Humanized antibodies to LIV-1 and use of the same to treat cancer

Abstract

To provide humanized antibodies that specifically bind to LIV-1.SOLUTION: A humanized antibody specifically binding to LIV-1 comprises a mature heavy chain variable region having a specific amino acid sequence, and a mature light chain variable region having a specific amino acid sequence. The antibodies are useful for treatment and diagnosis of various cancers as well as detection of LIV-1.SELECTED DRAWING: Figure 27

Description

[Technical field]

[0001] The present invention relates to humanized antibodies against LIV-1 and their use for the treatment of cancer.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a nonprovisional application which claims the benefit of 61 / 420,291 filed December 6, 2010 and 61 / 446,990 filed February 25, 2011, each of which is incorporated by reference in its entirety for all purposes. [Background technology]

[0003] LIV-1 is a member of the LZT (LIV-1-ZIP zinc transporter) subfamily of zinc transporter proteins (Taylor et al., Biochim. Biophys. Acta 1611:16-30 (2003)). Computational analysis of the LIV-1 protein reveals a potential metalloprotease motif that matches the consensus sequence for the catalytic zinc-binding site motif of zinc metalloproteases. LIV-1 mRNA is expressed primarily in breast, prostate, pituitary and brain tissues.

[0004] The LIV-1 protein has also been associated with certain cancerous conditions, such as breast and prostate cancer. Detection of LIV-1 has been associated with estrogen receptor-positive breast cancer (McClelland et al., Br. J. Cancer 77:1653-1656 (1998)) and the metastatic spread of these cancers to regional lymph nodes (Manninget et al., Eur. J. Cancer 30A:675-678 (1994)). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Taylor et al., Biochim. Biophys. Acta 1611:16-30 (2003) [Non-Patent Document 2] McClelland et al., Br. J. Cancer 77:1653-1656 (1998) [Non-Patent Document 3] Manninget et al., Eur. J. Cancer 30A:675-678 (1994) Summary of the Invention

[0006] Summary of the claimed invention The invention provides a humanized antibody comprising a mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO:53, where position H27 is occupied by L, position H29 is occupied by I, position H30 is occupied by E, and position H94 is occupied by V, and a mature light chain variable region at least 90% identical to SEQ ID NO:60, where position L36 is occupied by Y, and position L46 is occupied by P. Optionally, the humanized antibody comprises the three CDRs of SEQ ID NO:53 and the three CDRs of SEQ ID NO:60. The CDRs are depicted in FIG. 16. Optionally, position H76 is occupied by N. Optionally, the humanized antibody comprises a mature heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:53, and a mature light chain variable region at least 95% identical to SEQ ID NO:60. Optionally, the mature heavy chain variable region is fused to a heavy chain constant region and the mature light chain constant region is fused to a light chain constant region. Optionally, the heavy chain constant region is a mutated form of the native human constant region that exhibits reduced binding to Fc gamma receptors compared to the native human constant region. Optionally, the heavy chain constant region is of the IgG1 isotype. Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 44 and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 42. Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 46 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 42. In some such humanized antibodies, any differences in the CDRs of the mature heavy chain variable region and mature light chain variable region from SEQ ID NO: 52 and 60, respectively, are present at positions H60-H65. In some such humanized antibodies, the mature heavy chain variable region has an amino acid sequence set forth in SEQ ID NO: 52 or 53 and the mature light chain variable region has an amino acid sequence set forth in SEQ ID NO: 59 or 60. In some such humanized antibodies, the mature heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 53 and the mature light chain variable region has the amino acid sequence set forth in SEQ ID NO: 60. Some such humanized antibodies may be conjugated to a cytotoxic or cytostatic agent.Some such humanized antibodies have a titer of 0.5-2×10. 9 M -1 , and has an association constant for human or cynomolgus monkey LIV-1.

[0007] The invention also provides humanized antibodies comprising a mature heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO:52, where position H27 is occupied by L, position H29 is occupied by I, position H30 is occupied by E, position H76 is occupied by N and position H94 is occupied by V, and a mature light chain variable region comprising the three Kabat CDRs of SEQ ID NO:60, where position L36 is occupied by Y and position L46 is occupied by P. The invention also provides nucleic acids encoding the mature heavy chain variable region and / or the mature light chain variable region of any of the above humanized antibodies.

[0008] The invention further provides a method of treating a patient having or at risk of having cancer, comprising administering to said patient an effective form of any of the above-described humanized antibodies. The cancer may be, for example, breast cancer, cervical cancer, melanoma or prostate cancer.

[0009] The present invention further provides a pharmaceutical composition comprising the humanized antibody.

[0010] The present invention further provides a method for treating a subject suffering from melanoma that expresses LIV-1 protein by administering to the subject an amount of a LIV-1 specific antibody or a LIV-1 antibody-drug conjugate sufficient to inhibit the growth of the melanoma cancer cells.

[0011] The present invention further provides a method for treating a subject suffering from cervical cancer that expresses LIV-1 protein by administering to the subject an amount of a LIV-1 specific antibody or a LIV-1 antibody-drug conjugate sufficient to inhibit the growth of the cervical cancer cells.

[0012] The invention further provides a humanized antibody comprising a mature heavy chain variable region having an amino acid sequence at least 90% identical to HB (SEQ ID NO: 10) and a mature light chain variable region at least 90% identical to LB (SEQ ID NO: 15). Optionally, the antibody comprises a mature heavy chain variable region having an amino acid sequence at least 95% identical to HB and a mature light chain variable region at least 95% identical to LB. Optionally, in any such antibody, positions H29, H30 and H76 are occupied by I, E and N, and position L36 is occupied by Y. Optionally, any difference between SEQ ID NO: 10 and the variable region framework of the mature heavy chain variable region is selected from the group consisting of H27 occupied by F, H28 occupied by N, H48 occupied by I, H66 occupied by K, H67 occupied by A, H71 occupied by A, H76 occupied by N, H93 occupied by N, H94 occupied by V, L37 occupied by L, L39 occupied by K, L45 occupied by K, and L46 occupied by L. Optionally, the three CDRs of the mature heavy chain variable region are those of SEQ ID NO: 10 and the three CDRs of the mature light chain variable region are those of SEQ ID NO: 15. The CDRs are shown in FIG. 1. Optionally, the mature heavy chain variable region is fused to a heavy chain constant region and the mature light chain constant region is fused to a light chain constant region. Optionally, the heavy chain constant region is a mutated form of the native human constant region that exhibits reduced binding to Fc gamma receptors compared to the native human constant region. Optionally, the heavy chain constant region is of the IgG1 isotype. Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:6 and the light chain constant region has an amino acid sequence comprising SEQ ID NO:4. Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:8 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO:4. Optionally, any differences in the CDRs of the mature heavy chain variable region and mature light chain variable region from SEQ ID NO:10 and 15, respectively, are present at positions H60-H65.Optionally, the mature heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 10, and the mature light chain variable region has an amino acid sequence comprising SEQ ID NO: 15. Optionally, the antibody is conjugated to a cytotoxic or cytostatic agent. A preferred humanized antibody has greater affinity for LIV-1 than antibody BR2-14a. In another embodiment, the humanized antibody has a mAb of 0.5-2×10. 9 M -1 It has an association constant for human or cynomolgus monkey LIV-1 of .

[0013] The present invention further provides a humanized antibody comprising a mature heavy chain variable region comprising the three CDRs of SEQ ID NO: 10, in which positions H29, H30 and H76 are occupied by I, E and N, respectively, and a mature light chain variable region comprising the three CDRs of SEQ ID NO: 15, in which position L36 is occupied by Y.

[0014] The present invention further provides nucleic acids encoding the mature heavy chain variable region and / or the mature light chain variable region of any of the above-mentioned humanized antibodies.

[0015] The invention further provides a method of treating a patient having or at risk of having cancer, comprising administering to said patient an effective form of any of the above-described humanized antibodies. Optionally, the cancer may be breast cancer, cervical cancer, melanoma or prostate cancer.

[0016] The present invention further provides a pharmaceutical composition comprising the humanized antibody.

[0017] The invention further provides a method of treating a patient having or at risk of having triple-negative breast cancer, comprising administering to the patient an effective form of an antibody that specifically binds to LIV-1, optionally wherein the antibody is conjugated to a cytotoxic or cytostatic agent. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 shows the alignment of the amino acid sequence of the parental murine mAb (designated BR2-14a) to the humanized LIV-1 heavy chain variable region (top two panels) and light chain variable region (bottom two panels). [Diagram 2] FIG. 2 shows the binding curves for the humanized LIV-1 mAb and the parental murine antibody (designated BR2-14a). [Diagram 3] Figure 3 shows the results of a competitive binding study of the humanized LIV-1 mAb and the parental murine antibody (designated BR2-14a). The numbers in parentheses after each variant indicate the number of backmutations. [Figure 4] Figure 4 shows the results of saturation binding studies on MCF7 cells. BR2-14a-AF refers to the AF-labeled parent mouse antibody. hLIV-14 refers to the AF-labeled HBLB antibody, a humanized antibody that specifically binds to LIV-1. [Diagram 5] Figure 5 shows the results of competitive binding studies on CHO cells expressing recombinant LIV-1 protein. BR2-14a refers to the parental mouse antibody. hLIV-14 HBLB WT refers to the HBLB antibody. hLIV-14 HBLB S239C refers to the HBLB antibody with serine to cysteine ​​substitutions at each position in the heavy chain. [Figure 6] FIG. 6 shows analysis of LIV-1 protein expression by IHC on breast cancer patient samples after hormone treatment. [Figure 7] FIG. 7 shows analysis of LIV-1 protein expression by IHC on hormone-refractory metastatic prostate cancer patient samples. [Figure 8] FIG. 8 shows analysis of LIV-1 protein expression by IHC on triple negative breast cancer patient samples. [Figure 9]Figure 9 shows the results of cytotoxicity assays for hLIV-14 antibody drug conjugates (i.e., HBLB mAb conjugated to vcMMAE(1006) or mcMMAF(1269)) and control mouse (mIgG) and human (hIgG) antibody conjugates. hLIV-14-SEA-1006 refers to the non-fucosylated form of HBLB mAb conjugated to vcMMAE(1006). [Figure 10] Figure 10 shows the results of an in vitro ADCC assay on MCF7 cells using human NK cells (donor 1; V / V). hLIV-14 WT refers to HBLB mAb. hLIV-14 SEA refers to the non-fucosylated form of HBLB mAb. hLIV-1 mcMMAF refers to the antibody drug conjugate of HBLB mAb conjugated to mcMMAF. hLIV-14 vcMMAE refers to the antibody drug conjugate of HBLB mAb conjugated to vcMMAE. hLIV-14 SEA vcMMAE refers to the non-fucosylated form of the HBLB mAb-vcMMAE antibody drug conjugate. [Figure 11]FIG. 11 shows the results of an in vitro ADCC assay on MCF7 cells using human NK cells (donor 2). hLIV-14 WT refers to HBLB mAb. hLIV-14 SEA refers to the non-fucosylated form of HBLB mAb. cLIV-14 SEA refers to the non-fucosylated form of the chimeric parental mouse antibody. hLIV-14 mcF(4) refers to the antibody drug conjugate of HBLB mAb with an average of 4 mcMMAF drug linker molecules per antibody. hLIV-14 vcE(4) refers to the antibody drug conjugate of HBLB mAb with an average of 4 vcMMAE drug linker molecules per antibody. hLIV-14 vcE(4) SEA refers to the non-fucosylated form of HBLB mAb-vcMMAE antibody drug conjugate with an average of 4 vcMMAE drug linker molecules per antibody. hIgG refers to control human IgG. H00-mcF(4) refers to a control antibody drug conjugate of a non-conjugated antibody with an average of 4 mcMMAF drug linker molecules per antibody. H00-vcE(4) refers to a control antibody drug conjugate of a non-conjugated antibody with an average of 4 vcMMAE drug linker molecules per antibody. [Figure 12] Figure 12 shows the results of a xenograft study of the MCF7 breast cancer line in nude mice. cLIV-14 mcMMAF(4) refers to an antibody drug conjugate in chimeric form of the parent mouse antibody with an average of 4 mcMMAF drug linker molecules per antibody. cLIV-14-vcMMAE(4) refers to an antibody drug conjugate in chimeric form of the parent mouse antibody with an average of 4 vcMMAE drug linker molecules per antibody. H00-mcMMAF(4) refers to an antibody drug conjugate of a non-binding control antibody with an average of 4 mcMMAF drug linker molecules per antibody. H00-vcMMAE(4) refers to an antibody drug conjugate of a non-binding control antibody with an average of 4 vcMMAE drug linker molecules per antibody. The dose and time of administration are indicated in the figure. [Figure 13]Figure 13 shows the results of a xenograft study of the PC3 prostate cancer line in male nude mice. cLIV-14-vcMMAE(4) refers to an antibody drug conjugate in chimeric form of the parent mouse antibody with an average of 4 vcMMAE drug linker molecules per antibody. hBU12-vcMMAE(4) refers to an antibody drug conjugate of an anti-CD19 antibody with an average of 4 vcMMAE drug linker molecules per antibody. Dosage and time of administration are indicated in the figure. [Figure 14] Figure 14 shows the results of a xenograft study of the MCF7 breast cancer line in nude mice. hLIV-14-vcMMAE(4) refers to an antibody drug conjugate of an HBLB antibody with an average of 4 vcMMAE drug linker molecules per antibody. hLIV-14d-vcMMAE(2) refers to an antibody drug conjugate of an HBLB antibody with an average of 2 vcMMAE drug linker molecules per antibody, each conjugated at the S239C position of each heavy chain. H00-vcMMAE(4) refers to an antibody drug conjugate of a non-binding control antibody with an average of 4 vcMMAE drug linker molecules per antibody. The dose and time of administration are indicated in the figure. [Figure 15]Figure 15 shows the results of a xenograft study of the PC3 prostate cancer line in male nude mice. hLIV-14-vcMMAE(4) refers to an antibody drug conjugate of an HBLB antibody with an average of 4 vcMMAE drug linker molecules per antibody. hLIV-14-mcMMAF(4) refers to an antibody drug conjugate of an HBLB antibody with an average of 4 mcMMAF drug linker molecules per antibody. hLIV-14d-vcMMAE(2) refers to an antibody drug conjugate of an HBLB antibody with an average of 2 vcMMAE drug linker molecules per antibody, each conjugated at the S239C position of each heavy chain. hLIV-14d-mcMMAF(2) refers to an antibody drug conjugate of an HBLB antibody with an average of 2 mcMMAF drug linker molecules per antibody, each conjugated at the S239C position of each heavy chain. H00-vcMMAE(4) refers to an antibody drug conjugate of a non-conjugated control antibody with an average of 4 vcMMAE drug linker molecules per antibody. H00-mcMMAF(4) refers to an antibody drug conjugate of a non-conjugated control antibody with an average of 4 mcMMAF drug linker molecules per antibody. Dosage and time of administration are indicated in the figure. [Figure 16A] Figures 16A and 16B show the alignment of the humanized heavy chain (Figure 16A) and light chain (Figure 16B) mature variable regions to those of murine BR2-22a. [Figure 16B] Figures 16A and 16B show the alignment of the humanized heavy chain (Figure 16A) and light chain (Figure 16B) mature variable regions to those of murine BR2-22a. [Figure 17] Figure 17 shows a competitive binding assay of various permutations of the humanized heavy chain HA-HF and the humanized light chain LA-LF derived from the mouse monoclonal anti-LIV-1 antibody BR2-22a. The total number of mouse back mutations in each light or heavy chain is shown in parentheses. Only HELF showed sufficient retention of binding. [Figure 18]Figure 18 shows systematic mutations of the HE and LF chains to test the contribution of individual back mutations to antigen binding. Potential somatic hypermutation sites are indicated in brackets. Mouse residues are underlined. The remaining residues are human germline residues. [Figure 19] Figure 19 shows competitive binding of LF mutants at the top of the figure. The backmutations tested are shown at the bottom of the figure. The murine residues are underlined. The remaining residues are human germline residues. [Figure 20] Figure 20 shows competitive binding of HE mutants at the top of the figure. The backmutations tested are shown at the bottom of the figure. The mouse residues are underlined. The remaining residues are human germline residues. [Figure 21] FIG. 21 shows the competitive binding of HE, HF, HG and various permutations of LF and LG. [Figure 22] FIG. 22 shows saturation binding of humanized LIV14 and LIV22 antibodies on human and cynomolgus LIV-1 expressed from CHO cells. [Diagram 23] Figure 23 shows the cytotoxic activity of humanized LIV22-vcMMAE on MCF-7 cells after 144 hours of treatment. h00-1006 is a control drug-conjugated antibody. [Figure 24] Figure 24 shows the cytotoxic activity of hLIV22-mcMMAF on MCF-7 cells after 144 hours of treatment. h00-1269 is a control drug-conjugated antibody. [Diagram 25] Figure 25 shows the activity of hLIV22 antibody in the PC3(DSMZ) prostate cancer model in nude female mice. Days of treatment are indicated by triangle symbols on the x-axis. [Figure 26] FIG. 26 shows the activity of hLIV22 antibody in MCF7 (NCI) breast cancer tumors in nude mice. [Figure 27] FIG. 27 compares the activity of hLIV22 and hLIV in the same model as FIG. [Figure 28]FIG. 28 shows analysis of LIV-1 protein expression by IHC on melanoma cancer patient samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition Monoclonal antibodies are typically provided in isolated form. This means that the antibody is typically at least 50% (w / w) pure from inhibitory proteins and other contaminants from its production or purification, and does not exclude the possibility that the monoclonal antibody may be combined with an excess of pharma- ceutically acceptable carriers or other vehicles intended to facilitate its use. Sometimes, the monoclonal antibody is at least 60%, 70%, 80%, 90%, 95% or 99% (w / w) pure from inhibitory proteins and contaminants from its production or purification.

