cells in 1995 and named regulatory T cells (see, for example, NPL 4). In 2003, the Foxp3 gene was identified as a major gene specifically expressed in regulatory T cells and regulating their development and function. Foxp3 acts as a transcription factor that regulates the expression of various immune response-related genes. In particular, Foxp3 is involved in the constitutive expression of CTLA-4 in regulatory T cells and is considered to play an important role in the immunosuppressive function of regulatory T cells (see, for example, NPL 5).

Regulatory T cell infiltration into tumor tissue is thought to lead to a weakening or suppression of immune surveillance mechanisms against tumors. Indeed, increased regulatory T cells have been revealed in many human cancers (see, for example, NPL 6), and localized regulatory T cell infiltration into tumors has been reported as a poor prognostic factor for cancer patients. Conversely, if regulatory T cells can be removed or reduced from tumor tissue, it is hoped that this could lead to enhanced anti-tumor immunity. Currently, the development of cancer immunotherapies targeting regulatory T cells is progressing rapidly.

Administration of the anti-CTLA-4 antibody ipilimumab enhances anti-tumor immunity, but it has been reported to cause autoimmune diseases because it systemically enhances immune reactivity. In one clinical trial, adverse events were observed in 60% of patients administered ipilimumab, many of which were skin or gastrointestinal autoimmune diseases. Similarly, in another clinical trial, approximately half of the patients administered ipilimumab developed similar autoimmune diseases. To suppress such side effects, immunosuppressants have been administered to patients already receiving ipilimumab in some cases. The development of novel drugs that can maintain anti-tumor immune responses while suppressing the side effects of immune checkpoint inhibitors is anticipated.

The cytotoxic effector functions, antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent phagocytosis (ADCP) activities of IgG antibodies have attracted attention as promising means of achieving antitumor effects via antibodies (see, for example, NPL 7 and 8). These effector functions are induced by the binding of the Fc region of the IgG antibody to antibody receptors (FcγRs) or various complement components present on the surface of effector cells, such as natural killer cells and macrophages. To date, numerous variants of the Fc region have been studied, and variants with enhanced properties such as FcγR binding activity compared to the wild type have been obtained (see, for example, PTL 5 and 6, and NPL 9 and 10). Furthermore, it has been reported that the Fc region of antibodies binds to FcγRs in a 1:1 ratio and asymmetrically recognizes FcγRs in the lower hinge region and CH2 region (see, for example, NPL 11). Based on this, methods for optimizing the interaction with FcγR by introducing different changes into the two polypeptide chains that make up the Fc region of the antibody and generating asymmetric Fc region variants have also been reported (see, for example, PTL 7, 8, 9 and 10).

Ideally, when therapeutic antibodies are administered to living organisms, their target antigens should be specifically expressed only at the site of injury. However, in many cases, the same antigens are also expressed in non-lesion sites, in normal tissues, and this can cause undesirable side effects in treatment. For example, while antibodies against tumor antigens can exert cytotoxic activity against tumor cells via ADCC, they can also damage normal cells when the same antigens are also expressed in normal tissues. To address these issues, techniques have been developed that focus on the phenomenon where a large number of specific compounds are present in the target tissue (e.g., tumor tissue) and produce antigen-binding molecules whose antigen-binding activity varies depending on the concentration of the compound (see, for example, PTL 11).

[List of Citations]

[Patent Literature]

[PTL 1]WO 2000/037504

[PTL 2]WO 2001/014424

[PTL 3]WO 2012/120125

[PTL 4]WO 2016/196237

[PTL 5]WO 2000/042072

[PTL 6]WO 2006/019447

[PTL 7]WO 2012/058768

[PTL 8]WO 2012/125850

[PTL 9]WO 2013/002362

[PTL 10]WO 2014/104165

[PTL 11]WO 2013/180200

[Non-patent literature]

[NPL 1]Pardoll, Nat Rev Cancer(2012)12:252-264

[NPL 2]Brunet et al., Nature(1987)328:267-270

[NPL 3]Leach et al., Science(1996)271:1734-1736

[NPL 4]Sakaguchi et al., J Immunol (1995) 155:1151-1164

[NPL 5]Takahashi et al., J Exp Med(2000)192:303-310

[NPL 6]Nishikawa&Sakaguchi, Int J Cancer(2010)127:759-767

[NPL 7] Clynes et al., Proc Natl Acad Sci U S A (1998) 95:652-656

[NPL 8]Clynes et al., Nat Med(2000)6:443-446

[NPL 9] Lazar et al., Proc Natl Acad Sci U S A (2006) 103:4005-4010

[NPL 10]Chu et al., Mol Immunol(2008)45:3926-3933

[NPL 11] Radaev et al., J Biol Chem (2001) 276:16469-16477

[Invention Summary]

[Technical Issues]

This invention provides an anti-CTLA-4 antibody and a method of using said antibody. This invention also provides a polypeptide comprising a variant Fc region and a method of generating said polypeptide.

[Problem Solving]

More specifically, the present invention provides the following [1] to [47].

[1] An anti-CTLA-4 antibody having CTLA-4 binding activity dependent on the concentration of an adenosine-containing compound, wherein said antibody has at least one feature selected from (a) to (i):

(a) The binding activity in the presence of 100 μM of adenosine-containing compound is two or more times that in the absence of adenosine-containing compound;

(b) The KD value is 5 x ^10⁻⁷ M or lower in the presence of 100 μM of adenosine-containing compounds;

(c) The KD value is 1× ^10⁻⁶ M or higher when no adenosine-containing compounds are present;

(d) Forms a ternary complex with adenosine-containing compounds and CTLA-4;

(e) The region of human CTLA-4 from amino acid 97 to amino acid 106 (extracellular domain, SEQ ID NO: 28);

(f) Competes with ABAM004 (VH, SEQ ID NO:10; and VL, SEQ ID NO:11) to bind to CTLA-4;

(g) Epitopes that bind to the same epitopes as those that bind to ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11);

(h) shows cytotoxic activity against cells expressing CTLA-4; and

(i) It binds to human and mouse-derived CTLA-4.

[2][1] antibodies, wherein the antibodies are monoclonal antibodies.

[3][1] or [2] antibodies, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

The antibody described in any one of [4][1] to [3], wherein the antibody is an antibody fragment that binds to CTLA-4.

The antibody of any one of [5][1] to [4], wherein the antibody comprises: (a) HVR-H1 (SEQ ID NO:223) comprising the amino acid sequence SX ₁ TMN, wherein X ₁ is H, A, R, or K; (b) HVR-H2 (SEQ ID NO:224) comprising the amino acid sequence SISX ₁ X ₂ SX ₃ YIYYAX ₄ SVX ₅ G, wherein X ₁ is S or T, X ₂ is R or Q, X ₃ is G or H, X ₄ is D, E, or R, and X ₅ is K or R; and (c) HVR-H3 (SEQ ID NO:225) comprising the amino acid sequence YGX ₁ REDMLWVFDY, wherein X ₁ is K or A.

[6][5] antibodies, further comprising: (a) HVR-L1 (SEQ ID NO:226) comprising the amino acid sequence _{X1 GX2 STX3 VGDYX4 X5 VX6} , wherein _X1 is T, D, Q or E, _X2 is T or P, _X3 is D or G, _X4 is N or T, _X5 is Y or W, and _X6 is S or H; (b) HVR-L2 (SEQ ID NO:227) comprising the amino acid sequence _{X1 TX2 X3 KPX4} , wherein _X1 is E, F or Y, _X2 is S or I, _X3 is K or S, and _X4 is S, E, or K; and (c) HVR-L3 (SEQ ID NO:228) comprising the amino acid sequence _{X1 TYAAPLGPX2} , wherein _X1 is S or Q and _X2 is M or T.

[7][5] The antibody further comprises: a heavy chain variable domain FR1, which comprises the amino acid sequence of any one of SEQ ID NO: 229 to 232; FR2, which comprises the amino acid sequence of SEQ ID NO: 233; FR3, which comprises the amino acid sequence of SEQ ID NO: 234; and FR4, which comprises the amino acid sequence of SEQ ID NO: 235.

[8][6] The antibody further comprises: a light chain variable domain FR1, which comprises the amino acid sequence of any one of SEQ ID NO: 236 to 238; FR2, which comprises the amino acid sequence of any one of SEQ ID NO: 240 and 241; FR3, which comprises the amino acid sequence of any one of SEQ ID NO: 242 to 244; and FR4, which comprises the amino acid sequence of any one of SEQ ID NO: 245 and 246.

The antibody of any one of [9][1] to [4] comprises: (a) a VH sequence having at least 95% sequence identity with an amino acid sequence of any one of SEQ ID NO: 83 to 86, 98 and 135 to 141; (b) a VL sequence having at least 95% sequence identity with an amino acid sequence of any one of SEQ ID NO: 88 to 95, 97, 99, 134 and 144 to 149; or (c) a VH sequence having an amino acid sequence of any one of SEQ ID NO: 83 to 86, 98 and 135 to 141 and a VL sequence having an amino acid sequence of any one of SEQ ID NO: 88 to 95, 97, 99, 134 and 144 to 149.

The antibody is any one of [10][1] to [3] and [5] to [9], which is a full-length IgG1 antibody.

[11][10] antibodies, wherein the Fc region is a variant Fc region containing amino acid changes, and wherein the variant Fc region has enhanced binding activity with at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa compared with the natural Fc region.

[12] The isolated nucleic acid that encodes an antibody for any one of [1] to [11].

[13] Host cells containing nucleic acids of [12].

[14] A method for producing antibodies, wherein the method includes culturing host cells of [13] in order to produce antibodies.

[15] A pharmaceutical preparation comprising an antibody of any one of [1] to [11] and a pharmaceutically acceptable carrier.

[16][15] pharmaceutical formulations, wherein the antibody is an immunoconjugate.

[17][15] or [16] pharmaceutical formulations, wherein the pharmaceutical formulation is used in combination with at least one selected from immune checkpoint inhibitors, EGFR inhibitors, HER2 inhibitors and chemotherapeutic agents.

A pharmaceutical preparation of any one of [18][15] to [17], wherein the pharmaceutical preparation is used to treat tumors.

[19][18] pharmaceutical formulations, wherein the tumor is a solid tumor in which regulatory T (Treg) cells have infiltrated.

A pharmaceutical preparation of any one of [20][15] to [17], wherein the pharmaceutical preparation is used to damage cells.

A pharmaceutical preparation of any one of [21][15] to [17], wherein the pharmaceutical preparation is used to damage Treg cells.

[22][20] Drug formulations in which cell damage is caused by ADCC activity, CDC activity or ADCP activity.

[23][20] or [21] drug formulations in which immunity is activated by damage to Treg cells.

A pharmaceutical preparation of any one of [24][15] to [17], wherein the pharmaceutical preparation is used to activate immunity.

[25][24] Drug formulations in which immune activation is T cell activation.

[26][24] or [25] drug formulations in which the immune system in tumor tissue is activated.

A pharmaceutical formulation of any one of [27][24] to [26], wherein the level of immune activation in non-tumor tissues is lower compared with a pharmaceutical formulation containing a control anti-CTLA-4 antibody.

A pharmaceutical formulation of any one of [28][24] to [27], wherein the level of side effects is lower compared with a pharmaceutical formulation containing a control anti-CTLA-4 antibody.

The pharmaceutical formulation of any one of [29][24] to [28], wherein the control anti-CTLA-4 antibody is an anti-CTLA-4 antibody that does not have CTLA-4 binding activity dependent on the concentration of an adenosine compound.

[30][29] pharmaceutical formulations, wherein the side effect is an autoimmune disease.

A pharmaceutical preparation of any one of [31][18], [19] and [26] to [30], wherein the tumor is breast cancer or liver cancer.

[32] A polypeptide comprising a variant Fc region containing an amino acid alteration in a parental Fc region, wherein the parental Fc region consists of two polypeptide chains, and wherein the variant Fc region contains an amino acid alteration at the following positions:

(i) The first polypeptide in the Fc region of the parental parent at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 according to EU numbers; and

(ii) The second polypeptide in the parental Fc region at positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering.

[33][32] peptides, wherein the variant Fc region is further contained in the first peptide of the parent Fc region according to an amino acid change at position 332 of the EU number.

[34][32] or [33] polypeptides, wherein the variant Fc region is further contained in a second polypeptide of the parent Fc region with an amino acid change at position 332 according to EU number.

The polypeptide of any one of [35][32] to [34], wherein the variant Fc region is further contained in a second polypeptide of the parent Fc region with an amino acid change at position 330 according to the EU number.

The polypeptide of any one of [36][32] to [35], wherein the variant Fc region is further contained in the first polypeptide of the parent Fc region according to the amino acid change at position 356 of the EU number.

The polypeptide of any one of [37][32] to [36], wherein the variant Fc region is further contained in the first polypeptide of the parent Fc region according to the amino acid change at position 366 of the EU number.

The polypeptide of any one of [38][32] to [37], wherein the variant Fc region is further contained in a second polypeptide of the parent Fc region with an amino acid change at position 439 according to the EU number.

The polypeptide of any one of [39][32] to [38], wherein the variant Fc region is further contained in a second polypeptide of the parent Fc region with amino acid changes at positions 366, 368 and 407 according to EU numbers.

The polypeptide of any one of [40][32] to [39] comprises at least one amino acid alteration selected from the following amino acid alterations:

(i) In the first polypeptide of the parental Fc region, according to EU number, Tyr or 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, Asp at position 326, Glu at position 332, Cys at position 349, Lys at position 356, and Trp at position 366; and

(ii) In the second polypeptide of the parental Fc region, according to EU number, 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, Cys at position 356, Ser at position 366, Ala at position 368, Val at position 407, and Glu at position 439.

The polypeptide of any one of [41][32] to [40], wherein the variant Fc region further comprises any one of the amino acid changes of (a) to (d) below in the first polypeptide and/or the second polypeptide of the parental Fc region:

(a) According to the EU designation, at location 434, Ala;

(b) According to EU numbers, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440;

(c) According to EU designations, Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; and

(d) According to EU number, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440.

The polypeptide of any one of [42][32] to [41], wherein the variant Fc region has enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa compared to the parental Fc region.

[43][42] peptides, wherein the binding activity of the variant Fc region to FcγRIIa and FcγRIIIa is enhanced compared to the parental Fc region.

The polypeptide of any one of [44][32] to [43], wherein the selectivity between the activating Fcγ receptor and the inhibitory Fcγ receptor in the variant Fc region is increased compared with that of the parental Fc region.

[45][44] of a polypeptide, wherein the activating Fcγ receptor is at least one Fcγ receptor selected from FcγRIa, FcγRIIa and FcγRIIIa, and wherein the inhibitory Fcγ receptor is FcγRIIb.

A polypeptide of any one of [46][32] to [45], wherein the polypeptide containing the variant Fc region, is an antibody.

[47] A method for generating a polypeptide comprising a variant Fc region, the method comprising introducing an amino acid alteration into a parental Fc region, wherein the parental Fc region comprises two polypeptide chains, and wherein the amino acid alteration is introduced at the following position:

(i) The first polypeptide in the Fc region of the parental parent at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 according to EU numbers; and

(ii) The second polypeptide in the parental Fc region at positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering.

[Image Description]

Figure 1 shows the binding activity of the anti-CTLA-4 antibody ABAM004 to CTLA-4, which depends on the concentration of ATP, ADP, or AMP as described in Examples 1-9.

Figure 2 shows the binding activity of the anti-CTLA-4 antibody ABAM004 on cells expressing CTLA-4, which depends on the AMP concentration described in Examples 1-10.

Figure 3 shows the ADCC activity of the anti-CTLA-4 antibody ABAM004 on cells expressing CTLA-4 in the presence and absence of AMP, as described in Examples 1-11.

Figure 4 shows the binding mechanism of the ABAM004 Fab fragment to AMP, as described in Examples 2-13. In the figure, the heavy chain of the antibody is represented in black, the light chain in gray, and AMP is represented by a ball-and-stick model. The amino acid residues that interact with AMP are represented by a rod-shaped model. The dashed lines and their values represent the distance between each amino acid residue and AMP.

Figure 5 shows the binding mode of the ABAM004 Fab fragment, AMP, and human CTLA4 (hCTLA4), as described in Examples 2-14. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, hCTLA4 in white, and AMP is represented by a ball-and-stick model. Amino acid residues of hCTLA4 containing one or more non-hydrogen atoms located within any part of the antibody or AMP are considered epitopes and represented by a stick model.

Figure 6 shows a plot of the epitopes of the ABAM004 Fab fragment in the hCTLA4 amino acid sequence as described in Examples 2-14. In the figure, the amino acid residues indicated in black are those of hCTLA4, which contain one or more non-hydrogen atoms located within any part of the crystal structure at a distance from ABAM004 or AMP. The amino acid residues indicated in gray show residues whose models were not constructed because they are disordered in the crystal structure.

Figure 7 is a superimposed diagram of antibody and AMP structures extracted from the crystal structures of the ABAM004 Fab fragment alone, the complex of the ABAM004 Fab fragment and AMP, and the ternary complex of the ABAM004 Fab fragment, AMP, and CTLA4, as described in Examples 2-15. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and AMP is represented by a ball-and-stick model. The structure of the ABAM004 Fab fragment alone is represented by a thin line, the structure of the binary complex with AMP is represented by a medium-thick line, and the structure of the ternary complex is represented by a thick line.

Figure 8 shows the binding activity of the anti-CTLA-4 antibody ABAM004 and its variants 04H0150/04L0072 to CTLA-4, which depends on the concentrations of ATP, ADP, or AMP described in Examples 3-2. As shown in the figure, WT and H150L072 represent ABAM004 and 04H0150/04L0072, respectively.

Figure 9 shows the neutralizing activity of the anti-CTLA-4 antibody SW1077 against CTLA-4, which depends on the ATP concentration described in Examples 3-6.

Figure 10 shows the antitumor effect of anti-CTLA-4 antibody mNS-mFa55 (control antibody) in a mouse model transplanted with FM3A cell line, as described in Examples 3-7-4. The antibody was administered via tail vein at doses of 0.01 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg, and 100 mg/kg. Each point represents the average tumor volume for a group, n = 4.

Figure 11 shows the antitumor effect of anti-CTLA-4 antibody SW1208-mFa55 (switch antibody) in a mouse model transplanted with FM3A cell line, as described in Examples 3-7-4. The antibody was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg. Each dot represents the average tumor volume for a group, n = 4.

Figure 12 shows the changes in the proportion of effector Treg cells in tumors after administration of anti-CTLA-4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody) in a mouse model transplanted with FM3A cells, as described in Example 3-7-7. mNS-mFa55 was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 100 mg/kg, while SW1208-mFa55 was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg. Tumors were collected six days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the ratio of effector Tregs (CD4 ⁺ FoxP3 ⁺ KLRG1 ⁺ ) to CD45 ⁺ cells. Mean values are shown for n=3.

Figure 13 shows the change in the proportion of activated helper T cells in the spleen in a mouse model transplanted with the FM3A cell line when administered anti-CTLA-4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody), as described in Examples 3-7-8. mNS-mFa55 was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 100 mg/kg, and SW1208-mFa55 was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg. Spleens were collected six days after administration, and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis represents the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) to CD45 ⁺ cells. Mean values are shown for n=3.

Figure 14 shows the antitumor effect of anti-CTLA-4 antibody SW1389-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Examples 4-3-5. The antibody was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 100 mg/kg. Each point represents the average tumor volume of a group, n = 4.

Figure 15 shows the antitumor effect of anti-CTLA-4 antibody hNS-mFa55 (control antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Examples 4-3-5. The antibody was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg. Each point represents the average tumor volume of a group, n = 4.

Figure 16 shows the changes in the proportion of effector Treg cells in tumors after administration of anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line, as described in Examples 4-3-8. hNS-mFa55 was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg, and SW1389-mFa55 was administered at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg. Tumors were collected six days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the ratio of effector Tregs (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) to CD45 ⁺ cells. Mean values are shown for n=3.

Figure 17 shows the change in the proportion of activated helper T cells in the spleen in a mouse model transplanted with the Hepa1-6/hGPC3 cell line, following administration of either anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switch antibody), as described in Examples 4-3-9. hNS-mFa55 was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg, while SW1389-mFa55 was administered at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg. Spleens were collected six days after administration, and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis represents the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) to CD45 ⁺ cells. Mean values are shown for n=3.

Figure 18 shows the antitumor effect of anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Examples 5-4-5. The antibody was administered via tail vein at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Each point represents the average tumor volume of a group, n = 5.

Figure 19 shows the antitumor effect of anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Examples 5-4-5. The antibody was administered via tail vein at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Each dot represents the average tumor volume of a group, n = 5.

Figure 20 shows the antitumor effect of anti-CTLA-4 antibody SW1615-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Examples 5-4-5. The antibody was administered via tail vein at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Each dot represents the average tumor volume of a group, n = 5.

Figure 21 shows the changes in the proportion of effector Treg cells in tumors after administration of anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, or SW1615-mFa55 (all switch antibodies) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line, as described in Examples 5-4-8. SW1610-mFa55 was administered via tail vein at 50 mg/kg, 100 mg/kg, and 200 mg/kg; SW1612-mFa55 at 50 mg/kg, 100 mg/kg, and 200 mg/kg; SW1615-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, and 400 mg/kg; and the negative control antibody KLH-mFa55 was administered at 400 mg/kg. Tumors were collected six days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the ratio of effector Tregs (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) to CD45 ⁺ cells. The mean is shown for n=3.

Figure 22 shows the changes in the proportion of activated helper T cells in the spleen when administered anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, or SW1615-mFa55 (all switch antibodies) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line, as described in Examples 5-4-9. SW1610-mFa55 was administered via tail vein at 50 mg/kg, 100 mg/kg, and 200 mg/kg; SW1612-mFa55 at 50 mg/kg, 100 mg/kg, and 200 mg/kg; SW1615-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, and 400 mg/kg; and the negative control antibody KLH-mFa55 was administered at 400 mg/kg. Spleens were collected six days after administration, and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis represents the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) to CD45 ⁺ cells. The mean value is shown for n=3.

Figure 23 shows a comparison of the in vitro ADCC activity of antibodies with various modified constant regions, which exhibit enhanced binding to FcγR as described in Examples 6-2. As shown, IgG1 represents MDX10D1H-G1m/MDX10D1L-k0MT, GASDALIE represents MDX10D1H-GASDALIE/MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461/MDX10D1H-Hl443/MDX10D1L-k0MT. Here, IgG1 is an antibody with a control constant region, GASDALIE is an antibody with the constant region described in the prior art literature, and ART6 and ART8 are antibodies with the modified constant regions produced in Examples 6-1.

Figure 24 shows a comparison of the in vitro ADCP activities of antibodies with various modified constant regions, which exhibit enhanced binding to FcγR as described in Examples 6-3. As shown, IgG1 represents MDX10D1H-G1m/MDX10D1L-k0MT, GASDIE represents MDX10D1H-GASDIE/MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461/MDX10D1H-Hl443/MDX10D1L-k0MT. Here, IgG1 is an antibody with a control constant region, GASDIE is an antibody with the constant region described in the prior art literature, and ART6 and ART8 are antibodies with the modified constant regions produced in Examples 6-1.

Figure 25 shows the in vitro ADCC activity of the anti-CTLA4 switch antibody SW1389-ART6 with a modified constant region, which exhibits enhanced binding to FcγR, as described in Examples 6-4.

Figure 26 shows the in vitro ADCC activity of the anti-CTLA4 switch antibody SW1610-ART6 with a modified constant region, which exhibits enhanced binding to FcγR, as described in Examples 6-4.

Figure 27 shows the in vitro ADCC activity of the anti-CTLA4 switch antibody SW1612-ART6 with a modified constant region, which exhibits enhanced binding to FcγR, as described in Examples 6-4.

Figure 28 shows the neutralizing activity of the anti-CTLA4 switch antibody SW1389 against CTLA4 (eliminating the activity of CTLA4 signaling that acts in an inhibitory manner on effector cell activation), as described in Examples 6-5.

Figure 29 shows the neutralizing activity of the anti-CTLA4 switch antibody SW1610 against CTLA4 (eliminating the activity of CTLA4 signaling that acts in an inhibitory manner on effector cell activation), as described in Examples 6-5.

Figure 30 shows the neutralizing activity of the anti-CTLA4 switch antibody SW1612 against CTLA4 (eliminating the activity of CTLA4 signaling that acts in an inhibitory manner on effector cell activation), as described in Examples 6-5.

Figure 31 shows the neutralizing activity of the anti-CTLA4 switch antibody SW1615 against CTLA4 (eliminating the activity of CTLA4 signaling that acts in an inhibitory manner on effector cell activation), as described in Examples 6-5.

Figure 32 shows the in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1389-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Examples 6-6.

Figure 33 shows the in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1389-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Examples 6-6.

Figure 34 shows the in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1610-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Examples 6-6.

Figure 35 shows the in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1610-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Examples 6-6.

[Implementation Plan Description]

The techniques and procedures described or cited herein are generally well understood by those skilled in the art and are typically used with conventional methodologies, such as Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor, eds., (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, editor (1987)); Oligonucleotide Synthesis (M.J. Gait, editor, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, editor, 1998) Academic Press; Ani mal Cell Culture (R.I. Freshney), editor, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, editors, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, editors); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, editors, 1987); PCR: The Polymerase Chain Reaction (Mullis et al., editors, 1994); Current Protocols in Immunology (J.E. Coligan et al., editors, 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 The widely used methodologies described in *The Antibodies Approach* (D. Catty, ed., IRL Press, 1988–1989); *Monoclonal Antibodies: A Practical Approach* (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); *Using Antibodies: A Laboratory Manual* (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); *The Antibodies* (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and *Cancer: Principles and Practice of Oncology* (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

I. Definition

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

For the purposes of this specification, the following definitions apply, and, where appropriate, terms used in the singular include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall prevail.

For the purposes of this document, a “recipient human frame” is a frame comprising the amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from the human immunoglobulin frame or the human common frame, as defined below. A recipient human frame “derived” from the human immunoglobulin frame or the human common frame may contain the same amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL recipient human frame is sequence-identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins, binding to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), enable these cytotoxic effector cells to specifically bind antigens to target cells and subsequently kill the target cells with cytotoxins. Primary NK cells, which mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of target molecules, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337, or U.S. Patent No. 6,737,056 (Presta). Useful effector cells for such assays include PBMCs and NK cells. Alternatively, the ADCC activity of the target molecule can be evaluated in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

"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 in which an antibody binds to an antigen present on the cell surface of a target cell, and effector cells further bind to the antibody, thereby causing the effector cells to damage the target cells. Whether a target antibody has ADCC activity and whether a target antibody has CDC activity can be determined by known methods (e.g., Current Protocols in Immunology, Chapter 7, Immunologic Studies in Humans, edited by Coligan et al. (1993)).

"Neutralizing activity" refers to the ability of an antibody to inhibit a biological activity by binding to a molecule involved in that activity. In some embodiments, the biological activity is caused by the binding between a ligand and a receptor. In other embodiments, the antibody inhibits the binding between a ligand and a receptor by binding to either a ligand or a receptor. Antibodies exhibiting this neutralizing activity are called neutralizing antibodies. The neutralizing activity of a test substance can be determined by comparing its biological activity in the presence and absence of a ligand.

The term “antibody-dependent phagocytosis” or “ADCP” refers to a process in which all or part of the cells covered by antibodies are incorporated into phagocytic immune cells (such as macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulins.

The term "binding activity" refers to the strength of the sum of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In this document, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). For example, when members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, binding activity is the sum of the strengths of each binding type. The binding activity of molecule X with its partner Y is typically expressed as a dissociation constant (KD) or "the amount of analyte bound per unit amount of ligand". Binding activity can be determined by methods commonly known in the art, including those described herein. Specific illustrative and exemplary embodiments for determining binding activity are described below.

"Affinity-mature" antibodies are those that have one or more alterations in one or more hypervariable regions (HVRs) compared to parental antibodies that do not have this alteration, resulting in increased affinity of the antibody for the antigen.

The terms "anti-CTLA-4 antibody" and "CTLA-4-binding antibody" refer to antibodies that bind to CTLA-4 with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting CTLA-4. In one embodiment, the anti-CTLA-4 antibody binds to less than about 10% of the antibody's binding to CTLA-4, as determined by, for example, radioimmunoassay (RIA). In some embodiments, the CTLA-4-binding antibody has a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., ^10⁻⁸ M or less, e.g., from ^10⁻⁸ M to ^10⁻¹³ M, e.g., from ^10⁻⁹ M to ^10⁻¹³ M). In some implementations, the anti-CTLA-4 antibody binds to an epitope of CTLA-4 that is conserved in CTLA-4 from different species.

The term “antibody” is used in the broadest sense in this article and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

"Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

"Antibodies that bind to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by, for example, 50% or more, in a competitive assay, and/or the reference antibody blocks the binding of the antibody to its antigen by, for example, 50% or more, in a competitive assay. Exemplary competitive assays are provided herein.

"Autoimmune diseases" refer to non-malignant diseases or conditions caused by and targeting the individual's own tissues. This article specifically excludes malignant or cancerous diseases or conditions, particularly B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloid leukemia. Examples of autoimmune diseases or conditions include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndromes (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic diseases such as eczema and asthma, and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome. e) Juvenile diabetes mellitus; and immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, commonly seen in tuberculosis, sarcoidosis, polymyositis, granulomatous diseases, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte exudation; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndromes; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrine disorders; Reiter's disease; stiff-person syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include breast cancer and liver cancer.

The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of an antibody binding to a target activates a series of enzymatic reactions, leading to the formation of pores in the cell membrane of the target cell. Typically, antigen-antibody complexes formed on target cells bind to and activate complement component C1q, which in turn activates the complement cascade and leads to target cell death. Furthermore, complement activation can also lead to the deposition of complement components on the surface of target cells, resulting in binding to complement receptors (e.g., CR3) on leukocytes, thereby promoting ADCC.

"Chemotherapy agents" refer to compounds that can be used to treat cancer. Examples of chemotherapy agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN, a registered trademark); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridine derivatives such as benzodopa, carboquone, meturedopa, and uredopa; and ethyleneimine and hydroxymethyl melamine, including melamine, triethylenemelamine, and trichloroethylenemelamine. Ethylenephosphamide, triethylenethiophosphamide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); δ-9-tetrahydrocannabinol (dronabinol, MARINOL (registered trademark)); β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogues topotecan (HYCAMTIN (registered trademark)) and CPT-11) (irinotecan, CAMPTOSAR (registered trademark)), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid (p odophyllinicacid); teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; docamycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; carnitine; spongistatin; nitrogen mustard. Mustards, such as chlorambucil, chlorophosphamide, estramustine, ifosfamide, nitrogen mustard, oxynitric acid mustard, melphalan, novobichin, phenesterine, prednimustine, trazophosphatide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, and flumustine. Mustine, lomustine, nimustine, and ranimustine; antibiotics, such as acetylenic antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ΩI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral α-4 integrin inhibitor; dynemicins, including dynemicin A; esperamicin; and new carcinogen chromophores and related chromopeptide chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin C, carrubicin, carminomycin, carzinophilin, chromomycins, dactinomycin D, daunorubicin, and dimethicone. Detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN (registered trademark)), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin hydrochloride liposome injection (DOXIL (registered trademark)), liposome doxorubicin TLC D-99 (MYOCET (registered trademark)), PEGylated liposome doxorubicin (CAELYX (registered trademark)), deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (acid), nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate, gemcitabine (GEMZAR (registered trademark)), tegafur (UFTORAL (registered trademark)), capecitabine ( XELODA (registered trademark), epothilone, and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimethotropic acid; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgen Hormones such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenergics such as aminoglutamine, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolactone; aldehyde phosphoramide glycoside; aminolevulinic acid; enuracil; acridine; ammoniated; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; and elliptinium acetate. ate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; PSK (registered trademark) polysaccharide complex (JHS) Natural Products (Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine); urethane; vindesine (ELDISINE (registered trademark), FILDESIN (registered trademark)); dacarbazine; mannomustine; mitobronitol; mitolactalol; pipebroman; cytosine; cytarabine (“Ara-C”); thiotepa; taxoids, such as paclitaxel (TAXOL (registered trademark)), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE ^™) . Docetaxel (TAXOTERE, a registered trademark); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin (e.g., ELOXATIN, a registered trademark), and carboplatin; vincas, which prevent tubulin polymerization to form microtubules, including vincaine (VELBAN, a registered trademark) and vincristine (ONCOVIN, a registered trademark). The following are listed: trademarks; vinorelbine (ELDISINE (registered trademark), FILDESIN (registered trademark)); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS. 2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid, including bexarotene (TARGRETIN); Bisphosphonates such as clodronate (e.g., BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID), or risedronate (ACTONEL); Trasatabine (1,3-dioxolane-cytosine analog); Antisense oligonucleotides In particular, those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE (registered trademark) vaccines and gene therapy vaccines, such as ALLOVECTIN (registered trademark) vaccines, LEUVECTIN (registered trademark) vaccines, and VAXID (registered trademark) vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN (registered trademark)); rmRH (e.g., ABARELIX (registered trademark)); BA Y439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT (registered trademark), Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); bortezomib (VELCADE (registered trademark)); CCI-779; telbifab (tipif arnib (R11577); sorafenib (ABT510); Bcl-2 inhibitors, such as oblimersen sodium (GENASEENSE (registered trademark)); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE (registered trademark)); farnesyltransferase inhibitors, such as lonafarnib (SCH 6636, SARASAR ^™ ); and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances; and combinations of two or more of the above, such as CHOP (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN ^™ ) in combination with 5-FU and leucovorin).

The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and/or light chain is derived from a specific source or species, while the remainder of the heavy chain and/or light chain is derived from a different source or species.

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

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., radioisotopes of ^211At , ^131I , ^125I , ^90Y , ^186Re , ^188Re , ^153Sm , ^212Bi , ^32P , ^212Pb , and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and their fragments, such as lysozymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including their fragments and/or variants; and many of the chemotherapeutic agents disclosed above.

"Effective cells" refer to leukocytes that express one or more FcRs and perform effector functions. In some 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 blood. In some embodiments, effector cells may be human effector cells.

"Effective functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotypes. 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 receptors); and B cell activation.

The term "epitope" encompasses any determinant that can be bound by an antibody. An epitope is an antigenic region that an antibody targeting an antigen binds to, comprising a specific amino acid that directly contacts the antibody. Epitope determinants can include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural features and/or specific charge features. Typically, antibodies specific to a particular target antigen preferentially recognize epitopes on target antigens in complex mixtures of proteins and/or macromolecules.

The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is a receptor that binds to IgG antibodies (γ receptors) and includes subclasses of FcγRI, FcγRII, and FcγRIII, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activating motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. (See, for example, Annu. Rev. Immunol. 15:203-234 (1997)). For example, FcR is outlined in 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” in this paper encompasses other FcRs, including those to be identified in the future.

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, for example, 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.)).

The in vivo binding of human FcRn and the serum half-life of human FcRn-binding peptides with high affinity can be determined, for example, in transgenic mice expressing human FcRn or in transfected human cell lines, or in primates to which they have been administered peptides with variant Fc regions. WO 2000/42072 (Presta) describes antibody variants that enhance or reduce binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

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

The term "antibody containing the Fc region" refers to an antibody that contains the Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Therefore, a composition containing an antibody having the Fc region according to the invention can contain an antibody having G446-K447, an antibody having G446 and not having K447, an antibody with all G446-K447 removed, or a mixture of the above three types of antibodies.

The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, each containing four conserved frame regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to specific antigens can be isolated using the VH or VL domains of the antibody binding the antigen, allowing for screening of libraries with complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

"Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences in VH (or VL) generally appear in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing the Fc region as defined herein.

A “functional Fc region” possesses the “effective function” of a native Fc region. Exemplary “effective functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require the Fc region to bind to a binding domain (e.g., antibody variable domain) and can be assessed using, for example, a variety of assays disclosed in the definition herein.

"Human antibody" is an antibody having an amino acid sequence corresponding to that of a non-human antibody produced by a human or human cell or derived from a human antibody library or other human antibody encoding sequence. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

The “human common framework” represents the framework of amino acid residues that most frequently occur in the selection of the human immunoglobulin VL or VH framework sequence. Typically, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al., ibid. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al., ibid.

A “humanized” antibody is a chimeric antibody comprising amino acid residues from a nonhuman HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of the nonhuman antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody. The humanized antibody may optionally comprise at least a portion of the antibody constant region derived from the human antibody. An antibody, such as a “humanized form” of a nonhuman antibody, refers to an antibody that has undergone humanization.

As used herein, the term “hypervariant region” or “HVR” refers to each region of an antibody variable domain that is sequence-hypervariant (“complementarity-determining region” or “CDR”) and/or forms a structure-defined loop (“hypervariant loop”) and/or contains antigen contact residues (“antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs as described herein include:

(a) Hypervariable rings appearing at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J.Mol.Biol.196:901-917(1987));

(b) CDRs appearing at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) Antigen contacts appearing at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J.Mol.Biol.262:732-745(1996)); and

(d) Combinations of (a), (b) and/or (c), including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).