[0020] The specific binding of a monoclonal antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 or 10 10 M -1 Specific binding refers to an affinity for a specific antibody. Specific binding is detectably higher in magnitude and can be distinguished from non-specific binding occurring against at least one unrelated target. Specific binding can be the result of the formation of bonds between specific functional groups or a specific spatial fit (e.g., lock-and-key type). Non-specific binding, on the other hand, is usually the result of van der Waals forces. However, specific binding does not necessarily imply that a monoclonal antibody binds to one and only one target.

[0021] The basic antibody structural unit is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. This variable region is first expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region refers to a light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain is a constant region that is primarily responsible for effector function.

[0022] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. In light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids (see generally, Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7, which is incorporated herein by reference in its entirety for all purposes).

[0023] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, a complete antibody has two binding sites. Except in the case of bifunctional or bispecific antibodies, the two binding sites are identical. The chains all exhibit the same general structure of relatively conserved framework regions (FR) linked by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions to allow binding to a specific epitope. Both light and heavy chains contain the following domains from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain follows Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also describes a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or different light chains are assigned the same number.

[0024] The term "antibody" includes intact antibodies and binding fragments thereof. Typically, antibody fragments are fragments of separate heavy chains, light chains, Fab, Fab', F(ab')2, F(ab)2, and / or Fab'-binding fragments that compete with the intact antibody from which they are derived for specific binding to a target. c These include diabodies, Dabs, nanobodies and Fvs. Fragments may be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also refers to diabodies (homodimeric Fv fragments) or minibodies (VFv fragments). L -V H -C H3), bispecific antibodies, etc. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). The term "antibody" includes an antibody by itself (naked antibody) or an antibody conjugated to a cytotoxic or cytostatic drug.

[0025] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically comprises at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris, (1996).

[0026] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can be determined by identifying contact residues by X-ray crystallography of the antibody bound to the corresponding antigen. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0027] Competition between antibodies is determined by an assay in which the antibody under test inhibits the specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold or 10-fold) inhibits the binding of the reference antibody by at least 50%, preferably 75%, 90% or 99%, as measured in a competitive binding assay. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are close enough to the epitope bound by the reference antibody to cause steric hindrance.

[0028] The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0029] For purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are classified as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions are the exchange of a member of one of these classes for a member of another class.

[0030] Percent sequence identity is determined for antibody sequences aligned to the fullest extent according to the Kabat numbering convention. After alignment, when a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared with the same region of a reference antibody, the percent sequence identity between the subject and reference antibody regions is the number of positions occupied by identical amino acids in both the subject and reference antibody regions divided by the total number of aligned positions of the two regions (gaps not being counted), multiplied by 100 to convert to a percentage.

[0031] A composition or method "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition that includes an antibody may contain only the antibody or other components in combination with it.

[0032] The representation of a range of values ​​includes all integers within or defining the range.

[0033] Antibody effector function refers to a function provided by the Fc domain of Ig. Such functions may include, for example, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, or complement-dependent cytotoxicity. Such functions may be provided, for example, by binding of the Fc effector domain to Fc receptors on immune cells with phagocytic or cytolytic activity, or by binding of the Fc effector domain to components of the complement system. Typically, the effect provided by the Fc-binding cells or complement components causes the inhibition and / or loss of LIV-1 targeted cells. The Fc region of an antibody recruits receptor (FcR)-expressing cells and brings them into juxtaposition with antibody-coated target cells. Cells expressing surface FcRs for IgG, including FcγRIII (CD16), FcγRII (CD32), and FcγRIII (CD64), may act as effector cells for the destruction of IgG-coated cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils, and eosinophils. Engagement of FcγR by IgG activates antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). ADCC activates CD16 through the secretion of pore-forming proteins and proteases. + Phagocytosis is triggered by effector cells, whereas phagocytosis is triggered by CD32 + and CD64 + Initiated by effector cells (Fundamental Immunology, 4 thed., Paul, Lippincott-Raven, NY, 1997, Chapters 3, 17 and 30; Uchida et al., 2004, J. Exp. Med. 199:1659-69; Akewanlop et al., 2001, Cancer Res. 61:4061-65; Watanabe et al., 1999, Breast Cancer Res. Treat. 53:199-207). In addition to ADCC and ADCP, the Fc region of cell-bound antibodies can also activate the classical complement pathway, initiating complement-dependent cytotoxicity (CDC). C1q of the complement system binds to the Fc region of antibodies when they are complexed with antigen. Binding of C1q to cell-bound antibodies can initiate a cascade of events, including the proteolytic activation of C4 and C2 to produce C3 convertase. Cleavage of C3 to C3b by C3 convertase allows activation of the terminal complement components, including C5b, C6, C7, C8, and C9. Taken together, these proteins form membrane attack complex pores on antibody-coated cells. These pores compromise the integrity of the cell membrane, killing the target cell (Immunobiology, 6 th ed., Janeway et al., Garland Science, NY, 2005, Chapter 2).

[0034] The term "antibody-dependent cellular cytotoxicity" or ADCC is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells with immune cells (also called effector cells) that have cytolytic activity. Such effector cells include natural killer cells, monocytes / macrophages and neutrophils. The effector cells bind to the Fc effector domain of Ig bound to the target cell via the antigen binding site. The effector cell activity results in the death of the antibody-coated target cell.

[0035] The term "antibody-dependent cellular phagocytosis" or ADCP refers to the process by which antibody-coated cells are totally or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to the Fc effector domain of Ig.

[0036] The term "complement dependent cytotoxicity" or CDC refers to a mechanism for inducing cell death in which the Fc effector domain of a target-binding antibody activates a series of enzymatic reactions that ultimately form holes in the target cell membrane. Typically, antigen-antibody complexes (e.g., on antibody-coated target cells) bind and activate complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation can also lead to the deposition of complement components on the target cell surface, which promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0037] "Cytotoxic (cytotoxic) effect" refers to the reduction, elimination and / or killing of a target cell. "Cytotoxic agent" refers to an agent that produces a cytotoxic effect on a cell. The cytotoxic agent can be conjugated to the antibody or can be administered together with the antibody.

[0038] "Cytostatic effect" refers to the inhibition of cell proliferation. "Cytostatic agent" refers to an agent that exerts a cytostatic effect on cells, inhibiting the growth and / or proliferation of a specific subset of cells. The cytostatic agent can be conjugated to an antibody or can be administered together with an antibody.

[0039] The term "pharmaceutical acceptable" means approved or capable of being approved by a regulatory agency of a federal or state government for use in animals, or more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. The term "pharmaceutical acceptable ingredient" refers to a pharmaceutical acceptable diluent, adjuvant, excipient, or vehicle used in conjunction with an anti-LIV-1 antibody.

[0040] The term "pharmaceutical acceptable salt" refers to a pharmaceutical acceptable organic or inorganic salt of an anti-LIV-1 antibody or a conjugate thereof, or a substance administered with an anti-LIV-1 antibody. Exemplary salts include sulfate, citrate, acetate, oxalate, chloride, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinic acid, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'methylene bis-(2 hydroxy 3 naphthoate)). A pharma- ceutically acceptable salt may include the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharma- ceutically acceptable salt may have more than one charged atom in its structure. An example in which multiple charged atoms are part of a pharma- ceutical acceptable salt may have multiple counterions. Thus, a pharma- ceutical acceptable salt may have one or more charged atoms and / or one or more counterions.

[0041] Unless otherwise clear from the context, the term "about" includes values ​​within the standard deviation of the stated value.

[0042] Detailed Description I. Summary The present invention provides monoclonal antibodies that specifically bind to LIV-1, which are useful for treating and diagnosing various cancers and detecting LIV-1.

[0043] II. target molecule Unless otherwise indicated, LIV-1 refers to human LIV-1. An exemplary human sequence is assigned Swiss Prot accession number Q13433. Q13433 is included herein as SEQ ID NO: 83. Three variant isoforms and one polymorphism are known. A second form of human LIV-1 protein, accession number AAA96258.2, is included herein as SEQ ID NO: 84. The four extracellular domains are delimited by residues 29-325, 377-423, 679-686, and 746-755 of Q13433, respectively.

[0044] Unless otherwise clear from the context, reference to LIV-1 refers to at least the extracellular domain of the protein, and usually to the entire protein except for the cleavable signal peptide (amino acids 1-28 of Q13433).

[0045] III. Antibodies of the Invention A. Binding specificity and functional properties The present invention provides humanized antibodies derived from two mouse antibodies, BR2-14a and BR2-22a. Unless otherwise indicated, the present disclosure relates to both antibodies. The two mouse antibodies show 94% and 91% sequence identity to each other in the mature heavy and light chain variable regions. The two antibodies bind to the same or overlapping epitopes on human LIV-1. However, as shown in Figure 22, the BR2-22a antibody has about 10-fold higher affinity to human LIV-1 and about 3-fold higher affinity to cynomolgus LIV-1 than BR2-14a.

[0046] The affinity (i.e., Ka) of the humanized form of the mouse BR2-14a antibody is preferably within 5-fold (a factor of 5) or 2-fold (a factor of 2) of the mouse antibody BR2-14a for human LIV-1. The humanized BR2-14a antibody, like the mouse antibody from which it is derived, specifically binds to human LIV-1 in its native form and / or recombinantly expressed from CHO cells. Preferred humanized BR2-14a antibodies have an affinity for human LIV-1 that is the same as or greater (i.e., greater than within the margin of measurement error) than BR2-14a (e.g., 1.1-5-fold, 1.1-3-fold, 1.5-3-fold, 1.7-2.3-fold, or 1.7-2.1-fold, or about 2-fold the affinity of BR2-14a). Preferred humanized BR2-14a antibodies bind to the same epitope and / or compete with BR2-14a for binding to human LIV-1. Preferred humanized BR2-14a antibodies also bind to a cyno-homolog of LIV-1, allowing for preclinical testing in non-human primates.

[0047] The affinity (i.e., Ka) of the humanized form of the murine BR2-22a antibody for human LIV-1, either naturally expressed or expressed from CHO cells, is preferably within 5-fold (a factor of 5) or 2-fold (a factor of 2) of that of the murine antibody BR2-22. Some humanized BR2-22a antibodies have association constants that are substantially the same (i.e., within experimental error) as that of BR2-22a. Some humanized BR2-22a antibodies have association constants within 0.5-1 or 0.5-1.5 times that of the BR2-22a antibody. A preferred humanized BR2-22a antibody has an association constant of 5×10 8 M -1 Larger, or 0.5 to 2 x 10 9 M -1 or approximately 0.8 × 10 9 M -1The preferred humanized BR2-22a antibody binds to the same epitope and / or competes with BR2-22a for binding to human LIV-1. The human BR2-22a antibody binds not only to human LIV-1 but also to the cyno-homolog of LIV-1. The preferred humanized BR2-22a antibody binds to human and cynomolgus LIV-1, both expressed from CHO cells, with substantially the same association constant (within experimental error), enabling and enhancing the predictive accuracy of clinical trials in non-human primates.

[0048] As shown in cancer cells grown in culture, in animal models, or in clinical trials, the preferred antibodies (both humanized BR2-14a and humanized BR2-22a) inhibit cancer (e.g., cell growth, metastasis, and / or organism lethality). Animal models can be generated by implanting LIV-1-expressing human tumor cell lines into appropriate immune-deficient rodent strains (e.g., athymic nude mice or SCID mice). These tumor cell lines can be established in immune-deficient rodent hosts as solid tumors by subcutaneous injection or as disseminated tumors by intravenous injection. Once established in the host, these tumor models can be applied to evaluate the therapeutic efficacy of anti-LIV-1 antibodies or conjugated forms thereof, as described in the Examples.

[0049] B. Humanized antibodies A humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted into a human "acceptor" antibody sequence (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. In the case of the heavy chain, the preferred acceptor sequence is a germline V H Exon V H 1-2 (also referred to in the literature as HV 1-2) (Shin et al., 1991, EMBO J. 10:3641-3645), and the hinge region (J H ) in the case of exon J H -6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred acceptor sequence is exon VK2-30 (also referred to in the literature as KV2-30), and for the hinge region, exon J κ-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Thus, a humanized antibody is an antibody with some or all CDRs completely or substantially derived from a donor antibody and variable region framework sequences and constant regions (if present) completely or substantially derived from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody heavy chain and heavy chain variable region framework sequences and heavy chain constant regions (if present) substantially derived from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody light chain and light chain variable region framework sequences and light chain constant regions (if present) substantially derived from human light chain variable region framework and constant region sequences. Unlike nanobodies and dAbs, humanized antibodies comprise a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is said to be substantially derived from a corresponding CDR in a non-human antibody if at least 60%, 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is said to be substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95% or 100% of the corresponding residues are identical as defined by Kabat.

[0050] Humanized antibodies often contain all six CDRs (preferably as defined by Kabat) from the murine antibody, although they can have fewer than all CDRs (e.g., at least three, four or five) from the murine antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0051] Certain amino acids from the human variable region framework residues may be selected for substitution based on their possible effects on CDR conformation and / or binding to antigen, by modeling, by examination of the characteristics of the amino acid at a particular position, or by experimental observation of the effect of substitution or mutagenesis of a particular amino acid.