Unless otherwise stated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered in this document in accordance with Kabat et al., ibid.

"Immune conjugates" are antibodies conjugated with one or more heterologous molecules, including but not limited to cytotoxic agents.

"Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some embodiments, antibodies are purified to a purity greater than 95% or 99% by means of, 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 antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

"Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally present in cells containing said nucleic acid molecules, but which are present outside the chromosome or at a chromosomal location different from their natural chromosomal location.

"Isolated nucleic acid encoding antibody" refers to one or more nucleic acid molecules that encode the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or in a separate vector, and such nucleic acid molecules are present at one or more locations in the host cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are effectively linked. Such vectors are referred to herein as "expression vectors."

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells do not need to be identical in nucleic acid content to the parent cells, but may contain mutations. Mutant progeny cells with the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical and/or bind to the same epitopes, except for possible variant antibodies, which, for example, contain naturally occurring mutations or are generated during the production of a monoclonal antibody formulation, where these variants are typically present in small amounts. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

"Natural antibodies" are 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 linked by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain. Based on the amino acid sequence of their constant domains, the light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ).

The “natural sequence Fc region” contains the same amino acid sequence as the Fc region found in nature. The natural sequence human Fc region includes the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region and its naturally occurring variants.

The “variant Fc region” comprises an amino acid sequence that differs from the native 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 compared to the native Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions, more preferably about 1 to about 5 amino acid substitutions, in the native Fc region or the Fc region of the parent polypeptide. The variant Fc region described herein preferably shares at least about 80% homology with the native Fc region and/or with the Fc region of the parent polypeptide, more preferably at least about 90% homology, and most preferably at least about 95% homology.

The "percentage (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope 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 sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared.

The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and will not change.

When using ALIGN-2 for amino acid sequence comparison, the percentage amino acid sequence identity of a given amino acid sequence A with (to, with, or against) a given amino acid sequence B (or, as can be expressed, a given amino acid sequence A that has or contains a specific amino acid sequence identity with given amino acid sequence B) is calculated as follows:

100 multiplied by the fraction X/Y

Where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the procedural alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A and B will not be equal to the % amino acid sequence identity of B and A. Unless otherwise specified, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a formulation in such a form that the biological activity of the active ingredient contained therein is effective, and that it does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, 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 some embodiments, the individual or subject is a human.

"Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are non-toxic to the subject, except for the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The “effective amount” of a drug (such as a pharmaceutical preparation) refers to the amount that effectively achieves the desired therapeutic or preventative outcome within the necessary dosage and time period.

The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings for using such a therapeutic product.

Unless otherwise stated, the term "CTLA-4" as used herein refers to any naturally occurring CTLA-4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed CTLA-4 as well as any form of CTLA-4 produced through cellular processing. The term also includes naturally occurring CTLA-4 variants, such as splice variants or allelic variants. Exemplary amino acid sequences of human CTLA-4 are shown in SEQ ID NO:214, mouse CTLA-4 in SEQ ID NO:247, monkey CTLA-4 in SEQ ID NO:248, and the extracellular domain of human CTLA-4 in SEQ ID NO:28. CTLA-4 may also be described herein as CTLA4.

The term "regulatory T (Treg) cells" refers to a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune diseases. These cells typically suppress or downregulate the induction and proliferation of effector T cells. The best-understood Treg cells are those that express CD4, CD25, and Foxp3 (CD4 ⁺ CD25 ⁺ Treg cells). These Tregs differ from helper T cells. Several different methods are used to identify and monitor Treg cells. When defined by CD4 and CD25 expression (CD4 ⁺ CD25 ⁺ cells), Treg cells constitute approximately 5% to approximately 10% of the mature CD4 ⁺ T cell subset in mice and humans, while approximately 1% to approximately 2% of Tregs can be detected in whole blood. Identification and monitoring can be performed by further assaying Foxp3 expression (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ cells). Additionally, the absence or low level of CD127 expression can be used in combination with the presence of CD4 and CD25 as another marker. Treg cells also express high levels of CTLA-4 and GITR. Tregs can also be identified using the methods described in the following examples.

As used herein, the terms “substantially similar,” “substantially equal,” or “substantially identical” mean a high degree of similarity between two values (e.g., one related to the antibody of the present invention and the other to a reference/comparison antibody) such that a person skilled in the art would consider that the difference between the two values has little or no biological and/or statistical significance in the context of the biological characteristics measured by said values (e.g., KD values).

As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention aimed at altering the natural processes of the individual being treated, and can be used for prevention or in the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of disease or slow its progression.

The term "tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "proliferative disorder," "proliferative lesion," and "tumor" are not mutually exclusive in this document.

The term "tumor tissue" refers to tissue containing at least one type of tumor cell. Tumor tissue typically consists of a population of tumor cells that form the main solid (parenchyma) of the tumor, and connective tissue and blood vessels present between these cells and supporting the tumor ("stromal"). In some cases, the distinction between the two is clear, while in others they are mixed. Tumor tissue may be infiltrated by immune cells, etc. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue within a living organism. Healthy/normal tissue not in a disease state is a typical example of non-tumor tissue.

II. Compositions and Methods

On one hand, this invention is based in part on anti-CTLA-4 antibodies and their uses. In some embodiments, antibodies that bind to CTLA-4 are provided. The antibodies of this invention can be used, for example, for the diagnosis or treatment of cancer.

A. Exemplary anti-CTLA-4 antibody

On one hand, the present invention provides isolated antibodies that bind to CTLA-4. In some embodiments, the anti-CTLA-4 antibody of the present invention has CTLA-4 binding activity dependent on the concentration of the adenosine-containing compound. In some embodiments, the binding activity to CTLA-4 is higher in the presence of an adenosine-containing compound than in the absence of an adenosine-containing compound. In another embodiment, the binding activity to CTLA-4 is higher in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of an adenosine-containing compound. In a further embodiment, the difference in binding activity with CTLA-4 is, for example, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1×10 ³ times or more, 2×10 ³ times or more, 3×10 ³ times or more, 5×10 ³ times or more, 1×10 ^{4 times} or more, 2×10 ^{4 times} or more, 3×10 ⁴ times or more, 5×10 ⁴ times or more, or 1×10 ⁵ times or more.

In some embodiments, the binding activity of the anti-CTLA-4 antibody can be represented by a KD (dissociation constant) value. In a further embodiment, the KD value of the anti-CTLA-4 antibody in the presence of an adenosine-containing compound is less than the KD value in the absence of an adenosine-containing compound. Alternatively, in another embodiment, the KD value of the anti-CTLA-4 antibody in the presence of a high concentration of an adenosine-containing compound is less than the KD value in the presence of a low concentration of an adenosine-containing compound. In a further embodiment, the KD value of the anti-CTLA-4 antibody varies, for example, by 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1x10 ³ times or more, 2x10 ³ times or more, 3x10 ³ times or more, 5x10 ³ times or more, 1x10 ⁴ times or more, 2x10 ⁴ times or more, 3x10 ⁴ times or more, 5x10 ⁴ times or more, or 1x10 ⁵ times or more. In the presence of adenosine-containing compounds or in the presence of high concentrations of adenosine-containing compounds, the KD value of the anti-CTLA-4 antibody can be, for example, ^9x10⁻⁷ M or lower, ^8x10⁻⁷ M or lower, ^7x10⁻⁷ M or lower, ^6x10⁻⁷ M or lower, ^5x10⁻⁷ M or lower, ^4x10⁻⁷ M or lower, ^3x10⁻⁷ M or lower, ^2x10⁻⁷ M or lower, ^1x10⁻⁷ M or lower, ^9x10⁻⁸ M or lower, ^8x10⁻⁸ M or lower, ^7x10⁻⁸ M or lower, ^6x10⁻⁸ M or lower, ^5x10⁻⁸ M or lower, ^4x10⁻⁸ M or lower, ^3x10⁻⁸ M or lower, ^2x10⁻⁸ M or lower, 1x10⁻⁸ M or lower, ^9x10⁻⁹ M or lower, ^8x10⁻⁹ M or lower, ^7x10⁻⁹ M or lower, 6 ... ^9x10⁻⁹ M or lower, 8x10⁻⁹ M or lower ^-9 M or lower, 5x10 ^-9 M or lower, 4x10 ^-9 M or lower, 3x10 ^-9 M or lower, 2x10 ^-9 M or lower, 1x10 ^-9 M or lower, 9x10 ^-10 M or lower, 8x10 ^-10 M or lower, 7x10 ^-10 M or lower, 6x10 ^-10 M or lower, 5x10 ^-10 M or lower, 4x10 ^-10 M or lower, 3x10 ^-10 M or lower, 2x10 ^-10 M or lower, or ^{1x10 -10 M} or lower. In the absence of adenosine-containing compounds or in the presence of low concentrations of adenosine-containing compounds, the KD value of anti-CTLA-4 antibodies can be, for example, ^1x10⁻⁸ M or higher, ^2x10⁻⁸ M or higher, ^3x10⁻⁸ M or higher, ^4x10⁻⁸ M or higher, ^5x10⁻⁸ M or higher, ^6x10⁻⁸ M or higher, ^7x10⁻⁸ M or higher, ^8x10⁻⁸ M or higher, ^9x10⁻⁸ M or higher, ^1x10⁻⁷ M or ^higher , ^2x10⁻⁷ M or higher, ^3x10⁻⁷ M or higher, ^4x10⁻⁷ M or higher, ^5x10⁻⁷ M or higher, ^6x10⁻⁷ M or higher, ^7x10⁻⁷ M or higher, ^8x10⁻⁷ M or higher, ^9x10⁻⁷ M or higher, ^1x10⁻⁶ M or higher, ^2x10⁻⁶ M or higher, ^3x10⁻⁶ M or higher, ^{4x10⁻⁶ ...} M or higher, ^5x10⁻⁶ M or higher, ^6x10⁻⁶ M or higher, ^7x10⁻⁶ M or higher, ^8x10⁻⁶ M or higher, or ^9x10⁻⁶ M or higher.

In another implementation, the binding activity of the anti-CTLA-4 antibody can be represented by the kd (dissociation rate constant) value instead of the KD value.

In another embodiment, the binding activity of the anti-CTLA-4 antibody can be expressed as the amount of CTLA-4 bound per unit volume of antibody. For example, in surface plasmon resonance assays, the amount of antibody bound to a sensor chip and the amount of antigen bound thereto are each measured as resonance units (RUs). The value obtained by dividing the amount of antigen bound by the amount of antibody bound can be defined as the amount of antigen bound per unit volume of antibody. Specific methods for determining and calculating this binding amount are described in the following examples. In some embodiments, the amount of CTLA-4 bound in the presence of an adenosine-containing compound is greater than the amount bound in the absence of an adenosine-containing compound. Alternatively, in another embodiment, the amount of CTLA-4 bound in the presence of a high concentration of an adenosine-containing compound is greater than the amount bound in the presence of a low concentration of an adenosine-containing compound. In a further embodiment, the binding amount of CTLA-4 varies, for example, by 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1x10 ³ times or more, 2x10 ³ times or more, 3x10 ³ times or more, 5x10 ³ times or more, 1x10 ⁴ times or more, 2x10 ⁴ times or more, 3x10 ⁴ times or more, 5x10 ⁴ times or more, or 1x10 ⁵ times or more. The binding amount of CTLA-4 in the presence of adenosine-containing compounds or in the presence of high concentrations of adenosine-containing compounds can be, for example, 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, or 1 or more. In the absence of adenosine-containing compounds or in the presence of low concentrations of adenosine-containing compounds, the binding amount of CTLA-4 can be, for example, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

In some implementations, the KD value, kd value, bonding amount, etc. described herein are determined or calculated by performing surface plasmon resonance measurements at 25°C or 37°C (see, for example, Example 3 herein).

Any concentration of adenosine-containing compound can be selected, as long as a difference in binding activity to the anti-CTLA-4 antibody is detected. In some embodiments, high concentrations may include, for example, 1 nM or higher, 3 nM or higher, 10 nM or higher, 30 nM or higher, 100 nM or higher, 300 nM or higher, 1 μM or higher, 3 μM or higher, 10 μM or higher, 30 μM or higher, 10 μM or higher, 30 μM or higher, 100 μM or higher, 300 μM or higher, 1 mM or higher, 3 mM or higher, 10 mM or higher, 30 mM or higher, 100 mM or higher, 300 mM or higher, 100 mM or higher, 300 mM or higher, and 1 M or higher. Alternatively, the high concentration here can be an amount sufficient to allow each anti-CTLA-4 antibody to exhibit maximum binding activity. In one embodiment, 1 μM, 10 μM, 100 μM, 1 mM, or an amount sufficient to allow each anti-CTLA-4 antibody to exhibit maximum binding activity can be selected as the high concentration here. In some embodiments, low concentrations may include, for example, 1 mM or less, 300 μM or less, 100 μM or less, 30 μM or less, 10 μM or less, 3 μM or less, 1 μM or less, 1 μM or less, 300 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, 1 nM or less, 300 pM or less, 100 pM or less, 30 pM or less, 10 pM or less, 3 pM or less, 10 pM or less, 3 pM or less, and 1 pM or less. Alternatively, low concentrations here may be the concentration at which each anti-CTLA-4 antibody exhibits minimal binding activity. Alternatively, a case where the substantial concentration is zero (no adenosine compound is present) can be selected as the low-concentration implementation. In one implementation, the concentrations of 1 mM, 100 μM, 10 μM, 1 μM, or the absence of adenosine compound can be selected here as the low concentrations, where each anti-CTLA-4 antibody exhibits minimal binding activity. In another embodiment, the following values can be selected as the ratio of high concentration to low concentration: for example, 3 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, 1x10 ³ times or more, 3x10 ³ times or more, 1x10 ⁴ times or more, 3x10 ⁴ times or more, 1x10 ⁵ times or more, 3x10 ⁵ times or more, 1x10 ⁶ times or more, 3x10 ⁶ times or more, 1x10 ⁷ times or more, 3x10 ⁷ times or more, 1x10 ⁸ times or more, 3x10 ⁸ times or more, 1x10 ⁹ times or more, 3x10 ⁹ times or more, 1x10 ¹⁰ times or more, 3x10 ¹⁰ times or more, 1x10 ¹¹ times or more, 3x10 ¹¹ times or more, or 1x10 ¹² times or more.

In another embodiment, the anti-CTLA-4 antibody of the present invention also has binding activity with an adenosine-containing compound. The amount of adenosine-containing compound bound per unit amount of the anti-CTLA-4 antibody can be calculated using the method described above and used as the binding activity of the antibody with the adenosine-containing compound. Specific methods for determining and calculating this binding amount are described in the following examples. The value of the amount of adenosine-containing compound bound per unit amount of the anti-CTLA-4 antibody of the present invention can be, for example, 0.0001 or more, 0.0002 or more, 0.0003 or more, 0.0004 or more, 0.0005 or more, 0.0006 or more, 0.0007 or more, 0.0008 or more, 0.0009 or more, 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, or 0.01 or more.

In another embodiment, the anti-CTLA-4 antibody of the present invention forms a ternary complex with an adenosine-containing compound and CTLA-4. In one embodiment, the anti-CTLA-4 antibody binds to the adenosine-containing compound via heavy chains CDR1, CDR2, and CDR3. In one embodiment, the anti-CTLA-4 antibody has a binding motif for the adenosine-containing compound. The binding motif for the adenosine-containing compound may consist of at least one amino acid, for example, present at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c according to Kabat numbers. In a further embodiment, the anti-CTLA-4 antibody binds to the adenosine-containing compound, for example, via at least one amino acid selected from positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c according to Kabat numbers. In some embodiments, the anti-CTLA-4 antibody has at least one amino acid selected from the group consisting of: Thr (position 33), Ser (position 52), Ser (position 52a), Arg (position 53), Tyr (position 56), Tyr (position 58), Tyr (position 95), Gly (position 96), Met (position 100a), Leu (position 100b), and Trp (position 100c). CTLA-4 can further bind to a complex formed by the anti-CTLA-4 antibody and an adenosine-containing compound. Furthermore, the adenosine-containing compound can be present at the interface of the interaction between the anti-CTLA-4 antibody and CTLA-4, and can bind to both. The formation of a ternary complex between the anti-CTLA-4 antibody, the adenosine-containing compound, and CTLA-4 can be confirmed by techniques such as crystal structure analysis described below (see Examples).

In another embodiment, the anti-CTLA-4 antibody of the present invention binds to at least one amino acid selected from the 3rd (Met), 33rd (Glu), 35th (Arg), 53rd (Thr), 97th (Glu), 99th (Met), 100th (Tyr), 101st (Pro), 102nd (Pro), 103rd (Pro), 104th (Tyr), 105th (Tyr), and 106th (Leu) amino acids of human CTLA-4 (extracellular domain; SEQ ID NO: 28). These amino acids can constitute the epitopes of the anti-CTLA-4 antibody of the present invention. In another embodiment, the anti-CTLA-4 antibody of the present invention binds to the region from amino acid 97 (Glu) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28). In another embodiment, the anti-CTLA-4 antibody of the present invention binds to the region from amino acid 99 (Met) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28).

In another embodiment, the anti-CTLA-4 antibody of the present invention competitively binds to CTLA-4 with ABAM004 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 11; HVR-H1, SEQ ID NO: 100; HVR-H2, SEQ ID NO: 101; HVR-H3, SEQ ID NO: 102; HVR-L1, SEQ ID NO: 113; HVR-L2, SEQ ID NO: 114; HVR-L3, SEQ ID NO: 115). In another embodiment, the anti-CTLA-4 antibody of the present invention binds to the same epitope as ABAM004. When an excess of anti-CTLA-4 antibody is present, the binding of ABAM004 to CTLA-4 can be reduced by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Exemplary competitive assays are provided herein.

In another embodiment, the anti-CTLA-4 antibody of the present invention exhibits cytotoxic activity against cells expressing CTLA-4. When CTLA-4 is expressed on the surface of target cells and the anti-CTLA-4 antibody binds to it, the cells are damaged. Damage to the cells may be caused by effector cells bound to the antibody, such as antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP) activities, or it may be caused by complement bound to the antibody, such as complement-dependent cytotoxicity (CDC) activities. Alternatively, damage may be caused by cytotoxic agents conjugated to the antibody (e.g., radioisotopes or chemotherapeutic agents), such as immunoconjugates. Cytotoxicity here can include effects such as inducing cell death, inhibiting cell proliferation, and impairing cell function. When anti-CTLA-4 antibody is present in sufficient quantities, it can cause damage to cells expressing CTLA-4, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. This cytotoxic activity can be measured by comparison with measurements in the absence of the antibody or in the presence of a negative control antibody. Exemplary cytotoxicity assays are provided herein.

In another embodiment, the anti-CTLA-4 antibody of the present invention exhibits neutralizing activity against CTLA-4. CTLA-4 is known to function by interacting with its ligands CD80 (B7-1) or CD86 (B7-2). In some embodiments, the anti-CTLA-4 antibody inhibits the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2). When the anti-CTLA-4 antibody is present in sufficient amounts, it can inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2), for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. This inhibitory activity can be measured by comparing it with measurements taken in the absence of antibodies or in the presence of negative control antibodies. This article provides a specific method for measuring neutralizing activity.

In another embodiment, the anti-CTLA-4 antibody of the present invention binds to CTLA-4 derived from a variety of animal species. Exemplary animal species may include mammals such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cattle, goats, horses, sheep, camels, dogs, and cats. In some embodiments, the anti-CTLA-4 antibody binds to CTLA-4 derived from humans and non-humans (e.g., monkeys, mice, and rats). The amino acid sequence of human CTLA-4 is shown in SEQ ID NO:214, the amino acid sequence of ape CTLA-4 is shown in SEQ ID NO:247, and the amino acid sequence of mouse CTLA-4 is shown in SEQ ID NO:248. The amino acid sequences of CTLA-4 derived from other animal species may also be appropriately determined by methods known to those skilled in the art.

In some embodiments, the adenosine-containing compounds of the present invention may include, for example, adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), deoxyadenosine (dADO), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), and adenosine (γ-thio) triphosphate (ATPγS).

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:223; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:224; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:225. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:223; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:224; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:225.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO:226; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO:227; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:228. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO:226; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO:227; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:228.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO:223, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO:224, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO:225; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO:226, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO:227, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:228.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:223; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:224; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:225; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:226; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:227; and (f) HVR-L3, comprising an amino acid sequence selected from SEQ ID NO:228.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 115.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO:100, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO:101, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO:113, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO:114, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:115.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:100; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:101; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:113; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:114; and (f) HVR-L3, comprising an amino acid sequence selected from SEQ ID NO:115.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 115.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO:100, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO:104, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO:116, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:115.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:100; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:104; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:116; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising an amino acid sequence selected from SEQ ID NO:115.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 105, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 106, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:105; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:106; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:122; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising the amino acid sequence selected from SEQ ID NO:133.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 108; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 108; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO:121; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO:123; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:153. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO:121; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO:123; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:153.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO:108, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO:121, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO:123, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO:153.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:108; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:121; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:123; and (f) HVR-L3, comprising an amino acid sequence selected from SEQ ID NO:153.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 110, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:110; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:122; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising the amino acid sequence selected from SEQ ID NO:133.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:112; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:128; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising the amino acid sequence selected from SEQ ID NO:133.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 152.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:111; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:152; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:128; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising the amino acid sequence selected from SEQ ID NO:133.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:112; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:129; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising the amino acid sequence selected from SEQ ID NO:133.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 152.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:111; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:152; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:129; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising the amino acid sequence selected from SEQ ID NO:133.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 109; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 109; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 109, and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 130, (ii) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133.

In another aspect, the present invention provides an antibody comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:109; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:130; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3, comprising the amino acid sequence selected from SEQ ID NO:133.

In some embodiments, any one or more amino acids of the anti-CTLA-4 antibody provided above are substituted at the following HVR positions:

- In HVR-H1 (SEQ ID NO: 223): Position 2

- In HVR-H2 (SEQ ID NO:224): positions 4, 5, 7, 13 and 16

- In HVR-H3 (SEQ ID NO:225): Position 3

- In HVR-L1 (SEQ ID NO:226): positions 1, 3, 6, 11, 12 and 14

- In HVR-L2 (SEQ ID NO:227): positions 1, 3, 4 and 7

- In HVR-L3 (SEQ ID NO:228): Positions 1 and 10

In some embodiments, the substitution is a conservative substitution as provided herein. In some embodiments, any one or more of the following substitutions may be carried out in any combination:

- In HVR-H1 (SEQ ID NO: 100): H2A, R, or K

- In HVR-H2 (SEQ ID NO: 101): S4T; R5Q; G7H; D13E or R; K16R

- In HVR-H3 (SEQ ID NO:102): K3A

- In HVR-L1 (SEQ ID NO:113): T1D, Q or E; T3P; D6G; N11T; Y12W; S14H

- In HVR-L2 (SEQ ID NO:114): E1F or Y; S3I; K4S; S7E or K

- In HVR-L3 (SEQ ID NO:115): S1Q; M10T

For HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, all possible combinations of the above substitutions are contained in the common sequences of SEQ ID NO: 223, 224, 225, 226, 227, and 228, respectively.

In any of the above embodiments, the anti-CTLA-4 antibody is humanized. In one embodiment, the anti-CTLA-4 antibody comprises an HVR as in any of the above embodiments, and further comprises a recipient human framework, such as a human immunoglobulin framework or a human common framework. In another embodiment, the anti-CTLA-4 antibody comprises an HVR as in any of the above embodiments, and further comprises a VH or VL containing an FR sequence. In a further embodiment, the anti-CTLA-4 antibody comprises the following heavy chain and/or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises any amino acid sequence of SEQ ID NO: 229 to 232, FR2 comprises the amino acid sequence of SEQ ID NO: 233, FR3 comprises the amino acid sequence of SEQ ID NO: 234, and FR4 comprises the amino acid sequence of SEQ ID NO: 235; for the light chain variable domain, FR1 comprises any amino acid sequence of SEQ ID NO: 236 to 238, FR2 comprises any amino acid sequence of SEQ ID NO: 240 to 241, FR3 comprises any amino acid sequence of SEQ ID NO: 242 to 244, and FR4 comprises any amino acid sequence of SEQ ID NO: 245 to 246.

In another aspect, the anti-CTLA-4 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising this sequence retains its ability to bind CTLA-4. In some embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids in SEQ ID NO: 10 are substituted, inserted, and/or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-CTLA-4 antibody comprises the VH sequence of SEQ ID NO: 10, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 100, (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 101, and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 102. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain to pyroglutamic acid via pyroglutamylation.

In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-CTLA-4 antibody containing this sequence retains its ability to bind CTLA-4. In some embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids in SEQ ID NO: 11 are substituted, inserted, and/or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-CTLA-4 antibody comprises the VL sequence of SEQ ID NO: 11, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 113, (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 115. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain to pyroglutamic acid via pyroglutamylation.

In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:149. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-CTLA-4 antibody comprising this sequence retains its ability to bind CTLA-4. In some embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids in SEQ ID NO:149 are substituted, inserted, and/or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-CTLA-4 antibody comprises the VL sequence of SEQ ID NO: 149, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 130, (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 133. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain to pyroglutamic acid via pyroglutamylation.

In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises VH and VL as provided in any of the embodiments described above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:10 and SEQ ID NO:11, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:98 and SEQ ID NO:99, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:83 and SEQ ID NO:97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:86 and SEQ ID NO:134, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:136 and SEQ ID NO:95, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:140 and SEQ ID NO:146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:141 and SEQ ID NO:146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:140 and SEQ ID NO:147, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:141 and SEQ ID NO:147, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:136 and SEQ ID NO:149, respectively, including post-translational modifications of those sequences. In another aspect, a heteropolymer anti-CTLA-4 antibody is provided, wherein the antibody comprises at least two distinct variable regions selected from variable regions comprising the VH and VL sequences provided above. In one embodiment, the antibody comprises VH and VL sequences as shown in SEQ ID NO:140 and SEQ ID NO:146, respectively, and VH and VL sequences as shown in SEQ ID NO:141 and SEQ ID NO:146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises VH and VL sequences as shown in SEQ ID NO:140 and SEQ ID NO:147, respectively, and VH and VL sequences as shown in SEQ ID NO:141 and SEQ ID NO:147, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain to pyroglutamic acid via pyroglutamylation.

When the N-terminal amino acid of the heavy or light chain of the anti-CTLA-4 antibody provided in this article is glutamine, this amino acid can be replaced by glutamic acid.

In another aspect, the present invention provides antibodies that bind to the same epitopes as the anti-CTLA-4 antibodies provided herein. For example, in some embodiments, antibodies that bind to the same epitopes as any of the antibodies listed in Tables 4, 9, 14, and 19 are provided. In some embodiments, antibodies that bind to epitopes within a CTLA-4 fragment are provided, the fragment comprising at least one amino acid selected from the group consisting of amino acids at positions 3 (Met), 33 (Glu), 35 (Arg), 53 (Thr), 97 (Glu), 99 (Met), 100 (Tyr), 101 (Pro), 102 (Pro), 103 (Pro), 104 (Tyr), 105 (Tyr), and 106 (Leu) of SEQ ID NO:28 are provided. In some embodiments, antibodies that bind to epitopes within a CTLA-4 fragment are provided, the fragment comprising amino acids at positions 97 (Glu) to 106 (Leu) of SEQ ID NO:28 are provided. In some embodiments, an antibody is provided that binds to an epitope within a CTLA-4 fragment consisting of amino acids from position 99 (Met) to position 106 (Leu) of SEQ ID NO:28.

In other aspects of the invention, the anti-CTLA-4 antibody according to any of the above embodiments is a monoclonal antibody, including chimeric antibodies, humanized antibodies, or human antibodies. In one embodiment, the anti-CTLA-4 antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody, a complete IgG4 antibody, or another antibody class or isotype as defined herein.

In other aspects, the anti-CTLA-4 antibody of the present invention comprises an Fc region. In other aspects, the anti-CTLA-4 antibody of the present invention comprises a constant region. The constant region may be a heavy chain constant region (including the Fc region), a light chain constant region, or both. In some embodiments, the Fc region is the Fc region of a natural sequence. Exemplary heavy chain constant regions derived from natural antibodies may include, for example, heavy chain constant regions such as human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), and human IgG4 (SEQ ID NO: 252). Furthermore, other exemplary heavy chain constant regions may include the heavy chain constant regions of SEQ ID NO: 82 and 158. Exemplary light chain constant regions derived from natural antibodies may include, for example, light chain constant regions such as human κ chain (SEQ ID NO: 33, 63, and 159) and human λ chain (SEQ ID NO: 53 and 87).

In another embodiment, the Fc region is a variant Fc region created by adding amino acid alterations to the native Fc region. In some embodiments, the variant Fc region exhibits enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to the native Fc region. In other embodiments, the variant Fc region exhibits enhanced binding activity to FcγRIIa and FcγRIIIa compared to the native Fc region. Examples of heavy chain constant regions containing such variant Fc regions include, for example, the heavy chain constant regions listed in Tables 26 to 30 and the heavy chain constant regions of SEQ ID NOs: 31, 32, 41 to 46, 65, 66, 81, 207, 239, 253 to 271, 276, 277, and 278.

The Fc region of a natural sequence is typically a homodimer composed of two identical polypeptide chains. In some embodiments, the variant Fc region can be a homodimer composed of polypeptide chains having the same sequence or a heterodimer composed of polypeptide chains having different sequences. Similarly, the heavy chain constant region containing the Fc region can be a homodimer composed of polypeptide chains having the same sequence or a heterodimer composed of polypeptide chains having different sequences. Examples of heteropolymer heavy chain constant regions include, for example, heavy chain constant regions of polypeptide chains comprising SEQ ID NO: 31 and 32; heavy chain constant regions of polypeptide chains comprising SEQ ID NO: 43 and 44; heavy chain constant regions of polypeptide chains comprising SEQ ID NO: 45 and 46; heavy chain constant regions of polypeptide chains comprising SEQ ID NO: 254 and 256; heavy chain constant regions of polypeptide chains comprising SEQ ID NO: 257 and 258; heavy chain constant regions of polypeptide chains comprising SEQ ID NO: 259 and 260; heavy chain constant regions of polypeptide chains comprising SEQ ID NO: 261 and 263; and polypeptide chains comprising SEQ ID NO: 262 and 264. The heavy chain constant region of the polypeptide chain containing SEQ ID NO: 265 and 267; the heavy chain constant region of the polypeptide chain containing SEQ ID NO: 266 and 268; the heavy chain constant region of the polypeptide chain containing SEQ ID NO: 269 and 270; the heavy chain constant region of the polypeptide chain containing SEQ ID NO: 271 and 81; the heavy chain constant region of the polypeptide chain containing SEQ ID NO: 65 and 66; the heavy chain constant region of the polypeptide chain containing SEQ ID NO: 239 and 207; the heavy chain constant region of the polypeptide chain containing SEQ ID NO: 259 and 276; and the heavy chain constant region of the polypeptide chain containing SEQ ID NO: 65 and 278.

In other respects, the anti-CTLA-4 antibody according to any of the above embodiments can bind any feature, alone or in combination, as described in sections 1-7 below:

1. Antibody binding activity

In some embodiments, the binding activity of the antibody provided herein is a dissociation constant (KD) of 10 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, ^0.01 nM or less, or ^0.001 nM or less (e.g., ^10⁻⁸ M or less, e.g., 10⁻⁸ M to ^10⁻¹³ M, e.g., ^10⁻⁹ M to ^10⁻¹³ M).

In one embodiment, the binding activity of the antibody is determined by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the target antibody and its antigen. For example, the solution binding affinity of Fab to the antigen is determined by equilibrating Fab with a minimum concentration of ( ¹²⁵ I) labeled antigen in the presence of a titration series of unlabeled antigen, followed by capturing the bound antigen with a plate coated with anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, MICROTITER (trademark) multiwell plates (Thermo Scientific) are coated overnight in 50 mM sodium carbonate (pH 9.6) with 5 μg/ml capture anti-Fab antibody (Cappel Labs) and subsequently blocked at room temperature (approximately 23°C) with 2% (w/v) bovine serum albumin in PBS for 2 to 5 hours. In a non-absorbent plate (Nunc#269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen was mixed with serial dilutions of the target Fab (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab was then incubated overnight; however, incubation may be prolonged (e.g., about 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plate was dry, 150 μl/well of scintillation agent (MICROSCINT-20 ^™ ; Packard) was added and the plate was counted for 10 minutes on a TOPCOUNT ^™ gamma counter (Packard). The concentration of each Fab that produces a maximum binding of less than or equal to 20% is selected for competitive binding assays.

In one embodiment, antibody binding activity is determined by, for example, a ligand capture assay using surface plasmon resonance assay as the assay principle, employing a BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare, Uppsala, Sweden). BIACORE (registered trademark) control software is used for device operation. In one embodiment, an amine conjugation kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions, and molecules for ligand capture, such as anti-tag antibodies, anti-IgG antibodies, and a sensor chip immobilized with carboxymethyl dextran (GE Healthcare, Uppsala, Sweden), are used. The molecules for ligand capture are diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. The assay buffer contains 0.05% polysorbate 20 (also known as TWEEN (registered trademark)-20) and is used at a flow rate of 10-30 μL/min, preferably at an assay temperature of 25°C or 37°C. When the assay is performed using an antibody that acts as a ligand, allowing molecular capture for ligand capture, a series of dilutions of the antigen or Fc receptor prepared with the assay buffer (analyte) are injected after the target amount of antibody is captured by injection. When the assay is performed using an antigen or Fc receptor that acts as a ligand, a series of dilutions of the antibody prepared with the assay buffer (analyte) are injected after the target amount of antigen or Fc receptor is captured by injection.

In one implementation, the BIACORE (registered trademark) evaluation software is used to analyze the assay results. Kinetic parameters are calculated by simultaneously fitting binding and dissociation sensor maps using a 1:1 binding model, and the binding rate (kon or ka), dissociation rate (koff or kd), and equilibrium dissociation constant (KD) can be calculated. When binding activity is weak, especially when dissociation is rapid and kinetic parameters are difficult to calculate, a steady-state model can be used to calculate the equilibrium dissociation constant (KD). As another parameter of binding activity, the "analyte binding per unit amount of ligand" can be calculated by dividing the amount of analyte bound (RU) at a specific concentration by the amount of ligand captured (RU).

2. Antibody fragments

In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

Biantibodies are antibody fragments with two antigen-binding sites, and can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triantibodies and tetraantibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1).

As described herein, antibody fragments can be prepared using a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production from recombinant host cells (e.g., Escherichia coli or bacteriophages).

3. Chimeric and humanized antibodies

In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In other instances, a chimeric antibody is a “class-switching” antibody, wherein the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, a nonhuman antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent nonhuman antibody. Typically, a humanized antibody contains one or more variable domains, wherein the HVR, such as the CDR, (or a portion thereof) is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may optionally also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the nonhuman antibody (e.g., an antibody from which HVR residues are derived), for example, to restore or enhance antibody specificity or affinity.

Humanized antibodies and their preparation methods are summarized, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36 :25-34 (2005) (Description of specific region-determining (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (Description of "resurfacing"); Dall’Acqua et al., Methods 36:43-60 (2005) (Description of "FR resurfacing"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (Description of "guided selection" technique to FR resurfacing).

Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151: 2296 (1993)); frame regions derived from specific subgroups of human antibody consortium sequences derived from the light chain or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)). 3)); human maturation (somatic mutation) framework region or human germline framework region (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

4. Human antibodies

In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE ^™ technology; U.S. Patent No. 5,770,429 describing HuMab (registered trademark) technology; U.S. Patent No. 7,041,870 describing KM MOUSE (registered trademark) technology; and U.S. Patent Application Publication No. US 2007/0061900 describing VelociMouse (registered trademark) technology. Human variable regions of intact antibodies produced from such animals can be further modified, for example, by combining them with different human constant regions.

Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines used to produce human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-191 (2005).

Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. These variable domain sequences can then be combined with desired human constant domains. The technique for selecting human antibodies from antibody libraries is described below.

5. Library-derived antibodies

The antibodies of the present invention can be isolated by screening for antibodies with the desired activity in a combinatorial library. For example, various methods are known in the art for generating phage display libraries and screening for antibodies with the desired binding properties in such libraries. Such methods are summarized, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O’Brien et al., editor, Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology. ular Biology 248:161-175 (Lo, editor, Human Press, Totowa, NJ, 2003); Sidhu et al., J.Mol.Biol.338(2):299-310 (2004); Lee et al., J.Mol.Biol.340(5):1073-1093 (2004); Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472 (2004); and Lee et al., J.Immunol.Methods 284(1-2):119-132 (2004).

In some phage display methods, repertoires of the VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library. The phage library can then be screened for antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-stranded Fv (scFv) fragments or Fab fragments. Libraries derived from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization. Finally, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), a primitive library encoding a highly variable CDR3 was also prepared by cloning an unrearranged V gene fragment from stem cells and synthesizing it using PCR primers containing random sequences, thus enabling in vitro rearrangement. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publications Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments in this paper.

6. Multispecific antibodies

In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In some embodiments, one binding specificity is against CTLA-4, while the other is against any other antigen. In some embodiments, a bispecific antibody can bind to two different epitopes of CTLA-4. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing CTLA-4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93/08829, and Trauneckerd et al., EMBO J.10:3655 (1991)), and "knob-in-hole" modification (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by: modifying the electrostatic steering effect of the Fc-heterodimeric molecule used to prepare the antibody (WO 2009/089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); or using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)). ); preparation of bispecific antibody fragments using “dual antibody” technology (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and preparation of trispecific antibodies using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and preparation of trispecific antibodies, for example, as described in Tutt et al., J. Immunol. 147:60 (1991).

This article also includes modified antibodies with three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006/0025576A1).

The antibodies or fragments described herein also include “dual-acting Fab” or “DAF”, which contain an antigen-binding site that binds to CTLA-4 as well as another different antigen (see, for example, US 2008/0069820).

7. Antibody variants

In some embodiments, amino acid sequence variants of the antibodies provided herein are considered. For example, it may be necessary to enhance the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

a) Substitution, insertion, and deletion variants

In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Conserved substitutions are shown in Table 1 under the heading “Preferred Substitutions”. Further essential changes are provided in Table 1 under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into target antibodies, and desired activities of the product can be screened, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[Table 1]

Amino acids can be divided into several groups based on their common side chain properties:

(1) Hydrophobic: Leucine, Methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu) and Isoleucine (Ile);

(2) Neutral and hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn) and Glutamine (Gln);

(3) Acidity: Aspartic acid (Asp) and glutamic acid (Glu);

(4) Alkaline: Histidine (His), Lysine (Lys) and Arginine (Arg);

(5) Residues affecting chain orientation: glycine (Gly) and proline (Pro); and

(6) Aromatics: tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe).

Non-conservative substitution refers to replacing members of one of these categories with members of another category.

One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further research will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain some of the biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

Modifications (e.g., substitutions) can be made in the HVR, for example, to improve antibody affinity. Such modifications can be made in HVR “hotspots,” i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact the antigen, where the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by constructing and reselecting from a secondary library has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O’Brien et al., editor, Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variant with the desired affinity. Another approach to introducing diversity involves HVR-directed methods, where several HVR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scan mutagenesis or modeling can be used to specifically identify HVR residues involved in antigen binding. CDR-H3 and CDR-L3 are particularly frequently targeted.

In some embodiments, substitution, insertion, or deletion may occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., the conserved substitutions provided herein) may be made in the HVRs that do not substantially reduce binding affinity. For example, such changes may be made outside the antigen-contacting residues in the HVRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

As described by Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is called "alanine scan mutagenesis." In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include fusion of the amino and/or carboxyl ends of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionine residue. Other insertion variants of antibody molecules include fusion of an enzyme (e.g., for ADEPT) or a peptide that increases the plasma half-life of the antibody with the N-terminus or C-terminus.

b) Glycosylation variants

In some implementations, the antibodies provided herein are modified to increase or decrease the degree to which the antibodies are glycosylated. The addition or deletion of glycosylation sites on the antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

In the case of an antibody containing an Fc region, the carbohydrates linked to it can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via N-linked bonds. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

In one embodiment, an antibody variant is provided having a carbohydrate structure lacking (directly or indirectly) fucose linked to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all sugar structures (e.g., complex, heterozygous, and high-mannose structures) linked to Asn 297 as determined by MALDI-TOF mass spectrometry, for example, as described in WO 2008/077546. Asn297 refers to an asparagine residue (EU number of Fc region residues) located approximately at position 297 in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may also be located approximately +/- 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/ 0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/03 1140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylation antibodies include Lec13 CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312A1, Adams et al., especially in Example 11), and knockout cell lines, such as α-1,6-fucosylation gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO 2003/085107).

Antibody variants also provide bipartite oligosaccharides, for example, wherein the bitendril oligosaccharide linked to the Fc region of the antibody is bipartitely divided by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc region variants

In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby creating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In some embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them ideal candidates for applications where the antibody's half-life in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm a reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary NK cells, which mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACT1 ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Or, alternatively, the ADCC activity of the target molecule can be assessed in vivo, for example in the animal models disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, WO 2006/029879 and WO C1q and C3c binding ELISA in 2005/100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 replaced by alanine (US Patent No. 7,332,581).

Certain antibody variants that bind to FcR with increased or decreased binding are described (see, for example, U.S. Patent No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

In some embodiments, the antibody variant comprises one or more amino acid substitutions with improved ADCC, such as substituted Fc regions at positions 298, 333, and/or 334 (EU numbers of residues) in the Fc region.

In some implementations, alterations are made in the Fc region that result in altered (i.e., increased or decreased) Clq binding and/or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn) are described in US2005/0014934A1 (Hinton et al.), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)). These antibodies contain Fc regions with one or more substitutions that increase the binding of the Fc regions to the FcRn. Such Fc variants include those with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, such as the substitution of Fc region residue 434 (US Patent No. 7,371,826).

Other examples involving Fc region variants can be found in Duncan & Winter, Nature 322:738-740 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94/29351.

d) Cysteine-modified antibody variants

In some embodiments, it is desirable to generate cysteine-modified antibodies, such as "thioMAb," wherein one or more residues of the antibody are replaced by cysteine residues. In a particular embodiment, the substituted residues are located at an accessible site on the antibody. By replacing those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site on the antibody and can be used to conjugate the antibody to other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to generate an immunoconjugate, as further described herein. In some embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; S400 (EU number) of the Fc region of the heavy chain. Cysteine-modified antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

e) Antibody derivatives

In some embodiments, the antibodies provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties for derived antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for treatment under specific conditions.

In another embodiment, a conjugate of the antibody and non-protein fraction is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein fraction is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein fraction to near the temperature at which the antibody-non-protein fraction kills cells.

B. Recombination methods and compositions

Antibodies can be generated using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the anti-CTLA-4 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH (e.g., the light chain and/or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., having been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp2/O cells). In one embodiment, a method for preparing an anti-CTLA-4 antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody provided above under conditions suitable for expressing anti-CTLA-4, and optionally recovering the antibody from the host cell (or host cell culture medium).

For the recombinant generation of anti-CTLA-4 antibodies, as described above, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody.

Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, for example, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, editor, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*). After expression, the antibody can be separated from the bacterial cell pellet in a soluble fraction and can be further purified.

Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," thereby producing antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells.

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 (description of PLATNIBODIES ^™ technology for generating antibodies in transgenic plants).

Vertebrate cells can also serve as hosts. For example, mammalian cell lines adapted for suspension growth can be important. Other examples of important mammalian host cell lines include monkey kidney CV1 cell lines transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK); mouse supporting cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); and monkey kidney cells (CV1). 1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT060562); TRI cells, as described by Matherd et al., Annals N.Y.Acad.Sci.383:44-68 (1982); MRC 5 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, NSO, and Sp2/0. For reviews of certain mammalian host cell lines applicable to antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C.Lo, editor, Humana Press, Totowa, NJ), pp. 255-268 (2003).

Polyclonal antibodies are preferably produced in animals via multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen with an immunogenic protein in the species to be immunized, such as keyhole hemocyanin, serum albumin, _bovine thyroglobulin, or a soybean ^trypsin inhibitor, using bifunctional or derivatizing agents, such as maleimide benzoyl sulfosuccinimide (via cysteine residue conjugation), N-hydroxysuccinimide (via lysine residue conjugation), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR (where R and ^R1 are different alkyl groups).

Animals (typically non-human mammals) are immunized against an antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and administering the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 of the initial amount of peptide or conjugate in Freund's complete adjuvant via subcutaneous injection at multiple sites. Blood is collected from the animals 7 to 14 days later, and serum antibody titers are determined. Boosting continues until the titer plateaus. Preferably, animals are boosted with the same antigen but conjugated to different proteins and/or via conjugates with different cross-linking agents. Conjugates can also be prepared as protein fusions in recombinant cell cultures. Furthermore, agglutinating agents such as alum are also suitable for enhancing the immune response.

Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical, except for possible small amounts of naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation). Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies.

For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro.

Immunosuppressants typically consist of an antigen protein or a fusion variant thereof. Peripheral blood lymphocytes (PBLs) are usually used if human-derived cells are required, and spleen cells or lymph node cells are used if non-human mammalian-derived cells are required. The lymphocytes are then fused with immortalized cell lines using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells thus prepared are seeded and cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium used for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that inhibit the growth of HGPRT-deficient cells.

Preferred immortalized myeloma cells are those that are efficiently fused, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Among these, mouse myeloma lines, such as MOPC-21 and MPC-11 mouse tumors derived from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 cells (and their derivatives, e.g., X63-Ag8-653) available from the American Type Culture Collection (Manassas, Virginia, USA). Human myeloma and mouse-human heterologous myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J. Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

The production of monoclonal antibodies against antigens in the culture medium in which hybridoma cells are grown is determined. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis by Munson, Anal. Biochem. 107(1):220-239 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, clonal subclones can be cultured using a limiting dilution procedure and standard methods (Goding, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 media. Furthermore, hybridoma cells can grow as tumors in mammals.

Monoclonal antibodies secreted by subclones are appropriately separated from culture medium, ascites, or serum using routine immunoglobulin purification procedures, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Antibodies can be generated by immunizing an appropriate host animal against an antigen. In one embodiment, the antigen is a polypeptide containing full-length CTLA-4. In one embodiment, the antigen is a polypeptide containing soluble CTLA-4. In one embodiment, the antigen is a polypeptide containing a region (extracellular domain, SEQ ID NO: 28) of amino acids corresponding to positions 97 (Glu) to 106 (Leu) of human CTLA-4. In one embodiment, the antigen is a polypeptide containing a region (extracellular domain, SEQ ID NO: 28) of amino acids corresponding to positions 99 (Met) to 106 (Leu) of human CTLA-4. The invention also includes antibodies generated by immunizing an animal against an antigen. As described above in “Exemplary Anti-CTLA-4 Antibodies,” antibodies can bind any feature alone or in combination.

C. Measurement

The anti-CTLA-4 antibodies described herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities using a variety of assays known in the art.

1. Combining measurements with other measurements

On one hand, the antigen-binding activity of the antibodies of the present invention can be tested, for example, by known methods such as ELISA, Western blotting, and surface plasmon resonance assay.

On the other hand, competitive assays can be used to identify antibodies that compete with the anti-CTLA-4 antibodies described herein (e.g., the anti-CTLA-4 antibodies described in Tables 4, 9, 14, and 19) for binding to CTLA-4. In some embodiments, if such a competitive antibody is excessively present, the binding of the reference antibody to CTLA-4 is blocked (e.g., reduced) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some instances, binding is blocked by at least 80%, 85%, 90%, 95%, or more. In some embodiments, this competitive antibody binds to the same epitope (e.g., a linear or conformational epitope) as the anti-CTLA-4 antibody described herein (e.g., the anti-CTLA-4 antibody described in Tables 4, 9, 14, and 19). A detailed exemplary method for mapping epitopes of antibody binding is provided in Morris (1996), "Epitope Mapping Protocols," Methods in Molecular Biology vol 66 (Humana Press, Totowa, NJ).

In an exemplary competitive assay, immobilized CTLA-4 is incubated in a solution containing a first labeled antibody that binds to CTLA-4 and a second unlabeled antibody that is being tested for its ability to competitively bind to CTLA-4 against the first antibody. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CTLA-4 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CTLA-4, excess unbound antibody is removed, and the amount of labeling bound to the immobilized CTLA-4 is measured. If the amount of labeling bound to the immobilized CTLA-4 in the test sample is significantly reduced compared to the control sample, it indicates that the second antibody is competitively binding to CTLA-4 against the first antibody. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity Assay

On one hand, an assay is provided for identifying anti-CTLA-4 antibodies that possess biological activity. Biological activity may include, for example, cell proliferation inhibition activity, cytotoxic activity (e.g., ADCC/CDC activity and ADCP activity), immunostimulatory activity, and CTLA-4 inhibitory activity. Antibodies possessing this biological activity in vivo and/or in vitro are also provided.

In some embodiments, the antibodies of the present invention are tested for such biological activity.

In some embodiments, the ability of the antibodies of the present invention to inhibit cell growth or proliferation in vitro was tested. Assays for inhibiting cell growth or proliferation are well known in the art. Using the “cell killing” assay example described herein, some assays for cell proliferation measure cell viability. One such assay is the CellTiter-Glo ^™ Luminescent Cell Viability Assay, commercially available from Promega (Madison, WI). This assay determines the number of viable cells in a culture based on the quantification of present ATP, an indicator of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-88, US Pat. No. 6602677. This assay can be performed in 96-well or 384-well form, making it suitable for automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure involves the direct addition of a single reagent (CellTiter-Glo (registered trademark) reagent) to the cultured cells. This leads to cell lysis and the generation of a luminescent signal via a luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which in turn is proportional to the number of live cells present in the culture. Data can be recorded using a photometer or a CCD camera imaging device. The luminescence output is expressed as relative light units (RLU).

Another assay used for cell proliferation is the “MTT” assay, a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. Like the CellTiter-Glo ^™ assay, this assay indicates the number of metabolically active cells present in the cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhang et al. (2005) Cancer Res. 65:3877-3882.

Cells used for any of the above in vitro assays include cells or cell lines that naturally express CTLA-4 or have been engineered to express CTLA-4. This class of cells also includes cell lines that express CTLA-4 and cell lines that do not normally express CTLA-4 but have been transfected with nucleic acids encoding CTLA-4.

On one hand, the ability of the anti-CTLA-4 antibody to inhibit cell growth or proliferation in vivo is tested. In some embodiments, the ability of the anti-CTLA-4 antibody to inhibit tumor growth in vivo is tested. In vivo model systems, such as xenograft models, can be used for such testing. In exemplary xenograft systems, human tumor cells are introduced into appropriate immunocompromised nonhuman animals, such as athymic "naked" mice. The antibody of the present invention is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is determined. In some embodiments of the above-described xenograft systems, the human tumor cells are tumor cells derived from human patients. Such xenograft models are commercially available from Oncotest GmbH (Frieberg, Germany). In some embodiments, human tumor cells are introduced into appropriate immunocompromised nonhuman animals by subcutaneous injection or by transplantation to a suitable site, such as a mammary fat pad.

It should be understood that the immunoconjugates of the present invention can be used in place of anti-CTLA-4 antibodies or any of the above assays except for anti-CTLA-4 antibodies.

A typical assay for determining ADCC activity of therapeutic antibodies is based on a ^51Cr release assay and includes the following steps: labeling target cells with [ ^51Cr ] _Na₂CrO₄ ; opsonizing the target cells _, which express antigens on their cell surface, with an antibody; combining the opsonized radiolabeled target cells with effector cells in an appropriate ratio in a microtiter plate, with or without the test antibody; incubating the cell mixture preferably at 37°C for 16 to 18 hours; collecting the supernatant; and analyzing the radioactivity in the supernatant sample. The cytotoxicity of the test antibody is then determined, for example, by the following equation: Specific cytotoxicity (%) = (Radioactivity in the presence of antibody - Radioactivity in the absence of antibody) / (Maximum radioactivity - Radioactivity in the absence of antibody) x 100. A graph can be generated by varying the ratio of target cells to effector cells or the antibody concentration.

To assess complement activation, complement-dependent cytotoxicity (CDC) assays can be performed as described, for example, in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Briefly, multiple concentrations of peptide variants and human complement are diluted with buffer. Cells expressing antigens that bind to the peptide variants are diluted to a density of approximately 1 × ^10⁶ cells/ml. A mixture of the peptide variant, diluted human complement, and antigen-expressing cells is added to flat-bottomed 96-well tissue culture plates and incubated at 37°C and 5% _CO₂ for 2 hours to promote complement-mediated cell lysis. Then, 50 μl of AlamarBlue (Accumed International) is added to each well and incubated overnight at 37°C. Absorbance is measured using a 96-well fluorometer with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Results are expressed in relative fluorescence units (RFU). Sample concentrations can be calculated from a standard curve, and the percentage of activity compared to the non-variant peptide for the target peptide variant is reported.

An exemplary method for determining ADCP activity may include the following: coating target biological particles, such as Escherichia coli labeled with FITC (molecular probe) or Staphylococcus aureus-FITC, with a test antibody; forming opsonization particles; adding the opsonization particles to THP-1 effector cells (monocyte cell lines, available from ATCC) at ratios of 1:1, 10:1, 30:1, 60:1, 75:1, or 100:1 to induce F… cγR-mediated phagocytosis; preferably, cells and *E. coli*-FITC/antibody are incubated at 37°C for 1.5 hours; after incubation, trypan blue is added to the cells (preferably left at room temperature for two to three minutes) to quench the fluorescence of bacteria that have not yet been incorporated into the cells and have already attached to the cell surface; the cells are transferred to FACS buffer (e.g., 0.1% BSA and 0.1% sodium azide in PBS) to measure the fluorescence of THP-1 cells using FACS (e.g., BDFACS Calibur). To determine the extent of ADCP, it is preferable to set a gating system on THP-1 cells and measure the median fluorescence intensity. In the most preferred embodiment, *E. coli*-FITC in culture medium (control); *E. coli*-FITC and THP-1 cells (used for FcγR-independent ADCP activity); and *E. coli*-FITC, THP-1 cells, and test antibody (used for FcγR-dependent ADCP activity) are used for ADCP measurement.

The cytotoxic activity of antibodies typically involves the binding of the antibody to the cell surface. Whether an antigen is expressed on the target cell surface can be appropriately confirmed by methods known to those skilled in the art, such as FACS.

Immune activation can be detected using cellular or humoral immune responses as indicators. Specifically, immune activation includes increased expression levels of cytokines (e.g., IL-6, G-CSF, IL-12, TNFα, and IFNγ) or their receptors, promoting the proliferation, increased activation status, enhanced function, and increased cytotoxic activity of immune cells (e.g., B cells, T cells, NK cells, macrophages, and monocytes). In particular, T cell activation can be detected by measuring elevated expression of activation markers such as CD25, CD69, and ICOS. For example, patients given the anti-CTLA-4 antibody ipilimumab are known to have increased ICOS ⁺ CD4 ⁺ T cells in their peripheral blood after administration, which is considered an activation effect of the anti-CTLA-4 antibody on the systemic immune status (Cancer Immunol. Res. (2013) 1(4):229-234).

T cell activation requires stimulation not only through antigen receptors (TCRs) but also through co-stimulation via CD28. When CD28 on the surface of T cells binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells, a co-stimulatory signal is transmitted to the T cell, and the T cell is then activated. On the other hand, CTLA-4 is expressed on the surface of activated T cells. Because CTLA-4 binds to CD80 and CD86 with a stronger affinity than to CD28, it preferentially interacts with CD80 and CD86, leading to inhibition of T cell activation.

Based on this mechanism of action, the inhibitory activity against CTLA-4 can be measured as the inhibition of CTLA-4 binding to CD80 or CD86. In one embodiment, the assay for measuring the inhibitory activity against CTLA-4 includes the following steps: binding purified CTLA-4 protein to a support such as a microtiter plate or magnetic beads; adding a test antibody and labeled soluble CD80 or CD86; washing away unbound components; and quantifying the bound labeled CD80 or CD86. Whether the test antibody cross-reacts with CD28 can be confirmed by using a similar assay in which CD28 is used instead of CTLA-4. Furthermore, in another embodiment, the functional assay for detecting T cell activation as described above can also be used to measure the inhibitory activity against CTLA-4. For example, when a test antibody with CTLA-4 inhibitory activity is added to a system (where T cell activation is measured by stimulating a T cell population with cells expressing CD80 or CD86), T cell activation is further enhanced.

D. Immunoconjugates

The present invention also provides immunoconjugates comprising the anti-CTLA-4 antibody described herein conjugated with one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzyme-active toxins, or fragments thereof), or radioisotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) wherein the antibody is conjugated to one or more drugs, including but not limited to maytansine (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0425 235B1); auristatin, such as the monomethyl auristatin drug portions DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298). Dolasatine; Galiciacin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)). Anthracyclines, such as doxorubicin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. U. SA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vinorelbine; taxanes such as docetaxel, paclitaxel, larotaxel, tessetaxel, and oxadecane; trichosporon toxins; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or a fragment thereof, said enzymatically active toxin or fragment thereof including, but not limited to, diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleuritesfordii protein, dianthin, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, localized aspergillin, phenolmycin, enoxacin, and trichosporine.

In another embodiment, the immunoconjugate comprises an antibody, as described herein, conjugated with a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to generate the radioconjugate. Examples include radioisotopes of ^211At , ^131I , ^125I , ^90Y , ^186Re , ^188Re , ^153Sm , ^212Bi , ^32P , ^212Pb , and Lu. When the radioconjugate is used for detection, it can contain radioactive atoms used for scintillation studies, such as Tc-99m or ^123I , or spin labels used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridinedithio)propionate (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imino esters (such as dimethyl diimide adipate HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94/11026. The adapter can be a “cleavable adapter” that promotes the release of toxic drugs from cells. For example, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, or adapters containing disulfide bonds can be used (Chari et al., Cancer Res. 52:127-131 (1992); US Patent No. 5,208,020).

The immunoconjugates or ADCs mentioned herein are explicitly considered, but not limited to, such conjugates prepared with crosslinking agents, including but not limited to commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, and sulfon-SMPB and SVSB (succinimide-(4-vinyl sulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

E. Methods and compositions for diagnosis and detection

In some embodiments, any anti-CTLA-4 antibody provided herein can be used to detect the presence of CTLA-4 in biological samples. As used herein, the term "detection" includes quantitative or qualitative detection. In some embodiments, biological samples comprise cells or tissues such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, breast milk, colostrum, mammary gland secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, ocular fluid, and mucus.

In one embodiment, an anti-CTLA-4 antibody is provided for use in diagnostic or detection methods. In other aspects, methods for detecting the presence of CTLA-4 in a biological sample are provided. In some embodiments, the method includes contacting a biological sample with the anti-CTLA-4 antibody described herein under conditions that allow the anti-CTLA-4 antibody to bind to CTLA-4, and detecting whether a complex is formed between the anti-CTLA-4 antibody and CTLA-4. This method may be an in vitro or in vivo method. In one embodiment, the anti-CTLA-4 antibody is used to select subjects suitable for treatment with the anti-CTLA-4 antibody, for example, where CTLA-4 is a biomarker used for patient selection.

The antibodies of this invention can be used, for example, to examine the state of the immune response and to diagnose immune system dysfunction.

In some embodiments, labeled anti-CTLA-4 antibodies are provided. Labeling includes, but is not limited to, labels or portions for direct detection (such as fluorescence, chromophores, electron-dense, chemiluminescence, and radioactive labels), as well as portions for indirect detection, such as enzymes or ligands, detected by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes ^32P , ^14C , ^125I , ^3H , and ^131I ; fluorophores such as rare earth chelates or luciferin and their derivatives; rhodamine and its derivatives; dansyl, umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucosylamylase; lysozyme; sugar oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase; those coupled to enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase, or microperoxidase); biotin/antibiotin protein; spin labeling; phage labeling; stable free radicals; etc.

F. Pharmaceutical preparations

The pharmaceutical formulation of the anti-CTLA-4 antibody described herein is prepared by mixing such an antibody of desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; chlorhexidine diammonium; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residuals). (Based on) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO 2006/044908, the latter of which comprises histidine-acetate buffer.

If necessary for the specific indications of treatment, the formulations described herein may also contain more than one active ingredient, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

The active ingredient can be encapsulated in microcapsules, for example, prepared by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed. 1980).

Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a molded article, such as a film or microcapsule.

Formulations intended for internal administration are typically sterile. Sterility can be easily achieved, for example, through filtration using a sterile filter membrane.

G. Treatment methods and compositions

Any anti-CTLA-4 antibodies provided in this article can be used for treatment.

In one aspect, an anti-CTLA-4 antibody is provided for use as a drug. In other aspects, an anti-CTLA-4 antibody is provided for treating tumors. In some embodiments, an anti-CTLA-4 antibody is provided for a treatment method. In some embodiments, the present invention provides an anti-CTLA-4 antibody in a method for treating an individual with a tumor, the method comprising administering an effective amount of the anti-CTLA-4 antibody to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one other therapeutic agent to the individual, such as those described below. In other embodiments, the present invention provides an anti-CTLA-4 antibody for destroying cells. In some embodiments, the present invention provides an anti-CTLA-4 antibody in a method for destroying cells in an individual, the method comprising administering an effective amount of the anti-CTLA-4 antibody to the individual to destroy the cells. In other embodiments, the present invention provides an anti-CTLA-4 antibody for immune activation. In some embodiments, the present invention provides an anti-CTLA-4 antibody in a method for activating immunity in an individual, the method comprising administering an effective amount of the anti-CTLA-4 antibody to the individual to activate immunity. The "individual" according to any of the above embodiments is preferably a human being.

In some embodiments, the tumor is a solid tumor. In solid tumors, tumor cells typically proliferate to form a colony, and tumor tissue is primarily formed by these cells. Furthermore, tumor tissue in a living organism is frequently infiltrated by immune cells such as lymphocytes, which also constitute part of the tumor tissue. In some embodiments, the tumor tissue is infiltrated by immune cells, particularly regulatory T (Treg) cells. In one embodiment, damage to the cells is triggered by ADCC activity, CDC activity, or ADCP activity. In one embodiment, cells expressing CTLA-4 on their cell surface are damaged. In other embodiments, the cells to be damaged are Treg cells. In some embodiments, Treg cells that have already infiltrated the tumor tissue are damaged. In one embodiment, immunity is activated by damage to Treg cells (immunosuppression of Treg cells is canceled). In other embodiments, immunity (particularly anti-tumor immunity) in the tumor tissue is activated. In some embodiments, immune activation is T cell activation.

In other aspects, the extent of action of the anti-CTLA-4 antibody of the present invention as a drug varies depending on the individual's tissue. In some embodiments, this extent of action varies according to the concentration of the adenosine-containing compound in the tissue. In other embodiments, the effect is increased in tissues with high concentrations of adenosine-containing compounds compared to tissues with low concentrations of adenosine-containing compounds. Tissues with high concentrations of adenosine-containing compounds include, for example, tumor tissues. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as normal tissues. In some embodiments, immunity is more strongly activated in tumor tissues than in non-tumor tissues. This difference in response does not need to be observed for all doses of the anti-CTLA-4 antibody, but only for a specific dose range. In another embodiment, immunity is activated in tumor tissues at a lower dose than in non-tumor tissues. Furthermore, in another embodiment, a therapeutic effect is observed at a dose lower than that observed for side effects. In some embodiments, the therapeutic effect is the expression of an anti-tumor effect (e.g., tumor regression and cell death induction or inhibition of tumor cell growth), and the side effect is the development of autoimmune diseases (including damage to normal tissues due to excessive immune responses).

On the other hand, the extent of the drug-like activity produced by the anti-CTLA-4 antibody of the present invention varies depending on whether it has CTLA-4 binding activity dependent on the concentration of the adenosine-containing compound (i.e., varies according to the concentration of the adenosine-containing compound). In some embodiments, the anti-CTLA-4 antibody of the present invention is an antibody whose CTLA-4 binding activity increases with increasing concentration of the adenosine-containing compound. In some embodiments, the control anti-CTLA-4 antibody is an antibody that does not have CTLA-4 binding activity dependent on the concentration of the adenosine-containing compound. In some embodiments, an antibody that does not have CTLA-4 binding activity dependent on the concentration of the adenosine-containing compound refers to an antibody in which the difference in CTLA-4 binding activity in the presence or absence of the compound is, for example, less than 2-fold, less than 1.8-fold, less than 1.5-fold, less than 1.3-fold, less than 1.2-fold, or less than 1.1-fold. It is desirable that the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody have substantially the same CTLA-4 binding activity in the presence of sufficient amounts of the adenosine-containing compound.

In some aspects, the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody differ in their effects as a drug produced by each antibody. In some embodiments, they differ in their effects as a drug in tissues having low concentrations of adenosine-containing compounds. Tissues having low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as normal tissue. The anti-CTLA-4 antibody may also be provided as a pharmaceutical formulation comprising the antibody. In some embodiments, in tissues having low concentrations of adenosine-containing compounds, the anti-CTLA-4 antibody of the present invention exhibits a low level of immune activation compared to the control anti-CTLA-4 antibody. In some embodiments, in tissues having low concentrations of adenosine-containing compounds, the dose of the anti-CTLA-4 antibody of the present invention required for immunization is high compared to the control anti-CTLA-4 antibody. In some embodiments, in tissues having low concentrations of adenosine-containing compounds, the anti-CTLA-4 antibody of the present invention has a low level of side effects compared to the control anti-CTLA-4 antibody. In some embodiments, the dose of the present invention's anti-CTLA-4 antibody, at which side effects are observed, is higher than that of a control anti-CTLA-4 antibody in tissues with low concentrations of adenosine-containing compounds. This difference in response does not need to be observed in all tissues (e.g., all tissues with low concentrations of adenosine-containing compounds), but only in some tissues. In some embodiments, the side effects are autoimmune diseases (including damage to normal tissues due to an excessive immune response).

In some aspects, the anti-CTLA-4 antibody and the control anti-CTLA-4 antibody of the present invention produce substantially the same effect as drugs. In some embodiments, they produce substantially the same effect as drugs in tissues having high concentrations of adenosine-containing compounds. Tissues having high concentrations of adenosine-containing compounds include, for example, tumor tissue. The anti-CTLA-4 antibody may also be provided as a pharmaceutical formulation containing the antibody. In some embodiments, in tissues having high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody exhibit substantially equal levels of immune activation. In some embodiments, in tissues having high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody are substantially equal in the dose required to activate immunity. In some embodiments, in tissues having high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody have substantially the same level of therapeutic effect. In some embodiments, in tissues containing high concentrations of adenosine compounds, the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody are substantially equal in dose at which therapeutic effects are observed. In some embodiments, the therapeutic effect is the expression of an anti-tumor effect (e.g., tumor regression and induction of cell death or inhibition of tumor cell growth).

In some implementations, the tumor is selected from breast cancer and liver cancer.

In other aspects, the present invention provides the use of anti-CTLA-4 antibodies in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical medicament is used to treat a tumor. In other embodiments, the method of using the pharmaceutical medicament to treat a tumor includes administering an effective amount of the pharmaceutical medicament to an individual suffering from a tumor. In one such embodiment, the method further includes administering an effective amount of at least one other therapeutic agent to the individual, for example, as described below. In other embodiments, the pharmaceutical medicament is used to destroy cells. In other embodiments, the method of using the pharmaceutical medicament to destroy cells in an individual includes administering an effective amount of the pharmaceutical medicament to the individual to destroy the cells. In other embodiments, the pharmaceutical medicament is used to activate immunity. In other embodiments, the method of using the pharmaceutical medicament to activate immunity in an individual includes administering an effective amount of the pharmaceutical medicament to the individual to activate immunity. An “individual” according to any of the above embodiments can be a human being.

In other aspects, the present invention provides a method for treating tumors. In one embodiment, the method includes administering an effective amount of an anti-CTLA-4 antibody to an individual suffering from such a tumor. In one such embodiment, the method further includes administering an effective amount of at least one other therapeutic agent to the individual, as described below. The "individual" according to any of the above embodiments can be a human being.

In other aspects, the present invention provides methods for destroying cells in an individual. In one embodiment, the method includes administering an effective amount of anti-CTLA-4 antibody to the individual to destroy cells. In other aspects, the present invention provides methods for activating immunity in an individual. In one embodiment, the method includes administering an effective amount of anti-CTLA-4 antibody to the individual to activate immunity. In one embodiment, "individual" is a human being.

In other aspects, the present invention provides pharmaceutical formulations comprising any anti-CTLA-4 antibody provided herein, for example, for any of the above-described treatment methods. In one embodiment, the pharmaceutical formulation comprises any anti-CTLA-4 antibody provided herein and a pharmaceutically acceptable carrier. In one embodiment, the present invention provides a pharmaceutical formulation for treating tumors. In one embodiment, the present invention provides a pharmaceutical formulation for destroying cells. In one embodiment, the present invention provides a pharmaceutical formulation for activating immunity. In another embodiment, the pharmaceutical formulation comprises any anti-CTLA-4 antibody provided herein and at least one additional therapeutic agent, for example, as described below.

In other aspects, the present invention provides a method for preparing a medicament or pharmaceutical formulation (e.g., for any of the above-described therapeutic methods), comprising mixing any anti-CTLA-4 antibody provided herein with a pharmaceutically acceptable carrier. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

The antibodies of the present invention can be used alone or in combination with other agents in treatment. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, or a chemotherapeutic agent. Immune checkpoint inhibitors may include, for example, anti-CTLA-4 inhibitors, anti-PD-1 inhibitors, anti-PD-L1 inhibitors, anti-PD-L2 inhibitors, anti-TIM-3 inhibitors, anti-LAG-3 inhibitors, anti-TIGIT inhibitors, anti-BTLA inhibitors, and anti-VISTA inhibitors. Anti-CTLA-4 inhibitors may include, for example, ipilimumab and tremelimumab. Anti-PD-1 inhibitors may include, for example, nivolumab and pembrolizumab. Anti-PD-L1 inhibitors may include, for example, atezolizumab, durvalumab, and avelumab. Anti-PD-L2 inhibitors may include, for example, anti-PD-L2 inhibitory antibodies. Anti-TIM-3 inhibitors may include, for example, anti-TIM-3 inhibitory antibodies. Anti-LAG-3 inhibitors may include, for example, anti-LAG-3 inhibitory antibodies. Anti-TIGIT inhibitors may include, for example, anti-TIGIT inhibitory antibodies. Anti-BTLA inhibitors may include, for example, anti-BTLA inhibitory antibodies. Anti-VISTA inhibitors may include, for example, anti-VISTA inhibitory antibodies. EGFR inhibitors may include, for example, cetuximab, panitumumab, nimotuzumab, necitumumab, and zalutumumab. HER2 inhibitors may include, for example, trastuzumab, trastuzumab emtansine, and pertuzumab.

Such combination therapies include combined administration (where two or more therapeutic agents are contained in the same or separate formulation) and single administration, in which case the antibody of the present invention may be administered before, simultaneously with, and/or after the administration of additional one or more therapeutic agents or agents. In one embodiment, the administration of the anti-CTLA-4 antibody and the administration of additional therapeutic agents occur within about one month, or about one, two, or three weeks, or about one, two, three, four, five, or six days. The antibody of the present invention may also be used in combination with radiotherapy.

The antibodies (and any other therapeutic agents) of this invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if necessary, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be made by any suitable route, such as by injection, like intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing regimens are considered herein, including but not limited to single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

The antibodies of this invention will be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the physician. The antibody does not need to be formulated, but optionally may be, with one or more agents currently used for the prevention or treatment of the condition in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These are generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and route determined empirically/clinically as appropriate.

For the prevention or treatment of disease, the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is for preventive or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for administration to the patient once or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) may be an initial candidate dose for administration to the patient, whether, for example, by single or multiple administrations alone, or by continuous infusion. Depending on the factors described above, a typical daily dose may vary in the range of about 1 μg/kg to 100 mg/kg or higher. For repeated administration over several days or longer, depending on the condition, treatment typically continues until the required suppression of disease symptoms is achieved. An exemplary dose of the antibody will be in the range of about 0.05 mg/kg to about 10 mg/kg. Therefore, patients can be administered one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof). Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20, or for example about 6 doses of antibody). An initial higher loading dose can be administered, followed by one or more lower doses. Progression of this treatment can be easily monitored using routine techniques and assays.

It should be understood that the immunoconjugates of the present invention can be used in place of anti-CTLA-4 antibodies or any of the above-described formulations or treatments other than anti-CTLA-4 antibodies.

H. Products

In another aspect of the invention, articles containing materials for treating, preventing, and/or diagnosing the aforementioned conditions are provided. The articles include a container and a label on the container or a packaging insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV (intravenous) solution bags, etc. The containers can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition effective for treating, preventing, and/or diagnosing the condition, and the container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or packaging insert indicates that the composition is used to treat the selected condition. Furthermore, the articles may comprise (a) a first container containing the composition, wherein the composition contains the antibody of the present invention; and (b) a second container containing the composition, wherein the composition contains other cytotoxic agents or other therapeutic agents. The articles in this embodiment of the invention may also include a packaging insert indicating that the composition is suitable for treating a specific condition. Alternatively, the product may include a second (or third) container containing pharmaceutically acceptable buffer solutions, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials required from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

It should be understood that any of the above-described products may include the immunoconjugates of the present invention in place of anti-CTLA-4 antibodies or in addition to anti-CTLA-4 antibodies.