[0052] For example, where an amino acid differs between the murine variable region framework residue and the selected human variable region framework residue, the amino acid is (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR regions; (3) interacts with the CDR regions (e.g., is within about 6 angstroms of a CDR region) unless the following substitutions are made: (4) The human framework amino acids may be replaced with the equivalent framework amino acids from the mouse antibody if they are reasonably expected to mediate interactions between the heavy and light chains.

[0053] The present invention provides humanized forms of the murine BR2-14a antibody, comprising five exemplary humanized heavy chain mature variable regions (HA-HE) and six exemplary humanized light chain mature variable regions (LA-LF). The permutations of these chains that show the strongest binding (lowest EC50) are HBLB, HBLF, HCLB, HCLF, HDLB, HDLF, HELE, and HELF. Of these permutations, HBLB (also known as hLIV14) is preferred, as it shows the strongest binding, approximately two-fold stronger than the murine donor antibody, and has the fewest back mutations (four).

[0054] The present invention provides variants of HBLB humanized antibodies, in which the humanized heavy chain mature variable region exhibits at least 90%, 95% or 99% sequence identity to SEQ ID NO: 10 and the humanized light chain mature variable region exhibits at least 90%, 95% or 99% sequence identity to SEQ ID NO: 15. Preferably, in such antibodies, some or all of the back mutations in HBLB are retained. In other words, at least one, two or preferably all three of positions H29, H30 and H76 of the heavy chain are occupied by I and E and N, respectively. Similarly, position L36 is preferably occupied by Y. The CDR regions of such humanized antibodies are substantially identical to the CDR regions of HBLB, which are preferably the same as those of the mouse donor antibody. The CDR regions may be defined by any conventional definition (e.g. Chothia), but are preferably defined by Kabat. In one embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO: 10 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO: 10. In another embodiment, the humanized antibody comprises a light chain comprising the three CDRs of SEQ ID NO: 15 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO: 15. In another embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO: 10 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO: 10, and a light chain comprising the three CDRs of SEQ ID NO: 15 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO: 15.

[0055] If the humanized antibody shows any mutations from the exemplified HBLB humanized antibody, one of the possibilities for such additional mutations is additional back mutations in the variable region framework. It may also occur at any or all of the back mutated positions in the other exemplified humanized heavy or light chain mature variable regions (i.e., 1, 2, 3, 4, 5, 6, 7, 8 or all 9 of H27 occupied by F, H28 occupied by N, H48 occupied by I, H66 occupied by K, H67 occupied by A, H71 occupied by A, H76 occupied by N, H93 occupied by N and H94 occupied by V in the heavy chain, and 1, 2, 3, 4 or all 5 of L37 occupied by L, L39 occupied by K, L45 occupied by K and L46 occupied by L in the light chain). However, such additional back mutations are not preferred. This is because they generally do not improve affinity and the introduction of more murine residues may lead to an increased risk of immunogenicity.

[0056] The present invention provides a humanized form of the murine BR2-22a antibody, comprising three exemplary humanized heavy chain mature variable regions (HE, HF, and HG) and two exemplary humanized light chains (LF and LG), which can be combined in various permutations by appropriate linking (see FIG. 21). Of these permutations, HGLG (also known as hLIV22) is preferred, since it has the best combination of binding characteristics (substantially the same as the murine BR2-22a antibody within experimental error) and the fewest back mutations (7).

[0057] The present invention provides variants of HGLG humanized antibodies, in which the humanized heavy chain mature variable region exhibits at least 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:53, and the humanized light chain mature variable region exhibits at least 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:60. Preferably, in such antibodies, some or all of the back mutations in HGLG are retained. In other words, at least one, two, three, four or preferably all five of positions H27, H29, H30, H76 and H94 of the heavy chain are occupied by L, I, E, N and V (wherein Kabat numbering is used to indicate positions in the mature variable heavy and light chain variable regions as described elsewhere in this application). Of these back mutations, H94 contributes most to retention of binding affinity, and H76 contributes least. Similarly, positions L36 and L46 are preferably occupied by Y and P, respectively. The CDR regions of such humanized antibodies are preferably substantially identical to the CDR regions of HGLG, which are the same as those of the mouse donor antibody. The CDR regions may be defined by any conventional definition (e.g., Chothia), but are preferably defined by Kabat. In one embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO:53 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO:53. In another embodiment, the humanized antibody comprises a light chain comprising the three CDRs of SEQ ID NO:60 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO:60. In another embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO:53 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO:53, and a light chain comprising the three CDRs of SEQ ID NO:60 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO:60.

[0058] If the humanized BR2-22a antibody shows any mutations from the exemplified HGLG humanized antibody, one of the possibilities for such additional mutations is additional back mutations in the variable region framework. They may also occur at any or all of the backmutated positions in the other exemplified humanized heavy or light chain mature variable regions (i.e., 1, 2, 3, 4, 5 or all 6 of H28 occupied by N, H48 occupied by I, H66 occupied by K, H67 occupied by A, H71 occupied by A, H93 occupied by T in the heavy chain, and 1 or 2 of L37 occupied by L and L45 occupied by K in the light chain). However, such additional back mutations are not preferred because they generally do not improve affinity and the introduction of a larger number of mouse residues may lead to increased risk of immunogenicity.

[0059] Another possible mutation is that certain residues in the CDRs of the mouse antibody are replaced with corresponding residues from human CDR sequences, typically from the CDRs of the human acceptor sequences used in the design of the exemplified humanized antibody. In some antibodies, only a portion of the CDRs, i.e., a subset of the CDR residues required for binding (called SDRs), are required to retain binding in the humanized antibody. CDR residues that do not contact the antigen and are not present in the SDRs can be identified based on previous studies (e.g., residues H60-H65 in CDR H12 are often not required), from regions of the Kabat CDRs located outside the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and / or experimentally, or as described in Gonzales et al., Mol. Immunol. 41: 863 (2004). In such humanized antibodies, at positions where one or more donor CDR residues are absent or where the entire donor CDR is omitted, the amino acid occupying the position may be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor to donor amino acids in the CDRs to be included reflects a balance of competing considerations. Such substitutions are potentially advantageous to reduce the number of mouse amino acids in the humanized antibody and, consequently, to reduce potential immunogenicity. However, substitutions may also cause changes in affinity, and significant reductions in affinity are preferably avoided. In another mutation, one or more residues in the CDRs of the humanized BR2-22a antibody (which would otherwise be the same as the CDRs of the mouse BR2-22a antibody) may be replaced by the corresponding residues from the CDRs from the mouse BR2-14a antibody (or vice versa). The positions for substitution in the CDRs and the amino acids to be replaced may also be selected empirically.

[0060] Although not preferred, other amino acid substitutions can be made, for example, in framework residues that do not contact CDRs, or even in some potential CDR contact residue amino acids within CDRs. Often, the substitutions made in the variant humanized sequence are conservative with respect to the HBLB amino acid (in the case of humanized BR2-14a) or HGLG amino acid (in the case of humanized BR2-22) that are replaced. Preferably, the substitutions made to HBLB or HGLG (whether conservative or not) do not substantially affect the binding affinity or potency of the humanized mAb, i.e., its ability to bind to human LIV-1 and inhibit the growth of cancer cells.

[0061] The variants typically differ from the HBLB (hLIV14) or HGLG (hLIV22) heavy and light chain mature variable region sequences in a small number of substitutions, deletions or insertions (e.g., typically no more than 1, 2, 3, 5 or 10 in either or both of the light or heavy chain mature variable regions).

[0062] C. Selection of constant regions The heavy and light chain variable regions of the humanized antibody may be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 exhibit potent complement-dependent cytotoxicity, human isotype IgG2 exhibits weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cellular effector functions than human IgG2 and IgG4. The light chain constant region may be lambda or kappa. Antibodies may be expressed as tetramers containing two light chains and two heavy chains, or as separate heavy and light chains, or as Fab, Fab', F(ab)2 and Fv, or as single chain antibodies in which the heavy and light chain variable domains are linked via a spacer.

[0063] Human constant regions exhibit allotypic and isoallotypic variation among different individuals, i.e., the constant region may differ at one or more polymorphic positions among different individuals. Isoallotypes differ from allotypes in that sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes.

[0064] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains (e.g., the C-terminal lysine of the heavy chain) can be deleted or derivatized in some or all of the molecules. Substitutions can be made in the constant region to reduce or enhance effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006) or to extend half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0065] Exemplary substitutions include amino acid substitutions of natural amino acids with cysteine ​​residues, introduced at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332, preferably the S239C mutation in human IgG1 isotype (US 20100158909). The presence of an additional cysteine ​​residue allows interchain disulfide bond formation. Such interchain disulfide bond formation can cause steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. Cysteine ​​residues introduced at or near the Fc region of the IgG constant region can also serve as sites for conjugation to therapeutic agents (i.e., coupling of cytotoxic drugs using thiol-specific reagents such as maleimide derivatives of the drug). The presence of a therapeutic agent can cause steric hindrance, thereby further reducing the affinity of the Fc region-FcγR binding interaction. Other substitutions at any of positions 234, 235, 236 and / or 237 reduce affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., US 6,624,821, US 5,624,821).

[0066] The in vivo half-life of an antibody can also affect its effector function. The half-life of an antibody can be increased or decreased to modify its therapeutic activity. FcRn is a receptor structurally similar to MHC class I antigens that non-covalently binds to β2-microglobulin. FcRn regulates the catabolism of IgG and its transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). IgG-FcRn interaction occurs at pH 6.0 (pH of intracellular vesicles) but not at pH 7.4 (pH of blood). This interaction allows IgG to return to the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The regions on human IgG1 involved in FcRn binding have been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitutions at Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules containing these substitutions have a longer serum half-life. Thus, these modified IgG1 molecules may be able to perform their effector functions for a longer period of time compared to unmodified IgG1, thus resulting in their therapeutic efficacy. Other exemplary substitutions to enhance binding to FcRn include Gln at position 250 and / or Leu at position 428. EU numbering is used for all positions within the constant region.

[0067] The oligosaccharide covalently attached to the conserved Asn297 is responsible for the ability of the Fc region of IgG to bind to FcγR (Lund et al., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 1997, Trends Biotechnol. 15:26-31). Engineering this glycoform on IgG can significantly improve IgG-mediated ADCC. The addition of branching N-acetylglucosamine modifications to this glycoform (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotech. Bioeng. 74:288-94) or removal of fucose from this glycoform (Shields et al., 2002, J. Biol. Chem. 277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer Res. 64:2127-33) are two examples of IgG Fc engineering that improves IgG Fc binding to FcγR and enhances Ig-mediated ADCC activity.

[0068] Systematic substitution of solvent-exposed amino acids in the human IgG1 Fc region has yielded IgG variants with altered FcγR binding affinity (Shields et al., 2001, J. Biol. Chem. 276:6591-604). A subset of these variants, including substitutions of Ala at Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 / Lys334, show both enhanced binding affinity to FcγR and ADCC activity when compared to the parent IgG1 (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49).

[0069] The complement fixation activity (both C1q binding and CDC activity) of antibodies can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166:2571-2575). The same substitutions on the human IgG2 backbone can convert an antibody isotype that does not bind C1q well and has severely deficient complement activation activity into an antibody isotype that can both bind C1q and mediate CDC (Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other methods have also been applied to improve the complement fixation activity of antibodies. For example, grafting the 18 amino acid carboxy-terminal tail fragment of IgM to the carboxy terminus of IgG significantly enhances their CDC activity. This is also observed in IgG4, which normally has undetectable CDC activity (Smith et al., 1995, J. Immunol. 154:2226-36). Also, substitution of Ser444, located near the carboxy terminus of the IgG1 heavy chain, with Cys induced tail-to-tail dimerization of IgG1 with a 200-fold increase in CDC activity compared to monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148:2918-22). Bispecific diabody constructs with specificity for C1q also confer CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

[0070] Complement activity can be reduced by mutating at least one of amino acid residues 318, 320 and 322 of the heavy chain to a residue with a different side chain, such as Ala. Other alkyl-substituted non-ionic residues, such as Gly, Ile, Leu or Val, or aromatic non-polar residues, such as Phe, Tyr, Trp and Pro, at any of these three residues also reduce or eliminate C1q binding. Ser, Thr, Cys and Met can be used at residues 320 and 322, but not at residue 318, to reduce or eliminate C1q binding activity. Substitution of residue 318 (Glu) with a polar residue can modify but not eliminate C1q binding activity. Substitution of residue 297 (Asn) with Ala results in the elimination of cytolytic activity but only slightly reduces affinity for C1q (approximately 3-fold weaker). This modification destroys the glycosylation site and the presence of carbohydrate required for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combination thereof also reduce C1q binding: D270A, K322A, P329A and P311S (see WO 06 / 036291).

[0071] Reference to a human constant region includes constant regions containing any naturally occurring allotypes, or constant regions containing any permutation of residues occupying polymorphic positions in the naturally occurring allotypes, and there may be one, two, five or up to ten mutations to the native human constant region, such as those set forth above to reduce Fc gamma receptor binding or enhance binding to Fc RNA.

[0072] D. Expression of recombinant antibodies Humanized antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically contain expression control sequences operably linked to the coding sequences of the antibody chains, including naturally associated or heterologous promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and collection and purification of cross-reactive antibodies.

[0073] Mammalian cells are preferred hosts for expressing nucleotide fragments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody producing myelomas (including Sp2 / 0 and NS0). Preferably, the cells are non-human cells. Expression vectors for these cells can include expression control sequences, such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0074] Once expressed, antibodies can be purified by standard methods in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0075] IV. nucleic acid The present invention further provides a nucleic acid encoding any of the above humanized heavy and light chains. Typically, the nucleic acid also encodes a signal peptide fused to the mature heavy and light chains. The coding sequence on the nucleic acid may be operably linked to regulatory sequences, such as promoters, enhancers, ribosome binding sites, transcription termination signals, etc., to ensure expression of the coding sequence. The nucleic acids encoding the heavy and light chains may be present in isolated form or may be cloned into one or more vectors. The nucleic acids may be synthesized, for example, by PCR or solid-phase synthesis of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be linked as one continuous nucleic acid, for example in an expression vector, or may be separate (for example, each may be cloned into its own expression vector).

[0076] V. Antibody Drug Conjugates The anti-LIV-1 antibody can be conjugated to a cytotoxic or cytostatic moiety (including its pharma- ceutically acceptable salts) to obtain an antibody-drug conjugate (ADC). Particularly suitable moieties for conjugation to the antibody are cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes or compounds, or toxins (collectively, these moieties are referred to as therapeutic agents). For example, the anti-LIV-1 antibody can be conjugated to a cytotoxic agent, such as a chemotherapeutic agent, or a toxin (e.g., a cytostatic or cytocidal agent, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin).

[0077] The anti-LIV-1 antibody can be conjugated to a prodrug-converting enzyme. The prodrug-converting enzyme can be recombinantly fused to the antibody or chemically conjugated thereto using known methods. Exemplary prodrug-converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, beta-glucosidase, nitroreductase and carboxypeptidase A.

[0078] Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (Robinson et al., eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al., eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT Publication WO 89 / 12624).

[0079] The therapeutic agent may be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, antibody degradation, or a cleaving agent). Such a therapeutic agent is attached to the antibody using a cleavable linker that is susceptible to cleavage in the intracellular environment of the LIV-1-expressing cancer cell, but substantially insensitive to the extracellular environment, such that the conjugate is cleaved from the antibody when the conjugate is internalized by the LIV-1-expressing cancer cell (e.g., in an endosome, or, e.g., by pH- or protease-sensitive, or in a lysosomal environment, or in a caveolae environment).