<Polypeptide containing variant Fc region>

In one aspect, the present invention provides isolated polypeptides comprising a variant Fc region. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In some embodiments, the variant Fc region contains at least one amino acid residue change (e.g., substitution) compared to the corresponding sequence in the Fc region of the natural sequence or a reference variant sequence (collectively referred to herein as the "parental" Fc region). The Fc region of the natural sequence typically consists of a homodimer composed of two identical polypeptide chains. The amino acid change in the variant Fc region of the present invention can be introduced into either one of the two polypeptide chains of the parental Fc region, or into both of the two polypeptide chains.

In some aspects, the present invention provides variant Fc regions whose function has been modified compared to the parental Fc region. In some aspects, the variant Fc regions of the present invention exhibit enhanced binding activity to Fcγ receptors compared to the parental Fc region. In some embodiments, the variant Fc regions of the present invention exhibit enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to the parental Fc region. In some embodiments, the variant Fc regions of the present invention exhibit enhanced binding activity to FcγRIIa. In some embodiments, the variant Fc regions of the present invention exhibit enhanced binding activity to FcγRIIIa. In other embodiments, the variant Fc regions of the present invention exhibit enhanced binding activity to both FcγRIIa and FcγRIIIa.

In some embodiments, the variant Fc region of the present invention contains at least one amino acid change at at least one position selected from EU numbers 234, 235, 236, 298, 330, 332, and 334. Alternatively, amino acid changes such as those described in WO 2013/002362 and WO 2014/104165 can be similarly used in the present invention.

In some embodiments, the binding activity of the parental Fc region and the variant Fc region can be expressed using KD (dissociation constant) values. In one embodiment, the ratio of [KD value of parental Fc region to FcγRIIa] to [KD value of variant Fc region to FcγRIIa] is, for example, 1.5 or higher, 2 or higher, 3 or higher, 4 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, 15 or higher, 20 or higher, 25 or higher, 30 or higher, 40 or higher, or 50 or higher. In other embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the ratio of [binding activity of the parental Fc region to FcγRIIIa] to [binding activity of the variant Fc region to FcγRIIIa] is, for example, 2 or higher, 3 or higher, 5 or higher, 10 or higher, 20 or higher, 30 or higher, 50 or higher, 100 or higher, 200 or higher, 300 or higher, 500 or higher, ¹ x 10³ or higher, ² x 10³ or higher, ³ x 10³ or higher, or 5 x ^10³ or higher. In other embodiments, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

In one embodiment, the KD value of the variant Fc region relative to FcγRIIa is, for example, 1.0 x ^10⁻⁶ M or lower, 5.0 ^{x 10⁻⁷} M or lower, 3.0 x ^10⁻⁷ M or lower, 2.0 x ^10⁻⁷ M or lower, 1.0 ^{x 10⁻⁷ M} or lower, 5.0 ^{x 10⁻⁸} M or ^lower , 3.0 x ^10⁻⁸ M or lower, 2.0 x ^10⁻⁸ M or lower, 1.0 x ^10⁻⁸ M or lower, 5.0 x ^10⁻⁹ M or lower, 3.0 x ^10⁻⁹ M or lower, 2.0 x ^10⁻⁹ M or lower, or 1.0 x ^10⁻⁹ M or lower. In other embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. In one implementation, the KD value of the variant Fc region for KDFcγRIIIa is, for example, 1.0 x ^10⁻⁶ M or lower, 5.0 x ^10⁻⁷ M or lower, 3.0 x ^10⁻⁷ M or lower, 2.0 x ^10⁻⁷ M or lower, 1.0 x ^10⁻⁷ M or lower, 5.0 x ^10⁻⁸ M or lower, 3.0 x ^10⁻⁸ M or lower, 2.0 x ^10⁻⁸ M or lower, 1.0 x ^10⁻⁸ M or lower, 5.0 x ^10⁻⁹ M or lower, 3.0 x ^10⁻⁹ M or lower, 2.0 x ^10⁻⁹ M or lower, 1.0 x ^10⁻⁹ M or lower, 5.0 x ^10⁻¹⁰ M or lower, 3.0 x ^10⁻¹⁰ M or lower, 2.0 x ^10⁻¹⁰ M or lower, or 1.0 x ^10⁻¹⁰ M or lower. In other embodiments, FcγRIIIa can be FcγRIIIa F or FcγRIIIa V, or both.

In another implementation, the binding activity of the parent and variant Fc regions can be represented by the kd (dissociation rate constant) value instead of the KD value.

In another embodiment, the binding activity of the parental and variant Fc regions can be expressed as the amount of binding of each unit amount of Fc region to the Fcγ receptor. For example, in surface plasmon resonance assays, the amount of binding of the Fc region immobilized on the sensor chip and the amount of binding of the Fcγ receptor thereto are each measured as resonance units (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 each unit amount of Fc region to the Fcγ receptor. Specific methods for measuring and calculating this amount of binding are described in the following embodiments. In some embodiments, the ratio of [the amount of binding of the variant Fc region to FcγRIIa]/[the amount of binding of the parental Fc region to FcγRIIa] is, for example, 1.5 or higher, 2 or higher, 3 or higher, 4 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, 15 or higher, 20 or higher, 25 or higher, 30 or higher, or 40 or higher, or 50 or higher. In some implementations, the ratio of [the amount of binding of the variant Fc region to FcγRIIIa] to [the amount of binding of the parent Fc region to FcγRIIIa] is, for example, 2 or higher, 3 or higher, 5 or higher, 10 or higher, 20 or higher, 30 or higher, 50 or higher, 100 or higher, 200 or higher, 300 or higher, 500 or higher, ¹ x 10³ or higher, ² x 10³ or higher, ³ x 10³ or higher, or 5 x ^10³ or higher.

In some embodiments, the KD value, kd value, binding amount value, etc. described herein are determined or calculated by performing surface plasmon resonance measurements at 25°C or 37°C (see, for example, Example 6 herein).

In some aspects, the variant Fc region of the present invention exhibits enhanced selectivity between activating and inhibitory Fcγ receptors compared to the parental Fc region. In other words, the variant Fc region of the present invention shows significantly enhanced binding activity to activating Fcγ receptors compared to inhibitory Fcγ receptors. In some embodiments, the activating Fcγ receptor is at least one Fcγ receptor selected from FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. In some embodiments, the variant Fc region of the present invention exhibits enhanced selectivity between FcγRIIa and FcγRIIb. In some embodiments, the variant Fc region of the present invention exhibits enhanced selectivity between FcγRIIIa and FcγRIIb. In other embodiments, the variant Fc region of the present invention has enhanced selectivity between FcγRIIa and FcγRIIb and between FcγRIIIa and FcγRIIb.

In some embodiments, the variant Fc region of the present invention contains at least one amino acid change at at least one position selected from positions 236, 239, 268, 270 and 326 according to EU numbers. Alternatively, the amino acid changes described in WO 2013/002362 and WO 2014/104165 can be similarly used in the present invention.

In some embodiments, the binding activity of the parent and variant Fc regions can be represented by KD (dissociation constant) values. Embodiments of the binding activity for FcγRIIa and FcγRIIIa are as described above. In one embodiment, the ratio of [KD value of parental Fc region to FcγRIIb]/[KD value of 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 kd (dissociation rate constant) values instead of KD values.

In another embodiment, the binding activity of the parental and variant Fc regions can be represented by the amount of binding of each unit amount of Fc region to the Fcγ receptor 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 parental 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.

In some respects, the variant Fc region of the present invention exhibits improved stability compared to the parental Fc region. In some embodiments, stability is thermodynamic stability. For example, the thermodynamic stability of the peptide can be determined by using the Tm value as an indicator. The Tm value can be determined using techniques known to those skilled in the art, such as circular dichroism (CD), differential scanning calorimetry (DSC), and differential scanning fluorescence (DSF). In one embodiment, compared to the parental Fc region, the Tm value of the CH2 region in the variant Fc region of the present invention is increased by 0.1 degrees or more, 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, or 10 degrees or more.

In some embodiments, the variant Fc region of the present invention contains at least one amino acid change at at least one position selected from positions 250 and 307 according to EU numbers. Alternatively, the amino acid changes described in WO 2013/118858 can be similarly used in the present invention.

In some aspects, the variant Fc region of this invention consists of two polypeptide chains with different sequences. In other aspects, heterodimerization between the first and second polypeptides has been promoted in the variant Fc region of this invention. When heterodimeric proteins are generated using recombinant methods, it is preferable that the different polypeptide chains bind preferentially to form heterodimers, rather than that the same polypeptide chains bind to form homodimers. For example, homodimers and heterodimers can be separated from the generated variant Fc region by techniques such as chromatography, and by determining the proportions of each component, it can be determined whether heterodimerization in the variant Fc region has been promoted.

In some embodiments, the variant Fc region of the present invention contains at least one amino acid change at at least one position selected from positions 349, 356, 366, 368, 407, and 439 according to EU numbers. Alternatively, the amino acid changes described in WO 2006/106905 and WO1996/027011 can be similarly used in the present invention.

In some aspects, the variant Fc region of the present invention exhibits enhanced binding activity to FcRn at acidic pH. In some embodiments, acidic pH refers to pH 4.0 to 6.5. In other embodiments, acidic pH is at least one pH selected from 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 some embodiments, acidic pH is pH 5.8.

In some embodiments, the variant Fc region of the present invention contains at least one amino acid change at at least one position selected from positions 428, 434, 436, 438, and 440 according to EU numbers. Alternatively, the amino acid changes described in WO 2016/125495 can be similarly used in the present invention.

In one aspect, the variant Fc region of the present invention contains at least one amino acid change at at least one position selected from 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 numbers.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 334 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, and 334 according to EU numbers in the second polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 332 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 332 according to EU number in the first polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 356 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 356 according to EU number in the first polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 366 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 366 according to EU number in the first polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 349 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 349 according to EU number in the first polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 332 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 332 according to EU number in the second polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 330 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 330 according to EU number in the second polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 439 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 439 according to EU number in the second polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes amino acid changes at positions 366, 368, and 407 according to EU numbers. In another aspect, the variant Fc region includes amino acid changes at positions 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In another aspect, the variant Fc region of the present invention further includes an amino acid change at position 356 according to EU number. In another aspect, the variant Fc region includes an amino acid change at position 356 according to EU number in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 349, 356, 366, 368, and 407 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 334, 356, 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 356, and 439 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 356 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 334, and 439 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, and 407 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 356, and 439 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 356 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, and 439 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349, and 366 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 356, and 439 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 356 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 332, 334, and 439 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 366, 368, and 407 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 366 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 366, 368, and 407 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 366 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 332, 334, 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, and 407 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In some aspects, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407 according to EU numbers. In other aspects, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349, and 366 according to EU numbers in the first polypeptide of the parental Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407 according to EU numbers in the second polypeptide of the parental Fc region.

In other embodiments, the variant Fc region of the present invention comprises at least one amino acid change selected from the group consisting of: (i) Tyr or 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, Asp at position 326, Glu at position 332, Cys at position 349, and Pro at position 356 in the first polypeptide according to EU numbering in the parental Fc region. Lys at position 366 and Trp at position 366; 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, Cys at position 356, Ser at position 366, Ala at position 368, Val at position 407, and Glu at position 439 in the second polypeptide of the parental Fc region according to EU number.

In other respects, the variant Fc region of the present invention further comprises any one of the following amino acid changes (a) to (d):

(a) According to Ala at position 434 of the EU number;

(b) According to EU number Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440;

(c) Based on the EU number at position 428 (Leu), position 434 (Ala), position 436 (Thr), position 438 (Arg), and position 440 (Glu); and

(d) Based on the EU number at position 428 (Leu), position 434 (Ala), position 438 (Arg), and position 440 (Glu).

In other embodiments, the present invention provides a polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO: 43 to 46, 65, 66, 81, 207, 239, 253 to 271, 276, 277 and 278.

The “Fcγ receptor” (referred to as Fcγ receptor, FcγR or FcgR in this article) refers to the receptor that can bind to the Fc region of IgG1, IgG2, IgG3 and IgG4 monoclonal antibodies, and in fact refers to any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isotypes FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including isotypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered FcγR, FcγR isotypes, or allotypes of FcγR, but not limited to these. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. Additionally, a splice variant named FcγRIIb3 has been reported (J Exp Med, 1989, 170:1369-1385). Besides these splice variants, human FcγRIIb includes all splice variants registered in the NCBI, namely NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. In addition, 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)), and each genetic polymorphism to be reported in the future.

In FcγRIIa, there are two allotypes: one in which the amino acid at position 131 of FcγRIIa is histidine (H type), and the other in which the amino acid at position 131 is replaced by arginine (R type) (Warrmerdam, J.Exp.Med.172:19-25(1990)).

FcγRs include, but are not limited to, those derived from humans, mice, rats, rabbits, and monkeys, and can be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isotype.

The amino acid sequence of human FcγRI is shown in SEQ ID NO:131 (NP_000557.1); the amino acid sequence of human FcγRIIa is shown in SEQ ID NO:132 (AAH20823.1), SEQ ID NO:142, SEQ ID NO:143 or SEQ ID NO:150; the amino acid sequence of human FcγRIIb is shown in SEQ ID NO:151 (AAI46679.1), SEQ ID NO:169 or SEQ ID NO:172; the amino acid sequence of human FcγRIIIa is shown in SEQ ID NO:174 (AAH33678.1), SEQ ID NO:175, SEQ ID NO:176 or SEQ ID NO:177; and the amino acid sequence of human FcγRIIIb is shown in SEQ ID NO:178 (AAI28563.1).

Unlike the Fcγ receptor, which belongs to the immunoglobulin superfamily, human FcRn is structurally similar to polypeptides of the major histocompatibility complex (MHC) class I, exhibiting 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-chain 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 SEQ ID NO:179 (NP_004098.1), and the amino acid sequence of β2 microglobulin is shown in SEQ ID NO:180.

As used herein, “parental Fc region” refers to the Fc region prior to the introduction of the amino acid alterations described herein. In some embodiments, the parental Fc region is the Fc region of a natural 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 may be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Natural antibodies may include naturally occurring mutations. Multiple allotypes of IgG due to genetic polymorphism are described in “Sequences of protein of immunological interest,” NIH Publication No. 91-3242, and any of them may be used in this invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU number) may be DEL or EEM. In some embodiments, the parental Fc region is an Fc region derived from the heavy chain constant region of human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), or human IgG4 (SEQ ID NO: 252). In another embodiment, the parental Fc region is an Fc region derived from the heavy chain constant region of SEQ ID NO: 82 or SEQ ID NO: 158. In other embodiments, the parental Fc region may be an Fc region generated by adding amino acid changes other than those described herein to the Fc region of the native sequence (the Fc region of the reference variant sequence). The Fc region of the native sequence is typically configured as a homodimer composed of two identical polypeptide chains.

Furthermore, amino acid alterations made for other purposes can be combined in the variant Fc region described herein. For example, amino acid substitutions that enhance FcRn binding activity can be added (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 to improve antibody heterogeneity or stability (WO 2009/041613). Alternatively, peptides with antigen-promoting clearance properties described in WO 2011/122011, WO 2012/132067, WO2013/046704, or WO 2013/180201, peptides with target tissue-specific binding properties described in WO 2013/180200, and peptides with repeatable binding properties to multiple antigen molecules described in WO 2009/125825, WO 2012/073992, or WO2013/047752 may be combined with the variant Fc region described herein. Alternatively, for the purpose of conferring binding ability to other antigens, the amino acid changes disclosed in EP1752471 and EP1772465 may be combined in the CH3 region of the variant Fc region described herein. Alternatively, for the purpose of increasing plasma retention, the amino acid change that reduces pI in the constant region (WO 2012/016227) may be combined in the variant Fc region described herein. Alternatively, for the purpose of promoting uptake into cells, an amino acid alteration that increases the pI in the constant region (WO 2014/145159) may be incorporated into the variant Fc region described herein. Alternatively, for the purpose of promoting the elimination of target molecules from plasma, an amino acid alteration that increases the pI in the constant region (WO 2016/125495) may be incorporated into the variant Fc region described herein. In one embodiment, such alteration may include, for example, substitution at at least one position selected from positions 311, 343, 384, 399, 400, and 413 according to EU numbers. In other embodiments, such substitution may be a replacement of an amino acid of Lys or Arg at each position.

In addition, the heterodimerization antibody generation technology described in WO 2011/028952, which uses the association of antibodies CH1 and CL and the association of VH and VL, can also be used.

Using the methods described in WO 2008/119353 and WO 2011/131746, a technique can also be used to generate heterodimerized antibodies by pre-generating two types of homodimerized antibodies, incubating the antibodies under reducing conditions to dissociate them, and allowing them to recombine.

In addition, the technique of generating heterodimerized antibodies by adding alterations to the CH2 and CH3 domains can also be used, as described in WO 2012/058768.

When two polypeptides containing variant Fc regions with different amino acid sequences are expressed simultaneously, polypeptides containing homologous variant Fc regions are often produced as impurities in order to generate polypeptides containing heterologous variant Fc regions. In this case, polypeptides containing heterologous variant Fc regions can be efficiently obtained by separating and purifying them from polypeptides containing homologous variant Fc regions using known techniques. Methods for the efficient separation and purification of heterodimerized antibodies from homodimerized antibodies have been reported using ion-exchange chromatography, which introduces amino acid alterations into the variable regions of the heavy chains of the two types of antibodies to create isoelectric point differences between the homodimerized and heterodimerized antibodies (WO 2007/114325). Another method has been reported to purify heterodimerized antibodies using protein A chromatography by constructing heterodimerized antibodies containing two types of heavy chains: mouse IgG2a (bound by protein A) and rat IgG2b (not bound by protein A) (WO 1998/050431 and WO1995/033844).

In addition, different protein A binding affinities can be generated by replacing amino acid residues at positions 435 and 436 (EU number) of the protein A binding site on the antibody heavy chain with amino acids such as Tyr or His, and protein A chromatography can be used to effectively purify heterodimerized antibodies.

In this invention, amino acid alteration refers to any one or a combination of substitution, deletion, addition, insertion, and modification. In this invention, amino acid alteration can be restated as amino acid mutation.

The number of amino acid changes introduced into the Fc region is not limited. In some 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, or 20 or less.

In one aspect, the present invention provides a method for generating a polypeptide comprising a variant Fc region. In other aspects, the present invention provides a method for generating a polypeptide comprising a variant Fc region whose function has been modified. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In some embodiments, those methods include introducing at least one amino acid alteration into the parental Fc region. In some embodiments, those methods include: (i) providing a polypeptide comprising the parental Fc region; and (ii) introducing at least one amino acid alteration into the parental Fc region. In some embodiments, those methods may further include (iii) determining the function of the polypeptide comprising the variant Fc region. A native Fc region typically consists of two identical polypeptide chains. An amino acid alteration of the parental Fc region may be introduced into one of the two polypeptide chains of the parental Fc region, or both of the two polypeptide chains.

In another embodiment, a method for generating a polypeptide containing a variant Fc region includes: (i) providing one or more nucleic acids encoding a polypeptide containing a parental Fc region; (ii) introducing at least one mutation into a region encoding the parental Fc region in the nucleic acid; (iii) introducing the nucleic acid generated in (ii) into a host cell; and (iv) culturing the cell described in (iii) to express the polypeptide containing the variant Fc region. In some embodiments, the above method may further include (v) collecting the polypeptide containing the variant Fc region from a host cell culture described in (iv).

In some implementations, the nucleic acid generated in (ii) may be contained in one or more vectors (e.g., expression vectors).

In some embodiments, the amino acid changes used in the production method of the present invention are selected from: any single change, a combination of single changes, or a combination of changes listed in Tables 26 to 30 that may be included in the Fc region of the above variants.

The Fc region can be obtained by partially digesting IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies with a protease such as pepsin and then eluting the fractions adsorbed on the protein A column. The protease is specifically limited to the ability to digest full-length antibodies so that Fab and F(ab')2 are produced in a restrictive manner by appropriately setting the enzyme reaction conditions, such as pH; examples include pepsin and papain.

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

The assay methods described herein, or many other assay 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.

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

In one embodiment, the binding activity of a peptide containing a variant Fc region with a member of the FcR family can be determined using surface plasmon resonance assays. For example, various FcRs can be used as analytes to interact with peptides containing variant Fc regions, which are immobilized or captured on a sensor chip using known methods and reagents (e.g., protein A, protein L, protein A/G, protein G, anti-λ chain antibody, anti-κ chain antibody, antigenic peptide, and antigenic protein). Alternatively, FcRs can be immobilized or captured on a sensor chip, and the peptide containing the variant Fc region can be used as an analyte. As a result of this interaction, binding sensing maps are obtained, and by analyzing them, the dissociation constant (KD) value of this binding can be calculated. Furthermore, the difference in resonance unit (RU) values in the sensing maps before and after interaction with an FcR (i.e., the amount of FcR bound) can be used as an indicator of the binding activity of the peptide containing the variant Fc region with the FcR. Furthermore, a correction value obtained by dividing the aforementioned binding amount of FcR (i.e., the binding amount of the polypeptide containing the variant Fc region) by the difference in RU values in the sensing map before and after the polypeptide containing the variant Fc region is immobilized or captured on the sensor chip (i.e., the correction value is the binding amount of FcR per unit amount of polypeptide containing the variant Fc region) can be used as an indicator of binding activity.

In other respects, the present invention provides pharmaceutical formulations comprising peptides, said peptides containing the variant Fc region provided herein. In one embodiment, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.

[Example]

Examples of the methods and compositions of the present invention are shown below. It should be understood that, in view of the above general description, many other embodiments can be implemented.

[Example 0] Concept of a switch antibody that exerts antibody-dependent cytotoxic activity only on cell surface markers of regulatory T cells in the cancer microenvironment.

Ipilimumab is thought to exert its antitumor effect by inhibiting the activation of effector T cells through CTLA4 expressed on the surface of effector T cells; however, antibody-dependent cytotoxic activity (ADCC activity) against CTLA4-expressing T cells has recently been reported to be important, and the removal of regulatory T cells and ADCC activity in tumors have been found to be important mechanisms of the antitumor effect of anti-CTLA4 antibodies.

Furthermore, it is known that the ADCC activity of IgG1 antibodies is the result of cytotoxic activity induced by the binding of the antibody constant region to FcγR of NK cells and macrophages, and antibodies with constant regions modified to enhance this binding induce stronger cytotoxic activity and exert antitumor effects.

On the other hand, it has been reported that the removal of systemic regulatory T cells causes an autoimmune-like systemic response, and it is believed that the regulation of the balance between cytotoxic activity used to exert antitumor effects and systemic response is important.

More specifically, antibodies that can strongly bind to regulatory T cells or depleted T cells in the cancer microenvironment are expected to exert more effective cytotoxic activity and suppress systemic responses, exerting potent antitumor effects by removing regulatory T cells or by depleting T cells through cytotoxic activity, and limiting responses only to the cancer microenvironment. Antibodies with this mechanism of action have not been reported. Therefore, we actually generated and validated such an antibody (CTLA4 switch antibody) that acts only on CTLA4 locally on the tumor and contains a constant region that enhances binding to FcγR(s) expressed on NK cells and macrophages.

[Example 1] Using phage display technology, antibodies that bind to antigens in the presence of ATP or its metabolites were obtained from a naive library and a rationally designed antibody library.

(1-1) Prepare antigens to obtain antibodies that bind to the antigens in the presence of small molecules.

Biotinylated mouse CTLA4 extracellular domain (mCTLA4), human CTLA4 extracellular domain (hCTLA4), and Abatacept were prepared as antigens. Specifically, regarding the hCTLA4 extracellular domain, the hCTLA4-His-BAP gene (SEQ ID NO: 1) was synthesized, wherein the His-tag and BAP-tag were fused to the C-terminus of the hCTLA4 extracellular domain, and the gene was inserted into an animal expression plasmid. The antigen protein was expressed and purified using the following method. The prepared plasmid was introduced into the FreeStyle 293-F cell line (Invitrogen) derived from human embryonic kidney cells via lipid transfection. After suspending the cell line in FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33 x ^10⁶ cells/mL, the cells were seeded in flasks. Three hours after plasmid introduction, biotin was added to a final concentration of 100 μM, and the mixture was incubated for 4 days in a _CO2 incubator (37°C, 8% _CO2 , 125 rpm). The antigen was purified from the culture supernatant using methods known to those skilled in the art. The absorbance of the purified antigen solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antigen was calculated from the measured values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423). On the other hand, mCTLA4-His (Sino Biologics Inc. 50503-M08H, Accession No. NP_033973.2) (where the His-tag is fused to the extracellular region of mCTLA4) and Abatacept (Alfresa Corporation) (where the human IgG1 constant region is fused to hCTLA4) were biotinylated by amine coupling (PIERC ECAT. No. 21329).

(1-2) In the presence of small molecules, mouse CTLA4 antibodies were obtained from the initial human antibody library by bead panning. bound antibodies

Using methods known to those skilled in the art, a human antibody phage display library composed of multiple phages is constructed using templates such as polyA RNA prepared from human PBMCs or commercially available human polyA RNA, wherein the phages exhibit Fab domains of human antibody sequences that are distinct from each other.

From a constructed initial human antibody phage display library, antibodies exhibiting altered binding activity to the extracellular region of mouse CTLA4 (mCTLA4) in the presence and absence of the small molecule were screened. More specifically, phages presenting antibodies that showed binding activity to mCTLA4 captured on beads in the presence of the small molecule were collected. In the absence of the small molecule, the phages were recovered from the phage eluent eluted from the beads. In this acquisition method, biotin-labeled mCTLA4 (mCTLA4-His-Biotin) was used as the antigen.

Phages generated from *E. coli* carrying constructive phage particles for phage display were purified using standard methods. A phage library solution for TBS dialysis was then obtained. Antigens were panned using magnetic beads immobilized on magnetic beads. NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.

To effectively obtain small molecule-dependent small molecule switch antibodies that can act as switches in cancerous tissue, panning was performed according to the method shown in existing patent document WO 2013/180200. This panning method enriches antibodies that bind to antigens in the presence of 5'-adenosine triphosphate (ATP) and ATP metabolites, but do not bind to antigens in the absence of ATP.

(1-3) Assess binding activity by phage ELISA in the presence and absence of small molecules.

According to standard methods (Methods Mol. Biol. (2002) 178, 133-145), culture supernatants containing bacteriophages were recovered from single E. coli colonies obtained in (1-2). The culture supernatants recovered using a NucleoFast 96 (MACHERY-NAGEL) were ultrafiltered. 100 μL of each culture supernatant was added to each well of the NucleoFast 96 and centrifuged at 4,500 g for 45 min to remove flowthrough. 100 μL of _H₂O was added, and the mixture was washed again by centrifugation at 4,500 g for 30 min. Then 100 μL of TBS was added, and the mixture was allowed to stand at room temperature for 5 min before recovering the bacteriophage solution contained in the supernatant.

ELISA was performed on purified phages supplemented with TBS using the following procedure: A StreptaWell 96 microtiter plate (Roche) was coated overnight with 100 μL of TBS containing mCTLA4-His-Biotin. After washing each well with TBST to remove unbound mCTLA4-His-Biotin, the wells were blocked with 250 μL of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, the prepared purified phage was added to each well, and the plate was incubated at room temperature for 1 hour to allow the antibody-presenting phage to bind to the mCTLA4-His-Biotin present in each well, with or without ATP. After washing each well with TBST or ATP/TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS or ATP/TBS was added, and the plate was incubated for 1 hour. After washing with TBST or ATP/TBST, the colorimetric reaction of the solutions in each well containing a single TMB solution (ZYMED) was terminated by adding sulfuric acid, and then the colorimetric reaction was measured by absorbance at 450 nm. The results confirmed the presence of multiple antibodies that bind to mCTLA4 only in the presence of ATP. The results of the phage ELISA are shown in Table 2. Here, clones with absorbance greater than 0.2 in the presence of ATP were identified as positive, and clones with an absorbance ratio greater than 2 in the presence/absence of ATP were identified as clones with ATP-dependent antigen-binding ability (switch clones). In this example, SM can be used as an abbreviation for small molecules/low-weight molecules (such as ATP).

[Table 2]

total Number of ELISA clones 192 Number of positive clones (absorbance > 0.2) 103 Number of switch clones (SM+/- ratio > 2) 28

(1-4) Using ATP or its metabolites from a rationally designed library to obtain antigens in the presence of small molecules. bound antibodies

Antibodies exhibiting antigen-binding activity in the presence of ATP or ATP metabolites (e.g., ADP, AMP, adenosine (ADO), etc.) were obtained from a rationally designed antibody phage display library constructed from existing patent document WO 2015/083764. For antibody acquisition, phage-presented antibodies exhibiting the ability to bind to antigens captured on beads in the presence of ATP or ATP metabolites were collected, and then the phages were recovered from the eluent eluted from the beads in the absence of ATP or ATP metabolites.

Phages were generated from *E. coli* carrying phage particles for phage display using standard methods. A phage library solution was obtained by diluting the phage population with TBS, wherein the phage population was precipitated by adding 2.5 M NaCl/10% PEG to the culture solution of the *E. coli* that produced the phages. Next, BSA was added to the phage library solution to bring its final concentration to 4%. Panning was performed using antigens immobilized on magnetic beads. NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads. Biotinylated Abatacept (Abatacept-Biotin) was used as the antigen.

In order to effectively obtain small molecule-dependent small molecule switch antibodies that can act as switches in cancerous tissues, panning is performed according to the method shown in existing patent document WO 2015/083674 to enrich antibodies that bind to antigens in the presence of 5'-adenosine triphosphate (ATP) or ATP metabolites, but do not bind to antigens in the absence of ATP or ATP metabolites.

(1-5) Assess binding activity in the presence and absence of ATP or its metabolites using phage ELISA.

According to standard methods (Methods Mol. Biol. (2002) 178, 133-145), culture supernatant containing bacteriophages was recovered from single colonies of *E. coli* obtained by the above method. The recovered culture supernatant was ultrafiltered using NucleoFast 96 (MACHEREY-NAGEL). Flowthrough was removed by centrifugation (4,500 g, 45 min) with 100 μL of culture supernatant added to each well. NucleoFast 96 with 100 μL of _H₂O added to each well was washed again by centrifugation (4,500 g, 30 min). Finally, 100 μL of TBS was added, and the bacteriophage solution contained in the supernatant of each well of NucleoFast 96, which had been allowed to stand at room temperature for 5 min, was recovered.

TBS or TBS containing ATP or its metabolites (SM/TBS) was added to purified phage, and the phage was subjected to ELISA using the following procedure. A StreptaWell 96 microtiter plate (Roche) was coated overnight with 100 μL of TBS containing the biotinylated antigen (Abatacept-Biotin) prepared in Examples 1-1. After removing free Abatacept-Biotin by washing each well with TBST, the wells were blocked with 250 μL of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, the prepared purified phage was added to each well, and the plate was incubated at 37°C for 1 hour, allowing the phage to present antibodies that bind to the Abatacept-Biotin present in each well, whether in the absence or presence of ATP or its metabolites. After washing each well of the plate with TBST or TBST containing ATP or its metabolites (SM/TBST), HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS or SM/TBST was added, and the plate was incubated for 1 hour. After washing the wells with TBST or SM/TBST, the colorimetric reaction of the solutions in the wells containing TMB single solution (ZYMED) was terminated by adding sulfuric acid, and then the colorimetric reaction was measured by absorbance at 450 nm. The results confirmed that the binding activity of various antibodies with Abatacept was altered in the presence and absence of ATP or its metabolites. The results of the phage ELISA are shown in Table 3. Here, clones with an absorbance S/N ratio greater than 2 in the presence of ATP or its metabolites were identified as positive, and clones with an absorbance ratio greater than 2 in the presence/absence of ATP or its metabolites were identified as clones with antigen-binding ability dependent on ATP or its metabolites (switch clones).

[Table 3]

(1-6) Switch antibodies whose antigen-binding activity varies depending on the presence or absence of ATP and its metabolites. Sequence Analysis

The nucleotide sequences of genes amplified from clones using specific primers lacPF (SEQ ID NO:2) and G1seqR (SEQ ID NO:3), which were determined to have antigen-binding activity by phage ELISA in the presence of ATP and its metabolites, were analyzed. As a result of the analysis, clones ABADh11-4_020, ABADh11-4_086, ABADh12-4_014, ABADh12-5_001, ABADh12-5_046, and ABADh5_041 were determined to have binding activity against biotin-labeled abatacept in the presence of ATP and its metabolites. The clone names were reassigned as ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, respectively (Table 4).

[Table 4]

clone name Antibody name VH SEQ ID NO: VL SEQ ID NO: ABADh11-4_020 ABAM001 4 5 ABADh11-4_086 ABAM002 6 7 ABADh12-4_014 ABAM003 8 9 ABADh12-5_001 ABAM004 10 11 ABADh12-5_046 ABAM005 12 13 ABADh5-5_041 ABAM006 14 15

(1-7) Switch antibodies whose antigen-binding activity varies depending on the presence or absence of ATP and its metabolites. Expression and purification

Genes encoding the variable regions of ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, obtained from a rationally designed human phage library, were inserted into the animal expression plasmid human IgG1/Lambda. Antibodies were expressed using the following method: The prepared plasmid was introduced into a FreeStyle 293-F line (Invitrogen) derived from human embryonic kidney cells via lipid transfection. This line was suspended in FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33 x ^10⁶ cells/mL and seeded at 3 mL/well in each well of a 6-well plate. Antibodies were purified from the culture supernatant after 4 days of culture in a _CO₂ incubator (37°C, 8% _CO₂ , 90 rpm) using rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) according to methods known to those skilled in the art. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The concentration of purified antibody was calculated from the obtained measurements using the extinction coefficient obtained by the PACE method (Protein Science (1995) 4, 2411-2423).

(1-8) The antibody response to hCTLA4 obtained in the presence and absence of AMP was assessed by IgG ELISA. Synergistic activity

The six antibodies obtained, ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, were used for IgG ELISA. The buffers shown in Table 5 were prepared appropriately. Biotin-labeled human CTLA4 (hCTLA4-His-Biotin) was used as the antigen.

[Table 5]

First, coat a StreptaWell 96 microtiter plate (Roche) with 100 μL of TBS containing hCTLA4-His-Biotin at room temperature for 1 hour or longer. After washing each well with wash buffer to remove unbound hCTLA4-His-Biotin, block the wells with 250 μL of blocking buffer for 1 hour or longer. Add 100 μL of various purified IgGs prepared to 2.5 μg/mL in sample buffer containing 1 mM AMP to each well after removing the blocking buffer, and incubate the plate at room temperature for 1 hour to allow each IgG to bind to the hCTLA4-His-Biotin present in each well. After washing with wash buffer containing 1 mM AMP, add HRP-conjugated anti-human IgG antibody (BIOSOURCE), diluted with sample buffer, to each well and incubate for 1 hour. After washing with a washing buffer containing each small molecule, the colorimetric reaction of the solutions in each well containing a single TMB solution (ZYMED) was terminated by adding sulfuric acid. The colorimetric reaction was then measured by absorbance at 450 nm. The buffers containing the compositions shown in Table 5 were used as buffers.

The results are shown in Table 6. Wells with overflow values are assumed to be 5.00. The results indicate that in all clones of ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, the absorbance in the absence of AMP was significantly lower than that in the presence of AMP. This confirms that all clones of ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006 possess the property of altering their binding to the antigen based on the presence or absence of the small molecule.

[Table 6]

(1-9) Evaluation of the effects of ATP and its metabolites on binding to human CTLA4 by surface plasmon resonance.

ABAM004 was further evaluated as a CTLA4 switch antibody.

The interaction between ABAM004 and hCTLA4-His-BAP antigen-antibody reaction was analyzed using a Biacore T200 (GE Healthcare). ABAM004 was allowed to be captured by a sensor chip CM5 (GE Healthcare) with an appropriate amount of protein A/G (Pierce) immobilized thereon via amine coupling, and interaction with the antigen hCTLA4-His-BAP prepared in Example 1-1 was permitted. TBS was used as the running buffer, and 10 mM Glycine-HCl (pH 1.5) was used as the regeneration solution.

After capturing 1 μg/mL ABAM004 suspended in TBS, a solution containing 500 nM hCTLA4-His-BAP and 10 concentrations of ATP, ADP, or AMP diluted at a common ratio from 4000 μM, and 2 mM MgCl2 was injected into _each flow cell at a flow rate of 10 μL/min for 3 minutes. This 3-minute period served as the binding phase for hCTLA4-His-BAP. After the binding phase, the injection was switched to run buffer for 2 minutes, which served as the dissociation phase for hCTLA4-His-BAP. After the dissociation phase, the regeneration solution was injected at a flow rate of 30 μL/min for 30 seconds. This cycle was used as the ABAM004 binding activity assay cycle. The amount of hCTLA-4-His-BAP bound to ABAM004 during the binding phase was corrected for the amount of antibody captured. BiacoreT200 evaluation software version 2.0 and Microsoft Excel 2013 (Microsoft) are used for data analysis and plotting.

Figure 1 shows the binding amounts of ABAM004 and hCTLA4-His-BAP obtained by this assay in the presence of ATP and its metabolites.

As shown in Figure 1, ABAM004 was confirmed to utilize not only ATP but also ATP metabolites as a switch to bind to hCTLA4. Furthermore, this antibody exhibited the strongest binding activity, especially in the presence of AMP.

(1-10) Evaluation of antibody binding to human CTLA4-expressing cells

Flow cytometry was used to assess how the antigen-antibody interaction between ABAM004 and human CTLA4 changed in the presence and absence of AMP. Stable human CTLA4-expressing CHO cells (hCTLA4-CHO cells) were prepared at appropriate concentrations. PBS (FACS buffer) containing 0.1% BSA was used for resuscitation. Antibody was added to 100 μL of cell solution to a final concentration of 10 mg/mL, followed by the addition of AMP to final concentrations of 0, 0.4, 4, 40, 200, and 1000 μM, and the cells were incubated at 4°C for 30 min. The cell lines were then washed with FACS buffer containing 0, 0.4, 4, 40, 200, and 1000 μM AMP, followed by the addition of FITC-labeled secondary antibody (Goat F(ab'2) Anti-Human IgG Mouseads-FITC, Beckman, 732598) and incubated again at 4°C for 30 min in the dark. After a second washing operation, the samples were measured and analyzed using flow cytometry (FACS CyAn ^™ ADP). The results are shown in Figure 2.

The above results indicate that ABAM004 exhibits AMP concentration-dependent binding activity to hCTLA4-expressing cells, and shows AMP concentration-dependent binding activity not only for soluble antigens but also for membrane-type antigens.

(1-11) Using human peripheral blood mononuclear cells as effector cells to test antibody ADCC activity

The antibody concentration-dependent ADCC activity of antibodies that bind to antigens in an ATP-dependent manner was determined using the following method. Human peripheral blood mononuclear cells (hereinafter referred to as human PBMCs) were used as effector cells, and the ADCC activity of the test antibodies was measured as follows.

First, human PBMC solution was prepared. 50 mL of peripheral blood was collected from healthy volunteers (adult males) using a syringe pre-filled with 200 μL of 1000 IU/mL heparin solution (Novo-heparin injection 5000 IU, Novo Nordisk). The peripheral blood was diluted 2-fold with PBS (-) and divided into four aliquots, which were then added to Leucosep lymphocyte separation tubes (Greiner Bio-One) that had been pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. The separation tubes containing peripheral blood were centrifuged at 2150 rpm for 10 minutes at room temperature, and then the monocyte fractions were separated. After washing the cells contained in the fractions once with RPMI-1640 (Nacalai Tesque) containing 10% FBS (hereinafter referred to as 10% FBS/RPMI), the cells were resuspended in 10% FBS/RPMI to a cell density of 1 x ^10⁷ cells/mL. This cell suspension was used as the human PBMC solution for subsequent experiments.

Next, as target cells, hCTLA4-CHO cells, prepared by forcibly expressing the extracellular domain of human CTLA4 in CHO cells, were suspended and prepared in 10% FBS/RPMI to achieve 2 × ^10⁵ cells/mL. Furthermore, AMP (Sigma) diluted to 4 mM using RPMI was used as the AMP solution for subsequent tests.

ADCC activity was assessed using LDH (lactate dehydrogenase) release. First, 50 μL of antibody solutions prepared at various concentrations (0, 0.04, 0.4, 4, 40 μg/mL) were added to each well of a 96-well U-shaped plate, each seeded with 50 μL of target cells (1 x ^10⁴ cells/well). Additionally, 50 μL of AMP solution was added to each well, and the mixture was incubated at room temperature for 15 minutes. Then, 50 μL ( ⁵ x 10⁵ cells/well) of human PBMC solution was added to each well, the plate was centrifuged, and then incubated at 37°C for 4 hours in a 5% CO₂ incubator. After the reaction was complete, 100 μL of the culture supernatant was collected and transferred to a 96-well plate for assay. The catalyst (C) and dye solution (D) attached to the LDH detection kit (TaKaRa) were mixed at a 1:45 ratio, and 100 μL of this mixture was added. After reacting for 15 minutes at room temperature, 50 μL of 1N HCl was added to stop the reaction. The absorbance at 492 nm was measured, and ADCC activity was determined by LDH release. ADCC activity was determined based on the following formula.

ADCC activity (%)={(A-D)-(C-D)}x100/{(B-D)-(C-D)}

In the above formula, A represents the average LDH activity (OD 492nm) in wells containing each test antibody. B represents the average LDH activity (OD 492nm) in wells containing 10 μL of 20% Triton-X aqueous solution after the reaction. C represents the average LDH activity (OD 492nm) in wells containing 150 μL of 10% FBS/RPMI or 100 μL of 10% FBS/RPMI and 50 μL of AMP solution in target cells. D represents the average LDH activity (OD 492nm) in wells containing only 10% FBS/RPMI. The tests were performed in duplicate, and the average ADCC activity (%) reflecting the ADCC activity of the test antibody was calculated. The results are shown in Figure 3.

The results above show that antibody ABAM004 has antigen-binding activity in the presence of AMP and has the ability to kill target cells by exerting ADCC activity.

[Example 2] Crystal structure analysis of an anti-CTLA4 antibody with ATP-dependent binding properties

(2-1) X-ray crystal structure analysis of the anti-CTLA4 binding antibody ABAM004 using AMP as a switch

For the hCTLA4 binding antibody ABAM004 obtained from the library of Example 1 using AMP as a switch, the crystal structures of the Fab fragment of ABAM004 alone, the complex of the Fab fragment of ABAM004 and AMP, and the complex of the Fab fragment of ABAM004, AMP and the extracellular domain of hCTLA4 were analyzed.

(2-2) Preparation of full-length ABAM004 antibody for crystallization

The full-length ABAM004 antibody for crystallization was prepared and purified using methods known to those skilled in the art.

(2-3) Preparation of Fab fragments for crystal structure analysis of ABAM004 Fab fragments

The ABAM004Fab fragment was prepared using a standard method of restriction digestion with rLys-C (Promega, catalog number V1671), and then loaded onto a protein A column (MabSelect SuRe, GE Healthcare), a cation exchange column (HiTrapSP HP, GE Healthcare), and a gel filtration column (SuperdeX200 16/60, GE Healthcare) to remove the Fc fragment. Fractions containing the Fab fragment were pooled and stored at -80°C.

(2-4) Generation of ABAM004 Fab fragment crystals

The Fab fragment of ABAM004 purified by method 2-3 for crystallization was concentrated to approximately 13 mg/mL and crystallized at 20 °C using a static drop vapor-phase diffusion technique. The reservoir solution consisted of 0.1 M MES, pH 6.5, and 25% w/v polyethylene glycol 4000. The resulting crystals were immersed in a solution of 0.08 MES, pH 6.5, 20% w/v polyethylene glycol 4000, and 20% ethylene glycol.

(2-5) X-ray diffraction data collection and structure determination of ABAM004 Fab fragment crystal

X-ray diffraction data were measured using the BL-17A radiation facility of the Photon Factory at the High Energy Accelerator Research Organization. During the measurements, the crystal was kept frozen at -178°C under a nitrogen flow and a Quantum 270 CCD detector (ADSC) connected to the beamline was used, with the crystal rotating 0.5° at a time to collect a total of 360 X-ray diffraction images. Cellular parameters were determined, diffraction points were indexed, and diffraction data were processed from the diffraction images using the Xia2 program (J. Appl. Cryst. 43:186-190 (2010)), the XDS package (Acta. Cryst. D66:125-132 (2010)), and Scala (Acta. Cryst. D62:72-82 (2006)), ultimately yielding high-resolution diffraction intensity data. Crystallographic data statistics are shown in Table 7.

The structure was determined by molecular substitution using the Phaser program (J. Appl. Cryst. (2007) 40, 658-674). The Fab fragment search model source published the Fab crystal structure (PDB code: 4NKI). The model was constructed using the Coot program (Acta Cryst. D66:486-501 (2010)) and refined using the Refmac5 program (Acta Cryst. D67:355-467 (2011)). The crystallographic reliability factor (R) of the diffraction intensity data was 16.92%, and the Free R value was 21.22%. The structural refinement statistics are shown in Table 7.

[Table 7]

a；R _merge ＝∑hk1∑j|Ij(hk1)-<I(hk1)>|/∑hk1∑j|Ij(hk1)|, where Ij(hk1) and <I(hk1)> are the intensity of the measured j and the average reflectance intensity of the fertility index hk1, respectively.

b; R _factor = ∑hk1|F _calc (hk1)|-|F _obs (hk1)|/∑hk1|F _obs (hk1)|, where F _obs and F _calc are the observed and calculated amplitudes of the structure factor, respectively.

c;R _free is calculated from 5% reflections that are not used randomly.

(2-6) Prepare ABAM004 Fab fragment from full-length antibody for use in the complexation of ABAM004 Fab fragment and AMP. Crystal structure analysis of the ABAM004 Fab fragment, AMP and hCTLA4 complex.

The Fab fragment of ABAM004 was prepared using a standard method of restriction digestion with papain (Roche Diagnostics, catalog number 1047825). The fragment was then loaded onto a protein A column (MabSelect SuRe, GE Healthcare), a cation exchange column (HiTrap SP HP, GE Healthcare), and a gel filtration column (SuperdeX20016/60, GE Healthcare) to remove the Fc fragment. Fractions containing the Fab fragment were pooled and stored at -80°C.

(2-7) Crystal formation of the complex of ABAM004 Fab fragment and AMP

The Fab fragment of ABAM004 purified using methods 2-6 for crystallization was concentrated to approximately 13 mg/mL. AMP was added to bring the final concentration to 2 mM, and crystallization was performed at 20 °C using a sitting-drop vapor-diffusion technique. The reservoir solution consisted of 0.1 M Morpheus buffer 2, pH 7.5, 37.5% w/v MPD_P1K_P3350, and 10% Morpheus carboxylic acid (Morpheus, Molecular Dimensions).

(2-8) X-ray diffraction data collection and structural determination of crystals of ABAM004 Fab fragment and AMP complex

X-ray diffraction data were measured at the Photon Factory BL-1A radiation facility of the High Energy Accelerator Research Organization. During the measurements, the crystal was kept frozen at -178°C under a nitrogen flow and 720 X-ray diffraction images were collected using a Pilatus 2M detector (DECTRIS) connected to the beamline, while rotating the crystal 0.25° at a time. Cellular parameters were determined, diffraction points were indexed, and diffraction data were processed from the diffraction images using the Xia2 program (J. Appl. Cryst. 43:186-190 (2010)), the XDS package (Acta. Cryst. D66:125-132 (2010)), and Scala (Acta. Cryst. D62:72-82 (2006)), ultimately yielding high-resolution diffraction intensity data. Crystallographic data statistics are shown in Table 7.

The structure was determined using the molecular substitution method with the Phaser program (J. Appl. Cryst. (2007) 40, 658-674). The Fab fragment search model was derived from published Fab crystal structures (PDB code: 4NKI). The model was constructed using the Coot program (ActaCryst. D66:486-501 (2010)) and refined using the Refmac5 program (Acta Cryst. D67:355-467 (2011)). The crystallographic reliability factor (R) of the diffraction intensity data was 19.97%, and the FreeR value was 25.62%. The structural refinement statistics are shown in Table 7.

( 2-9) Preparation of the extracellular domain of hCTLA4

The extracellular domain of hCTLA4 was prepared using a standard method involving restriction digestion of abatacept with the endopeptidase Lys-C (Roche, catalog number 11047825001). The fractions were then loaded onto a protein A column (MabSelect SuRe, GE Healthcare) and a gel filtration column (SuperdeX200 10/300, GE Healthcare) to remove the Fc fragment. Fractions containing the hCTLA4 extracellular domain were pooled and stored at -80°C.

(2-10) Preparation of a complex of ABAM004 Fab fragment, AMP and hCTLA4 extracellular domain

The extracellular domain of hCTLA4 purified by method 2-9 was mixed with the Fab fragment of ABAM004 purified by method 2-6 at a molar ratio of 1.5:1, and AMP was added to bring the final concentration to 2 mM. The complex was purified by gel filtration chromatography (SuperdeX200 10/300, GE Healthcare) using a column equilibrated with 25 mM HEPES, pH 7.5, 100 mM NaCl, and 2 mM AMP.

(2-11) Crystallization of the complex of ABAM004 Fab fragment, AMP and hCTLA4 extracellular domain

The purified complex was concentrated to approximately 8 mg/mL and crystallized at 20 °C using a combination of seated drop vapor diffusion and seeding. The storage solution consisted of 0.1 M Morpheus buffer 1, pH 6.5, 37.5% w/v M1K3350, and 10% halogen (Morpheus, Molecular Dimensions).

(2-12) X-ray diffraction of a crystal of a complex of ABAM004 Fab fragment, AMP, and hCTLA4 extracellular domain. Data collection and structural determination

X-ray diffraction data were obtained using the SPring-8 spectrometer on a BL32XU. During the measurements, the crystal was kept frozen at -178°C under a nitrogen atmosphere, and a total of 180 X-ray diffraction images were collected using an MX-225HS CCD detector (RAYONIX) connected to the beamline, while rotating the crystal 1.0° at a time. Cellular parameters were determined, diffraction points were indexed, and diffraction data was processed from the diffraction images using the Xia2 program (J.Appl.Cryst.43:186-190(2010)), the XDS package (Acta.Cryst.D66:125-132(2010)), and Scala (Acta.Cryst.D62:72-82(2006)). High-resolution diffraction intensity data were ultimately obtained. Crystallographic data statistics are shown in Table 7.

The structure was determined using the molecular substitution method with the Phaser program (J. Appl. Cryst. (2007) 40, 658-674). The Fab fragment search model was derived from the published Fab crystal structure (PDB code: 4NKI), and the hCTLA4 extracellular domain search model was derived from the published human hCTLA4 crystal structure (PDB code: 3OSK, J. Biol. Chem. 286: 6685-6696 (2011)). The model was constructed using the Coot program (Acta Cryst. D66: 486-501 (2010)) and refined using the Refmac5 program (Acta Cryst. D67: 355-467 (2011)). The crystallographic reliability factor (R) of the diffraction intensity data was 23.49%, and the Free R value was 31.02%. The structural refinement statistics are shown in Table 7.

(2-13) Identification of the interaction sites between ABAM004 and AMP

The crystal structure shows that AMP is mainly recognized by the heavy chain of the antibody.

The adenine ring moiety of AMP is recognized by heavy chain CDR1 and CDR3, while the ribose and phosphate moiety are recognized by CDR1 and CDR2.

Specifically, as shown in Figure 4, the adenine ring moiety of AMP is recognized by the side chains of T33 (belonging to heavy chain CDR1) and Y95L, 100B, and W100C (belonging to CDR3), as well as the main chains of the antibody G96 and M100A. In particular, it was found that the carbonyl oxygen in the main chains of G96 and M100A forms a hydrogen bond with N at position 6 of AMP, and the amide NH group in the main chain of W100C forms a hydrogen bond with N at position 1. Furthermore, the side chains of Y95, L100B, and W100C interact using the π electrons of the adenine ring moiety, thus enabling the antibody to strongly recognize the adenine ring moiety. The ribosome is recognized by each side chain of T33 (belonging to heavy chain CDR1) and Y56 and Y58 (belonging to CDR2) through van der Waals interactions and through the π electrons of Y56. Furthermore, the phosphate moiety is identified by each of the side chains of T33 (belonging to heavy chain CDR1) and S52, S52A, and R53 (belonging to CDR2), as well as the main chain of S52A. In particular, hydrogen bonds formed between the side chain of T33 and the amide NH group of the main chain of S52A and the phosphate moiety, as well as van der Waals interactions of S52 and R53, are considered to play important roles in the identification of the phosphate moiety. The amino acid residue numbering of Fab is based on the Kabat numbering scheme.

(2-14) Identification of the ABAM004 epitope

In Figures 5 and 6, the epitopes of the ABAM004 Fab contact region are mapped in the hCTLA4 crystal structure and amino acid sequence, respectively. The epitopes contain amino acid residues of hCTLA4, which contain one or more non-hydrogen atoms located within any part of the ABAM004 Fab or AMP crystal structure.

The crystal structure clearly shows that at least the antigen's M3, E33, R35, T53, E97, M99, Y100, P101, P102, P103, Y104, Y105, and L106 are recognized by the antibody's heavy chain CDR2 and CDR3, as well as its light chain CDR1 and CDR3, and AMP. In particular, the ring formed by the antigen's M99 to Y104 is strongly recognized by the antibody in a manner embedded within the antibody's CDR ring, and this ring is considered to play a major role in the recognition of the antigen by the antibody.

(2-15) AMP-dependent antigen binding mechanism

The variable regions of antibodies were extracted from the crystal structures of ABAM004 Fab alone, the crystal structure of the ABAM004 Fab and AMP complex, and the crystal structure of the ternary complex composed of ABAM004 Fab, AMP, and CTLA4. Figure 7 is a superimposed diagram of the variable regions extracted with the heavy chain as the center. For AMP-dependent antigen binding, not only the direct interaction between AMP and CTLA4 shown in Examples 2-14, but also the structural changes of the antibody associated with AMP binding are considered important.

As shown in Figure 7, comparing the crystal structure of the antibody alone with that of the antibody bound to AMP revealed changes in the ring structures of the heavy chain CDR3 and light chain CDR3, as well as the torsion angle between the heavy and light chains in the antibody's variable region. Furthermore, comparing the crystal structure of the antibody bound to AMP with that of the ternary complex composed of antibody, AMP, and CTLA4 revealed further alterations in the structures of the heavy chain CDR3 and light chain CDR1 due to antigen-binding, exhibiting antigen-dependent structural changes. On the other hand, since the ring structure of the light chain CDR3 and the torsion angle between the heavy and light chains remained unchanged, it is believed that AMP binding may have altered the antibody structure to a state close to that of antigen binding. Therefore, the structural changes associated with AMP binding are considered necessary for forming a suitable structure for antigen binding and play an important role in AMP-dependent antigen binding.

[Example 3] Generation and activity evaluation of modified CTLA4 antibody

(3-1) Generation of CTLA4-enhanced variants of ABAM004 antibody

The amino acid sequence (VH SEQ ID NO:10, VL SEQ ID NO:11) of ABAM004 obtained from a rationally designed human phage library described in Example 1 was modified to reduce the CTLA4 binding activity of the sequence in the absence of ATP analogs, enhance human CTLA4 binding activity in the presence of ATP analogs, and enhance binding to ATP and ATP analogs. To achieve this, variants with point mutations in the residues expected to participate in binding were generated based on the co-crystal structures of ABAM004 and AMP obtained by the method described in Example 2, as well as the co-crystal structures of ABAM004, AMP, and human CTLA4. Furthermore, variants in which each amino acid contained in the CDR was replaced by Ala or Pro were also generated. The binding activity of the point mutation variants to human CTLA4 (Abatacept and hCTLA4-His-BAP) in the absence of ATP and in the presence of ATP, ADP, or AMP was determined using a Biacore T200 or Biacore 4000 (GE Healthcare) to screen for mutations that enhance binding activity. Variants were prepared by combining mutations that enhance binding activity, and KD values were calculated using Biacore. The results clearly show that introducing H32A and S52aT substitutions into the heavy chain of ABAM004 and introducing T24D, T26P, and E50F into the light chain (according to Kabat numbering) enhances the binding properties of ABAM004. This variant is designated 04H0150/04L0072 (VH SEQ ID NO:47, VL SEQ ID NO:48).

(3-2) The effects of ATP and its metabolites on ABAM004 and O4H0150/O4L0072 were determined by surface plasmon resonance. Effect of human CTLA4 binding activity

First, a chip was created for the Biacore T200 assay. The Biacore T200 was set to 25°C and the flow rate to 10 μL/min. HBS-EP+ was used as the run buffer. An equal volume of a mixture of NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) was added to the sensor chip CM5 (GE Healthcare) at a flow rate of 10 μL/min for 10 minutes to activate the flow cell. Next, 25 μg/mL protein A/G (Pierce) suspended in 10 mM sodium acetate at pH 4.0 was added, and binding was allowed at 10 μL/min for 30 minutes. Then, 1 M ethanolamine-HCl was added at 10 μL/min for 10 minutes to block excess active groups on the flow cell.

Next, the effect of ATP and its metabolites on the binding of the target antibody to human CTLA4 was determined. The temperature was set at 25°C, and TBS was used as the run buffer. 10 mM Glycine-HCl (pH 1.5) was used as the regeneration solution. After allowing the antibody suspended in TBS to be captured, each flow cell was injected at a flow rate of 10 μL/min with a TBS solution containing 500 nM hCTLA4-His-BAP, 10 concentrations of ATP, ADP or AMP diluted at a common ratio of ₄ from 4000 μM, and 2 mM MgCl2 for 3 minutes. This 3-minute period was used as the binding phase of hCTLA4-His-BAP. After the binding phase, the injection was switched to the run buffer for 2 minutes, which served as the dissociation phase. After the dissociation phase, the regeneration solution was injected at a flow rate of 30 μL/min for 30 seconds. This was the cycle for the binding activity assay. The binding amount of hCTLA4-His-BAP interacting with ABAM004 or 04H0150/04L0072 during the binding phase was corrected for the amount of captured antibody. The results are shown in Figure 8. Additionally, in the ABAM004 binding assay with 04H0150/04L0072, the small molecule concentration during the binding phase was maintained at 62.5 μM or 1 mM, and eight concentrations of hCTLA4-His-BAP diluted from 2000 nM at a common ratio of 2 were used for the binding phase. The KD values obtained from analyzing the binding amount of hCTLA4-His-BAP are shown in Table 8. Biacore T200 assessment software version 2.0 and Microsoft Excel 2013 (Microsoft) were used for data analysis and plotting. A steady-state affinity model was used to calculate the KD values.

[Table 8]

SM stands for the small molecule (ATP, ADP, or AMP) used in each assay.

(3-3) Enhance integration capabilities by introducing comprehensive changes

To generate better anti-CTLA4 antibodies, amino acid alterations were comprehensively introduced into 04H0150/04L0072, which is the variable region of the anti-human CTLA4 antibody prepared in Example 3-1. Variants were generated using methods known to those skilled in the art, such as PCR, wherein every 18 amino acids, except for cysteine, were substituted in all CDRs of 04H0150 and 04L0072. Approximately 1200 variants generated for binding to human CTLA4 were assayed using a Biacore 4000. Protein A/G (Thermo Fisher Scientific) was immobilized on a Series S Sensor Chip CM5 (GE Healthcare), and the antibodies were captured onto the chip by interaction with culture supernatant containing the antibody variants. Subsequently, human CTLA4 solutions with or without small molecules (ATP, ADP, or AMP) were allowed to interact with the antibodies to evaluate the binding affinity of the antibodies to human CTLA4 in the presence or absence of small molecules. The assay was performed at 25°C using Tris-buffered saline and 0.02% PS20 as the run buffer.

Combining alterations that enhance binding to human CTLA4 in the presence of small molecules and alterations that reduce binding to human CTLA4 in the absence of small molecules (alterations discovered using the methods described above) yields anti-human CTLA4 antibodies exhibiting a better spectrum. For the antibody heavy chain 04H0150-G1m (SEQ ID NO:209) gene (which has 04H0150 as the heavy chain variable region and G1m (SEQ ID NO:82) where the C-terminal Gly and Lys of human IgG1 have been removed as the heavy chain constant region), alterations discovered by introducing general changes and frame modifications are combined to prepare the antibody heavy chain gene. For the antibody light chain 04L0072-lam1 (SEQ ID NO:208) (which has 04L0072 as the light chain variable region and human λ chain lam1 (SEQ ID NO:87) as the light chain constant region), the discovered alterations are combined to generate the antibody light chain gene. Furthermore, variants were generated in which the framework and constant region of the light chain variable region were replaced by sequences of the human κ chain. For comparison, genes for antibody heavy chain MDX10D1H-G1m (SEQ ID NO: 210) with the heavy chain variable region MDX10D1H (SEQ ID NO: 154) of the existing anti-human CTLA4 antibody described in WO0114424 and genes for antibody light chain MDX10D1L-k0MT (SEQ ID NO: 211) with the light chain variable region MDX10D1L (SEQ ID NO: 155) were generated. Antibodies were expressed and purified by combining these genes to produce the target anti-CTLA4 antibody using methods known to those skilled in the art. Table 9 lists the SEQ ID NOs of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and hypervariable region of the generated antibodies.

The antibodies in this article are named according to the following rule: (heavy chain variable region) - (heavy chain constant region) / (light chain variable region) - (light chain constant region). For example, this means that if the antibody name is 04H0150-G1m/04L0072-lam1, then the heavy chain variable region of this antibody is 04H0150, the heavy chain constant region is G1m, the light chain variable region is 04L0072, and the light chain constant region is lam1.

The amino acid sequences of the heavy chain, light chain, and their hypervariable regions (represented by SEQ ID NO:).

[Table 9]

The Biacore T200 was used to determine the binding of the generated antibodies to human CTLA4. A run buffer of 20 mM ACES (pH 7.4), 150 mM NaCl, ₂ mM MgCl2, 0.05% Tween 20, and ATP added to the desired concentration was used, and the assay was performed at 37°C. First, protein G (CALBIOCHEM) was immobilized on a Series S Sensor Chip CM3 (GE Healthcare), and antibodies were captured by interacting the chip with an antibody solution prepared in an ATP-free run buffer. Next, the binding affinity of the antibodies to human CTLA4 was assessed in the presence or absence of ATP by interacting with a human CTLA4 solution prepared in a run buffer containing ATP to the desired concentration or in a run buffer without ATP. The chip was regenerated with 25 mM NaOH and 10 mM Glycine-HCl (pH 1.5), and assays were performed by repeatedly capturing antibodies. The dissociation constant of each antibody to CTLA4 was calculated using Biacore T200 evaluation software 2.0. Specifically, the binding rate constant ka (L/mol/s) and the dissociation rate constant kd (1/s) were calculated by globally fitting the sensor map obtained from the assay using a 1:1 Langmuir binding model. The dissociation constant KD (mol/L) was then calculated from these values. Alternatively, the dissociation constant KD (mol/L) was calculated using a steady-state model. Furthermore, the amount of CTLA4 bound per unit of antibody was calculated by correcting the amount of CTLA4 bound from the sensor map obtained from the assay with the amount of antibody captured on the chip surface. Table 10 shows the results of these assays.

Analysis of the binding of altered antibodies to human CTLA4

[Table 10]

The values for “Binding with Human CTLA4” in the table represent the amount of human CTLA4 bound per unit volume of antibody when human CTLA4 is allowed to interact at 1000 nM for each of the listed ATP concentrations. “KD(M) of Human CTLA4” represents the dissociation constant of human CTLA4 at each ATP concentration. KD values marked with * in the table were calculated using a steady-state model. It shows that all variants prepared using 04H0150-G1m/04L0072-lam1 as the parent antibody exhibit enhanced binding in the presence of ATP compared to 04H0150-G1m/04L0072-lam1. Furthermore, both 04H0150-G1m/04L0072-lam1 and these variants showed higher binding amounts in the presence of 10 μM ATP instead of 1 μM; and even higher binding amounts were observed in the presence of 100 μM, demonstrating an ATP concentration-dependent binding to human CTLA4. On the other hand, the comparative MDX10D1H-G1m/MDX10D1L-k0MT did not show this ATP concentration-dependent binding to human CTLA4. Although 04H1077-G1m/04L1305-k0MT, in which the light chain framework and constant region of 04H1077-G1m/04L1086-lam1 are replaced by the human κ chain, exhibited enhanced binding to human CTLA4 in the absence of ATP compared to 04H1077-G1m/04L1086-lam1, the ATP concentration-dependent binding was also enhanced. These results indicate that the ATP-dependent binding property to human CTLA4 is maintained even when the sequence is replaced with the human κ chain sequence. Among the antibodies generated in this study, 04H1077-G1m/04L1066-lam1, 04H1077-G1m/04L1305-k0MT, and 04H1207-G1m/04L1086-lam1 exhibited almost the same binding activity as the existing anti-human CTLA4 antibody MDX10D1H-G1m/MDX10D1L-k0MT in the presence of 100 μM ATP, while 04H1208-G1m/04L1407-k0MT exhibited stronger binding activity than MDX10D1H-G1m/MDX10D1L-k0MT in the presence of 10 μM or more ATP.

Next, the binding of the antibodies 04H1077-G1m/04L1086-lam1 and 04H1208-G1m/04L1407-k0MT to mouse CTLA4 was evaluated among the antibodies prepared in Table 10. For comparison, the genes for the antibody heavy chain hUH02-G1d (SEQ ID NO: 212) containing the heavy chain variable region hUH02 (SEQ ID NO: 156) of the anti-mouse CTLA4 antibody and the genes for the antibody light chain hUL01-k0 (SEQ ID NO: 213) containing the light chain variable region hUL01 (SEQ ID NO: 157) were generated, expressed, purified, and used. The assays were performed using a Biacore T200 under the same conditions as those used to determine binding to human CTLA4, except that mouse CTLA4 was used as the sample (Table 11). Mouse CTLA4 was prepared as follows.

A gene linking the His-tag (mCTLA4-His) (SEQ ID NO: 49) to the extracellular region of mouse CTLA4 was synthesized and inserted into an animal expression plasmid. The prepared plasmid was introduced into the FreeStyle 293-F cell line (Invitrogen) derived from human embryonic kidney cells via lipid transfection. The cell line was suspended in FreeStyle 293 expression medium (Invitrogen) and seeded in flasks at a cell density of 1.33 x ^10⁶ cells/mL. The absorbance of the purified antigen solution at 280 nm was measured using a spectrophotometer. From the obtained measurements, the concentration of the purified antigen was calculated using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

Analysis of altered antibody binding to mouse CTLA4

[Table 11]

N.D.; for detecting binding and its weakness.

The values for “Binding with mouse CTLA4” in the table represent the amount of mouse CTLA4 bound per unit volume of antibody when mouse CTLA4 is allowed to interact at 1000 nM under each of the listed ATP concentrations, and “KD(M) of mouse CTLA4” represents the dissociation constant of mouse CTLA4 under each ATP concentration. hUH02-G1d/hUL01-k0 showed the same degree of binding to mouse CTLA4 regardless of ATP concentration, while 04H1077-G1m-04L1086-lam1 and 04H1208-G1m/04L1407-k0MT both showed ATP concentration-dependent binding to mouse CTLA4. Compared with the binding ability to human CTLA4 shown in Table 10, 4H1077-G1m-04L1086-lam1 binds to mouse CTLA4 about 5 times weaker than human CTLA4. In the presence of 100 μM ATP, 04H1208-G1m/04L1407-k0MT binds to mouse CTLA4 about 2 times weaker than human CTLA4.

(3-4) Generation of anti-mCTLA4 control antibody and anti-mCTLA4 switch antibody

Anti-mCTLA4 control antibodies (hUH02-mFa55/hUL01-mk1, abbreviation: mNS-mFa55) and anti-CTLA4 switches (04H1077-mFa55/04L1086-ml0r, abbreviation: SW1077-mFa55; and 04H1208-mFa55/04L1407s-mk1, abbreviation: SW1208-mFa55) were generated. In the mNS-mFa55 antibody, the heavy chain variable region hUH02 (SEQ ID NO: 16) and the light chain variable region hUL01 (SEQ ID NO: 17) were used, and for the constant region, the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) and the wild-type mouse light chain constant region mk1 (SEQ ID NO: 19) were used. At this time, the mouse heavy chain constant region was modified to enhance binding to the Fcγ receptor. Antibodies are expressed and purified using methods known to those skilled in the art.

In the SW1077-mFa55 antibody, the heavy chain variable region 04H1077 (SEQ ID NO:20) and the light chain variable region 04L1086 (SEQ ID NO:21) are used, and for the constant region, the mouse heavy chain constant region mFa55 (SEQ ID NO:18) and the wild-type mouse light chain constant region ml0r (SEQ ID NO:22) are used. The mouse heavy chain constant region is modified to enhance binding to the Fcγ receptor. The antibody is expressed and purified using methods known to those skilled in the art.

In the SW1208-mFa55 antibody, the heavy chain variable region 04H1208 (SEQ ID NO:23) and the light chain variable region 04L1407s (SEQ ID NO:24) are used, and for the constant region, the mouse heavy chain constant region mFa55 (SEQ ID NO:18) and the wild-type mouse light chain constant region mk1 (SEQ ID NO:19) are used. The mouse heavy chain constant region is modified to enhance binding to the Fcγ receptor. The antibody is expressed and purified using methods known to those skilled in the art.

(3-6) Evaluation of the neutralizing activity of CTLA4 switch antibodies

The neutralizing activity of the anti-CTLA4 switch antibody (SW1077-mFa55) prepared in Examples 3-4 was evaluated using a competitive ELISA method. mCTLA4-Fc (SEQ ID NO: 25), in which the human constant region is linked to mCTLA4, was diluted to 5 μg/mL (55 nM) with 0.1 _M NaHCO3 and 0.05% _NaN3 to prepare an mCTLA4-Fc solution. 100 μL of the prepared mCTLA4-Fc solution was added to each well of a 96-well plate, and the plate was incubated overnight at 4°C to immobilize the mCTLA4-Fc on the plate surface. After washing three times with TBS, 0.1% Tween 20 was added to each well, diluted with TBS to 250 μL of BSA solution to block the plate surface. The plate was then washed three times. A mixture of mCD86-Fc-His (Sino Biologics Inc. 50068-M03H, accession number NP_062261.3) (in which the human constant region and His-tag are fused with mouse CD86 diluted to a final concentration of 55 nM with TBS), SW1077-mFa55 antibody solutions diluted to final concentrations of 6.25, 1.56, 0.390, 0.0977, 0.0061, and 0 μg/mL, and ATP solutions diluted to final concentrations of 0, 1, 10, and 100 μM were each mixed to a total volume of 100 μL. The mixture was added to each well, and the plate was incubated at 37°C for 1 hour. Each well was then washed three times with TBS and 0.1% Tween 20 to prepare a solution containing the same ATP concentration as the solution added to each well. Add 100 μL of anti-His-tag mAb-HRP-Direct (MBL Life Sciences) diluted 10,000 times with blocking buffer to each well to achieve the same ATP concentration as the solution added to each well, and incubate the plate at 37°C for 1 hour. Then wash each well three times with TBS and 0.1% Tween 20 to prepare a solution with the same ATP concentration as the solution added to each well. Add 100 μL of TMB solution to each well and incubate the plate at 37°C for 1 hour. Add 50 μL _of 1M _H₂SO₄ to each well to terminate the reaction, and measure the absorbance at 450 nm using a microplate reader (Wako Sunrise).

The absorbance values of the antibody-free wells under the same ATP concentration conditions were used as the 100% mCTLA4-mCD86 binding rate, and the extent to which the binding rate decreased after the addition of antibody was assessed. The results are shown in Figure 9.

These results show that the neutralizing activity of the SW1077 antibody against the mCTLA4-mCD86 interaction increases with increasing ATP concentration during the assay. These results confirm that the SW1077 antibody possesses ATP-dependent neutralizing activity.

(3-7) Efficacy of anti-CTLA4 switch antibodies in syngeneic tumor cell transplantation mouse models, and regulatory T cells in tumors. Increase/decrease of (Treg) cells and changes in systemic response markers in the spleen.

(3-7-1) Preparation of mouse models of cell line and syngeneic tumor line transplantation

The cells used were FM3A mouse breast cancer cell lines purchased from RIKEN. FM3A cells were maintained and passaged in RPMI 1640 medium (Sigma) containing 10% bovine serum (Thermo Fisher Scientific). C3H/HeN mice (7 weeks old, female) purchased from Charles River Laboratories, Japan were used as mice. FM3A cells were transplanted subcutaneously into the abdominal skin of mice, and the model was established when the transplanted tumor volume increased from approximately 150 ^mm³ to approximately 300 ^mm³ .

The volume of the transplanted tumor is calculated using the following formula.

Tumor volume = long diameter × short diameter × short diameter / 2

(3-7-2) Preparation of drugs for administration

The drugs to be administered to the FM3A cell transplantation model were the anti-mouse CTLA4 control antibody (mNS-mFa55) and the anti-CTLA4 switch antibody (SW1208-mFa55) prepared in Examples 3-4. Using His-buffered saline (20 mM His-HCl, 150 mM NaCl, pH 6.0), mNS-mFa55 was prepared at concentrations of 0.0005 mg/mL, 0.005 mg/mL, 0.0125 mg/mL, 0.05 mg/mL, 0.5 mg/mL, 1.5 mg/mL, and 5 mg/mL, respectively, and SW1208-mFa55 was prepared at concentrations of 0.005 mg/mL, 0.05 mg/mL, 0.5 mg/mL, 5 mg/mL, and 25 mg/mL.