[0080] Typically, the ADC comprises a linker region between the therapeutic agent and the anti-LIV-1 antibody. As described above, typically, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., in lysosomes or endosomes or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by intracellular peptidase or protease enzymes (including lysosomal or endosomal proteases). Typically, the peptidyl linker is at least 2 amino acids long or at least 3 amino acids long. Cleavage agents can include cathepsin B and D and plasmin (Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typically, the peptidyl linker is cleavable by enzymes present in LIV-1-expressing cells. For example, a peptidyl linker that can be cleaved by cathepsin-B, a thiol-dependent protease that is highly expressed in cancerous tissues, can be used (e.g., a linker that includes Phe-Leu or Gly-Phe-Leu-Gly peptides). Other such linkers are described, for example, in U.S. Pat. No. 6,214,345. In certain embodiments, the peptidyl linker that can be cleaved by intracellular proteases includes a Val-Cit linker or a Phe-Lys dipeptide (see, for example, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a Val-Cit linker). One advantage of utilizing intracellular proteolytic release of the therapeutic agent is that the agent is typically weakened when conjugated, and the serum stability of the conjugate is typically high.

[0081] The cleavable linker can be pH sensitive, i.e. sensitive to hydrolysis in a certain pH range.For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that can be hydrolyzed in lysosomes can be used (see, for example, U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661).Such linkers are relatively stable under neutral pH conditions such as in blood, but are unstable below pH 5.5 or 5.0 (approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (eg, a thioether attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929)).

[0082] Other linkers are cleavable under reducing conditions (eg, disulfide linkers). Disulfide linkers include those which can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (ed. C.W. Vogel, Oxford U. Press, 1987; see also U.S. Pat. No. 4,880,935).

[0083] The linker can also be a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304) or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0084] The linker may also be a non-cleavable linker, such as a maleimide-alkylene- or maleimide-aryl linker that is directly attached to a therapeutic agent (e.g., a drug) and the active drug-linker is released by degradation of the antibody.

[0085] Typically, the linker is not substantially sensitive to the extracellular environment. This means that when the ADC is present in an extracellular environment (e.g., in plasma), about 20% or less of the linker in the ADC sample is cleaved, typically about 15% or less, more typically about 10% or less, even more typically about 5% or less, 3% or less, or about 1% or less. Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating both (a) the ADC ("ADC sample") and (b) an equimolar amount of unconjugated antibody or therapeutic agent ("control sample") with plasma for a predetermined time (e.g., 2, 4, 8, 16 or 24 hours), and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample with the amount present in the control sample, for example, by measuring by high performance liquid chromatography.

[0086] The linker may also promote cellular internalization. The linker may promote cellular internalization when conjugated to a therapeutic agent (i.e., in the environment of a linker-therapeutic agent portion of an ADC or ADC derivative described herein). Alternatively, the linker may promote cellular internalization when conjugated to both a therapeutic agent and an anti-LIV-1 antibody (i.e., in the environment of an ADC described herein).

[0087] Various linkers that may be used in the present compositions are described in WO 2004-010957 and have the formula [ka]

[0088] (In the formula, -A- is an elongation unit; a is 0 or 1; Each -W- is independently an amino acid unit; W is independently an integer ranging from 0 to 12; -Y- is a spacer unit; and and y is 0, 1 or 2.

[0089] Representative extender units are shown within brackets in formulas (Ia) and (Ib; see below), where A-, -W-, -Y-, -D-, w and y are as defined above, R 1 HA-C1-C 10 Alkylene-, -C3-C8 carbocyclo-, -O-(C1-C8 alkyl)-, -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-, -C3-C8 heterocyclo-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C10 Alkylene-, -(CH2CH2O) r -and-(CHCHO) r -CH2-, and r is an integer ranging from 1 to 10. Ab is an antibody. [ka] JPEG2024178209000004.jpg2982

[0090] The drug loading is represented by p, which is the number of drug-linker molecules per antibody. Depending on the context, p may represent the average number of drug-linker molecules per antibody, also referred to as the average drug loading. P ranges from 1 to 20, preferably 1 to 8. In some preferred embodiments, when p represents the average drug loading, p ranges from about 2 to about 5. In some embodiments, p is about 2, about 3, about 4, or about 5. The average number of drugs per antibody in the preparation can be characterized by conventional means, such as mass spectrometry, ELISA assay, and HPLC. The amino acid unit (-W-) (if present) links the extender unit (-A-) to the spacer unit (-Y-) (if present), and in the absence of the spacer unit, links the extender unit to a cytotoxic or cytostatic agent (drug unit; D).

[0091] -W w -, when present, is preferably a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.

[0092] The spacer unit (-Y-), when present, links the amino acid unit to the drug unit. Spacer units are of two general types: self-immolative and non-self-immolative. A non-self-immolative spacer unit is one in which some or all of the spacer unit remains attached to the drug unit after enzymatic cleavage of the amino acid unit from the anti-LIV-1 antibody-linker-drug conjugate or drug-linker compound. Examples of non-self-immolative spacer units include (glycine-glycine) spacer units and glycine spacer units. When a glycine-glycine spacer unit or an anti-LIV-1 antibody-linker-drug conjugate containing a glycine spacer unit undergoes enzymatic cleavage by a tumor cell-associated protease, a cancer cell-associated protease or a lymphocyte-associated protease, the glycine-glycine-drug moiety or the glycine-drug moiety becomes an Ab-A a -W w - To release the drug, an independent hydrolysis reaction must occur within the target cell to cleave the glycine-drug unit bond.

[0093] Alternatively, anti-LIV-1 antibody drug conjugates containing a self-immolative spacer unit may release the drug (D) without the need for a separate hydrolysis step. In some of these embodiments, -Y- is linked to -W through the nitrogen atom of the PAB group. w- and a p-aminobenzyl alcohol (PAB) unit linked directly to -D via a carbonate, carbamate or ether group. Other examples of self-immolative spacers include aromatic compounds that are electrically equivalent to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (see, e.g., Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that can easily undergo cyclization upon amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Removal of amine-containing drugs that are substituted at the alpha position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of the self-immolative spacer method that can be applied to the anti-LIV-1 antibody-linker-drug conjugate. Alternatively, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional drugs.

[0094] Useful classes of cytotoxic agents for conjugating to anti-LIV-1 antibodies include, for example, antitubulin agents, DNA minor groove binders, DNA replication inhibitors, chemotherapy sensitizers, etc. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecins, duocarmycins, etoposide, maytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include auristatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin.

[0095] The cytotoxic agent may be a chemotherapeutic agent such as doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agent may also be a CC-1065 analog, a calicheamicin, a maytansine, an analog of dolastatin 10, rhizoxin, or palytoxin.

[0096] The cytotoxic agent can also be an auristatin. The auristatin can be an auristatin E derivative, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF and MMAE. The synthesis and structure of various auristatins are described, for example, in US 2005-0238649 and US2006-0074008.

[0097] The cytotoxic agent can be a DNA minor groove binder (see, e.g., U.S. Patent No. 6,130,237). For example, the minor groove binder can be a CBI compound or an enediyne (e.g., a calicheamicin).

[0098] The cytotoxic or cytostatic agent may be an antitubulin agent. Examples of antitubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel), T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine and vinorelbine) and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other suitable antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colchimide, estramustine, cryptophycin, cemadotin, maytansinoids, combretastatin, discodermolide and eleutherobin. [ka] JPEG2024178209000006.jpg207125JPEG2024178209000007.jpg176129

[0099] The cytotoxic agent may be a maytansinoid, another group of antitubulin agents. For example, the maytansinoid may be maytansine or a maytansine-containing drug linker, such as DM-1 or DM-4 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131).

[0100] Exemplary antibody drug conjugates include the vcMMAE and mcMMAF antibody drug conjugates as follows, where p and Ab are as previously described herein: [ka]

[0101] or a pharma- ceutically acceptable salt thereof.

[0102] VI. Other antibodies against LIV-1 Humanized forms of the BR2-14a and BR2-22a antibodies described above, as well as other antibodies that bind to the extracellular domain of LIV-1, may be used in some of the methods of the invention, particularly in the treatment of triple-negative breast cancer. A series of murine antibodies against LIV-1 are described in US20080175839. These antibodies include 1.1F10, 1.7A4, BR2-10b, BR2-11a, BR2-13a, BR2-14a, BR2-15a, BR2-16a, BR2-17a, BR2-18a, BR2-19a, BR2-20a, BR2-21a, BR2-22a, BR2-23a, BR2-24a and BR2-25a, of which BR2-14a and BR2-22a, as well as BR2-19a produced by hybridoma ATCC Accession No. PTA-5706 or BR2-23a produced by hybridoma ATCC Accession No. PTA-5707, are preferred. Humanized, chimeric or veneered forms of these antibodies may be produced by conventional methods as summarized below.

[0103] Other antibodies against LIV-1 can be produced de novo by immunization with LIV-1 or one or more extracellular domains thereof. Production of other non-human (e.g., mouse, guinea pig, primate, rabbit or rat) monoclonal antibodies against immunogens can be performed as described in Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988), which is incorporated herein by reference for all purposes. Such immunogens can be obtained from natural sources, by peptide synthesis, or by recombinant expression.

[0104] Humanized, chimeric or veneered forms of non-human antibodies can be produced. General methods for producing humanized antibodies are described in Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557. Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human (e.g., mouse) antibody are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the mouse antibody, with about two-thirds being human sequences. A veneered antibody is a type of humanized antibody that retains some of the non-human variable region framework and some, usually all, of the CDRs of a non-human antibody, but replaces other variable region framework residues that may contribute to B- or T-cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues from the corresponding positions in a human antibody sequence, resulting in an antibody in which the CDRs are completely or substantially derived from a non-human antibody and the variable region framework of the non-human antibody has been made more human-like by the replacement.

[0105] Human antibodies against LIV-1 can be obtained by various techniques, as described below. Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666; the use of transgenic mice containing human immunoglobulin genes (see, e.g., Lonberg et al., WO 93 / 12227 (1993); U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148, 1547-1553). (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991)) and phage display methods (see, for example, Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332).

[0106] Any of the antibodies can be selected, such as by competitive binding assays, to have the same or overlapping epitopic specificity as an exemplary antibody, such as the BR2-14a antibody.

[0107] VII. therapeutic use The humanized antibodies of the present invention, alone or as LIV-1 antibody drug conjugates thereof, can be used to treat cancer. Some such cancers exhibit detectable levels of LIV-1 as measured at the protein (e.g., by immunoassay using one of the exemplified antibodies) or mRNA level. Some such cancers exhibit elevated levels of LIV-1, preferably compared to non-cancerous tissues of the same type from the same patient. Typical levels of LIV-1 on cancer cells suitable for treatment are 5000-150000 LIV-1 molecules per cell, although higher or lower levels are also treatable. In some cases, the level of LIV-1 in the cancer is measured before treatment is administered.

[0108] Examples of cancers associated with LIV-1 expression and suitable for treatment include breast cancer, prostate cancer, ovarian cancer, endometrial cancer, cervical, liver, stomach, kidney and squamous cell carcinoma (e.g., bladder, head, neck and lung), skin cancer, such as melanoma, small cell lung cancer or lung carcinoid. The treatment can be applied to patients with these types of primary or metastatic tumors. The treatment can also be applied to patients who are resistant to conventional treatment (e.g., hormones, tamoxifen, herceptin) or who have relapsed after responding to such treatment. The method can also be used for triple-negative breast cancer. Triple-negative breast cancer is a terminology that refers to cancers that lack detectable estrogen and progesterone receptors and lack overexpression of HER2 / neu (when stained with antibodies against either of these receptors as described in the Examples). Staining can be performed against an irrelevant control antibody, and the lack of expression can be indicated by a background level of staining that is the same or similar to that of the control within experimental error. Similarly, lack of overexpression is indicated by the same or similar levels of staining, preferably within experimental error, of non-cancerous breast tissue obtained from the same patient. Alternatively or additionally, triple negative breast cancers are characterized by invasive behavior, characteristic metastatic patterns, and lack of responsiveness to hormones that interact with these receptors. hLIV14 antibodies may be used to treat cancers that express LIV-1. In one embodiment, hLIV14 antibodies are used to treat subjects with LIV-1 expressing breast cancer. In another embodiment, hLIV14 antibodies are used to treat subjects with LIV-1 expressing prostate cancer. In another embodiment, hLIV14 antibodies are used to treat subjects with LIV-1 expressing melanoma. In another embodiment, hLIV14 antibodies are used to treat subjects with LIV-1 expressing ovarian cancer. In another embodiment, hLIV14 antibodies are used to treat subjects with LIV-1 expressing endometrial cancer. In another embodiment, hLIV14 antibodies are used to treat subjects with LIV-1-expressing cervical cancer.In another embodiment, the hLIV14 antibody is used to treat a subject with a LIV-1 expressing liver cancer. In another embodiment, the hLIV14 antibody is used to treat a subject with a LIV-1 expressing gastric cancer. In another embodiment, the hLIV14 antibody is used to treat a subject with a LIV-1 expressing kidney cancer. In another embodiment, the hLIV14 antibody is used to treat a subject with a LIV-1 expressing squamous cell carcinoma (e.g., bladder, head, neck and lung cancer). In another embodiment, the hLIV14 antibody is used to treat a subject with a LIV-1 expressing breast cancer. In another embodiment, the hLIV14 antibody is used to treat a subject with a LIV-1 expressing skin cancer. In another embodiment, the hLIV14 antibody is used to treat a subject with a LIV-1 expressing small cell lung cancer or lung carcinoid. The hLIV22 antibody can be used to treat a cancer that expresses LIV-1. In one embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing breast cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing prostate cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing melanoma. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing ovarian cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing endometrial cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing cervical cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing liver cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing gastric cancer. In another embodiment, hLIV22 antibodies are used to treat subjects with LIV-1-expressing renal cancer.In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing squamous cell carcinoma (e.g., bladder, head, neck and lung cancer). In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing breast cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing skin cancer. In another embodiment, the hLIV22 antibody is used to treat a subject with a LIV-1 expressing small cell lung cancer or lung carcinoid. This application provides the first disclosure that the LIV-1 protein is expressed on the surface of melanoma cells. Thus, antibodies that bind to LIV-1 can be used to treat patients with melanomas that express LIV-1. Such antibodies include, but are not limited to, the antibodies disclosed herein, such as hLIV14 and hLIV22.

[0109] The humanized antibody, alone or as a conjugate thereof, is administered in an effective form, which means a dosage, route of administration and frequency of administration that delays the onset of cancer, reduces its severity, inhibits further progression, and / or improves at least one sign or symptom.If the patient is already suffering from cancer, the form may be referred to as a therapeutically effective form.If the patient has an elevated risk of cancer compared to the general population, but has not yet developed symptoms, the form may be referred to as a prophylactically effective form.In some cases, therapeutic or prophylactic efficacy may be observed in an individual patient by comparison with past controls or past experience in the same patient.In other cases, therapeutic or prophylactic efficacy may be shown in preclinical or clinical trials in a population of treated patients by comparison with a control population of untreated patients.

[0110] Typical dosages of monoclonal antibodies are 0.1 mg / kg to 50 mg / kg per kg of patient body weight, more preferably 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 12 mg / kg, or 1 mg / kg to 10 mg / kg, or 2 mg / kg to 30 mg / kg, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 12 mg / kg, or 2 mg / kg to 10 mg / kg, or 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg, 3 mg / kg to 12 mg / kg, or 3 mg / kg to 10 mg / kg. Typical dosages of the monoclonal antibody or antibody drug conjugate thereof are 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg, or 3 mg / kg to 7.5 mg / kg, or 0.1 to 20 or 0.5 to 5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg) or a flat dose of 10 to 1500 or 200-1500 mg per kg of subject's body weight. In some methods, the patient is administered a dose of at least 1.5 mg / kg, at least 2 mg / kg, or at least 3 mg / kg, at least once every three weeks. The dosage depends, among other things, on the frequency of administration, the patient's condition and response to previous treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0111] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Administration can also be directly localized within the tumor. Administration into the systemic circulation by intravenous or subcutaneous administration is preferred. Intravenous administration can be, for example, by infusion over a period of, for example, 30 to 90 minutes, or by a single bolus injection.