(3-7-3) Drug administration used to determine antitumor efficacy

On day 9 post-transplantation, mice were administered mNS-mFa55 at doses of 0.01 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg, and 100 mg/kg, respectively, and SW1208-mFa55 at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg, respectively. The prepared solution was administered via tail vein at a dose of 20 mL/kg.

Table 12 shows the details of drug treatment when the antitumor effect was measured.

Determination of antitumor effect in FM3A cell transplantation model

[Table 12]

Group Head count drug dose Application route Application route 1 4 His-buffer - tail vein 9 days post-transplant 2 4 mNS-mFa55 0.01 mg/kg tail vein 9 days post-transplant 3 4 mNS-mFa55 0.1 mg/kg tail vein 9 days post-transplant 4 4 mNS-mFa55 0.25mg/kg tail vein 9 days post-transplant 5 4 mNS-mFa55 1mg/kg tail vein 9 days post-transplant 6 4 mNS-mFa55 10mg/kg tail vein 9 days post-transplant 7 4 mNS-mFa55 30mg/kg tail vein 9 days post-transplant 8 4 mNS-mFa55 100mg/kg tail vein 9 days post-transplant 9 4 SW1208-mFa55 0.1 mg/kg tail vein 9 days post-transplant 10 4 SW1208-mFa55 1mg/kg tail vein 9 days post-transplant 11 4 SW1208-mFa55 10mg/kg tail vein 9 days post-transplant 12 4 SW1208-mFa55 100mg/kg tail vein 9 days post-transplant 13 4 SW1208-mFa55 500mg/kg tail vein 9 days post-transplant

(3-7-4) Evaluation of anti-tumor effects

The anti-tumor effect is assessed by calculating the tumor volume using the formula described in (3-7-1).

The tumor growth inhibition rate (TGI) value is calculated using the following formula.

TGI (%) = (1 - (mean tumor volume in the target group at the time of measurement - mean tumor volume in the target group at the time of initial administration) / (mean tumor volume in the control group at the time of measurement - mean tumor volume in the control group at the time of initial administration)) x 100

As a result, mNS-mFa55 at doses of 0.1 mg/kg or higher and SW1208-mFa55 at doses of 1 mg/kg or higher showed TGI of 60% or higher on day 13 (Figures 10 and 11).

(3-7-5) Drug administration for Treg cell assessment in tumors and verification of systemic effects in the spleen

On day 7 post-transplantation, anti-mouse CTLA4 control antibody (mNS-mFa55) was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 100 mg/kg, and anti-CTLA switch antibody (SW1208-mFa55) was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg. Table 13 shows the details of the drug treatments used to assess Treg cells in the tumor and to validate systemic effects in the spleen.

Validating intratumoral and systemic effects in FM3A cell transplantation models (mNS-mFa55 and SW1208-mFa55)

[Table 13]

(3-7-6) Tumor and spleen resection in FM3A cell transplantation model mice

Six days after antibody administration, mice were euthanized under anesthesia, and the tumor and spleen were removed. Cell suspensions were prepared from the removed spleen using RPMI-1640 medium (SIGMA) containing 10% FBS (SIGMA), followed by hemolysis using a Mouse Erythrocyte Lysing kit (R&D) to prepare splenocytes. The removed tumor was pulverized using a Tumor Dissociation Kt in Miltenyi mice. Splenic cells and fragmented tumors reacted with the following antibodies, and the immunocellular fractions present were analyzed by FACS analysis: anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: 145-2C11), anti-CD4 antibody (BD, clone: RM4-5), anti-FoxP3 antibody (eBioscience, clone: FJK-16s)), anti-ICOS antibody (eBioscience, clone: 7E17G9), and anti-KLRG1 antibody (Biolegend, clone: 2F1/KLRG1). FACS analysis was performed using a BD LSR Fortessa X-20 (BD).

(3-7-7) Evaluation of tumor Tregs in FM3A cell transplantation model

The changes in effector Treg cells (CD4 ⁺ FoxP3 ⁺ KLRG1 ⁺ ) in tumors after administration of anti-mouse CTLA4 control antibody (mNS-mFa55) or anti-CTLA4 switch antibody (SW1208-mFa55) were evaluated. Results showed that mNS-mFa55 and SW1208-mFa55 reduced the proportion of effector Tregs to less than 0.2% of CD45-positive cells at 1 mg/kg and higher doses (Figure 12).

(3-7-8) Evaluation of the systemic effects of spleen on the FM3A cell transplantation model

Changes in activated helper T cells (CD4+Foxp3-ICOS+) in the spleen after administration of mNS-mFa55 or SW1208-mFa55 were assessed using FACS analysis. Results showed that administration of mNS-mFa55 significantly increased the ratio of activated helper T cells to CD45-positive cells in the spleen, but even with increased doses of SW1208-mFa55, the ratio of activated helper T cells to CD45-positive cells in the spleen did not significantly increase (Figure 13). It has been confirmed that although the switch antibody exhibited the same efficacy as the control antibody, it did not elicit a response in tissues outside the tumor, and the concept of locally manifested activity only in the tumor was confirmed in mice.

[Example 4] Generation and activity evaluation of modified CTLA4 antibody

Further modifications and evaluations were conducted on the anti-CTLA4 switch antibody generated in Example 3.

(4-1) Enhanced binding affinity to CTLA4 through altered binding with ATP.

The structural analysis results from Examples 2-1 showed that the CDR2 of the antibody heavy chain interacts with the phosphate group of AMP. It is thought that when the small molecule is ATP, the γ-phosphate group may cause steric hindrance to the CDR2 of the heavy chain. Then, amino acids in this region were substituted to examine the enhanced ATP binding ability. Specifically, 04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 were generated by introducing changes to R53Q and G55H into the variable region of the heavy chain of 04H1207-G1m/04L1086-lam1 and 04H1208-G1m/04L1086-lam1 generated in Example 3. Furthermore, 04H1389-G1m/04L1305-k0MT was generated by substituting the light chain of 04H1389-G1m/04L1086-lam1 with the sequence of the human κ chain. Table 14 lists the SEQ ID NOs of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and hypervariable region of these antibodies.

The amino acid sequences of the heavy chain, light chain, and their hypervariable regions (represented by SEQ ID NO:).

[Table 14]

The Biacore T200 was used to evaluate the binding of the generated variants to ATP and human CTLA4. The ATP binding assay was performed at 37°C using a run buffer of 20 mM MACES (pH 7.4), 150 mM NaCl, ₂ mM MgCl2, and 0.05% Tween 20. First, Sure Protein A (GE Healthcare) was immobilized on a Series S Sensor Chip CM3 (GE Healthcare), and the antibody was captured onto the chip by interaction with the antibody solution prepared in the run buffer. Next, the binding affinity to the antibody was assessed by interaction with the ATP solution prepared in the run buffer. The chip was regenerated with 25 mM NaOH and 10 mM Glycine-HCl (pH 1.5), and the assay was performed by repeated antibody capture. The amount of ATP bound per unit volume of antibody was calculated by correcting the amount of ATP bound when injected at a concentration of 100 nM with the amount of antibody captured on the chip surface. The binding to human CTLA4 was determined using the Biacore T200 via the methods described in Examples 3-3. Table 15 shows the results of these determinations.

Analysis of binding with ATP and human CTLA4

[Table 15]

Compared with the parental antibodies 04H1207-G1m/04L1086-lam1 and 04H1208-G1m/04L1086-lam1, which did not introduce R53Q/G55H, 04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 showed enhanced ATP binding ability. Compared to the parental antibodies 04H1207-G1m/04L1086-lam1 and 04H1208-G1m/04L1086-lam1 without the introduction of R53Q/G55H, the binding affinity of 04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 to human CTLA4 was approximately 10-fold increased in the presence of 10 μM ATP. Comparison of binding amounts showed enhanced binding affinity to human CTLA4 at lower ATP concentrations. It was also shown that 04H1389-G1m/04L1305-k0MT (in which the light chain of 04H1389-G1m/04L1086-lam1 is replaced by the human κ chain sequence) has the same ATP-binding capacity and ATP-dependent human CTLA4-binding capacity as 04H1389-G1m/04L1086-lam1.

(4-2) Generation and evaluation of the binding ability of anti-human CTLA4 control antibody and anti-CTLA4 switch antibody

Anti-human CTLA4 control antibody (MDX10D1H-mFa55/MDX10D1L-mk1, abbreviation: hNS-mFa55) and anti-CTLA4 switch antibody (04H1389-mFa55/04L1305-mk1, abbreviation: SW1389-mFa55) were generated. In the hNS-mFa55 antibody, the heavy chain variable region MDX10D1H (SEQ ID NO:26) and the light chain variable region MDX10D1L (SEQ ID NO:27) were used, and for the constant region, the mouse heavy chain constant region mFa55 (SEQ ID NO:18) and the wild-type mouse light chain constant region mk1 (SEQ ID NO:19) were used. At this time, the mouse heavy chain constant region was modified to enhance binding to the Fcγ receptor. The antibody was expressed and purified using methods known to those skilled in the art.

In the SW1389-mFa55 antibody, the heavy chain variable region 04H1389 (SEQ ID NO:29) and the light chain variable region 04L1305 (SEQ ID NO:30) were used, and for the constant region, the mouse heavy chain constant region mFa55 (SEQ ID NO:18) and the wild-type mouse light chain constant region mk1 (SEQ ID NO:19) were used. The mouse heavy chain constant region was modified to enhance binding to the Fcγ receptor. The antibody was expressed and purified using methods known to those skilled in the art.

The binding of generated hNS-mFa55 and SW1389-mFa55 to human CTLA4 was evaluated. Run buffers of 20 mM ACES (pH 7.4), 150 mM NaCl, ₂ mM MgCl2, 0.05% Tween 20, and ATP added to the desired concentration were used, and assays were performed at 37 °C. First, rabbit anti-mouse IgG (Thermo Fisher Scientific) was immobilized on a Series SSensor Chips CM5 (GE Healthcare), and the antibody was captured onto the chip by interaction with an antibody solution prepared in an ATP-free run buffer. Next, the binding capacity of the antibody to human CTLA4, with or without ATP, was evaluated by interaction with a human CTLA4 solution prepared in a run buffer with ATP added to the desired concentration or in an ATP-free run buffer. The chip was regenerated with 25 mM NaOH and 10 mM Glycine-HCl (pH 1.5), and assays were performed by repeated antibody capture. The dissociation constant of each antibody against CTLA4 was calculated using Biacore T200 evaluation software 2.0. Specifically, the binding rate constant ka (L/mol/s) and dissociation rate constant kd (1/s) were calculated by globally fitting sensor maps obtained from assays using a 1:1 Langmuir binding model, and the dissociation constant KD (mol/L) was calculated from these values. Table 16 shows the results of these assays.

Binding analysis of variants with mouse constant regions to human CTLA4

[Table 16]

Both antibodies generated using the mouse constant region were confirmed to bind to human CTLA4. SW1389-mFa55 was also shown to bind to human CTLA4 in an ATP-dependent manner, similar to 04H1389-G1m/04L1305-k0MT generated using the same variable region and human constant region shown in Table 15.

(4-3) Anti-CTLA4 switch antibodies and anti-CTLA4 non-switch antibodies each have their own characteristics when using human CTLA4 knock-in and human CD3 transfection. Efficacy in syngeneic tumor cell transplantation models in genetically modified mice, increase/decrease of Treg cells in tumors and systemic Treg cells in the spleen Changes in reaction markers

(4-3-1) Cell line

Hepa1-6/hGPC3 cells were used. This cell line was obtained by purchasing mouse hepatocellular carcinoma line Hepa1-6 cells from ATCC, transfecting them with constitutive expression of the human Glypican 3 (hGPC3, SEQ ID NO: 181) gene, and then cloning them. Hepa1-6/hGPC3 cells were maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (SIGMA) and 600 μg/mL GENETICIN (Gibco).

(4-3-2) Preparation of syngeneic tumor line transplantation mouse model

The hCTLA4 KI hCD3 EDG-replacement mouse was used, which is a hybrid of the human CTLA4 knock-in mouse (Blood(2005)106(9):3127-3133) and the internally generated human CD3 EDG-replacement mouse (SciRep(2017)7:45839). Hepa1-6/hGPC3 cells were subcutaneously transplanted into the hCTLA4 KI hCD3 EDG-replacement mouse, and the model was established when the average volume of the transplanted tumor reached approximately 200 ^mm³ to approximately 400 ^mm³ .

The volume of the transplanted tumor is calculated using the following formula.

Tumor volume = long diameter × short diameter × short diameter / 2

(4-3-3) Preparation of drugs for administration

As drugs administered to the Hepa1-6/hGPC3 cell transplantation model, the anti-CTLA4 switch antibody (SW1389-mFa55) and the anti-human CTLA4 control antibody (hNS-mFa55) prepared in Examples 4-2 were prepared using His buffer (150mM NaCl/20mM His-HCl buffer, pH 6.0) at concentrations of 0.01, 0.1, 1, 5, 10, and 20 mg/mL, and 0.01, 0.1, 1, and 3 mg/mL, respectively.

(4-3-4) Drug administration for determining antitumor efficacy

On day 7 post-transplantation, mice were administered SW1389-mFa55 via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 100 mg/kg, and hNS-mFa55 at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg. Table 17 shows the details of the drug treatments used to determine the antitumor effects.

Determination of antitumor effect in Hepa1-6/hGPC3 cell transplantation model

[Table 17]

Group Head count drug dose Application route Application date 1 4 His buffer - tail vein Day 7 post-transplant 2 4 hNS-mFa55 0.1 mg/kg tail vein Day 7 post-transplant 3 4 hNS-mFa55 1mg/kg tail vein Day 7 post-transplant 4 4 hNS-mFa55 10mg/kg tail vein Day 7 post-transplant 5 4 hNS-mFa55 30mg/kg tail vein Day 7 post-transplant 6 4 SW1389-mFa55 0.1 mg/kg tail vein Day 7 post-transplant 7 4 SW1389-mFa55 1mg/kg tail vein Day 7 post-transplant 8 4 SW1389-mFa55 10mg/kg tail vein Day 7 post-transplant 9 4 SW1389-mFa55 100mg/kg tail vein Day 7 post-transplant

(4-3-5) Evaluation of anti-tumor effects

The antitumor effect was assessed by calculating the tumor volume using the formula described in (4-3-2). JMP 11.2.1 (SAS Institute Inc.) was used for statistical analysis.

The TGI (tumor growth inhibition) value is calculated using the following formula.

TGI = (1 - (average tumor volume in the target group at the time of measurement - average tumor volume before antibody administration) / (average tumor volume in the control group at the time of measurement - average tumor volume before antibody administration)) x 100

As a result, both hNS-mFa55 and SW1389-mFa55 showed TGI=60 or higher efficacy on day 18 after administration at doses of 1 mg/kg or higher (Figures 14 and 15).

(4-3-6) Drug administration for Treg cell assessment in tumors and verification of systemic effects in the spleen

On day 7 post-transplantation, SW1389-mFa55 was administered to mice via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg, and hNS-mFa55 was administered via tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg. Table 18 shows details of the drug treatments used to assess Treg cells in the tumor and to validate systemic effects in the spleen.

Validation of intratumoral and systemic effects in Hepa1-6/hGPC3 cell transplantation models (hNS-mFa55 and SW1389-mFa55)

[Table 18]

Group Head count drug dose Application route Application date 1 3 His buffer - tail vein Day 7 post-transplant 2 3 hNS-mFa55 0.1 mg/kg tail vein Day 7 post-transplant 3 3 hNS-mFa55 1mg/kg tail vein Day 7 post-transplant 4 3 hNS-mFa55 10mg/kg tail vein Day 7 post-transplant 5 3 hNS-mFa55 30mg/kg tail vein Day 7 post-transplant 6 3 SW1389-mFa55 0.1 mg/kg tail vein Day 7 post-transplant 7 3 SW1389-mFa55 1mg/kg tail vein Day 7 post-transplant 8 3 SW1389-mFa55 10mg/kg tail vein Day 7 post-transplant 9 3 SW1389-mFa55 100mg/kg tail vein Day 7 post-transplant 10 3 SW1389-mFa55 500mg/kg tail vein Day 7 post-transplant

(4-3-7) Tumor and spleen resection in Hepa1-6/hGPC3 cell transplantation model mice

Six days after antibody administration, mice were euthanized under anesthesia, and the tumor and spleen were removed. Cell suspensions were prepared from the removed spleen using RPMI-1640 medium (SIGMA) containing 10% FBS (SIGMA), followed by hemolysis using a Mouse Erythrocyte Lysing kit (R&D) to prepare splenocytes. The removed tumor was pulverized using a Tumor Dissociation Kt in Miltenyi mice. Splenic cells and fragmented tumors reacted with the following antibodies, and the immunocellular fractions present were analyzed by FACS analysis: anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RM4-5), anti-FoxP3 antibody (eBioscience, clone: FJK-16s), anti-ICOS antibody (eBioscience, clone: 7E17G9), anti-CCR7 antibody (Biolegend, clone: 4B12), and anti-KLRG1 antibody (Biolegend, clone: 2F1/KLRG1). FACS analysis was performed using a BD LSR Fortessa X-20 (BD).

(4-3-8) Evaluation of tumor Tregs in Hepa1-6/hGPC3 cell transplantation model

The changes in effector Treg cells (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) in tumors after administration of SW1389-mFa55 or hNS-mFa55 were evaluated. Results showed that hNS-mFa55 and SW1389-mFa55 at doses of 1 mg/kg or higher reduced the proportion of effector Treg cells to less than 0.2% of CD45-positive cells (Figure 16).

(4-3-9) Evaluation of systemic effects in the spleen in a Hepa1-6/hGPC3 cell transplantation model

Changes in activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) in the spleen after administration of hNS-mFa55 or SW1389-mFa55 were assessed using FACS analysis. Results showed that administration of hNS-mFa55 significantly increased the ratio of activated helper T cells to CD45-positive cells in the spleen, but administration of SW1389-mFa55 did not significantly increase the number of activated helper T cells in the spleen, even with increasing doses (Figure 17). This confirms that the switch antibody exhibits the same efficacy as the control antibody, but it does not elicit a response in tissues outside the tumor. Therefore, the concept of locally exhibiting activity only in the tumor was confirmed in human CTLA4 KI mice.

(4-4) Generation of anti-CTLA4 control antibody and anti-CTLA4 switch antibody for cynomolgus monkey toxicity testing

Anti-CTLA4 control antibody (MDX10D1H-Kn125/MDX10D1L-k0MT//MDX10D1H-Hl076/MDX10D1L-k0MT, abbreviation: NS-ART1) and anti-CTLA4 switch antibody (04H1389-Kn125/04L1305-k0MT//04H1389-Hl076/04L1305-k0MT, abbreviation: SW1389-ART1) were produced. In the NS-ART1 antibody, the heavy chain variable region MDX10D1H (SEQ ID NO:26) and the light chain variable region MDX10D1L (SEQ ID NO:27) are used, and for the constant region, the human heavy chain constant regions Kn125 (SEQ ID NO:31) and H1076 (SEQ ID NO:32), as described in prior patent document WO 2013/002362, and the human light chain constant region k0MT (SEQ ID NO:33) are used. In this case, the human heavy chain constant region is modified to enhance binding to the Fcγ receptor. The antibody is expressed and purified using methods known to those skilled in the art.

In the SW1389-ART1 antibody, the heavy chain variable region 04H1389 (SEQ ID NO: 29) and the light chain variable region 04L1305 (SEQ ID NO: 30) are used, and for the constant region, the human heavy chain constant region Kn125 (SEQ ID NO: 31) and the human heavy chain constant region H1076 (SEQ ID NO: 32), and the human light chain constant region k0MT (SEQ ID NO: 33) are used. In this case, the human heavy chain constant region is modified to enhance binding to the Fcγ receptor. The antibody is expressed and purified using methods known to those skilled in the art.

Heterodimerized antibodies (antibodies having two different heavy chain polypeptides and/or two different light chain polypeptides) are named in this paper according to the following rules: (first heavy chain variable region)-(first heavy chain constant region)/(first light chain variable region)-(first light chain constant region)//(second heavy chain variable region)-(second heavy chain constant region)/(second light chain variable region)-(second light chain constant region). For example, if the antibody name is 04H1389-Kn125/04L1305-k0MT//04H1389-Hl076/04L1305-k0MT, it means that in this antibody, the first heavy chain variable region is 04H1389, the first heavy chain constant region is Kn125, the first light chain variable region is 04L1305, the first light chain constant region is k0MT, the second heavy chain variable region is 04H1389, the second heavy chain constant region is Hl076, the second light chain variable region is 04L1305, and the second light chain constant region is k0MT.

(4-5) Implementation of toxicity testing on cynomolgus monkeys

To assess and compare toxicity, including systemic reactions, the NS-ART1 antibody or SW1389-ART1 antibody prepared in Examples 4-4 was administered to male cynomolgus monkeys at a dose of 60 mg/kg once a week (3 monkeys each time) for a total of 5 weeks. The administration was slow intravenous using an infusion pump, and general observation, weight measurement, blood/blood chemistry examination, bone marrow examination, pathological examination, and plasma drug concentration determination were performed.

Anti-drug antibodies were observed during administration of both antibodies, but exposure was maintained until the end of the administration period. Administration of the NS-ART1 antibody resulted in systemic autoimmune disease-like changes, such as the appearance of autoantibodies, inflammatory changes (vasculitis, inflammatory cell infiltration), anemic changes, and T cell activation, while these changes were not observed with administration of the SW1389-ART1 antibody. These results suggest that the SW1389-ART1 antibody has reduced in vivo toxicity.

[Example 5] Generation and activity evaluation of modified CTLA4 antibody

The anti-CTLA4 switch antibody prepared in Example 4 was further modified and evaluated.

(5-1) Antibody optimization through the introduction of a complete change and replacement of the framework

Amino acid alterations were comprehensively introduced into the CDR of 04H1389-G1m/04L1086-lam1, and alterations with better spectra were explored. Comprehensive introduction and evaluation of amino acid alterations were performed using the methods described in Examples 3-3. Variants were generated in which combinations of the alterations discovered herein were introduced and the frame was replaced. Table 19 lists the SEQ ID NOs of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and hypervariable region of these antibodies. The light chains 04L1594-lam1, 04L1581-lam1, 04L1610-lam1, 04L1612-lam1, and 04L1610-lam1 in Table 19 introduce alterations in both the CDR and frame compared to the parental antibody light chain 04L1086-lam1, and have frames and constant regions with germline sequences of the human λ chain. Furthermore, 04L1615-k0MT, 04L1616-k0MT, and 04L1617-k0MT have alterations introduced into the CDR of 04L1086-lam1 and possess the frame and constant regions of the human κ chain germline sequence. The heavy chain variable region 04H1389v373 is one of the alterations introduced into the CDR of the parental antibody's heavy chain variable region 04H1389. Heavy chain variable regions 04H1637, 04H1643, 04H1654, 04H1656, 04H1642, and 04H1735 have alterations introduced into the CDR of 04H1389, as well as substitutions of the frame sequence from different germlines.

Amino acid sequences of the heavy chain, light chain, and their hypervariable regions (represented by SEQ ID NO:)

[Table 19]

The binding activity of the resulting variants to human CTLA4 was evaluated using the methods described in Examples 3-3 (Table 20).

Analysis of binding with human CTLA4

[Table 20]

*: Determining KD value through static model

The KD values marked with * in the table are calculated using a steady-state model. All variants prepared using 04H1389-G1m/04L1086-lam1 as the parent antibody showed ATP-dependent binding to human CTLA4 and stronger binding ability than the parent antibody in the presence of 10 μM ATP, with a KD of 3.7 x ^10⁻⁸ M. It was also shown that all these antibodies had stronger binding ability than the existing anti-human CTLA4 antibody MDX10D1H-G1m/MDX10D1L-k0MT in the presence of 10 μM ATP.

The binding capacity of the generated variants to human CTLA4 in the presence of ADP or AMP was assessed using a Biacore T200 and compared with that in the presence of ATP. The binding capacity to human CTLA4 in the presence of ADP or AMP was determined using the methods described in Examples 3-3, as in assessing the binding capacity in the presence of ATP, but using ADP or AMP instead of ATP (Table 21).

Assessment of ATP, ADP and AMP dependence

[Table 21]

Existing human CTLA4 antibodies MDX10D1H-G1m/MDX10D1L-k0MT exhibit similar kinetic parameters regardless of the type and presence of small molecules. All ATP-dependent anti-CTLA4 antibodies bind to human CTLA4 not only in the presence of ATP but also in the presence of ADP or AMP, with higher binding capacity in the presence of these small molecules compared to the absence of the small molecules shown in Table 20. Therefore, these antibodies are shown to bind to CTLA4 in an ATP, ADP, and AMP-dependent manner. These antibodies exhibit the highest binding capacity in the presence of ATP, followed by the highest binding capacity in the presence of ADP, and the lowest binding capacity in the presence of AMP. In all cases, the binding capacity in the presence of ADP is approximately 3 times stronger than that in the presence of AMP, and the binding capacity in the presence of ATP is approximately 5 times stronger than that in the presence of AMP. The kA values are similar in the presence of any small molecule, but the kD values differ. Since dissociation is faster in the presence of ADP than in the presence of ATP, and further dissociation is faster in the presence of AMP than in the presence of ADP, the difference in KD values depending on the type of small molecule is due to the difference in dissociation rates.

Next, the binding affinity of several variants to mouse CTLA4 and cynomolgus monkey CTLA4 was evaluated. Binding activity with human CTLA4, mouse CTLA4, and cynomolgus monkey CTLA4 was assessed using a Biacore T200 apparatus, following the methods described in Examples 3-3 (Table 22). Cynomolgus monkey CTLA4 was prepared using the following method.

The gene CyCTLA4-His-BAP (SEQ ID NO:50), a fusion of the His-tag and BAP-tag at the C-terminus of the extracellular region of cynomolgus monkey CTLA4, was synthesized and inserted into an animal expression plasmid. The prepared plasmid was introduced into the FreeStyle 293-F line (Invitrogen) derived from human embryonic kidney cells via lipid transfection. This line was seeded into flasks after being suspended in FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33 x ^10⁶ cells/mL. Three hours after plasmid transfection, biotin was added to a final concentration of 100 μM, and the cells were cultured for 4 days in a _CO₂ incubator (37°C, 8% _CO₂ , 125 rpm). The antigen was purified from the culture supernatant of each sample using methods known to those skilled in the art. The absorbance of the purified antigen solution at 280 nm was measured using a spectrophotometer. The concentration of purified antibody was calculated from the obtained measurements using the extinction coefficient obtained by the PACE method (Protein Science (1995) 4, 2411-2423).

Analysis of binding with CTLA4 in humans, mice, and cynomolgus monkeys

[Table 22]

The results showed that all six small molecule-dependent CTLA4 antibodies evaluated not only bound to human CTLA4, but also to mouse CTLA4 and cynomolgus monkey CTLA4 in an ATP-dependent manner.

(5-2) Production of altered anti-CTLA4 switch antibodies and negative control antibodies

Modified anti-CTLA4 switch antibodies (04H1654-mFa55m2P1/04L1610-ml0r//04H1656-mFa55m2N1/04L1610-ml0r, abbreviation: SW1610-mFa55; 04H1654-mFa55m2P1/04L1612-ml0r//04H1656-mFa55m2N1/04L1612-ml0r, abbreviation: SW1612-mFa55; and 04H1389-mFa55/04L1615-mk1, abbreviation: SW1615-mFa55) and negative control antibodies (IC17Hdk-mFa55/IC17L-mk1, abbreviation: KLH-mFa55) were produced.

In the SW1615-mFa55 antibody, the heavy chain variable region 04H1389 (SEQ ID NO:29) and the light chain variable region 04L1615 (SEQ ID NO:34) were used, and for the constant region, the mouse heavy chain constant region mFa55 (SEQ ID NO:18) and the wild-type mouse light chain constant region mk1 (SEQ ID NO:19) were used. The mouse heavy chain constant region was modified to enhance binding to the Fcγ receptor. The antibody was expressed and purified using methods known to those skilled in the art.

In the SW1610-mFa55 antibody, as constant regions, one heavy chain variable region 04H1654 (SEQ ID NO:35) is linked to the mouse heavy chain constant region mFa55m2P1 (SEQ ID NO:36), another heavy chain variable region 04H1656 (SEQ ID NO:37) is linked to the mouse heavy chain constant region mFa55m2N1 (SEQ ID NO:38), and for the light chain variable region 04L1610 (SEQ ID NO:39), the wild-type mouse light chain constant region ml0r (SEQ ID NO:22) is used. The antibody was expressed and purified using methods known to those skilled in the art.

In the SW1612-mFa55 antibody, as constant regions, one heavy chain variable region 04H1654 (SEQ ID NO:35) is linked to the mouse heavy chain constant region mFa55m2P1 (SEQ ID NO:36), another heavy chain variable region 04H1656 (SEQ ID NO:37) is linked to the mouse heavy chain constant region mFa55m2N1 (SEQ ID NO:38), and for the light chain variable region 04L1612 (SEQ ID NO:40), the wild-type mouse light chain constant region ml0r (SEQ ID NO:22) is used. The antibody was expressed and purified using methods known to those skilled in the art.

In the negative control antibody, the heavy chain variable region IC17Hdk (SEQ ID NO: 51) is linked to the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) as the constant region, and for the light chain variable region IC17L (SEQ ID NO: 52), the wild-type mouse light chain constant region mk1 (SEQ ID NO: 19) is used. The antibody was expressed and purified using methods known to those skilled in the art.

(5-3) Evaluation of the binding ability of antibodies with mouse constant regions to human CTLA4

The ability of anti-CTLA4 antibodies with mouse constant regions to bind to the antigen was evaluated using the methods described in Examples 4-2 (Table 23). All of these antibodies with mouse constant regions showed the same ATP-dependent binding capacity as human CTLA4, as the antibodies with the same variable regions but human constant regions shown in Table 22.

Binding analysis of variants with mouse constant regions to human CTLA4

[Table 23]

(5-4) Anti-CTLA4 switch antibodies were used in the transfection of syngeneic tumor cells from human CTLA4 knock-in and human CD3 transgenic mice. Efficacy in the implantation model, increase/decrease of Treg cells in the tumor, and changes in systemic response markers in the spleen.

(5-4-1) Cell line

Hepa1-6/hGPC3 cells were used. This cell line was obtained by purchasing mouse hepatocellular carcinoma cell line Hepa1-6 from ATCC, transfecting it with constitutive expression of the human Glypican 3 (hGPC3) gene, and then cloning. Hepa1-6/hGPC3 cells were maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (Sigma) and 0.6 mg/mL G418 (Nacalai Tesque).

(5-4-2) Generation of syngeneic tumor line transplantation mouse models

The hCTLA4 KI hCD3 EDG-replacement mouse was used, which is a hybrid of the human CTLA4 knock-in mouse (Blood(2005)106(9):3127-3133) and the internally generated human CD3 EDG-replacement mouse (SciRep(2017)7:45839). Hepa1-6/hGPC3 cells were subcutaneously transplanted into the hCTLA4 KI hCD3 EDG-replacement mouse population, and the model was established when the average volume of the transplanted tumor reached approximately 200 ^mm³ to approximately 400 ^mm³ .

The volume of the transplanted tumor is calculated using the following formula.

Tumor volume = long diameter × short diameter × short diameter / 2

(5-4-3) Preparation of drugs for administration

The drugs to be administered to the Hepa1-6/hGPC3 cell transplantation model were the anti-CTLA4 switch antibodies (SW1610-mFa55, SW1612-mFa55, SW1615-mFa55) prepared in Examples 5-2. The drugs were prepared using His-buffered saline (20 mM His-HCl, 150 mM NaCl, pH 6.0) to achieve concentrations of 0.03 mg/mL, 0.1 mg/mL, and 0.3 mg/mL, respectively.

(5-4-4) Drug administration used to determine antitumor efficacy

On day 8 post-transplantation, three samples of anti-CTLA4 switch antibody were administered to mice via tail vein at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg, respectively. Table 24 shows details of the drug treatments used to determine the antitumor efficacy.

Antitumor efficacy was determined in a Hepa1-6/hGPC3 cell transplantation model (anti-CTLA4 switch antibody).

[Table 24]

Group Head count drug dose Application route Application date 1 5 His-buffer - tail vein Day 8 post-transplant 2 5 SW1610-mFa55 0.3 mg/kg tail vein Day 8 post-transplant 3 5 SW1610-mFa55 1mg/kg tail vein Day 8 post-transplant 4 5 SW1610-mFa55 3mg/kg tail vein Day 8 post-transplant 5 5 SW1612-mFa55 0.3 mg/kg tail vein Day 8 post-transplant 6 5 SW1612-mFa55 1mg/kg tail vein Day 8 post-transplant 7 5 SW1612-mFa55 3mg/kg tail vein Day 8 post-transplant 8 5 SW1615-mFa55 0.3 mg/kg tail vein Day 8 post-transplant 9 5 SW1615-mFa55 1mg/kg tail vein Day 8 post-transplant 10 5 SW1615-mFa55 3mg/kg tail vein Day 8 post-transplant

(5-4-5) Evaluation of anti-tumor effects

The anti-tumor effect is assessed by calculating the tumor volume using the formula described in (5-4-2).

The tumor growth inhibition rate (TGI) value is calculated using the following formula.

TGI (%) = (1 - (mean tumor volume in the target group at the time of measurement - mean tumor volume in the target group at the time of initial administration) / (mean tumor volume in the control group at the time of measurement - mean tumor volume in the control group at the time of initial administration)) x 100

As a result, SW1610-mFa55 and SW1612-mFa55 showed TGI of 60% or higher on day 16 after administration at doses of 1 mg/kg or higher, and SW1615-mFa55 at doses of 3 mg/kg or higher (Figures 18 to 20).

(5-4-6) Drug administration for Treg cell assessment in tumors and validation of systemic effects in the spleen

On day 10 post-transplantation, mice were administered SW1610-mFa55 at 50 mg/kg, 100 mg/kg, and 200 mg/kg via tail vein; SW1612-mFa55 at 50 mg/kg, 100 mg/kg, and 200 mg/kg; and SW1615-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, and 400 mg/kg. Additionally, for the control group, the negative control antibody IC17Hdk-mFa55/IC17L-mk1 (abbreviated: KLH-mFa55) was administered via tail vein at 400 mg/kg. Table 25 shows details of the drug treatments used to assess Treg cells in the tumor and to validate systemic effects in the spleen.

Validation of intratumoral and systemic effects in the Hepa1-6/hGPC3 cell transplantation model (anti-CTLA4 switch antibody)

[Table 25]

Group Head count drug dose Application route Application date 1 3 KLH-mFa55 400mg/kg tail vein Day 10 post-transplant 2 3 SW1610-mFa55 50mg/kg tail vein Day 10 post-transplant 3 3 SW1610-mFa55 100mg/kg tail vein Day 10 post-transplant 4 3 SW1610-mFa55 200mg/kg tail vein Day 10 post-transplant 5 3 SW1612-mFa55 50mg/kg tail vein Day 10 post-transplant 6 3 SW1612-mFa55 100mg/kg tail vein Day 10 post-transplant 7 3 SW1612-mFa55 200mg/kg tail vein Day 10 post-transplant 8 3 SW1615-mFa55 50mg/kg tail vein Day 10 post-transplant 9 3 SW1615-mFa55 100mg/kg tail vein Day 10 post-transplant 10 3 SW1615-mFa55 200mg/kg tail vein Day 10 post-transplant 11 3 SW1615-mFa55 400mg/kg tail vein Day 10 post-transplant

(5-4-7) Tumor and spleen resection in Hepa1-6/hGPC3 cell transplantation model mice

Six days after antibody administration, mice were euthanized under anesthesia, and the tumor and spleen were removed. Cell suspensions were prepared from the removed spleen using RPMI-1640 medium (SIGMA) containing 10% FBS (SIGMA), followed by hemolysis using a Mouse Erythrocyte Lysing kit (R&D) to prepare splenocytes. The removed tumor was pulverized using a Tumor Dissociation Kt in Miltenyi mice. Splenic cells and fragmented tumors reacted with the following antibodies, and the immunocellular fractions present were analyzed by FACS analysis: anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RM4-5), anti-FoxP3 antibody (eBioscience, clone: FJK-16s), anti-ICOS antibody (eBioscience, clone: 7E17G9), anti-CCR7 antibody (Biolegend, clone: 4B12), and anti-KLRG1 antibody (Biolegend, clone: 2F1/KLRG1). FACS analysis was performed using a BD LSR Fortessa X-20 (BD).

(5-4-8) Evaluation of tumor Tregs in Hepa1-6/hGPC3 cell transplantation model

The changes in effector Treg cells (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) in tumors after administration of anti-CTLA4 switch antibody were evaluated. Results showed that, across all administered doses, SW1610-mFa55, SW1612-mFa55, and SW1615-mFa55 reduced the proportion of effector Tregs to less than 0.2% of CD45-positive cells (Figure 21).