[0112] The frequency of administration depends, inter alia, on the half-life of the antibody or conjugate in circulation, the condition of the patient, and the route of administration. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals, depending on the change in the patient's condition or the progression of the cancer being treated. A typical frequency for intravenous administration is twice weekly to four times a year over the continuous course of treatment, although more or less frequent administration is also possible. Another typical frequency for intravenous administration is weekly to three times every four weeks over the continuous course of treatment, although more or less frequent administration is also possible. For subcutaneous administration, the typical frequency is daily to monthly, although more or less frequent administration is also possible.

[0113] The number of doses administered depends on the nature of the cancer (e.g., whether it presents an acute or chronic condition) and the response of the condition to treatment. In the case of acute disorders or acute exacerbations of chronic disorders, 1 to 10 doses are often sufficient. Sometimes, in the case of acute disorders or acute exacerbations of chronic disorders, a single bolus dose, optionally in divided form, is sufficient. In the case of recurrence of acute disorders or acute exacerbations, treatment can be repeated. In the case of chronic disorders, the antibody can be administered at regular intervals, for example, weekly, biweekly, monthly, quarterly, every 6 months, for at least 1, 5 or 10 years or for the life of the patient.

[0114] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., single-dose dosage form). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients or adjuvants. The formulation depends on the route of administration selected. For injection, the antibody may be formulated in an aqueous solution, preferably a physiologically acceptable buffer, such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulating agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the antibody may be in lyophilized form that is reconstituted with a suitable vehicle, such as pyrogen-free sterile water, before use. The concentration of the antibody in the liquid formulation may be, for example, 1 to 100 mg / mg, for example, 10 mg / ml.

[0115] Treatment with the antibodies of the invention may be combined with chemotherapy, radiation, stem cell therapy, surgery, or other treatments effective against the disorder being treated. Useful classes of other agents that may be administered with humanized antibodies against LIV-1 include, for example, antibodies against other receptors expressed on cancerous cells, antitubulin agents (e.g., auristatins), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, e.g., cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycin, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosoureas, platinol, pre-forming compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0116] Treatment with a humanized anti-LIV-1 antibody, either alone or as an antibody-drug conjugate, optionally in combination with any of the other substances or forms mentioned above, may increase the median progression-free survival or overall survival time of a patient, particularly one with a recurrent or refractory tumor (e.g., breast cancer, prostate cancer, melanoma), by at least 30% or 40%, preferably 50%, 60%-70%, or even 100% or more, compared to the same treatment (e.g., chemotherapy) without the anti-LIV-1 antibody (alone or as a conjugate). Additionally or alternatively, treatment (e.g., standard chemotherapy) including an anti-LIV-1 antibody (alone or as a conjugate) may increase the complete response rate, partial response rate, or objective response rate (complete + partial) of a patient with a tumor by at least 30% or 40%, preferably 50%, 60%-70%, or even 100%, compared to the same treatment (e.g., chemotherapy) without the anti-LIV-1 antibody.

[0117] Typically, in clinical trials (e.g., phase II, phase II / III or phase III trials), the increase in the semi-progression-free survival and / or response rate of patients treated with standard therapy and the humanized anti-LIV-1 antibody compared to a control group of patients receiving standard therapy alone (or in addition to a placebo) is statistically significant, for example at a level of p=0.05 or 0.01 or even 0.001. The complete and partial response rates are determined by objective criteria commonly used in cancer clinical trials (e.g., those listed or accepted by the National Cancer Institute and / or Food and Drug Administration).

[0118] VIII. Other uses The anti-LIV-1 humanized antibody can be used to detect LIV-1 in clinical diagnosis or therapy or research. Expression of LIV-1 on cancer provides an indication that the cancer is suitable for treatment with the antibody of the present invention. The antibody can be sold as a research reagent for laboratory research in detecting cells containing LIV-1 and their response to various stimuli. In such applications, the monoclonal antibody can be labeled with a fluorescent molecule, a spin-label molecule, an enzyme or a radioisotope, and can be provided in the form of a kit that includes all the necessary reagents to perform an assay for LIV-1. The antibodies BR2-14a, BR2-22a, and their humanized forms, such as hLIV14 and hLIV22, described herein, can be used to detect LIV-1 protein expression and to determine whether a cancer is suitable for treatment with LIV-1 ADC. As an example, BR2-14a, BR2-22a, and their humanized forms, such as hLIV14 and hLIV22, can be used to detect LIV-1 expression on breast cancer cells, melanoma cells, cervical cancer cells, or prostate cancer cells. The antibodies can also be used to purify LIV-1, for example, by affinity chromatography.

[0119] IX. Cynomolgus monkey LIV-1 The invention further provides the amino acid sequence of LIV-1 from cynomolgus monkey (CY LIV-1) in SEQ ID NO:85, with or without the signal peptide (which occupies about residues 1-28 of SEQ ID NO:85), and nucleic acids encoding the amino acid sequence. Variants that differ by up to 1, 2, 3, 4 or 5 substitutions, deletions or insertions are also included. However, CY variants do not include the native human LIV-1 sequence. As with human LIV-1, reference to CY-LIV-1 refers to the entire protein except at least the extracellular domain of the protein, usually the cleavable signal peptide (amino acids 1-28). The invention further provides antibodies that specifically bind to SEQ ID NO:85, with or without specific binding to human LIV-1 (i.e., binding to human LIV-1 at the level of a negative control irrelevant antibody). The invention further provides antibodies that bind preferentially to CY-LIV-1 over to human LIV-1 (or vice versa). Preferential binding means binding higher than experimental error, preferably at least 2, 3 or 4 fold higher. The present invention further provides antibodies that exhibit a binding profile to human and CY LIV-1 that is the same within experimental error as any of the exemplified antibodies described below. The present invention further provides methods for analyzing the binding of an antibody to CY LIV-1. Such methods include contacting an antibody with CY LIV-1 and determining whether the antibody specifically binds to CY LIV-1, and optionally determining a measure of the binding strength, such as an association constant.

[0120] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated herein in their entirety for all purposes as if each of them was specifically and individually indicated to be incorporated herein by reference. Where various forms of a sequence are associated with an accession number at different times, the form associated with the accession number at the effective filing date of this application is relevant. The effective filing date means the earlier of the actual filing date or, where applicable, the filing date of the priority application to which the accession number is referred. Similarly, where various forms of publications, websites, etc. are published at different times, the form most recently published at the effective filing date of this application is relevant, unless otherwise indicated. Any feature, step, element, embodiment or aspect of the invention may be combined with any other, unless otherwise indicated. The invention has been described in considerable detail by way of illustration and example for purposes of clarity and understanding, but it will be apparent that certain changes and modifications may be made within the scope of the appended claims. The present invention encompasses the following embodiments: 1. A humanized antibody that specifically binds to human LIV-1, comprising a mature heavy chain variable region having the amino acid sequence of SEQ ID NO: 52 or 53, and a mature light chain variable region having the amino acid sequence of SEQ ID NO: 59 or 60. 2. The humanized antibody of embodiment 1, wherein the mature heavy chain variable region is fused to a heavy chain constant region and the mature light chain variable region is fused to a light chain constant region. 3. The humanized antibody of embodiment 2, wherein said heavy chain constant region is a mutated form of the native human constant region that exhibits reduced binding to Fc gamma receptors compared to the native human constant region. 4. The humanized antibody of embodiment 2, wherein the heavy chain constant region is of the IgG1 isotype. 5. The humanized antibody of embodiment 2, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:44, and the light chain constant region has an amino acid sequence comprising SEQ ID NO:42. 6. The humanized antibody of embodiment 2, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:46, and the light chain constant region has an amino acid sequence comprising SEQ ID NO:42. 7. A humanized antibody according to any one of embodiments 1 to 6, wherein the mature heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 53, and the mature light chain variable region has the amino acid sequence shown in SEQ ID NO: 60. 8. The humanized antibody of any of embodiments 1 to 7, wherein the antibody is conjugated to a cytotoxic or cytostatic agent. 9. The humanized antibody of embodiment 8, which is conjugated to a cytotoxic agent, auristatin. 10. The humanized antibody of embodiment 8, wherein the cytotoxic agent is monomethylauristatin F (MMAF). 11. The humanized antibody of embodiment 8, wherein the cytotoxic or cytostatic agent is monomethylauristatin E (MMAE). 12. A nucleic acid encoding a mature heavy chain variable region and / or a mature light chain variable region according to any one of embodiments 1 to 11. 13. A pharmaceutical composition comprising the humanized antibody according to any one of embodiments 1 to 11 for use in treating a cancer expressing LIV-1. 14. The pharmaceutical composition of embodiment 13, wherein the cancer is triple negative breast cancer. 15. The pharmaceutical composition of embodiment 13, wherein the cancer is breast cancer, prostate cancer, cervical cancer or melanoma.

[0121] (Example) I. Humanization of BR2-14a material The cell lines described in the following examples were maintained in culture according to conditions specified by the American Type Culture Collection (ATCC), the National Cancer Institute (NCI) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ). Cell culture reagents were obtained from Invitrogen Corp. (Carlsbad, Calif.) or other suppliers.

[0122] method: Saturation binding assay 1×10 5 Antigen-expressing cells (MCF7 cells (ATCC) expressing human LIV-1, transfected CHO cell lines expressing human LIV-1, or transfected CHO cell lines expressing sino LIV-1) were aliquoted per well of a 96-well v-bottom plate. AlexaFluor-647-labeled mouse LIV-1 mAb, e.g., BR2-14a, was added at concentrations ranging from 0.66 pM to 690 nM and incubated on ice for 30 min. Cells were pelleted and washed three times with PBS / BSA. The cells were then pelleted and resuspended in 125 μL PBS / BSA. Fluorescence was analyzed by flow cytometry to determine percent binding using the percentage of saturated fluorescent signal, and the apparent Kd was then calculated.

[0123] Competitive binding assay 1 x 10 CHO cells in PBS / BSA expressing recombinant human LIV-1 51000 s was aliquoted into each well of a 96-well V-bottom plate on ice. The cells were incubated for 1 hour with 5 nM AlexaFluor-647 (AF)-labeled parental mouse LIV-1 mAb and increasing concentrations (from 0.038 nM to 600 nM) of unlabeled humanized LIV-1 mAb, a combination of humanized light chain LA-LF and humanized heavy chain HA-HE. The cells were pelleted and washed three times with PBS / BSA. The cells were pelleted and resuspended in 125 μL PBS / BSA. Fluorescence was analyzed by flow cytometry to determine the percentage of labeled mouse LIV-1 mAb bound using the percentage of saturated fluorescent signal, and the EC50 was then extrapolated by fitting the data to a sigmoidal dose-response curve with different slopes.

[0124] 1 × 10 MCF7 cells in PBS / BSA expressing LIV-1 5 1000g of each was aliquoted into each well of a 96-well V-bottom plate on ice. The cells were incubated for 1 hour with 5 nM AlexaFluor-647-labeled mouse LIV-1 mAb and increasing concentrations (from 0.038 nM to 600 nM) of unlabeled humanized LIV-1 mAb, a combination of humanized light chain LA-LF and humanized heavy chain HA-HE. The cells were pelleted and washed three times with PBS. The cells were pelleted and resuspended in 125 μL PBS / BSA. Fluorescence was analyzed by flow cytometry to determine the percentage of labeled mouse LIV-1 mAb bound using the percentage of saturated fluorescent signal, and the EC50 was then extrapolated by fitting the data to a sigmoidal dose-response curve with different slopes.

[0125] 1 x 10 CHO cells in PBS expressing recombinant Sino-LIV-1 51000g of each was aliquoted into each well of a 96-well V-bottom plate on ice. The cells were incubated for 1 hour with 5 nM AlexaFluor-647-labeled mouse LIV-1 mAb and increasing concentrations (from 0.038 nM to 600 nM) of unlabeled humanized LIV-1 mAb, a combination of humanized light chain LA-LF and humanized heavy chain HA-HE. The cells were pelleted and washed three times with PBS. The cells were pelleted and resuspended in 125 μL PBS / BSA. Fluorescence was analyzed by flow cytometry to determine the percentage of labeled mouse LIV-1 mAb bound using the percentage of saturated fluorescent signal, and the EC50 was then extrapolated by fitting the data to a sigmoidal dose-response curve with different slopes.

[0126] Quantitative flow cytometry analysis Quantitation of LIV-1 copy number on the cell surface was determined using mouse LIV-1 mAb as the primary antibody and the DAKO QiFiKit flow cytometry indirect assay according to the manufacturer's instructions (DAKO A / S, Glostrup, Denmark) and assessed using a Becton Dickinson FACS®can flow cytometer.

[0127] Cytotoxicity Assay Tumor cells were incubated with LIV-1 antibody drug conjugates for 96-144 hours at 37°C. Non-binding (H00) ADC was used as a negative control. Viability was measured with resazurin (Sigma) at a final concentration of 50 μM. Cells were incubated at 37°C for 4-6 hours. Fluorescence signal was measured on a Fusion HT fluorescent plate reader (Perkin Elmer, Waltham, MA). Results are expressed as IC50, the concentration of compound required to cause a 50% reduction in viability when compared to vehicle-treated cells (control = 100%).

[0128] Production of antibody drug conjugates The antibody drug conjugate of the LIV-1 antibody was prepared as described in US20050238649. Both drug linkers vcMMAE (also referred to as 1006) and mcMMAF (referred to as 1269) are described in US20050238649. The preparation of cysteine ​​mutants of IgG1 antibodies is generally described in US20100158919. US20050238649 and US20100158919 are incorporated herein by reference for all purposes.

[0129] Nonfucosylated anti-LIV-1 mAb CHO DG44 cell line producing humanized IgG1 anti-LIV-1 monoclonal antibody HBLB mAb (hLIV-14) was cultured at 3.0 × 10 in 30 mL of CHO medium at 37 °C, 5% CO2 with shaking at 100 RPM in a 125 mL shake flask. 5 The cells were cultured at 1000 ng / mL. The medium was supplemented with insulin-like growth factor (IGF), penicillin, streptomycin and 65 μM 2-fluorofucose peracetate (SGD-2084) (see US20090317869). On day 3, the culture was fed with 2% volume of feed medium. On day 4, the culture was split 1:4 into fresh medium. On days 5, 7, 9 and 10, the culture was fed with 6% volume of production feed medium. On day 13, conditioned medium was collected by passing the culture through a 0.2 μm filter. Antibody purification was performed by applying the conditioned medium to a Protein A column pre-equilibrated with 1× phosphate buffered saline (PBS) (pH 7.4).

[0130] After washing the column with 20 column volumes of 1x PBS, the antibody was eluted with 5 column volumes of Immunopure IgG Elution Buffer (Pierce Biotechnology, Rockford, IL). A 10% volume of 1M Tris (pH 8.0) was added to the eluted fraction. Samples were dialyzed overnight into 1x PBS.