(5-4-9) Evaluation of systemic effects in the spleen in a Hepa1-6/hGPC3 cell transplantation model

Changes in activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) in the spleen after administration of anti-CTLA4 switch antibodies were assessed using FACS analysis. Results showed no significant increase in the ratio of activated helper T cells to CD45-positive cells in the spleen at the assessed doses of SW1610-mFa55 and SW1612-mFa55 of 50 mg/kg and at the assessed dose of SW1615-mFa55 of 200 mg/kg or less. Dunnett's test was performed on the KLH-mFa55 administration group using JMP 11.2.1 (SAS Institute Inc.) for significance testing (Figure 22). This confirmed that while all switch antibodies showed efficacy, they did not elicit a response in tissues outside the tumor and possessed the characteristic of showing activity only locally in the tumor.

[Example 6] Generation of a variant Fc capable of enhancing ADCC/ADCP activity

To generate antibodies with enhanced ADCC and ADCP (which are cytotoxic effector functions), the preparation of Fc region variants with enhanced binding ability to activated FcγRs FcγRIIIa and FcγRIIa was investigated.

(6-1) Generation and evaluation of variants enhanced by binding to FcγR

Heterodimerized antibodies 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1 were generated, which have heavy chain constant regions Kn125 and H1076 with enhanced binding ability to FcγR as described in WO2013/002362, and have 04H1637 as a heavy chain variable region and 04L1610-lam1 as a light chain. Specifically, a gene for the antibody heavy chain 04H1637-Kn125 (SEQ ID NO:162) was generated, which contains 04H1637 (SEQ ID NO:138) as a heavy chain variable region and introduces L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in G1d (SEQ ID NO:158), removes Gly and Lys at the C-terminus of the human IgG1 heavy chain constant region, and also has altered Y349C/T366W in the CH3 region that promotes heterodimerization. Similarly, a gene for the antibody heavy chain 04H1637-H1076 (SEQ ID NO: 163) was generated, which includes 04H1637 (SEQ ID NO: 138) as another heavy chain variable region and has D270E/K326D/A330M/K334E introduced into the human IgG1 heavy chain constant region G1d (SEQ ID NO: 158), and also has altered D356C/T366S/L368A/Y407V in the CH3 region that promotes heterodimerization. Using 04L1610-lam1 (SEQ ID NO: 161) as the antibody light chain, heterodimers 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1 were generated by methods known to those skilled in the art. The antibody heavy chains 04H1637-Kn462 (SEQ ID NO:164), 04H1637-Hl441 (SEQ ID NO:165), 04H1637-Hl445 (SEQ ID NO:166), 04H1637-Kn461 (SEQ ID NO:167), and 04H1637-H1443 (SEQ ID NO:168) were generated. These genes, in addition to L235Q, G236W, S239M, H268D, and D270E, [were also found]. In addition to S298A, K326D, and K334E, there are modifications introduced in the CH2 region L234F and A330K as changes to the combination with FcγR, as reported in WO2013/002362; G236A, I332E, and I332D as reported in Mol. Cancer Ther., 2008, 7, 2517-2527 and WO2004/029207; and the combination of T250V and T307P as reported in WO 2013/118858 as changes to improve stability. In addition, a gene 04H1654-KT462 (SEQ ID NO:182) for antibody heavy chain was generated, which removed Gly and Lys from the C-terminus of human IgG1 (IGHG1*03), had the same changes as Kn462 in the CH2 region, had the E356K change in the CH3 region as described in WO 2006/106905 that promotes heterodimerization, and included 04H1654 (SEQ ID NO:140) as a heavy chain variable region. Similarly, the gene for antibody heavy chain 04H1656-HT441 (SEQ ID NO: 170) was generated, which removed the Gly and Lys from the C-terminus of human IgG1 (IGHG1*03), had the same alterations as Hl441 in the CH2 region, had the K439E alteration in the CH3 region that promotes heterodimerization as described in WO 2006/106905, and included 04H1656 (SEQ ID NO: 141) as a variable region of the heavy chain. Similarly, the genes for 04H1656-HT445 (SEQ ID NO: 171), 04H1654-KT461 (SEQ ID NO: 183), and 04H1656-HT443 (SEQ ID NO: 173) were generated. Furthermore, alterations were investigated to improve the hemodynamics of the antibodies, as described in Mabs, 2017, 9, 844-853. Specifically, a gene for 04H1654-KT473 (SEQ ID NO: 184) was generated, which introduced N434A/Y436T/Q438R/S440E into the CH3 region of 04H1654-KT462 (SEQ ID NO: 182), which was a combination of alterations that enhanced binding to human FcRn and reduced binding to rheumatoid factor under acidic conditions. Similarly, a gene for 04H1656-HT482 (SEQ ID NO: 185) was generated, which introduced N434A/Y436T/Q438R/S440E into 04H1656-HT445 (SEQ ID NO: 171). Similarly, antibody heavy chains 04H1654-KT461 (SEQ ID NO: 186) and 04H1656-HT443 were generated by introducing the same modifications into 04H1654-KT461 and 04H1656-HT498 (SEQ ID NO: 187), respectively. The desired heterodimerized antibodies were generated by combining these heavy chains and using either 04L1610-lam1 or 04L1612-lam1 (SEQ ID NO: 188) as the light chain.

The extracellular domains of FcγR were prepared using the following method. First, the genes for the extracellular domains of FcγR were synthesized using methods known to those skilled in the art. Then, the sequences of each FcγR were prepared based on information registered in NCBI. Specifically, FcγRI was prepared based on the sequence of NCBI accession number #NM_000566.3, FcγRIIa based on the sequence of NCBI accession number #NM_001136219.1, FcγRIIb based on the sequence of NCBI accession number #NM_004001.3, and FcγRIIIa based on the sequence of NCBI accession number #NM_001127593.1, with a His tag added to the C-terminus. Polymorphic sites of FcγRIIa were prepared with reference to J. Exp. Med., 1990, 172, 19-25, and polymorphic sites of FcγRIIIa were prepared with reference to J. Clin. Invest., 1997, 100, 1059-1070. Expression vectors were prepared by inserting the obtained gene fragments into animal cell expression vectors. The prepared expression vectors were transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic renal cell carcinoma to express the target protein. After collecting the culture supernatant, purification was performed in principle through a 0.22 μm filter using the following four steps: First, cation exchange column chromatography (SP Sepharose FF); second, His-tag affinity column chromatography (HisTrap HP); third, gel filtration column chromatography (SuperdeX200); and fourth, sterile filtration. However, for FcγRI, anion exchange column chromatography using Q Sepharose FF was performed in the first step. The concentration of the purified protein was calculated by measuring the absorbance at 280 nm using a spectrophotometer and using the extinction coefficient calculated from values obtained by methods such as PACE (Protein Science, 1995, 4, 2411-2423). Human FcRn was prepared using the method described in WO 2010/107110.

The interaction between the generated antibody and human FcγR was analyzed using a Biacore T200 as follows: 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween 20 (pH 7.4) were used as the run buffer, and the assay was performed at 25°C. For the sensor chip, a Series S SA (GE Healthcare) chip immobilized with CaptureSelect Human Fab-lambda Kinetics Biotin Conjugate (Thermo Fisher Scientific) was used. The target antibody was captured onto these chips, allowing each FcγR diluted in the run buffer to interact with them. The chips were regenerated with 10 mM Glycine-HCl (pH 1.5) and 25 mM NaOH, and the assay was performed by repeatedly capturing the antibody. Using Biacore T200 evaluation software 2.0, a 1:1 Langmuir binding model was used for FcγRIa and FcγRIIIa, and a steady-state affinity model was used for FcγRIIa to calculate the dissociation constant KD (mol/L) of each antibody to FcγR. For FcγRIIb, the amount of FcγRIIb bound per unit amount of antibody was calculated by correcting the amount of FcγRIIb bound obtained from the sensor map obtained by the assay with the amount of antibody captured onto the chip surface.

The interaction between the generated antibodies and human FcRn was analyzed using a Biacore T200 as follows: 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween 20 (pH 6) were used as the run buffer, and the assay was performed at 25°C. For the sensor chip, a Series S SA (GE Healthcare) chip with CaptureSelect Human Fab-lambda Kinetics Biotin Conjugate (Thermo Fisher Scientific) immobilized on it was used. The target antibodies were captured onto these chips, allowing FcRn diluted in the run buffer to interact with them. The chips were regenerated with 10 mM Mlycine-HCl (pH 1.5) and 25 mM NaOH, and the assay was performed by repeatedly capturing antibodies. The FcRn dissociation constant for each antibody was calculated using a steady-state model and Biacore T200 evaluation software 2.0.

Table 26 shows these measurement results.

Binding analysis of Fc region variants with human FcγR and FcRn

[Table 26]

The values of "KD(M) of hFcRn" and "KD(M) of hFcγRs" in the table represent the dissociation constants of hFcRn and each FcγR, respectively. "Binding Amount" shows the amount of FcγRIIb bound per unit volume of antibody when FcγRIIb is allowed to interact at 1000 nM. The "Relative Values of KD between G1m and hFcRn" and "Relative Values of KD between G1m and hFcγRs" are obtained by dividing the KD value of 04H1637-G1m/04L1610-lam1 for hFcRn and each FcγR by the KD value of each variant, respectively. "Relative Binding Amount" refers to the amount of FcγRIIb bound by each variant divided by the amount bound by 04H1637-G1m/04L1610-lam1. The amino acid sequences of the antibody heavy chain 04H1637-G1m and the antibody light chain 04L1610-lam1 are shown in SEQ ID NO: 160 and 161, respectively. This indicates that all the generated heterodimerized antibodies exhibit enhanced binding to FcγRIIa and FcγRIIIa compared to 04H1637-G1m/04L1610-lam1, which possesses the constant region of native human IgG1. Furthermore, compared to the Fc region variants 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1 reported in WO 2013/002362, all of the following variants showed enhanced binding to FcγRIIa: 04H1637-Kn462/04L1610-lam1//04H1637-Hl441/04L1610-lam1, 04H1637-Kn462/04L1610-lam1//04H1637-Hl445/04L1610-lam1, and 04H1637-Kn461/04L1610-lam1//04H1637-Hl443/04L1610-lam1. The binding affinity of 04H1637-Kn462/04L1610-lam1//04H1637-Hl445/04L1610-lam1 to FcγRIIIa was shown to be equal to that of 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1, and the binding affinity of 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1 to FcγRIIIa was enhanced compared to 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1. Similarly, in the constant region and ch3 region of IGHG1*03, there are different modifications for heterodimerization of 04H1654-KT462/04L1610-lam1//04H1656-HT445/04L1610-lam1 and 04H1654-KT462/04L1612-lam1//04H1656-HT445/04L1610-lam1 and 04H1637-Kn462/04L1610-lam1//04H1637-Hl445/04L1610 -lam1 shows a comparable FcγR binding spectrum, and 04H1654-KT461/04L1610-lam1//04H1656-HT443/04L1610-lam1 and 04H1654-KT461/04L1612-lam1//04H1656-HT443/04L1610-lam1 show FcγR binding spectra comparable to those of 04H1637-Kn461/04L1610-lam1//04H1637-Hl443/04L1610-lam1. Furthermore, compared to those antibodies that were modified to improve hemodynamics before the introduction of these modifications, the following antibodies, 04H1654-KT473/04L1610-lam1//04H1656-HT482/04L1610-lam1, 04H1654-KT481/04L1610-lam1//04H1656-HT498/04L1610-lam1, 04H1654-KT473/04L1612-lam1//04H1656-HT482/04L1612-lam1 and 04H1654-KT481/04L1612-lam1//04H1656-HT498/04L1612-lam1, showed enhanced binding to human FcRn and comparable binding to FcγRs.

Furthermore, the gene 04H1656-HT451 (SEQ ID NO:272) was generated, in which N434A/Y436T/Q438R/S440E was introduced into 04H1656-HT441, which is a combination of alterations that enhance binding to human FcRn and reduce binding to rheumatoid factor under acidic conditions. The amino acid sequence of the antibody heavy chain HT451 is shown in SEQ ID NO:276. Heterodimerized antibodies were generated by combining 04H1654-KT473 and 04H1656-HT451 and using 04L1610-lam1 as the antibody light chain. Table 27 shows the results of the interaction analysis of the generated antibodies with human FcRn and human FcγR.

Binding analysis of Fc region variants with human FcγR and FcRn

[Table 27]

Compared to 04H1656-G1m/04L1610-lam1, which possesses the constant region of natural human IgG1, the resulting heterodimerized antibodies 04H1654-KT462/04L1610-lam1//04H1656-HT441/04L1610-lam1 and 04H1654-KT473/04L1610-lam1//04H1656-HT451/04L1610-lam1 all showed enhanced binding to activated FcγRs FcγRIIa and FcγRIIIa. Furthermore, in these antibodies, binding to FcγRIIb, which acts as an inhibitory FcγR, was maintained at the same level in both antibodies as in 04H1656-G1m/04L1610-lam1. Compared to the pre-modification, 04H1654-KT473/04L1610-lam1//04H1656-HT451/04L1610-lam1 (where N434A/Y436T/Q438R/S440E were introduced into 04H1654-KT462/04L1610-lam1//04H1656-HT441/04L1610-lam1) showed enhanced binding affinity to human FcRn.

Next, the binding activity of FcγR binding-enhancing variants produced using different modifications for heterodimerization as described in Nat. Biotechnol., 1998, 16, 677-681, with respect to human FcRn and FcγR, was evaluated. Genes for antibody heavy chains 04H1389-Ks462 (SEQ ID NO:191) and 04H1389-Km462 (SEQ ID NO:199) were generated, which contain 04H1389 (SEQ ID NO:136) as the heavy chain variable region and have a heavy chain constant region (which has the same modification as KT462 in the CH2 region of the constant region, the constant region having the Gly and Lys of the C-terminus of human IgG1 (IGHG1*03) removed, and further having T366W introduced in the CH3 region as a modification for heterodimerization of 04H1389-Ks462, and Y349C/T366W used as a modification for heterodimerization of 04H1389-Km462). In addition, genes for antibody heavy chains 04H1389-Hs445 (SEQ ID NO:192) and 04H1389-Hm445 (SEQ ID NO:200) were generated, which have the same alterations as HT445 introduced into the CH2 region, and further have T366S/L368A/Y407V introduced in the CH3 region as alterations for heterodimerization of 04H1389-Hs445, and E356C/T366S/L368A/Y407V used as alterations for heterodimerization of 04H1389-Hm445. Similarly, 04H1389-Ks461 (SEQ ID NO:193), 04H1389-Km461 (SEQ ID NO:201), 04H1389-Hs443 (SEQ ID NO:194) and 04H1389-Hm443 (SEQ ID NO:202) were generated, which have the same changes as KT461 and HT443 in the CH2 region. In addition, antibody heavy chains 04H1389-Ks473 (SEQ ID NO:195), 04H1389-Hs482 (SEQ ID NO:196), 04H1389-Ks481 (SEQ ID NO:197), 04H1389-Hs498 (SEQ ID NO:198), 04H1389-Km473 (SEQ ID NO:203), and 04H1389-Hm482 (SEQ ID NO:198) were generated. Genes 04H1389-Km481 (SEQ ID NO: 205) and 04H1389-Hm498 (SEQ ID NO: 206) were introduced into the constant regions Ks462, Hs445, Ks461, Hs443, Km462, Hm445, Km461, and Hm443 of the antibody heavy chain, which enhanced hemodynamics (N434A/Y436T/Q438R/S440E) and had 04H389 as a variable region. 04L1615-k0MT (SEQ ID NO: 190) was used as the light chain, and the desired heterodimer was produced. For comparison, a homodimer 04H1389-G1m/04L16150k0MT with the SEQ ID NO: 189 was generated. The interaction between the generated antibody and human FcγRs was analyzed using a Biacore T200. 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween 20 (pH 7.4) were used as run buffers, and the assay was performed at 25°C. For the sensor chips, a Series S SA (GE Healthcare) chip immobilized with CaptureSelect Human Fab-lambda Kinetics Biotin Conjugate (Thermo Fisher Scientific) was used. The target antibody was captured onto these chips, allowing each FcγR diluted in the run buffer to interact with them. The chip was regenerated using 10 mM M Lycine-HCl (pH 1.5) and 25 mM NaOH, and assays were performed by repeatedly capturing antibodies. The dissociation constant KD (mol/L) for each antibody against FcγR was calculated using Biacore T200 evaluation software 2.0. A 1:1 Langmuir binding model was used for FcγRIa and FcγRIIIa, and a steady-state affinity model was used for FcγRIIa. For FcγRIIb, the binding amount of FcγRIIb per unit volume of antibody was calculated by correcting the amount of FcγRIIb bound from the sensor map obtained from the assay with the amount of antibody captured onto the chip surface. To determine the binding with FcRn, a run buffer of 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween 20 (pH 6.0) was used, and the dissociation constant KD (mol/L) was calculated using a steady-state model (Table 28). In Table 28, the dissociation constants of FcγRIIIa are represented by "*", and the values are calculated using the steady-state affinity model.

Analysis of the binding of Fc region variants to human FcγR and FcRn

[Table 28]

Compared to 04H1389-G1m/04L1615-k0MT, all heterodimerized antibodies generated here exhibit enhanced binding activity to FcγRIIa and FcγRIIIa. Furthermore, compared to the parental antibodies before the introduction of the alteration, all 04H1389-Ks473/04L1615-k0MT//04H1389-Hs482/04L1615-k0MT, 04H1389-Ks481/04L1615-k0MT//04H1389-Hs498/04L1615-k0MT, and 04H1389-Km473/04L1615- k0MT//04H1389-Hm482/04L1615-k0MT and 04H1389-Km481/04L1615-k0MT//04H1389-Hm498/04L1615-k0MT (which have hemodynamic alterations introduced to enhance antibody binding) exhibit enhanced binding activity with human FcRn, and for FcγR binding activity, they have the same binding spectrum as the parental antibody.

Furthermore, the 04H1389-Hm441 (SEQ ID NO: 273) gene was generated, which contains 04H1389 (SEQ ID NO: 136) as a heavy chain variable region, has the same alterations as HT441 introduced into the CH2 region of the heavy chain, and has Y349C/366W used as an alteration for heterodimerization in the CH3 region. Conversely, the 04H1389-Hm451 (SEQ ID NO: 274) gene was generated, in which N434A/Y436T/Q438R/S440E was introduced, which is a combination of alterations that enhance binding to human FcRn and reduce binding to rheumatoid factor under acidic conditions. The amino acid sequences of antibody heavy chains Hm441 and Hm451 are shown in SEQ ID NO: 277 and 278, respectively. Heterodimerized antibodies were generated using 04H1389-Km473, 04H1389-Hm451, or 04H1389-Hm482 as the antibody heavy chain and 04L1305-k0MT as the antibody light chain. Table 29 shows the results of the interaction analysis between the generated antibodies and human FcRn and human FcγR.

Analysis of the binding of Fc region variants to human FcγR and FcRn

[Table 29]

Compared to 04H1389-G1m/04L1305-k0MT, which possesses the constant region of native human IgG1, the resulting heterodimerized antibodies 04H1389-Km473/04L1305-k0MT//04H1389-Hm451/04L1305-k0MT and 04H1389-Km473/04L1305-k0MT//04H1389-Hm482/04L1305-k0MT both showed enhanced binding to activated FcγRs FcγRIIa and FcγRIIIa. Furthermore, both antibodies showed enhanced binding to human FcRn compared to 04H1389-G1m/04L1305-k0MT.

Next, the constant-region variants with enhanced binding to FcγR discovered here will be compared with existing FcγR-binding enhanced variants. Genes of antibody heavy chains MDX10D1H-Kn125 (SEQ ID NO:217), MDX10D1H-Hl076 (SEQ ID NO:218), MDX10D1H-Kn462 (SEQ ID NO:219), MDX10D1H-Hl445 (SEQ ID NO:220), MDX10D1H-Kn461 (SEQ ID NO:221), and MDX10D1H-Hl443 (SEQ ID NO:222) (which contain MDX10D1H (SEQ ID NO:154) as the heavy chain variable region and the heavy chain constant region listed in Table 26) and MDX10D1H-G1m (SEQ ID NO:210) containing the CH2 region of natural human IgG1 were generated. The gene for the antibody heavy chain MDX10D1H-GASDIE (SEQ ID NO: 215) was generated, which has the altered G236A/S239D/I332E in the CH2 region as described in Mol. Cancer Ther., 2008, 7, 2517-2527, as a variant with enhanced binding to FcγRIIa. Furthermore, the gene for the antibody heavy chain MDX10D1H-GASDALIE (SEQ ID NO: 216) was also generated, which has the altered G236A/S239D/A330L/I332E in the CH2 region as described in J. Struct. Biol., 2016, 194, 78-89, as a variant with enhanced binding to FcγRIIIa. MDX10D1L-k0MT (SEQ ID NO: 211) was used as the antibody light chain, and the target antibody was generated. The binding activity of these antibodies to human FcγR was determined using the CaptureSelect Human Fab-kappa Kinetics Biotin Conjugate method described above (Table 30).

Analysis of the binding of Fc variants to human FcγR

[Table 30]

The values for “KD(M) of hFcγR” in the table represent the dissociation constant of each listed FcγR, and “Binding Amount” represents the amount of FcγRIIb bound per unit volume of antibody when FcγRIIb is allowed to interact at 1000 nM. “Relative values of G1m and hFcγRn with respect to KD” are obtained by dividing the KD value of each FcγR by the KD value of each variant using MDX10D1H-G1m/MDX10D1L-k0MT, and “Relative Binding Amount” represents the amount of FcγRIIb bound by each variant by the amount bound to MDX10D1H-G1m/MDX10D1L-k0MT.

Compared to existing FcγR binding enhancing antibodies, the generated heterodimers MDX10D1H-Kn125/MDX10D1H-Hl076/MDX10D1L-k0MT, MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT, and MDX10D1H-Kn461/MDX10D1H-Hl443/MDX10D1L-k0MT all exhibit enhanced binding to FcγRIIIa. Furthermore, compared to the existing FcγRIIa enhancing antibody MDX10D1H-GASDIE/MDX10D1L-k0MT, MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT shows approximately a 2-fold enhanced binding to FcγRIIaH.

(6-2) Evaluation of in vitro ADCC activity of multiple antibodies with altered constant regions

The hFcγRIIIaV ADCC Reporter Bioassay, Core Kit (Promega) was used for in vitro ADCC activity assay. 25 μL of hCTLA4-CHO cells, prepared to a concentration of 2 x ^10⁶ /mL using culture medium, were added to each well of a 96-well plate as target cells, using Assay Buffer (90% RPMI 1640, 10% FBS) as the culture medium. Next, 25 μL of each antibody solution diluted with Assay Buffer was added to final concentrations of 0, 0.001, 0.01, 0.1, and 1 μg/mL. Finally, 25 μL of Jurkat cells expressing hFcγRIIIaV (included in the kit), prepared to a concentration of 6 x ^10⁶ /mL using culture medium, was added as the effector cell solution, bringing the total volume to 75 μL. The plate was incubated overnight at 37°C in a 5% _CO₂ incubator. The plate was then incubated at room temperature for 15 minutes, and 75 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-glo reagent. The luminescence of each well was then measured using a plate reader. The fold induction was defined as the luminescence value of each well divided by the luminescence value of the antibody-free well, which was used as an indicator to evaluate the ADCC of each antibody. The results are shown in Figure 23. In the figure, the fold induction is expressed as relative luminescence units (RLU).

These results show that antibodies with altered Fc exhibit stronger ADCC activity against hCTLA4-CHO cells than those against the constant region of wild-type human IgG1.

(6-3) Evaluation of in vitro ADCP activity of multiple antibodies with altered constant regions

The hFcγRIIaH ADCP Reporter Bioassay, Core Kit (Promega) was used for in vitro ADCC activity assay. 25 μL of hCTLA4-CHO cells, prepared to a concentration of 2 x ^10⁶ /mL using culture medium, were added to each well of a 96-well plate as target cells, using Assay Buffer (4% Low IgG serum in RPMI 1640) as the culture medium. Next, 25 μL of each antibody solution diluted with Assay Buffer was added to final concentrations of 0, 0.001, 0.01, 0.1, and 1 μg/mL. Finally, 25 μL of Jurkat cells expressing hFcγRIIIaH, included in the kit, was added as the effector cell solution, bringing the total volume to 75 μL. The plate was incubated overnight at 37°C in a 5% _CO₂ incubator. The cell density of the Jurkat cells expressing hFcγRIIaH was 8.25 x ^10⁵ /mL. The plate was then incubated at room temperature for 15 minutes, and 75 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The fold induction was defined as the luminescence value of each well divided by the luminescence value of the antibody-free well, and was used as an indicator to evaluate the ADCP of each antibody. The results are shown in Figure 24. In the figure, the fold induction is expressed as relative luminescence units (RLU).

The results showed that antibodies with altered Fc exhibited stronger ADCP activity against hCTLA4-CHO cells than those against the ADCP activity of the wild-type human IgG1 constant region.

(6-4) Evaluation of in vitro ADCC activity of anti-CTLA4 switch antibodies with altered Fc

Anti-CTLA4 switch antibodies with altered Fc were produced (04H1654-Kn462/04L1610-lam1//04H1656-Hl445/04L1610-lam1, abbreviation: SW1610-ART6; 04H1654-Kn462/04L1612-lam1//04H1656-Hl445/04L1612-lam1, abbreviation: SW1612-ART6; and 04H1389-Kn462/04L1305-k0MT//04H1389-Hl445/04L1305-k0MT, abbreviation: SW1389-ART6).

In the SW1610-ART6 antibody, one heavy chain variable region 04H1654 (SEQ ID NO:35) is linked to the human heavy chain constant region Kn462 (SEQ ID NO:43), another heavy chain variable region 04H1656 (SEQ ID NO:37) is linked to the human heavy chain constant region Hl445 (SEQ ID NO:44), which is a constant region, and for the light chain variable region 04L1610 (SEQ ID NO:39), the wild-type human light chain constant region lam1 (SEQ ID NO:53) is used. The antibody is expressed and purified using methods known to those skilled in the art.

In the SW1612-ART6 antibody, one heavy chain variable region 04H1654 (SEQ ID NO:35) is linked to the human heavy chain constant region Kn462 (SEQ ID NO:43), another heavy chain variable region 04H1656 (SEQ ID NO:37) is linked to the human heavy chain constant region Hl445 (SEQ ID NO:44), which is a constant region, and for the light chain variable region 04L1612 (SEQ ID NO:40), the wild-type human light chain constant region lam1 (SEQ ID NO:53) is used. The antibody is expressed and purified using methods known to those skilled in the art.

In the SW1389-ART6 antibody, two heavy chain variable regions, 04H1389 (SEQ ID NO:29), are linked to the human heavy chain constant region Kn462 (SEQ ID NO:43) and the human heavy chain constant region Hl445 (SEQ ID NO:44), respectively, and further, for the light chain variable region 04L1305 (SEQ ID NO:30), the wild-type human light chain constant region k0MT (SEQ ID NO:33) is used. The antibody is expressed and purified using methods known to those skilled in the art.

The hFcγRIIIaV ADCC Reporter Bioassay, Core Kit (Promega) was used for in vitro ADCC activity assays. 12.5 μL of hCTLA4-CHO cells, prepared to a concentration of 2 x ^10⁶ /mL with culture medium, were added to each well of a 96-well plate as target cells, using Assay Buffer (4% Low IgG serum in RPMI 1640) as the culture medium. Next, ATP solutions diluted with Assay Buffer to final concentrations of 0 and 100 μM were added sequentially, along with antibody solutions of SW1389-ART6, SW1610-ART6, and SW1612-ART6 diluted with Assay Buffer to final concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μg/mL. Finally, 25 μL of Jurkat cells expressing hFcγRIIIaV (included in the kit), prepared to a concentration of 3 x ^10⁶ /mL with culture medium, were added as the effector cell solution, bringing the total volume to 75 μL. The plate was incubated overnight at 37°C in a 5% _CO2 incubator. Then, the plate was incubated at room temperature for 15 minutes, and 75 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The fold induction was defined as the luminescence value of each well divided by the luminescence value of the antibody-free well, which was used as an indicator to evaluate the ADCC of each antibody. The results are shown in Figures 25 (SW1389-ART6), 26 (SW1610-ART6), and 27 (SW1612-ART6). In the figures, the fold induction is expressed as relative luminescence units (RLU).

These results confirm that the ADCC activity of anti-CTLA4 switch antibodies with altered Fc differs between the presence and absence of ATP in hCTLA4-CHO cells, and that they exhibit ATP-dependent cytotoxicity in hCTLA4-CHO cells.

(6-5) In vitro neutralizing activity assessment of anti-CTLA4 switch antibody

As a modified anti-CTLA4 switch antibody, a human antibody with variable regions of SW1389, SW1610, SW1612 and SW1615 was generated.

For the SW1389 antibody, 04H1389 (SEQ ID NO: 29) was used as the heavy chain variable region, and 04L1305 (SEQ ID NO: 30) was used as the light chain variable region. After the variable regions were linked to the human constant regions, the antibodies were expressed and purified using methods known to those skilled in the art.

For the SW1610 antibody, 04H1654 (SEQ ID NO: 35) and 04H1656 (SEQ ID NO: 37) were used as the heavy chain variable region, and 04L1610 (SEQ ID NO: 39) was used as the light chain variable region. After the variable regions were linked to the human constant region, the antibody was expressed and purified using methods known to those skilled in the art.

For the SW1612 antibody, 04H1654 (SEQ ID NO: 35) and 04H1656 (SEQ ID NO: 37) were used as the heavy chain variable region, and 04L1612 (SEQ ID NO: 40) was used as the light chain variable region. After the variable regions were linked to the human constant region, the antibody was expressed and purified using methods known to those skilled in the art.

For the SW1615 antibody, 04H1389 (SEQ ID NO: 29) was used as the heavy chain variable region, and 04L1615 (SEQ ID NO: 34) was used as the light chain variable region. After the variable regions were linked to the human constant regions, the antibodies were expressed and purified using methods known to those skilled in the art.

The CTLA-4 Blockade Bioassay (Promega) was used to determine in vitro neutralizing activity. To each well of a 96-well plate, add 25 μL of aAPC-Raji cells (prepared to a concentration of 1 x ^10⁶ /mL with the provided kit) as target cells, and add Assay Buffer (10% FBS in RPMI 1640) as the culture medium. Next, sequentially add ATP solutions diluted with Assay Buffer to final concentrations of 0 and 100 μM, followed by antibody solutions containing the variable regions SW1389, SW1610, SW1612, and SW1615, diluted with Assay Buffer to final concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μg/mL. Finally, add 25 μL of IL2-luc2-CTLA4-Jurkat cells (prepared to a concentration of 2 x ^10⁶ /mL with the provided kit) as the effector cell solution, bringing the total volume to 75 μL. The plate was incubated overnight at 37°C in a 5% _CO2 incubator. Then, the plate was incubated at room temperature for 15 minutes, and 75 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The fold induction was defined as the luminescence value of each well divided by the luminescence value of the antibody-free well, which was used as an indicator to assess the neutralizing activity of each antibody. The results are shown in Figures 28 (SW1389), 29 (SW1610), 30 (SW1612), and 31 (SW1615). In the figures, the fold induction is expressed as relative luminescence units (RLU).

These results confirm that the neutralizing activity of the anti-CTLA4 switch antibody on hCTLA4-expressing cells differs in the presence and absence of ATP, and that ATP-dependent neutralizing activity exists.

(6-6) Evaluation of the in vitro cytotoxic activity of anti-CTLA4 switch antibody against CTLA4-positive regulatory T cells

Anti-CTLA4 switch antibodies with altered Fc were produced (04H1654-KT473/04L1610-lam1//04H1656-HT451/04L1610-lam1, abbreviated: SW1610-ART5+ACT1; 04H1654-KT473/04L1610-lam1//04H1656-HT482/04L1610-lam1, abbreviated: SW1610). -ART6+ACT1; 04H1389-Km473/04L1305-k0MT//04H1389-Hm451/04L1305-k0MT, abbreviation: SW1389-ART5+ACT1; 04H1389-Km473/04L1305-k0MT//04H1389-Hm482/04L1305-k0MT, abbreviation: SW1389-ART6+ACT1).

For the SW1610-ART5+ACT1 antibody, 04H1654-KT473 (SEQ ID NO:184) was used as one heavy chain, 04H1656-HT451 (SEQ ID NO:272) was used as another heavy chain, and 04L1610-lam1 (SEQ ID NO:161) was used as the light chain. The antibody was expressed and purified using methods known to those skilled in the art.

For the SW1610-ART6+ACT1 antibody, 04H1654-KT473 (SEQ ID NO:184) was used as one heavy chain, 04H1656-HT482 (SEQ ID NO:185) was used as another heavy chain, and 04L1610-lam1 (SEQ ID NO:161) was used as the light chain. The antibody was expressed and purified using methods known to those skilled in the art.

For the SW1389-ART5+ACT1 antibody, 04H1389-Km473 (SEQ ID NO: 203) is used as one heavy chain, 04H1389-Hm451 (SEQ ID NO: 274) is used as another heavy chain, and 04L1305-k0MT (SEQ ID NO: 274) (SEQ ID NO: 275) is used as the light chain. The antibody is expressed and purified using methods known to those skilled in the art.

For the SW1389-ART6+ACT1 antibody, 04H1389-Km473 (SEQ ID NO:203) is used as one heavy chain, 04H1389-Hm482 (SEQ ID NO:204) is used as another heavy chain, and 04L1305-k0MT (SEQ ID NO:275) is used as the light chain. The antibody is expressed and purified using methods known to those skilled in the art.

The in vitro cytotoxic activity of the generated anti-CTLA4 switch antibody with altered Fc against CTLA4-positive regulatory T cells (CD3 ⁺ CD4 ⁺ CD25 ⁺ CD45RA ^- CTLA4 ⁺ ) was evaluated. First, human PBMCs (CTL Cryopreserved Human PBMCs, CTL) were freeze-thawed and suspended in 50 U/ml interleukin-2 (IL-2)/RPMI/10% FBS at a cell density of ² x 10⁶ cells/mL and cultured at 37°C for 4 days in a 5% _CO₂ incubator. After 4 days, cells were harvested and washed twice with RPMI/10% FBS, then seeded at 100 μL into each well of a 96-well U-shaped plate (8 x ^10⁵ cells/well or ⁵ x 10⁵ cells/well). 25 μL of KLH-G1m solution, adjusted to 8 mg/ml with RPMI/10% FBS, was added to each well. Then, 25 μL of each antibody solution prepared in RPMI/10% FBS at each well was added to each well of a 96-well U-shaped plate (0, 2.4, 8, 24, and 80 μg/mL, or 0, 0.8, 8, 80, and 800 μg/mL). Additionally, 50 μL of ATP solution adjusted to 0 or 400 μM in RPMI/10% FBS was added to fully suspend the mixture, and then the plate was incubated at 37°C for 6 hours in a _CO2 incubator (with 5 μL of ATP solution adjusted to 0 or 4000 μM added every 2 hours, for a total of 2 times). Six hours later, PBMCs were collected, washed twice with auto MACS Rinsing Solution (Miltenyi), reacted with the following antibodies, and the present immune cell fractions were analyzed by FACS analysis: reagents for determining viability (Biolegend, Zombie Aqua), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RPA-T4), anti-CD8 antibody (BD, clone: SK1), anti-CD45RA antibody (Biolegend, clone: HI100), anti-CD25 antibody (BD, clone: 2A3), anti-CD16 antibody (Biolegend, clone: 3G8), anti-CD56 antibody (Biolegend, clone: HCD56), and anti-CTLA4 antibody (Biolegend, clone: BNI3). FACS analysis was performed using a BD LSR Fortessa X-20 (BD). The proportion of CTLA4-positive regulatory T cells in viable cells was calculated at each antibody concentration. The relative value (with 0% as the value) was defined as the survival rate (%) of CTLA4-positive regulatory T cells, and this was used as an indicator to assess the cytotoxic activity of each antibody. The results are shown in Figures 32 (SW1389-ART5+ACT1), 33 (SW1389-ART6+ACT1), 34 (SW1610-ART5+ACT1), and 35 (SW1610-ART6+ACT1).

These results confirm that the cytotoxic activity of anti-CTLA4 switch antibodies with altered Fc against CTLA4-positive regulatory T cells differs in the presence and absence of ATP, and that they exhibit ATP-dependent cytotoxic activity against CTLA4-positive regulatory T cells.

Although the invention has been described in detail with reference to illustrations and embodiments for purposes of clarity, these descriptions and embodiments should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited throughout this document are expressly incorporated herein by reference.