[0131] Antibody-dependent cellular cytotoxicity (ADCC) Standard 51ADCC activity was measured using a Cr release assay. Briefly, the MCF-7 target tumor cells were treated with 100 μCi of Na 51 The cells were labeled with CrO4, washed, and preincubated with the test antibody before the addition of effector (natural killer, NK) cells. Immunomagnetic beads (EasySep, StemCell Technologies, Vancouver, BC, Canada) were used to isolate NK (CD16 + CD56 + ) cells were prepared. Viable NK cells were added to the target cells at an effector to target cell ratio of 10:1. Human IgG1κ (Ancell, Bayport, MN) was used as a negative control in this assay. After 4 hours of incubation, the supernatant was collected and dried overnight on a Luma plate. Gamma radiation emitted from lysed MCF-7 cells was then detected using a TopCount Microplate Scintillation and Luminescence Counter (Perkin Elmer, Waltham, Massachusetts). ADCC activity is shown as % specific cell lysis.

[0132] in vivo activity studies Nude (nu / nu) mice (7–8 per group) were implanted with the following tumor cells grown in culture: MCF-7 (5 × 10 in 25% Matrigel) from NCI; 6 cells), PC3 from ATCC (2.5 × 10 6 cells) and PC3 from DSMZ (5 × 10 5 For in vivo growth of MCF-7 cells, estrogen supplementation was also administered to female mice by implanting sustained release estrogen pellets (90-day release). 3When tumor volume reached ∼800 mm, dosing with chimeric or humanized LIV-1 ADC or non-binding control ADC (3 mg / kg) was initiated (q4d × 4 intraperitoneal injections). Tumor volume was monitored using calipers and was monitored until tumor volume reached ∼800 mm. 3 Animals were euthanized when tumor volume reached 0.01 mg / kg / day. Median tumor volume plots continued for each group until one or more animals were euthanized. All animal care was performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility and in accordance with protocols approved by the Institutional Animal Care and Use Committee.

[0133] LIV-1 immunohistochemical (IHC) staining method Tumor microarrays (TMA) and individual tumor samples were obtained from commercial sources. Tissue microarrays from normal or tumor formalin-fixed and paraffin-embedded (FFPE) tissues were purchased from US Biomax Inc. or Cybrdi. Frozen arrays were purchased from BioChain. Single sections were purchased from NDRI, Asterand, Tissue Solution or CHTN. A set of 25 paraffin-embedded samples of metastatic hormone-refractory prostate cancer (with corresponding bone and soft tissue metastatic sites) was provided by Dr. R. Vessella (University of Washington, Genitourinary Cancer Department). All samples were processed on a Bond-Max™ automated stainer (Leica).

[0134] IHC staining of FFPE tissue: TMAs sectioned on FFPE or glass slides were deparaffinized and rehydrated using Bond™ Dewax solution (Leica, cat # AR9222) at 72°C. Antigen retrieval was performed using EDTA-based Bond™ Epitope Retrieval Solution 2 (Leica, cat # AR9640) for 20 min at 95-100°C prior to incubation with primary mouse LIV-1 mAb (1-2 μg / ml) (30-45 min at 25°C). Isotype-matched mouse IgG1 (Sigma; cat # M5284) was used as a negative control for background staining. For automated IHC staining, the Refine DAB kit or an alkaline phosphatase-based detection kit: Bond™ Polymer AP Red Detection kit (Leica, cat # DS9305) was used. Slides were incubated with mouse monoclonal primary antibody against mouse LIV-1 mAb (1 μg / ml) for 45 min with a preliminary 30 min protein block (DAKO cat #X0909). After chromogen development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by a pathologist. Images were taken using a Zeiss Axiovert 200M microscope (Carl Zeiss, Inc., Thornwood, NY).

[0135] IHC on frozen tissue: 5 μm sections of frozen / OCT samples were fixed with acetone for 10 min, air-dried for 30 min, and pretreated with 1×Morphosave for 20 min at room temperature. The slides were loaded into a Bond-Max™ autostainer (Leica) and stained with primary antibodies for 45 min with a preliminary 30 min protein block (DAKO cat# X0909). Mouse IgG1 (BD Pharmingen cat #550878) was used as a negative control. For detection, a DAB-based Bond Polymer Refine kit (Leica, cat # DS9800) was used. After chromogen development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by a pathologist.

[0136] result 1. Mouse Antibody Binding Mouse LIV-1 monoclonal antibody BR2-14a antibody (US2004141983) K D was determined for human LIV-1 expressed as an endogenous protein in a human breast cancer cell line or as a recombinant protein in a CHO cell line. D was also determined for cyno LIV-1 expressed as a recombinant protein in a CHO cell line. MCF7 is a human breast cancer cell line. 293F is a human embryonic kidney cell line. Table 1 shows that the antibody had a dissociation constant about 5-fold lower for non-recombinant LIV-1 expressed from human cell lines than for recombinant LIV-1, both human (hLIV-1) and cynomolgus (cyLIV-1). [Table 1]

[0137] 2. Design and Testing of Humanized Antibodies The starting point for humanization in this example, i.e. the donor antibody, is the murine antibody BR2-14a produced by the hybridoma with ATCC accession number PTA-5705A and described in US2004141983. Suitable human acceptor sequences are the genomic sequences provided by VH1-02 and JH5 for the heavy chain and VK2-30 and Jk4 for the light chain. The human acceptor sequences show 68 and 85% identity to the donor sequence in the variable region framework. The light chain CDRs of the human acceptor sequence are of the same canonical type as the CDRs of the donor sequence. In contrast, the heavy chain CDRs of the human acceptor sequence were different in their canonical type (1-3 in the germline but 1-2 in the mouse donor).

[0138] Alignment of the donor sequences identified 11 positions in the heavy chain (H27, H28, H29, H30, H48, H66, H67, H71, H76, H93, and H94) and 5 positions in the light chain (L36, L37, L45, L46, and L39) where the human acceptor sequence differed from the donor sequence. These positions may affect antibody binding as a result of direct contact with the antigen, affect the conformation of the CDRs, or affect packing between the heavy and light chains. Five humanized heavy chains and six humanized light chains containing back mutations at various permutations of these positions were generated (Figure 1 (sequence alignment) and Table 2). [Table 2]

[0139] Humanized antibodies corresponding to each permutation of these chains (30 possibilities) of humanized heavy and light chains were then expressed. Binding curves for recombinant human LIV-1 expressed from CHO cells are shown in Figure 2. EC50s are summarized in Table 3 below. [Table 3]

[0140] These data show considerable variation in EC50 among the 30 humanized antibodies tested, of which HBLB and HELE show at least 2-fold better binding than the next humanized antibody, HBLF, and a larger margin than most of the humanized antibodies. The binding curves in Figure 2 show that HBLB and HELE provide stronger binding than the parent mouse antibody.

[0141] The HBLB antibody was selected as the best of the humanized antibodies because it (along with HELE) provides the strongest binding but has fewer back mutations than HELE (there are 4 back mutations in HBLB and 12 in HELE).

[0142] The EC50 for human LIV-1 mAbs binding to human LIV-1 expressed on CHO cells was determined against human LIV-1 expressed as native protein in the MCF7 cell line (Figure 3). Again, LIV-1 mAbs HBLB and HELE were determined to be the strongest binders. K on HBLB against human LIV-1 on MCF7 cells d was determined to be 1.5 nM from the average of several saturation binding curves, while the K d is 2.9 nM. In other words, the HBLB antibody has approximately twice the affinity for native human LIV-1 compared to the mouse antibody. The saturation binding curves shown in Figure 4 are representative.

[0143] The two forms of HBLB were compared for binding to human LIV-1 recombinantly expressed from CHO cells. One form was expressed with wild-type human IgG1 and kappa constant regions. The other form was the same except for the S239C mutation (EU numbering) in the IgG1 heavy chain, which reduced the binding of the antibody to Fc gamma receptors (referred to as LIV-14d or HBLB S239C). The binding curves and EC50 of these antibodies compared to the mouse donor antibody are shown in Figure 5. The EC50 of both forms of HBLB were similar to each other (within the error of the study) and both were more potent than the mouse antibody.

[0144] The EC50 for the humanized LIV-1 mAbs HBLB and HBLB S239C was also determined against sino LIV-1 expressed as recombinant protein in a CHO cell line, and both antibodies bound with equal affinity (better than the murine LIV-1 mAb).

[0145] Expression data for LIV-1 Mouse LIV-1 mAbs (at least two for matching) were used for immunohistochemical analysis of various tumor types using formalin-fixed paraffin-embedded tissues. [Table 4]

[0146] We observed lower LIV-1 IHC positivity in studies performed using tissue microarrays than in larger tissue sections. The difference in expression was highly significant, suggesting that analysis of LIV-1 expression in larger tissue sections is preferred. There was good concordance of expression when at least two different anti-LIV-1 mAbs were used. Figures 6 and 7 show high levels of LIV-1 expression in breast and prostate tumors after hormonal (tamoxifen or aromatase inhibitor) treatment, providing a strong rationale for targeting these tumors using LIV-1 ADCs. Figure 8 shows detectable LIV-1 expression in triple-negative (ER-, PgR-, Her2-) breast cancer tissues. The LIV-1 expression levels in triple-negative breast cancer by immunohistochemical staining were comparable to those in the PC3 animal model in which we demonstrated antitumor activity of LIV-1 ADCs. Therefore, triple negative breast cancers are a possible target population, especially those that are known to express LIV-1.

[0147] In Vitro Antitumor Activity of hLIV-14 mAb as ADC and Enhanced Effector Function mAb (SEA) The antitumor activity of LIV-1 ADC in vitro was measured using both cytotoxicity assays (Figure 9) and antibody-dependent cellular cytotoxicity (ADCC) (Figures 10 and 11). First, a survey of LIV-1 expression in different cell lines was performed by quantitative FACS analysis. The breast cancer cell line MCF-7 from ATCC had the highest level of LIV-1 binding sites / cell compared to MCF-7 cell lines from other sources (data not shown). This cell line was used for both in vitro assays. With respect to Figure 9, various hLIV-14 ADCs (HBLB antibodies conjugated to vcMMAE (designated 1006) or mcMMAF (designated 1269), both small molecules and / or linkers described in US20050238649) were highly effective in killing MCF-7 cells compared to non-binding and mouse control conjugates (mIgG-1006, mIgG-1269, hIgG-1006 and hIgG-1269). Cysteine ​​mutant LIV-14d ADCs, with an average of two drug linkers per antibody, were also highly effective in killing MCF-7 cells as measured by cytotoxicity assays. With respect to Figures 10 and 11, the activity of fucosylated / wild type (WT) mAbs and ADCs was compared to effector function-enhanced forms (non-fucosylated mAbs and ADCs designated SEA) in ADCC assays. The results showed that effector-function-enhanced LIV-1 mAbs and ADCs had better ADCC activity against MCF-7 cells compared to non-effector-function-enhanced mAbs or ADCs (e.g., compare hLIV SEA vcMMAE to hLIV-1 vcMMAE in FIG. 10). Referring again to FIG. 9, the effector-function-enhanced LIV-1 ADC (shown as SEA) also had similar levels of cytotoxic activity to the wild-type (non-fucosylated) ADC (compare hLIV-1 SEA 1006 (vcMMAE) to hLIV-1 1006 (vcMMAE)). Thus, cytotoxicity can be influenced by both effector function and conjugation.

[0148] In vivo antitumor activity of hLIV-14 ADC Breast (MCF-7) and prostate (PC-3) cancer models were used to measure the in vivo antitumor activity of LIV-1 ADC (chimeric and humanized (HBLB) mAbs with an average of 4 drugs per antibody) (Figures 12-15). LIV1 ADC conjugated to vcMMAE showed significant tumor retardation compared to untreated and control ADC. In all studies using LIV-1-vcMMAE at 3 mg / kg, at least one complete regression (CR) was observed, with some animals having static or slow-growing tumors compared to controls. With regard to Figure 12, the chimeric form of the parental murine antibody conjugated to vcMMAE caused complete regression in 3 out of 7 mice. With regard to Figure 13, the same chimeric ADC caused complete regression in 1 out of 8 mice. With respect to Figure 14, humanized ADC conjugated to vcMMAE (HBLB) (hLIV-14-vcMMAE(4)) caused complete regression in 1 of 8 mice. Also, a cysteine ​​mutated form of the HBLB antibody (vcMMAE drug linker conjugated to each heavy chain at position 239 to give a conjugate with an average drug loading of 2 drug linkers per antibody, designated hLIV-14d-vcMMAE(2)) showed similar activity to the 4-loaded form. With respect to Figure 15, humanized ADC conjugated to vcMMAE (HBLB) (hLIV-14-vcMMAE(4)) caused complete regression in 1 of 8 mice in a prostate cancer model. In contrast, activity of the two loaded cysteine ​​mutants was not significant in this model (compare hLIV-14-vcMMAE(4) with hLIV-14d-vcMMAE(2) and hLIV-14-mcMMAF(4) with hLIV-14d-mcMMAF(2). In summary, these studies demonstrate that LIV-1 ADCs can stop or slow the growth of LIV-1-expressing cancers, including breast and prostate cancers.

[0149] II. Humanization of BR2-22a BR2-22a, sometimes referred to as mAb2, is a murine monoclonal antibody of the isotype IgG1 kappa.

[0150] method: Unless otherwise indicated below, the methods described for humanizing and testing BR2-14a are also applicable to BR2-22.

[0151] Saturation binding assay 1×10 5 Antigen-expressing cells (MCF7 cells expressing human LIV-1, 293F cells expressing human LIV-1, transfected CHO cell lines expressing human LIV-1, or transfected CHO cell lines expressing sino LIV-1) were aliquoted per well in a 96-well v-bottom plate. AlexaFluor-647-labeled mouse BR2-22a was added at concentrations ranging from 0.66 pM to 690 nM and incubated on ice for 30 minutes. Cells were pelleted and washed three times with PBS / BSA. The cells were then pelleted and resuspended in 125 μL PBS / BSA. Fluorescence was analyzed by flow cytometry to determine percent binding using the percentage of saturated fluorescent signal, and the apparent Kd was then calculated.

[0152] Competitive binding assay 1 x 10 CHO cells in PBS expressing recombinant LIV-1 5100 ml of each of the 1000 μL aliquots were aliquoted into each well of a 96-well V-bottom plate on ice. The cells were incubated with 5 nM AlexaFluor-647 (AF)-labeled parental mouse BR2-22a and increasing concentrations (from 0.038 nM to 600 nM) of unlabeled humanized BR2-22a antibody (all combinations of humanized light chain LA-LG and humanized heavy chain HA-HG). The cells were pelleted and washed three times with PBS. The cells were then pelleted and resuspended in 125 μL PBS / BSA. Fluorescence was analyzed by flow cytometry to determine the percentage of labeled humanized BR2-22a antibody bound using the percentage of saturated fluorescent signal, and the EC50 was then extrapolated by fitting the data to a sigmoidal dose-response curve with different slopes.

[0153] in vivo activity studies Nude (nu / nu) mice (7–8 per group) were implanted with the following tumor cells grown in culture: MCF-7 (NCI) (5 × 10 6 cells), PC3 from ATCC (2.5 × 10 6 cells) and PC3 from DSMZ (5 × 10 5 For in vivo growth of MCF-7 cells, estrogen supplementation was also administered to female mice by implanting sustained release estrogen pellets (90-day release). 3 When tumor volume reached ∼800 mm, dosing with chimeric or humanized LIV-1 ADC or non-binding control ADC (3 mg / kg) was initiated (q4d × 4 intraperitoneal injections). Tumor volume was monitored using calipers and was monitored until tumor volume reached ∼800 mm. 3Animals were euthanized when tumor volume reached 0.01 mg / kg / day. Median tumor volume plots continued for each group until one or more animals were euthanized. All animal care was performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility and in accordance with protocols approved by the Institutional Animal Care and Use Committee.

[0154] Summary of Results and Discussion saturated bond BR2-22a shows 94% identity to BR2-14a in the mature heavy chain variable region and 91% identity in the mature light chain variable region. The KD of BR2-22a for mouse Liv1 (Table 5) was determined for human LIV-1 expressed as an endogenous protein in a human breast cancer cell line, 293F cells, or as a recombinant protein in a CHO cell line. The KD for BR2-22a was also determined for sino LIV-1 expressed as a recombinant protein in a CHO cell line. [Table 5]

[0155] Humanization Methods The BR2-22a antibody was humanized using the VH1-02 JH5 germline acceptor sequence for the heavy chain and the VK2-30 JK4 acceptor sequence for the light chain. These acceptor sequences were selected based on their highest sequence identity to the mature variable region frameworks of the BR2-22A heavy and light chains. First, five mutant heavy chains were constructed, each containing the three Kabat CDRs from the heavy chain of BR2-22a, and the chains differed in having between 0 (VA) and 11 (VE) backmutations. First, six mutant light chains were constructed, each containing the three Kabat CDRs from the light chain of BR2-22a and between 0 (LA) and 4 (LF) backmutations. These back mutations were selected as a result of modeling the BR2-22A antibody to identify positions that may directly interact with antigen and affect CDR conformation or affect the interface between the heavy and light chains, and based on previous experience in humanizing BR2-14a due to the high sequence identity between BR2-14a and BR2-22a. In fact, the same 11 positions in the heavy chain and the same 4 positions in the light chain were considered for back mutation in both BR2-14a and BR2-22a (L39 was not considered in BR2-22a because the mouse residue is the same as the human residue). The back mutations present in each variant of humanized BR2-22a are shown in Tables 6 and 7 below. [Table 6] [Table 7]

[0156] The complete sequence of the mature variable region of each mutant is shown in Figures 16A and 16B.

[0157] All five of these heavy and six light chain permutations were then tested in a competition assay compared to BR2-22a (see FIG. 17). Surprisingly, given the experience with the BR2-14a antibody, where improved binding compared to the murine antibody was obtained with only four backmutations and further backmutations did not necessarily improve binding affinity, the only combination of humanized chains that showed binding affinity approaching that of BR2-22a was HELF with 15 backmutations. The other permutations showed poor or no significant binding to LIV-1. The EC50s of these various permutations are shown in Table 8 below. [Table 8]

[0158] Although HELF shows satisfactory binding, the antibody contains a total of 15 back mutations, a number that is greater than ideal for potential immunogenicity. Therefore, the HE and LF chains were systematically varied to test the effect of removing individual back mutations. Figure 18 shows the mutants tested. LF-1 to LF-4 differ from LF in that each lacks a different back mutation present in LF. Similarly, HE-1 to HE-11 lack one of the back mutations present in HE. Figure 19 compares LF-1 to LF-4 (each paired with HE). Figure 19 shows that LF-2 and LF-3 lose substantial binding affinity compared to LF (shown as HELF progress control in the graph), whereas LF-1 and LF-4 do not. It was concluded that back mutations L36 and L46 contribute substantially to the retention of binding affinity, while back mutations at positions L37 and L45 can occur without significant effect on binding. Figure 20 shows similar binding curves for the HE mutants. Figure 20 shows that HE-11 lost most of its binding, indicating that the backmutation at H94 has the greatest effect on binding affinity of the backmutations tested. Loss of backmutations at H27, H29, and H30 also caused significant loss of affinity. The role of H30 can be rationalized by a murine residue that is the result of a somatic mutation. Loss of backmutation at H76 caused a significant loss of affinity. Other backmutations at H28, H48, H66, H67, H71, and H93 could be made with little or no effect on binding affinity.

[0159] With these experiments in mind, the heavy chains HF and HG were constructed, as was the light chain LG. HF contained back mutations at positions H27, H29, H30, and H94, and HG contained these mutations as well as a back mutation at position H76. LG contains back mutations at positions L36 and L46. Several permutations of HF, HG, LE, and LF were tested for competitive binding, as shown in Figure 21, and all showed binding within a factor of 3 of that of murine BR2-22a.

[0160] Considering this experiment, HGLG, which represents the best combination of binding affinity and minimal back mutations, was selected for further experiments. Hereafter, this antibody is referred to as hLIV22. The saturation binding affinity of hLIV22 for human and cyno LIV-1 expressed from CHO cells is shown in FIG. 22 in comparison with hLIV14. FIG. 22 shows that hLIV22 has approximately 4-fold higher affinity (reciprocal of dissociation constant) for human LIV-1 than hLIV14. Furthermore, the affinity of hLIV22 for human LIV-1 is the same, within experimental error, as its affinity for cyno LIV-1, while hLIV14 shows twice the affinity for cyno LIV-1 as it does for human LIV-1. The affinity of hLIV22 for human LIV-1 is the same, within experimental error, as the parent murine antibody BR2-22a.

[0161] In vitro antitumor activity of hLIV22 ADC The antitumor activity of hLIV22 ADCs in vitro was measured by cytotoxicity assay. First, LIV-1 expression in various cell lines was investigated by quantitative FACS analysis. The breast cancer cell line MCF-7 from ATCC had the highest level of LIV-1 binding sites / cell compared to MCF-7 cell lines from other sources (data not shown). This cell line was used for the in vitro assay. We observed that various hLIV22 ADCs conjugated to vcMMAE (designated 1006) or mcMMAF (designated 1269), both small molecules described in US2005-0238649, were highly effective in killing MCF-7 cells, as measured by the in vitro cytotoxicity assay. Figures 23 and 24 compare hLIV22 conjugated to 1006 or 1269 with a non-binding control antibody conjugated to 1006 or 1269.

[0162] In vivo antitumor activity of LIV-1 ADC As shown in Figures 25 and 26, the in vivo antitumor activity of hLIV22 ADC (with an average of 4 drugs per antibody) was measured using prostate cancer (PC-3) and breast cancer (MCF-7) models. The hLIV22 ADC conjugated to vcMMAE showed significant tumor retardation compared to untreated and control ADC. In the MCF-7 study using hLIV22-vcMMAE at 3 mg / kg, multiple complete regressions were observed. Also, in all studies, some animals had static or slow growing tumors compared to controls. These studies show that hLIV22 ADC can stop or slow the growth of LIV-1 expressing cancers, including breast and prostate cancers. Figure 27 compares the activity of hLIV22 and hLIV14 ADC in the MCF-7 model. Both antibodies were effective, but hLIV22 was slightly more effective. The hLIV22 ADC was also tested in a model of cervical cancer. A HeLA cell xenograft model was used in the assay. After tumors had grown to an appropriate size, animals were administered hLIV22 conjugated to vcMMAE at 3 mg / kg and 1 mg / kg. A control antibody conjugate was administered at 3 mg / kg. Complete and partial regressions were observed in animals administered 3 mg / kg of hLIV22 vc MMAE conjugate (data not shown). Thus, LIV-1 antibodies and antibody drug conjugates can be used to treat LIV-1-expressing cervical cancer.

[0163] III. Treatment of Skin Cancer Using Anti-LIV-1 Antibodies Expression of LIV-1 protein on melanoma tumor samples Melanoma samples from patients were evaluated for LIV-1 expression using IHC staining. FFPE slides were deparaffinized using Bond™ Dewax solution (Leica, cat # AR9222) at 72°C. Antigen retrieval was performed using EDTA-based Bond™ Epitope Retrieval Solution 2 (Leica, cat # AR9640) at 100°C for 20 minutes. For IHC staining, an alkaline phosphatase-based detection kit was used: Bond™ Polymer Refine Red Detection kit (Leica, cat # DS9390). Slides were incubated with mouse monoclonal primary antibody against LIV-1 (1 μg / ml) (BR2-14a) for 45 minutes with a preliminary 30-minute protein block (DAKO cat #X0909). Mouse IgG (Sigma, cat # M5284) was used as a negative control. After chromogen development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by a pathologist.

[0164] The results are shown in Figure 28. 72% (21 / 29) of the melanoma patient samples tested were positive for LIV-1 expression, indicating that LIV-1 inhibitors, such as anti-LIV-1 antibodies, can be used to treat melanoma cancer.

[0165] In vivo anti-melanoma activity of LIV-1 ADC Nude (nu / nu) mice (7–8 mice / group) were treated with 10 × 10 6 SK-MEL-5 cells (a melanoma tumor derived cell line) were implanted. The tumors were grown to a size of 100 mm, as measured using calipers. 3Grow in vivo until it reaches. Administer the humanized LIV-1 ADC, such as hLIV14 or hLIV22, at 3 mg / kg. The drug conjugate is, for example, vcMMAE or mcMMAF. Also, administer the control ADC to the control animals at 3 mg / kg. The ADC is administered as an intraperitoneal injection q4d×4. Use calipers to monitor the tumor volume, and euthanize the animals when the tumor volume reaches ~800 mm 3 Once reached. Administration of hLIV14 ADC or hLIV22 ADC significantly reduces tumor growth in animals compared to animals administered the control ADC.

[0166] Sequence Listing SEQ ID NO: 1 <LIV-1 mAb light chain leader; PRT / 1; mouse (mus musculus)> JPEG2024178209000017.jpg756

[0167] SEQ ID NO: 2 <LIV-1 mAb heavy chain leader; PRT / 1; mouse (mus musculus)> JPEG2024178209000018.jpg756

[0168] SEQ ID NO: 3 <Substituted heavy chain leader sequence; PRT / 1; mouse (mus musculus)> JPEG2024178209000019.jpg756

[0169] SEQ ID NO: 4 <Light chain constant region; PRT / 1; human (homo sapiens)> JPEG2024178209000020.jpg17145

[0170] SEQ ID NO: 5 <CH1-CH3; PRT / 1; human (homo sapiens)> JPEG2024178209000021.jpg38146

[0171] SEQ ID NO: 6 <heavy chain CH1 - CH3 (without c-terminal K); PRT / 1; human (homo sapiens)> JPEG2024178209000022.jpg39146

[0172] Accession number 7 <S239C heavy chain CH1 - CH3;PRT / 1;human (homo sapiens)> JPEG2024178209000023.jpg39146

[0173] Accession number 8 <S239C heavy chain CH1 - CH3 (without c-terminal K);PRT / 1;human (homo sapiens)> JPEG2024178209000024.jpg38146

[0174] Accession number 9 <hLIV-1 mAb HA;PRT / 1;artificial> JPEG2024178209000025.jpg17145

[0175] Accession number 10 <hLIV-1 mAb HB;PRT / 1;artificial> JPEG2024178209000026.jpg17145

[0176] Accession number 11 <hLIV-1 mAb HC;PRT / 1;artificial> JPEG2024178209000027.jpg17145

[0177] Accession number 12 <hLIV-1 mAb HD;PRT / 1;artificial> JPEG2024178209000028.jpg15125

[0178] Accession number 13 <hLIV-1 mAb HE;PRT / 1;artificial> JPEG2024178209000029.jpg17145

[0179] Accession number 14 <hLIV-1 mAb LA;PRT / 1;artificial> JPEG2024178209000030.jpg17145

[0180] Accession number 15 <hLIV-1 mAb LB;PRT / 1;artificial> JPEG2024178209000031.jpg17145

[0181] Accession number 16 <hLIV-1 mAb LC;PRT / 1;artificial> JPEG2024178209000032.jpg17145

[0182] Accession number 17 <hLIV-1 mAb LD;PRT / 1;artificial> JPEG2024178209000033.jpg17145

[0183] Accession number 18 <hLIV-1 mAb LE;PRT / 1;artificial> JPEG2024178209000034.jpg17146

[0184] Accession number 19 <hLIV-1 mAb LF;PRT / 1;artificial> JPEG2024178209000035.jpg17146

[0185] DNA sequence: Accession number 20 <LIV-1 mAb heavy chain leader;DNA;mouse (mus musculus)> JPEG2024178209000036.jpg11145

[0186] Accession number 21 <LIV-1 mAb light chain leader;DNA;mouse (mus musculus)> JPEG2024178209000037.jpg12145

[0187] Accession number 22 <replacement heavy chain leader sequence;DNA;mouse (mus musculus)> JPEG2024178209000038.jpg11145

[0188] SEQ ID NO:23 <light chain constant region; DNA; mouse (mus musculus)> JPEG2024178209000039.jpg38146

[0189] SEQ ID NO:24<CH1-CH3;DNA;ヒト(homo sapiens)> JPEG2024178209000040.jpg70146JPEG2024178209000041.jpg39146

[0190] SEQ ID NO:25<CH1-CH3 (w / o c-term K);DNA;ヒト(homo sapiens)> JPEG2024178209000042.jpg106146

[0191] SEQ ID NO:26<S239C CH1-CH3;DNA;artificial> JPEG2024178209000043.jpg40146JPEG2024178209000044.jpg70146

[0192] SEQ ID NO:27<S239C CH1-CH3 (w / o c-term K);DNA;artificial> JPEG2024178209000045.jpg107146

[0193] SEQ ID NO:28<hLIV-1 mAb HA;DNA;artificial> JPEG2024178209000046.jpg43145

[0194] SEQ ID NO:29<hLIV-1 mAb HB;DNA;artificial> JPEG2024178209000047.jpg43145

[0195] SEQ ID NO:30<hLIV-1 mAb HC;DNA;artificial> JPEG2024178209000048.jpg42145

[0196] SEQ ID NO:31<hLIV-1 mAb HD;DNA;artificial> JPEG2024178209000049.jpg43146

[0197] SEQ ID NO:32<hLIV-1 mAb HE;DNA;artificial> JPEG2024178209000050.jpg43146

[0198] SEQ ID NO:33<hLIV-1 mAb LA;DNA;artificial> JPEG2024178209000051.jpg38146

[0199] SEQ ID NO:34<hLIV-1 mAb LB;DNA;artificial> JPEG2024178209000052.jpg38146

[0200] SEQ ID NO:35<hLIV-1 mAb LC;DNA;artificial> JPEG2024178209000053.jpg39146

[0201] SEQ ID NO:36<hLIV-1 mAb LD;DNA;artificial> JPEG2024178209000054.jpg39145

[0202] SEQ ID NO:37<hLIV-1 mAb LE;DNA;artificial> JPEG2024178209000055.jpg38146

[0203] SEQ ID NO:38<hLIV-1 mAb LF;DNA;artificial> JPEG2024178209000056.jpg38146

[0204] Accession No. 39 <Liv1 mAb2 light chain leader; PRT / 1; mouse (mus musculus)> JPEG2024178209000057.jpg759

[0205] Accession No. 40 <Liv1 mAb2 heavy chain leader; PRT / 1; mouse (mus musculus)> JPEG2024178209000058.jpg756

[0206] Accession No. 41 <Replacement heavy chain leader sequence; PRT / 1; mouse (mus musculus)> JPEG2024178209000059.jpg757

[0207] Accession No. 42 <Light chain constant region; PRT / 1; human (homo sapiens)> JPEG2024178209000060.jpg17145

[0208] Accession No. 43 <CH1-CH3; PRT / 1; human (homo sapiens)> JPEG2024178209000061.jpg38145

[0209] Accession No. 44 <heavy chain CH1 - CH3 (without c-terminal K); PRT / 1; human (homo sapiens)> JPEG2024178209000062.jpg38146

[0210] Accession No. 45 <S239C heavy chain CH1 - CH3; PRT / 1; human (homo sapiens)> JPEG2024178209000063.jpg39146