[Industrial Applicability]

The anti-CTLA-4 antibody and its method of use disclosed herein can be used for the development, production, supply, and use of drugs with fewer side effects, because although the antibody of this disclosure has immune cell activating activity, cytotoxic activity, and/or antitumor activity, it has very low effects on non-tumor tissues such as normal tissues. Furthermore, the polypeptide containing the variant Fc region disclosed herein, as well as its production and method of use, can be used for the development, production, supply, and use of such drugs.

sequence list

<110> Chugai Pharmaceutical Co., Ltd.

<120> Anti-CTLA-4 antibodies and their uses

<130> C1-A1927P

<160> 278

<170> PatentIn version 3.5

<210> 1

<211> 188

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4

<400> 1

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe His His His Gly Gly Gly Gly Ser Gly Leu Asn

165 170 175

Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

180 185

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primers

<400> 2

cgcaacgcaa ttaatgtgag 20

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primers

<400> 3

tgagttccac gacaccgtca c 21

<210> 4

<211> 122

<212> PRT

<213> Homo sapiens

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Ile Lys Asn Lys Arg Asn Trp Val Leu Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 5

<211> 110

<212> PRT

<213> Homo sapiens

<400> 5

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Ala Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Gln Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Leu Tyr Thr Thr Arg

85 90 95

Pro Gln Leu Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 6

<211> 122

<212> PRT

<213> Homo sapiens

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Thr Tyr Ala His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Gly Arg Gly His Leu Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

<211> 110

<212> PRT

<213> Homo sapiens

<400> 7

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Thr Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Gln Gly Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Val Ser Gly Asp

85 90 95

Phe His Val Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 8

<211> 122

<212> PRT

<213> Homo sapiens

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Arg Tyr Ala Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Ile Lys Asn His Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 110

<212> PRT

<213> Homo sapiens

<400> 9

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Val Ser Thr

85 90 95

His Ser Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 10

<211> 122

<212> PRT

<213> Homo sapiens

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Gly Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 110

<212> PRT

<213> Homo sapiens

<400> 11

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 12

<211> 122

<212> PRT

<213> Homo sapiens

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ile Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Leu Asn His Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 110

<212> PRT

<213> Homo sapiens

<400> 13

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Ala Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Ser Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Leu Tyr Arg Tyr Ala

85 90 95

Gln Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 14

<211> 122

<212> PRT

<213> Homo sapiens

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Gly His Ala His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Arg Lys Lys Lys Arg Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 110

<212> PRT

<213> Homo sapiens

<400> 15

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Gln Asn Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Asn Arg Ala Ala

85 90 95

Arg Ser Val Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 16

<211> 116

<212> PRT

<213> Brown rat

<400> 16

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr Gly Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 17

<211> 113

<212> PRT

<213> Brown rat

<400> 17

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser

20 25 30

Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg His Thr Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 18

<211> 330

<212> PRT

<213> House Mouse

<400> 18

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Asp Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 19

<211> 107

<212> PRT

<213> House Mouse

<400> 19

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 20

<211> 122

<212> PRT

<213> Homo sapiens

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Arg

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 110

<212> PRT

<213> Homo sapiens

<400> 21

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 22

<211> 106

<212> PRT

<213> House Mouse

<400> 22

Gly Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp

20 25 30

Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro

35 40 45

Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu

65 70 75 80

Arg His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val

85 90 95

Glu Lys Ser Leu Ser Arg Ala Asp Cys Ser

100 105

<210> 23

<211> 122

<212> PRT

<213> Homo sapiens

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 111

<212> PRT

<213> Homo sapiens

<400> 24

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp

20 25 30

Tyr Asn Trp Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Tyr Thr Ser Ser Lys Pro Glu Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Thr Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 25

<211> 356

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4

<400> 25

Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His

1 5 10 15

Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu

35 40 45

Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp

50 55 60

Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Phe Asp

115 120 125

Ile Glu Gly Arg Met Asp Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

130 135 140

Glu Val Ser Asp Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

145 150 155 160

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

165 170 175

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

180 185 190

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

195 200 205

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

210 215 220

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

225 230 235 240

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

245 250 255

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

260 265 270

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

275 280 285

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

290 295 300

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

305 310 315 320

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

325 330 335

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

340 345 350

Ser Pro Gly Lys

355

<210> 26

<211> 118

<212> PRT

<213> Homo sapiens

<400> 26

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 27

<211> 108

<212> PRT

<213> Homo sapiens

<400> 27

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 28

<211> 127

<212> PRT

<213> Homo sapiens

<400> 28

Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Phe

115 120 125

<210> 29

<211> 122

<212> PRT

<213> Homo sapiens

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 30

<211> 111

<212> PRT

<213> Homo sapiens

<400> 30

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Asp Gly Pro Ser Thr Gly Val Gly Asp

20 25 30

Tyr Asn Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Met Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 31

<211> 328

<212> PRT

<213> Homo sapiens

<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 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> 32

<211> 328

<212> PRT

<213> Homo sapiens

<400> 32

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> 33

<211> 107

<212> PRT

<213> Homo sapiens

<400> 33

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> 34

<211> 111

<212> PRT

<213> Homo sapiens

<400> 34

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Gln Gly Pro Ser Thr Gly Val Gly Asp

20 25 30

Tyr Asn Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Met Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 35

<211> 122

<212> PRT

<213> Homo sapiens

<400> 35

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 36

<211> 330

<212> PRT

<213> House Mouse

<400> 36

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Asp Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Arg Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 37

<211> 122

<212> PRT

<213> Homo sapiens

<400> 37

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 38

<211> 330

<212> PRT

<213> House Mouse

<400> 38

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Asp Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Glu Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 39

<211> 110

<212> PRT

<213> Homo sapiens

<400> 39

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Trp Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 40

<211> 110

<212> PRT

<213> Homo sapiens

<400> 40

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Thr Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 41

<211> 328

<212> PRT

<213> Homo sapiens

<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 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 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> 42

<211> 328

<212> PRT

<213> Homo sapiens

<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 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> 43

<211> 328

<212> PRT

<213> Homo sapiens

<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 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> 44

<211> 328

<212> PRT

<213> Homo sapiens

<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 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> 45

<211> 328

<212> PRT

<213> Homo sapiens

<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

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> 46

<211> 328

<212> PRT

<213> Homo sapiens

<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 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> 47

<211> 122

<212> PRT

<213> Homo sapiens

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 110

<212> PRT

<213> Homo sapiens

<400> 48

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 49

<211> 168

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4

<400> 49

Met Ala Cys Leu Gly Leu Arg Arg Tyr Lys Ala Gln Leu Gln Leu Pro

1 5 10 15

Ser Arg Thr Trp Pro Phe Val Ala Leu Leu Thr Leu Leu Phe Ile Pro

20 25 30

Val Phe Ser Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala

35 40 45

Ser Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His

50 55 60

Asn Thr Asp Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln

65 70 75 80

Met Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly

85 90 95

Phe Leu Asp Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe His His His

165

<210> 50

<211> 188

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4

<400> 50

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Arg Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Arg Pro Tyr Thr Leu Leu Phe Ser Leu Leu Phe Ile Pro

20 25 30

Val Phe Ser Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Asn Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Met Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe His His His Gly Gly Gly Gly Ser Gly Leu Asn

165 170 175

Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

180 185

<210> 51

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 51

Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Tyr Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 52

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 52

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys

100 105

<210> 53

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> CL

<400> 53

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 54

<400> 54

000

<210> 55

<400> 55

000

<210> 56

<400> 56

000

<210> 57

<400> 57

000

<210> 58

<400> 58

000

<210> 59

<400> 59

000

<210> 60

<400> 60

000

<210> 61

<400> 61

000

<210> 62

<400> 62

000

<210> 63

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> CL

<400> 63

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> 64

<400> 64

000

<210> 65

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 65

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

Arg 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 Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 66

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 66

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

Arg 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

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 67

<400> 67

000

<210> 68

<400> 68

000

<210> 69

<400> 69

000

<210> 70

<400> 70

000

<210> 71

<400> 71

000

<210> 72

<400> 72

000

<210> 73

<400> 73

000

<210> 74

<400> 74

000

<210> 75

<400> 75

000

<210> 76

<400> 76

000

<210> 77

<400> 77

000

<210> 78

<400> 78

000

<210> 79

<400> 79

000

<210> 80

<400> 80

000

<210> 81

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 81

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

Arg 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 Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 82

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 82

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 Glu Glu

225 230 235 240

Met 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> 83

<211> 122

<212> PRT

<213> Homo sapiens

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Arg

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 84

<211> 122

<212> PRT

<213> Homo sapiens

<400> 84

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Arg

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 85

<211> 122

<212> PRT

<213> Homo sapiens

<400> 85

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 86

<211> 122

<212> PRT

<213> Homo sapiens

<400> 86

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 87

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> CL

<400> 87

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 88

<211> 110

<212> PRT

<213> Homo sapiens

<400> 88

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 89

<211> 110

<212> PRT

<213> Homo sapiens

<400> 89

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 90

<211> 110

<212> PRT

<213> Homo sapiens

<400> 90

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 91

<211> 110

<212> PRT

<213> Homo sapiens

<400> 91

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Trp Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 92

<211> 110

<212> PRT

<213> Homo sapiens

<400> 92

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Trp Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 93

<211> 110

<212> PRT

<213> Homo sapiens

<400> 93

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 94

<211> 110

<212> PRT

<213> Homo sapiens

<400> 94

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 95

<211> 111

<212> PRT

<213> Homo sapiens

<400> 95

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Asp Gly Pro Ser Thr Gly Val Gly Asp

20 25 30

Tyr Asn Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Met Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 96

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> CL

<400> 96

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> 97

<211> 110

<212> PRT

<213> Homo sapiens

<400> 97

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 98

<211> 122

<212> PRT

<213> Homo sapiens

<400> 98

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 99

<211> 110

<212> PRT

<213> Homo sapiens

<400> 99

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 100

<211> 5

<212> PRT

<213> Homo sapiens

<400> 100

Ser His Thr Met Asn

1 5

<210> 101

<211> 17

<212> PRT

<213> Homo sapiens

<400> 101

Ser Ile Ser Ser Arg Ser Gly Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 102

<211> 13

<212> PRT

<213> Homo sapiens

<400> 102

Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr

1 5 10

<210> 103

<211> 5

<212> PRT

<213> Homo sapiens

<400> 103

Ser Ala Thr Met Asn

1 5

<210> 104

<211> 17

<212> PRT

<213> Homo sapiens

<400> 104

Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 105

<211> 5

<212> PRT

<213> Homo sapiens

<400> 105

Ser Arg Thr Met Asn

1 5

<210> 106

<211> 17

<212> PRT

<213> Homo sapiens

<400> 106

Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Glu Ser Val Lys

1 5 10 15

Gly

<210> 107

<211> 5

<212> PRT

<213> Homo sapiens

<400> 107

Ser Lys Thr Met Asn

1 5

<210> 108

<211> 17

<212> PRT

<213> Homo sapiens

<400> 108

Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Arg Ser Val Lys

1 5 10 15

Gly

<210> 109

<211> 17

<212> PRT

<213> Homo sapiens

<400> 109

Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val Lys

1 5 10 15

Gly

<210> 110

<211> 17

<212> PRT

<213> Homo sapiens

<400> 110

Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val Lys

1 5 10 15

Gly

<210> 111

<211> 17

<212> PRT

<213> Homo sapiens

<400> 111

Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val Arg

1 5 10 15

Gly

<210> 112

<211> 17

<212> PRT

<213> Homo sapiens

<400> 112

Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val Arg

1 5 10 15

Gly

<210> 113

<211> 14

<212> PRT

<213> Homo sapiens

<400> 113

Thr Gly Thr Ser Thr Asp Val Gly Asp Tyr Asn Tyr Val Ser

1 5 10

<210> 114

<211> 7

<212> PRT

<213> Homo sapiens

<400> 114

Glu Thr Ser Lys Lys Pro Ser

1 5

<210> 115

<211> 10

<212> PRT

<213> Homo sapiens

<400> 115

Ser Thr Tyr Ala Ala Pro Leu Gly Pro Met

1 5 10

<210> 116

<211> 14

<212> PRT

<213> Homo sapiens

<400> 116

Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr Asn Tyr Val Ser

1 5 10

<210> 117

<211> 7

<212> PRT

<213> Homo sapiens

<400> 117

Phe Thr Ser Lys Lys Pro Ser

1 5

<210> 118

<211> 14

<212> PRT

<213> Homo sapiens

<400> 118

Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr Asn Tyr Val His

1 5 10

<210> 119

<211> 14

<212> PRT

<213> Homo sapiens

<400> 119

Asp Gly Pro Ser Thr Gly Val Gly Asp Tyr Asn Tyr Val Ser

1 5 10

<210> 120

<211> 14

<212> PRT

<213> Homo sapiens

<400> 120

Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr Asn Trp Val Ser

1 5 10

<210> 121

<211> 14

<212> PRT

<213> Homo sapiens

<400> 121

Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr Asn Trp Val His

1 5 10

<210> 122

<211> 14

<212> PRT

<213> Homo sapiens

<400> 122

Asp Gly Pro Ser Thr Gly Val Gly Asp Tyr Asn Tyr Val His

1 5 10

<210> 123

<211> 7

<212> PRT

<213> Homo sapiens

<400> 123

Tyr Thr Ser Ser Lys Pro Glu

1 5

<210> 124

<211> 14

<212> PRT

<213> Homo sapiens

<400> 124

Gln Gly Pro Ser Thr Gly Val Gly Asp Tyr Asn Trp Val His

1 5 10

<210> 125

<211> 7

<212> PRT

<213> Homo sapiens

<400> 125

Phe Thr Ser Lys Lys Pro Lys

1 5

<210> 126

<211> 14

<212> PRT

<213> Homo sapiens

<400> 126

Gln Gly Pro Ser Thr Gly Val Gly Asp Tyr Thr Trp Val His

1 5 10

<210> 127

<211> 7

<212> PRT

<213> Homo sapiens

<400> 127

Phe Thr Ile Lys Lys Pro Lys

1 5

<210> 128

<211> 14

<212> PRT

<213> Homo sapiens

<400> 128

Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr Asn Trp Val His

1 5 10

<210> 129

<211> 14

<212> PRT

<213> Homo sapiens

<400> 129

Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr Thr Tyr Val His

1 5 10

<210> 130

<211> 14

<212> PRT

<213> Homo sapiens

<400> 130

Gln Gly Pro Ser Thr Gly Val Gly Asp Tyr Asn Tyr Val His

1 5 10

<210> 131

<211> 374

<212> PRT

<213> Homo sapiens

<400> 131

Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln

1 5 10 15

Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser

20 25 30

Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu

35 40 45

Pro Gly Ser Ser Ser Thr Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln

50 55 60

Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser

65 70 75 80

Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile

85 90 95

Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg

100 105 110

Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys

115 120 125

Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe

130 135 140

Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile

145 150 155 160

Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr

165 170 175

Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro

180 185 190

Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val

195 200 205

Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln

210 215 220

Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn

225 230 235 240

Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly

245 250 255

Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg

260 265 270

Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro

275 280 285

Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu

290 295 300

Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys

305 310 315 320

Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys

325 330 335

Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys

340 345 350

Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys

355 360 365

Glu Pro Gln Gly Ala Thr

370

<210> 132

<211> 316

<212> PRT

<213> Homo sapiens

<400> 132

Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu

1 5 10 15

Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp

20 25 30

Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp

35 40 45

Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala

50 55 60

Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu

65 70 75 80

Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn

85 90 95

Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp

100 105 110

Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro

115 120 125

His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser

130 135 140

Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys

145 150 155 160

Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala

165 170 175

Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr

180 185 190

Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser

195 200 205

Met Gly Ser Ser Ser Pro Met Gly Val Ile Val Ala Val Val Ile Ala

210 215 220

Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys

225 230 235 240

Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala

245 250 255

Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln

260 265 270

Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met

275 280 285

Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu

290 295 300

Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn

305 310 315

<210> 133

<211> 10

<212> PRT

<213> Homo sapiens

<400> 133

Gln Thr Tyr Ala Ala Pro Leu Gly Pro Met

1 5 10

<210> 134

<211> 111

<212> PRT

<213> Homo sapiens

<400> 134

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp

20 25 30

Tyr Asn Trp Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Tyr Thr Ser Ser Lys Pro Glu Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 135

<211> 122

<212> PRT

<213> Homo sapiens

<400> 135

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 136

<211> 122

<212> PRT

<213> Homo sapiens

<400> 136

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 137

<211> 122

<212> PRT

<213> Homo sapiens

<400> 137

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 138

<211> 122

<212> PRT

<213> Homo sapiens

<400> 138

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 139

<211> 122

<212> PRT

<213> Homo sapiens

<400> 139

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 140

<211> 122

<212> PRT

<213> Homo sapiens

<400> 140

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 141

<211> 122

<212> PRT

<213> Homo sapiens

<400> 141

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 142

<211> 317

<212> PRT

<213> Homo sapiens

<400> 142

Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu

1 5 10 15

Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp

20 25 30

Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro

35 40 45

Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly

50 55 60

Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn

65 70 75 80

Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn

85 90 95

Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser

100 105 110

Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr

115 120 125

Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His

130 135 140

Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly

145 150 155 160

Lys Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln

165 170 175

Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly

180 185 190

Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro

195 200 205

Ser Met Gly Ser Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Ile

210 215 220

Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr

225 230 235 240

Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala

245 250 255

Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg

260 265 270

Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr

275 280 285

Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr

290 295 300

Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn

305 310 315

<210> 143

<211> 218

<212> PRT

<213> Homo sapiens

<400> 143

Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu

1 5 10 15

Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp

20 25 30

Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro

35 40 45

Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly

50 55 60

Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn

65 70 75 80

Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn

85 90 95

Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser

100 105 110

Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr

115 120 125

Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His

130 135 140

Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly

145 150 155 160

Lys Ser Gln Lys Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln

165 170 175

Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly

180 185 190

Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro

195 200 205

Ser Met Gly Ser Ser Ser Ser Pro Met Gly Ile

210 215

<210> 144

<211> 110

<212> PRT

<213> Homo sapiens

<400> 144

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Gln Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Trp Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Lys Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 145

<211> 110

<212> PRT

<213> Homo sapiens

<400> 145

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Gln Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Thr Trp Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ile Lys Lys Pro Lys Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 146

<211> 110

<212> PRT

<213> Homo sapiens

<400> 146

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Trp Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 147

<211> 110

<212> PRT

<213> Homo sapiens

<400> 147

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Thr Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 148

<211> 110

<212> PRT

<213> Homo sapiens

<400> 148

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Gln Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Trp Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 149

<211> 111

<212> PRT

<213> Homo sapiens

<400> 149

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Gln Gly Pro Ser Thr Gly Val Gly Asp

20 25 30

Tyr Asn Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Met Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 150

<211> 218

<212> PRT

<213> Homo sapiens

<400> 150

Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu

1 5 10 15

Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp

20 25 30

Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro

35 40 45

Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly

50 55 60

Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn

65 70 75 80

Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn

85 90 95

Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser

100 105 110

Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr

115 120 125

Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His

130 135 140

Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly

145 150 155 160

Lys Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln

165 170 175

Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly

180 185 190

Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro

195 200 205

Ser Met Gly Ser Ser Ser Ser Pro Met Gly Ile

210 215

<210> 151

<211> 291

<212> PRT

<213> Homo sapiens

<400> 151

Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp

1 5 10 15

Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr

20 25 30

Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro

35 40 45

Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu

50 55 60

Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp

65 70 75 80

Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln

85 90 95

Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr

100 105 110

Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val

115 120 125

Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu

130 135 140

Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu

145 150 155 160

Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg

165 170 175

Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly

180 185 190

Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys

195 200 205

Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile

210 215 220

Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala

225 230 235 240

Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Pro

245 250 255

Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn Thr Ile Thr

260 265 270

Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln

275 280 285

Asn Arg Ile

290

<210> 152

<211> 13

<212> PRT

<213> Homo sapiens

<400> 152

Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr

1 5 10

<210> 153

<211> 10

<212> PRT

<213> Homo sapiens

<400> 153

Gln Thr Tyr Ala Ala Pro Leu Gly Pro Thr

1 5 10

<210> 154

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 154

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 155

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 155

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 156

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 156

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr Gly Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 157

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 157

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser

20 25 30

Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg His Thr Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 158

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 158

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> 159

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> CL

<400> 159

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> 160

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 160

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 161

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> Lch

<400> 161

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Asn Trp Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 162

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 162

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 163

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 163

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Met Pro

325 330 335

Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 164

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 164

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 165

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 165

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 166

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 166

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro

325 330 335

Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 167

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 167

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 168

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 168

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 169

<211> 310

<212> PRT

<213> Homo sapiens

<400> 169

Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp

1 5 10 15

Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr

20 25 30

Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro

35 40 45

Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu

50 55 60

Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp

65 70 75 80

Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln

85 90 95

Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr

100 105 110

Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val

115 120 125

Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu

130 135 140

Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu

145 150 155 160

Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg

165 170 175

Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly

180 185 190

Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys

195 200 205

Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile

210 215 220

Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala

225 230 235 240

Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Leu Pro

245 250 255

Gly Tyr Pro Glu Cys Arg Glu Met Gly Glu Thr Leu Pro Glu Lys Pro

260 265 270

Ala Asn Pro Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn

275 280 285

Thr Ile Thr Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro

290 295 300

Asp Asp Gln Asn Arg Ile

305 310

<210> 170

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 170

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 171

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 171

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro

325 330 335

Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 172

<211> 217

<212> PRT

<213> Homo sapiens

<400> 172

Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp

1 5 10 15

Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr

20 25 30

Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro

35 40 45

Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu

50 55 60

Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp

65 70 75 80

Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln

85 90 95

Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr

100 105 110

Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val

115 120 125

Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu

130 135 140

Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu

145 150 155 160

Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg

165 170 175

Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly

180 185 190

Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys

195 200 205

Pro Val Thr Ile Thr Val Gln Ala Pro

210 215

<210> 173

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 173

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 174

<211> 254

<212> PRT

<213> Homo sapiens

<400> 174

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ser Val Ser Thr Ile Ser Phe Phe Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp

225 230 235 240

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys

245 250

<210> 175

<211> 254

<212> PRT

<213> Homo sapiens

<400> 175

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp

225 230 235 240

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys

245 250

<210> 176

<211> 208

<212> PRT

<213> Homo sapiens

<400> 176

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

195 200 205

<210> 177

<211> 208

<212> PRT

<213> Homo sapiens

<400> 177

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

195 200 205

<210> 178

<211> 233

<212> PRT

<213> Homo sapiens

<400> 178

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile

225 230

<210> 179

<211> 365

<212> PRT

<213> Homo sapiens

<400> 179

Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe

1 5 10 15

Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr

20 25 30

His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp

35 40 45

Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu

50 55 60

Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val

65 70 75 80

Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys

85 90 95

Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Gly Lys Gly Pro Tyr Thr

100 105 110

Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val

115 120 125

Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp

130 135 140

Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile

145 150 155 160

Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr

165 170 175

Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg

180 185 190

Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys

195 200 205

Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe

210 215 220

Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu

225 230 235 240

Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser

245 250 255

Phe His Ala Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His

260 265 270

Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val

275 280 285

Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val

290 295 300

Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu

305 310 315 320

Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg

325 330 335

Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp

340 345 350

Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala

355 360 365

<210> 180

<211> 119

<212> PRT

<213> Homo sapiens

<400> 180

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

20 25 30

His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

35 40 45

Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

50 55 60

Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

65 70 75 80

Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

85 90 95

Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

100 105 110

Val Lys Trp Asp Arg Asp Met

115

<210> 181

<211> 580

<212> PRT

<213> Homo sapiens

<400> 181

Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu

1 5 10 15

Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp

20 25 30

Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly

35 40 45

Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val

50 55 60

Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys

65 70 75 80

Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala

85 90 95

Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln

100 105 110

Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala

115 120 125

Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe

130 135 140

Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp

145 150 155 160

Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro

165 170 175

Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu

180 185 190

Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe

195 200 205

Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln

210 215 220

Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile

225 230 235 240

Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu

245 250 255

Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys

260 265 270

Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly

275 280 285

Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu

290 295 300

Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu

305 310 315 320

Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys

325 330 335

Asn Ala Gly Lys Leu Thr Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser

340 345 350

Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile

355 360 365

Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Thr Leu

370 375 380

Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser

385 390 395 400

Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala

405 410 415

Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr

420 425 430

Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His

435 440 445

Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp

450 455 460

Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys

465 470 475 480

Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly

485 490 495

Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly

500 505 510

Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr

515 520 525

Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro

530 535 540

Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser

545 550 555 560

Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe

565 570 575

Phe Leu Val His

580

<210> 182

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 182

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 183

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 183

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 184

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 184

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 185

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 185

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro

325 330 335

Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Glu Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 186

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 186

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Arg Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 187

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 187

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Glu Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 188

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> Lch

<400> 188

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Glu Gly Pro Ser Thr Gly Val Gly Asp Tyr

20 25 30

Thr Tyr Val His Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 189

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 189

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 190

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> Lch

<400> 190

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Gln Gly Pro Ser Thr Gly Val Gly Asp

20 25 30

Tyr Asn Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Met Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 191

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 191

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 192

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 192

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro

325 330 335

Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 193

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 193

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 194

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 194

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 195

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 195

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 196

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 196

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro

325 330 335

Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 197

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 197

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 198

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 198

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 199

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 199

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 200

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 200

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro

325 330 335

Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 201

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 201

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 202

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 202

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 203

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 203

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 204

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 204

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro

325 330 335

Asp Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 205

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 205

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln

225 230 235 240

Trp Gly Pro Met Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Asp Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 206

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 206

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Glu Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 207

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 207

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

Arg 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

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 208

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> Lch

<400> 208

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Asp Gly Pro Ser Thr Asp Val Gly Asp Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Ala Pro

85 90 95

Leu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 209

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 209

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Arg Ser Gly Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 210

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 210

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 211

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Lch

<400> 211

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 212

<211> 444

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 212

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr Gly Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gln Thr Gly Tyr 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 Leu 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 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> 213

<211> 220

<212> PRT

<213> Artificial Sequence

<220>

<223> Lch

<400> 213

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser

20 25 30

Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg His Thr Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 214

<211> 223

<212> PRT

<213> Homo sapiens

<400> 214

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 215

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 215

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 216

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 216

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 217

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 217

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Gln Trp Gly Pro Met

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 218

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 218

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Met Pro Ile Glu Glu Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 219

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 219

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 220

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 220

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Lys Pro Asp Glu Glu Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 221

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 221

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Gln Trp Gly Pro Met

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Glu Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Glu Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 222

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 222

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro Glu Glu Glu Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 223

<211> 5

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_Features

<222> (2)..(2)

<223> Xaa is His, Ala, Arg is Lys

<400> 223

Ser Xaa Thr Met Asn

1 5

<210> 224

<211> 17

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_Features

<222> (4)..(4)

<223> Xaa is Ser is Thr

<220>

<221> MISC_Features

<222> (5)..(5)

<223> Xaa is Arg is Gln

<220>

<221> MISC_Features

<222> (7)..(7)

<223> Xaa is Gly is His

<220>

<221> MISC_Features

<222> (13)..(13)

<223> Xaa is Asp, Glu is Arg

<220>

<221> MISC_Features

<222> (16)..(16)

<223> Xaa is Lys is Arg

<400> 224

Ser Ile Ser Xaa Xaa Ser Xaa Tyr Ile Tyr Tyr Ala Xaa Ser Val Xaa

1 5 10 15

Gly

<210> 225

<211> 13

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_Features

<222> (3)..(3)

<223> Xaa is Lys is Ala

<400> 225

Tyr Gly Xaa Arg Glu Asp Met Leu Trp Val Phe Asp Tyr

1 5 10

<210> 226

<211> 14

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_Features

<222> (1)..(1)

<223> Xaa is Thr, Asp, Gln, and Glu.

<220>

<221> MISC_Features

<222> (3)..(3)

<223> Xaa is Thr is Pro

<220>

<221> MISC_Features

<222> (6)..(6)

<223> Xaa is Asp is Gly

<220>

<221> MISC_Features

<222> (11)..(11)

<223> Xaa is Asn is Thr

<220>

<221> MISC_Features

<222> (12)..(12)

<223> Xaa is Tyr is Trp

<220>

<221> MISC_Features

<222> (14)..(14)

<223> Xaa is Ser is His

<400> 226

Xaa Gly Xaa Ser Thr Xaa Val Gly Asp Tyr Xaa Xaa Val Xaa

1 5 10

<210> 227

<211> 7

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_Features

<222> (1)..(1)

<223> Xaa is Glu, Phe is Tyr

<220>

<221> MISC_Features

<222> (3)..(3)

<223> Xaa is Ser is Ile

<220>

<221> MISC_Features

<222> (4)..(4)

<223> Xaa is Lys is Ser

<220>

<221> MISC_Features

<222> (7)..(7)

<223> Xaa is Ser, Glu is Lys

<400> 227

Xaa Thr Xaa Xaa Lys Pro Xaa

1 5

<210> 228

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_Features

<222> (1)..(1)

<223> Xaa is Ser is Gln

<220>

<221> MISC_Features

<222> (10)..(10)

<223> Xaa is Met is Thr

<400> 228

Xaa Thr Tyr Ala Ala Pro Leu Gly Pro Xaa

1 5 10

<210> 229

<211> 30

<212> PRT

<213> Homo sapiens

<400> 229

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 230

<211> 30

<212> PRT

<213> Homo sapiens

<400> 230

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 231

<211> 30

<212> PRT

<213> Homo sapiens

<400> 231

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 232

<211> 30

<212> PRT

<213> Homo sapiens

<400> 232

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 233

<211> 14

<212> PRT

<213> Homo sapiens

<400> 233

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 234

<211> 32

<212> PRT

<213> Homo sapiens

<400> 234

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 235

<211> 11

<212> PRT

<213> Homo sapiens

<400> 235

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 236

<211> 22

<212> PRT

<213> Homo sapiens

<400> 236

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys

20

<210> 237

<211> 22

<212> PRT

<213> Homo sapiens

<400> 237

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys

20

<210> 238

<211> 23

<212> PRT

<213> Homo sapiens

<400> 238

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys

20

<210> 239

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 239

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

Arg 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 Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 240

<211> 15

<212> PRT

<213> Homo sapiens

<400> 240

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr

1 5 10 15

<210> 241

<211> 15

<212> PRT

<213> Homo sapiens

<400> 241

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210> 242

<211> 32

<212> PRT

<213> Homo sapiens

<400> 242

Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

1 5 10 15

Leu Thr Val Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys

20 25 30

<210> 243

<211> 32

<212> PRT

<213> Homo sapiens

<400> 243

Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

1 5 10 15

Leu Thr Val Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

20 25 30

<210> 244

<211> 32

<212> PRT

<213> Homo sapiens

<400> 244

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210> 245

<211> 10

<212> PRT

<213> Homo sapiens

<400> 245

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

1 5 10

<210> 246

<211> 10

<212> PRT

<213> Homo sapiens

<400> 246

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 247

<211> 223

<212> PRT

<213> Mus musculus

<400> 247

Met Ala Cys Leu Gly Leu Arg Arg Tyr Lys Ala Gln Leu Gln Leu Pro

1 5 10 15

Ser Arg Thr Trp Pro Phe Val Ala Leu Leu Thr Leu Leu Phe Ile Pro

20 25 30

Val Phe Ser Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala

35 40 45

Ser Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His

50 55 60

Asn Thr Asp Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln

65 70 75 80

Met Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly

85 90 95

Phe Leu Asp Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Val Ala Val Ser Leu Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Val Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 248

<211> 223

<212> PRT

<213> Macaca fascicularis

<400> 248

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Arg Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Arg Pro Tyr Thr Leu Leu Phe Ser Leu Leu Phe Ile Pro

20 25 30

Val Phe Ser Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Asn Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Met Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 249

<211> 330

<212> PRT

<213> Homo sapiens

<400> 249

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 Gly Lys

325 330

<210> 250

<211> 326

<212> PRT

<213> Homo sapiens

<400> 250

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 Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 251

<211> 377

<212> PRT

<213> Homo sapiens

<400> 251

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His His Thr Cys Pro

100 105 110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

115 120 125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135 140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155 160

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

165 170 175

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

180 185 190

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

195 200 205

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

210 215 220

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

225 230 235 240

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

245 250 255

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

260 265 270

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

275 280 285

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

290 295 300

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

305 310 315 320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330 335

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

340 345 350

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

355 360 365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375

<210> 252

<211> 327

<212> PRT

<213> Homo sapiens

<400> 252

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 Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 253

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 253

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> 254

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 254

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

Arg 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 Tyr Thr Leu Pro Pro Ser Arg Lys Glu

225 230 235 240

Met 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> 255

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 255

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

Arg 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 Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro

325

<210> 256

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 256

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

Arg 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 Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro

325

<210> 257

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 257

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

Arg 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 Tyr Thr Leu Pro Pro Ser Arg Lys Glu

225 230 235 240

Met 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> 258

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 258

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

Arg 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 Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro

325

<210> 259

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 259

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

Arg 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 Tyr Thr Leu Pro Pro Ser Arg Lys Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 260

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 260

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

Arg 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 Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Glu Glu Leu Ser Leu Ser Pro

325

<210> 261

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 261

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

Arg 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 Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met 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> 262

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 262

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

Arg 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 Glu Glu

225 230 235 240

Met 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> 263

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 263

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

Arg 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 Glu Glu

225 230 235 240

Met 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> 264

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 264

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

Arg 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

Met 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> 265

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 265

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

Arg 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 Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met 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> 266

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 266

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

Arg 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 Glu Glu

225 230 235 240

Met 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> 267

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 267

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

Arg 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 Glu Glu

225 230 235 240

Met 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> 268

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 268

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

Arg 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

Met 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> 269

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 269

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

Arg 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 Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 270

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 270

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

Arg 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 Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 271

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 271

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

Arg 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 Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 272

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 272

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Glu Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 273

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 273

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 274

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Hch

<400> 274

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Lys

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gln Ser His Tyr Ile Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Arg Glu Asp Met Leu Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Ala Pro

325 330 335

Ile Glu Glu Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu

435 440 445

Ser Pro

450

<210> 275

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> Lch

<400> 275

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Asp Gly Pro Ser Thr Gly Val Gly Asp

20 25 30

Tyr Asn Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Phe Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

65 70 75 80

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Thr Tyr Ala Ala

85 90 95

Pro Leu Gly Pro Met Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 276

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 276

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

Arg 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 Glu Glu

225 230 235 240

Met 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 Ala His Thr Thr

305 310 315 320

Arg Glu Glu Leu Ser Leu Ser Pro

325

<210> 277

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 277

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

Arg 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

Met 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> 278

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 278

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

Arg 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

Met 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 Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

Claims (3)

1. Anti-CTLA-4 antibody, which contains: (1) (a) HVR-H1 composed of the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 composed of the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 composed of the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 composed of the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 composed of the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 composed of the amino acid sequence of SEQ ID NO: 133; (2) (a) HVR-H1 composed of the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 composed of the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 composed of the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 composed of the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 composed of the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 composed of the amino acid sequence of SEQ ID NO: 133; (3) (a) HVR-H1 composed of the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 composed of the amino acid sequence of SEQ ID NO: 106; (c) HVR-H3 composed of the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 composed of the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 composed of the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 composed of the amino acid sequence of SEQ ID NO: 133; (4) (a) HVR-H1 composed of the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 composed of the amino acid sequence of SEQ ID NO: 108; (c) HVR-H3 composed of the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 composed of the amino acid sequence of SEQ ID NO: 121; (e) HVR-L2 composed of the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 composed of the amino acid sequence of SEQ ID NO: 153; (5) (a) HVR-H1 composed of the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 composed of the amino acid sequence of SEQ ID NO: 110; (c) HVR-H3 composed of the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 composed of the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 composed of the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 composed of the amino acid sequence of SEQ ID NO: 133; (6) (a) HVR-H1 composed of the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 composed of the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 composed of the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 composed of the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 composed of the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 composed of the amino acid sequence of SEQ ID NO: 133; or (7) (a) HVR-H1 consisting of the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 consisting of the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 consisting of the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 consisting of the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 consisting of the amino acid sequence of SEQ ID NO: 133. 2. Anti-CTLA-4 antibody, which contains: (1) A first variable region comprising the VH sequence shown in SEQ ID NO: 140 and the VL sequence shown in SEQ ID NO: 146 and a second variable region comprising the VH sequence shown in SEQ ID NO: 141 and the VL sequence shown in SEQ ID NO: 146; (2) A first variable region comprising the VH sequence shown in SEQ ID NO: 140 and the VL sequence shown in SEQ ID NO: 147 and a second variable region comprising the VH sequence shown in SEQ ID NO: 141 and the VL sequence shown in SEQ ID NO: 147; (3) The VH sequence shown in SEQ ID NO: 140 and the VL sequence shown in SEQ ID NO: 146; (4) The VH sequence shown in SEQ ID NO: 141 and the VL sequence shown in SEQ ID NO: 146; (5) The VH sequence shown in SEQ ID NO: 140 and the VL sequence shown in SEQ ID NO: 147; (6) The VH sequence shown in SEQ ID NO: 141 and the VL sequence shown in SEQ ID NO: 147; (7) The VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 97; (8) The VH sequence shown in SEQ ID NO: 86 and the VL sequence shown in SEQ ID NO: 134; (9) The VH sequence shown in SEQ ID NO: 136 and the VL sequence shown in SEQ ID NO: 95; or (10) The VH sequence shown in SEQ ID NO: 136 and the VL sequence shown in SEQ ID NO: 149. 3. A pharmaceutical preparation comprising the antibody of claim 1 or 2 and a pharmaceutically acceptable carrier.