[0211] Accession Number 46 <S239C heavy chain CH1 - CH3 (without c-terminal K); PRT / 1; Homo sapiens> JPEG2024178209000064.jpg12146JPEG2024178209000065.jpg28145

[0212] Accession Number 47 <hLiv1 mAb2 HA; PRT / 1; artificial> JPEG2024178209000066.jpg17145

[0213] Accession Number 48 <hLiv1 mAb2 HB; PRT / 1; artificial> JPEG2024178209000067.jpg17144

[0214] Accession Number 49 <hLiv1 mAb2 HC; PRT / 1; artificial> JPEG2024178209000068.jpg17145

[0215] Accession Number 50 <hLiv1 mAb2 HD; PRT / 1; artificial> JPEG2024178209000069.jpg20170

[0216] Accession Number 51 <hLiv1 mAb2 HE; PRT / 1; artificial> JPEG2024178209000070.jpg20169

[0217] Accession Number 52 <hLiv1 mAb2 HF; PRT / 1; artificial> JPEG2024178209000071.jpg17145

[0218] Accession Number 53 <hLiv1 mAb2 HG; PRT / 1; artificial> JPEG2024178209000072.jpg17145

[0219] Accession number 54 <hLiv1 mAb2 LA;PRT / 1;artificial> JPEG2024178209000073.jpg18145

[0220] Accession number 55 <hLiv1 mAb2 LB;PRT / 1;artificial> JPEG2024178209000074.jpg17145

[0221] Accession number 56 <hLiv1 mAb2 LC;PRT / 1;artificial> JPEG2024178209000075.jpg17145

[0222] Accession number 57 <hLiv1 mAb2 LD;PRT / 1;artificial> JPEG2024178209000076.jpg17145

[0223] Accession number 58 <hLiv1 mAb2 LE;PRT / 1;artificial> JPEG2024178209000077.jpg20163

[0224] Accession number 59 <hLiv1 mAb2 LF;PRT / 1;artificial> JPEG2024178209000078.jpg21163

[0225] Accession number 60 <hLiv1 mAb2 LG;PRT / 1;artificial> JPEG2024178209000079.jpg17145

[0226] DNA sequence: Accession number 61 <Liv1 mAb2 heavy chain leader;DNA;mouse (mus musculus)> JPEG2024178209000080.jpg11145

[0227] Accession No. 62 <Liv1 mAb2 light chain leader; DNA; mouse (mus musculus)> JPEG2024178209000081.jpg11145

[0228] Accession No. 63 <replacement heavy chain leader sequence; DNA; mouse (mus musculus)> JPEG2024178209000082.jpg11145

[0229] Accession No. 64 <light chain constant region; DNA; human (homo sapiens)> JPEG2024178209000083.jpg38145

[0230] Accession No. 65 <CH1-CH3; DNA; human (homo sapiens)> JPEG2024178209000084.jpg65147JPEG2024178209000085.jpg43145

[0231] Accession No. 66 <CH1-CH3 (w / o c-term K); DNA; human (homo sapiens)> JPEG2024178209000086.jpg106146

[0232] Accession No. 67 <S239C CH1-CH3; DNA; artificial> JPEG2024178209000087.jpg33146JPEG2024178209000088.jpg75146

[0233] Accession No. 68 <S239C CH1-CH3 (w / o c-term K); DNA; artificial> JPEG2024178209000089.jpg106146

[0234] Accession No. 69 <hLiv1 mAb2 HA; DNA; artificial> JPEG2024178209000090.jpg43145

[0235] SEQ ID NO:70<hLiv1 mAb2 HB;DNA;artificial> JPEG2024178209000091.jpg42145

[0236] SEQ ID NO:71<hLiv1 mAb2 HC;DNA;artificial> JPEG2024178209000092.jpg43146

[0237] SEQ ID NO:72<hLiv1 mAb2 HD;DNA;artificial> JPEG2024178209000093.jpg43146

[0238] SEQ ID NO:73<hLiv1 mAb2 HE;DNA;artificial> JPEG2024178209000094.jpg43146

[0239] SEQ ID NO:74<hLiv1 mAb2 HF;DNA;artificial> JPEG2024178209000095.jpg43145

[0240] SEQ ID NO:75<hLiv1 mAb2 HG;DNA;artificial> JPEG2024178209000096.jpg43146

[0241] SEQ ID NO:76<hLiv1 mAb2 LA;DNA;artificial> JPEG2024178209000097.jpg43145

[0242] SEQ ID NO:77<hLiv1 mAb2 LB;DNA;artificial> JPEG2024178209000098.jpg43145

[0243] Accession No. 78 <hLiv1 mAb2 LC;DNA;artificial> JPEG2024178209000099.jpg43146

[0244] Accession No. 79 <hLiv1 mAb2 LD;DNA;artificial> JPEG2024178209000100.jpg44146

[0245] Accession No. 80 <hLiv1 mAb2 LE;DNA;artificial> JPEG2024178209000101.jpg23146JPEG2024178209000102.jpg23145

[0246] Accession No. 81 <hLiv1 mAb2 LF;DNA;artificial> JPEG2024178209000103.jpg39145

[0247] Accession No. 82 <hLiv1 BR2-22a LG;DNA;artificial> JPEG2024178209000104.jpg38146

[0248] Accession No. 83 <Q13433; Protein JPEG2024178209000105.jpg64124JPEG2024178209000106.jpg72124

[0249] Accession No. 84 <AAA96258.2; Protein JPEG2024178209000107.jpg107124JPEG2024178209000108.jpg29123

[0250] Accession No. 85 Cyno LIV-1 JPEG2024178209000109.jpg81146

[0251] [Sequence Listing] SEQUENCE LISTING <110> Seagen Inc. <120> Humanized Antibodies to LIV-1 and Use of Same to Treat Cancer <130> PA24-462 <150> US 61 / 420,291 <151> 2010-12-06 <150> US 61 / 446,990 <151> 2011-02-25 <160> 85 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 19 <212> PRT <213> Mus musculus <400> 1 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Val 1 5 10 15 Ser Thr Ser <210> 2 <211> 19 <212> PRT <213> Mus musculus <400> 2 Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Leu Gly 1 5 10 15 Ile Asn Ser <210> 3 <211> 19 <212> PRT <213> Mus musculus <400> 3 Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15 Gln Wing Wing <210> 4 <211> 106 <212> PRT <213> Homo sapiens <400> 4 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 5 <211> 330 <212> PRT <213> Homo sapiens <400> 5 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> 6 <211> 329 <212> PRT <213> Homo sapiens <400> 6 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 325 <210> 7 <211> 330 <212> PRT <213> Homo sapiens <400> 7 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 Cys 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> 8 <211> 329 <212> PRT <213> Homo sapiens <400> 8 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 Cys 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 325 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Thr Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 10 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Thr Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Thr Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Thr Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val His Asp Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Thr Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val His Asp Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 14 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Ile Arg Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 15 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Ile Arg Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 16 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 16 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Ile Arg Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 17 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 17 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Ile Arg Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 18 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 18 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Ile Arg Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 19 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 19 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Ile Arg Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 20 <211> 57 <212> DNA <213> Mus musculus <400> 20 atgaaatgca gctgggtcat cttcttcctg atggcagtgg ttctaggaat caattca 57 <210> 21 <211> 57 <212> DNA <213> Mus musculus <400> 21 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgtttc taccagt 57 <210> 22 <211> 57 <212> DNA <213> Mus musculus <400> 22 atggcttggg tgtggacctt gctattcctg atggcagctg cccaaagtgc ccaagca 57 <210> 23 <211> 318 <212> DNA <213> Mus musculus <400> 23 acggtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60 actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120 aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180 aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240 cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300 ttcaacaggg gagagtgt 318 <210> 24 <211> 990 <212> DNA <213> Homo sapiens <400> 24 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 25 <211> 987 <212> DNA <213> Homo sapiens <400> 25 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggt 987 <210> 26 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 26 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtgtgtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 27 <211> 987 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 27 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtgtgtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggt 987 <210> 28 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 28 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcaca gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg agaatggtga tactgaatat 180 gcccccacct tccagggcag ggtcaccatg accagggaca cctccatcag cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cagacatgat 300 gctcactatg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 29 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 29 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccattgaa gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg agaatggtga tactgaatat 180 gcccccacct tccagggcag ggtcaccatg accagggaca cctccatcaa cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cagacatgat 300 gctcactatg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 30 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 30 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcaca gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg agaatggtga tactgaatat 180 gcccccacct tccagggcaa ggccactatg actgcagaca cctccatcag cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cagacatgat 300 gctcactatg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 31 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 31 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggatt caccttcaca gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg agaatggtga tactgaatat 180 gcccccacct tccagggcag ggtcaccatg accagggaca cctccatcag cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cagacatgat 300 gctcactatg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 32 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 32 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggatt caacattgaa gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gattggatgg attgatcctg agaatggtga tactgaatat 180 gcccccacct tccagggcaa ggccactatg actgcagaca cctccatcaa cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtaa tgtccatgat 300 gctcactatg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 33 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 33 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcattata aggaatgatg gaaacaccta tttggaatgg 120 tttcagcaga ggccaggcca atctccaagg aggctaattt atagagtttc caacaggtttt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaaggttc acatgttccc 300 tacaccttg gaggagggac caaggtggag atcaaacgt 339 <210> 34 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 34 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcattata aggaatgatg gaaacaccta tttggaatgg 120 taccagcaga ggccaggcca atctccaagg aggctaattt atagagtttc caacaggtttt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaaggttc acatgttccc 300 tacaccttg gaggagggac caaggtggag atcaaacgt 339 <210> 35 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 35 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcattata aggaatgatg gaaacaccta tttggaatgg 120 tttctgcaga ggccaggcca atctccaagg aggctaattt atagagtttc caacaggtttt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaaggttc acatgttccc 300 tacaccttg gaggagggac caaggtggag atcaaacgt 339 <210> 36 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 36 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcattata aggaatgatg gaaacaccta tttggaatgg 120 tttcagcaga ggccaggcca atctccaaag aggctaattt atagagtttc caacaggtttt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaaggttc acatgttccc 300 tacaccttg gaggagggac caaggtggag atcaaacgt 339 <210> 37 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 37 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcattata aggaatgatg gaaacaccta tttggaatgg 120 tttcagcaga ggccaggcca atctccaagg ctcctaattt atagagtttc caacaggtttt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaaggttc acatgttccc 300 tacaccttg gaggagggac caaggtggag atcaaacgt 339 <210> 38 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 38 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcattata aggaatgatg gaaacaccta tttggaatgg 120 tacctgcaga aaccaggcca atctccaaag ctcctaattt atagagtttc caacaggtttt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaaggttc acatgttccc 300 tacaccttg gaggagggac caaggtggag atcaaacgt 339 <210> 39 <211> 20 <212> PRT <213> Mus musculus <400> 39 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Val 1 5 10 15 Ala Thr Create Create 20 <210> 40 <211> 19 <212> PRT <213> Mus musculus <400> 40 Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Ile Gly 1 5 10 15 Ile Asn Ser <210> 41 <211> 19 <212> PRT <213> Mus musculus <400> 41 Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15 Ala Gln Ala <210> 42 <211> 106 <212> PRT <213> Homo sapiens <400> 42 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 43 <211> 330 <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 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> 44 <211> 329 <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 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 325 <210> 45 <211> 330 <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 Leu Leu Gly Gly Pro Cys 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> 46 <211> 329 <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 Gly Gly Pro Cys 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 325 <210> 47 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 47 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 49 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 49 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 50 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 50 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Val His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 51 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 51 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Asn Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Val His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 52 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 52 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 53 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 53 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 54 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 55 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 55 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 56 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 56 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 57 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 57 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 58 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 58 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Pro Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 59 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 59 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Pro Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 60 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 60 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Pro Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 61 <211> 57 <212> DNA <213> Mus musculus <400> 61 atgaaatgca gctgggtcat cttcttcctg atggcagtgg ttataggaat caattca 57 <210> 62 <211> 57 <212> DNA <213> Mus musculus <400> 62 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgctac cagcagt 57 <210> 63 <211> 57 <212> DNA <213> Mus musculus <400> 63 atggcttggg tgtggacctt gctattcctg atggcagctg cccaaagtgc ccaagca 57 <210> 64 <211> 324 <212> DNA <213> Homo sapiens <400> 64 acgacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttag 324 <210> 65 <211> 990 <212> DNA <213> Homo sapiens <400> 65 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 66 <211> 987 <212> DNA <213> Homo sapiens <400> 66 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggt 987 <210> 67 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 67 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtgtgtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 68 <211> 987 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 68 gctagcacca agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtgtgtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggt 987 <210> 69 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 69 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcaca gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg aaaatggtga tactgaatat 180 ggcccgaagt tccagggcag ggtcaccatg accagggaca cctccatcag cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cagacataat 300 gctcactacg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 70 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 70 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccattgaa gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg aaaatggtga tactgaatat 180 ggcccgaagt tccagggcag ggtcaccatg accagggaca cctccatcaa cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cagacataat 300 gctcactacg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 71 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 71 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcaca gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg aaaatggtga tactgaatat 180 ggcccgaagt tccagggcaa ggccaccatg accgcagaca cctccatcag cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cagacataat 300 gctcactacg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 72 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 72 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggact caccttcaca gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg aaaatggtga tactgaatat 180 ggcccgaagt tccagggcag ggtcaccatg accagggaca cctccatcag cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtac tgtccataat 300 gctcactacg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 73 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 73 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggact caacattgaa gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gattggatgg attgatcctg aaaatggtga tactgaatat 180 ggcccgaagt tccagggcaa ggccaccatg accgcagaca cctccatcaa cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtac tgtccataat 300 gctcactacg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 74 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 74 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggact caccattgaa gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg aaaatggtga tactgaatat 180 ggcccgaagt tccagggcag ggtcaccatg accagggaca cctccatcag cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cgtccataat 300 gctcactacg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 75 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 75 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggact caccattgaa gactactata tgcactgggt gaggcaggcc 120 cctggacaag ggcttgagtg gatgggatgg attgatcctg aaaatggtga tactgaatat 180 ggcccgaagt tccagggcag ggtcaccatg accagggaca cctccatcaa cacagcctac 240 atggagctga gcaggctgag atctgatgac acagctgtgt attactgtgc cgtccataat 300 gctcactacg ggacctggtt tgcttactgg ggccaaggaa ccctggtcac agtctcctca 360 <210> 76 <211> 370 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 76 gatgttctgg attcctgcta ccagcagtga tgttgtgatg actcagtctc cactctccct 60 gcctgtcacc cttggacagc ctgcctccat ctcctgcaga tctagtcaga gccttttaca 120 cagtagtgga aacacctatt tagaatggtt tcagcagagg ccaggccaat ctccaaggag 180 gctaatttat aaaatttcca cccgattttc tggggtccca gacagattct ctggcagtgg 240 gtcaggcact gatttcacac tgaaaatcag cagggtggag gctgaggatg ttggggttta 300 ttactgcttt caaggttcac atgttcccta cacctttgga ggagggacca aggtggagat 360 caaacgtacg 370 <210> 77 <211> 370 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 77 gatgttctgg attcctgcta ccagcagtga tgttgtgatg actcagtctc cactctccct 60 gcctgtcacc cttggacagc ctgcctccat ctcctgcaga tctagtcaga gccttttaca 120 cagtagtgga aacacctatt tagaatggta ccagcagagg ccaggccaat ctccaaggag 180 gctaatttat aaaatttcca cccgattttc tggggtccca gacagattct ctggcagtgg 240 gtcaggcact gatttcacac tgaaaatcag cagggtggag gctgaggatg ttggggttta 300 ttactgcttt caaggttcac atgttcccta cacctttgga ggagggacca aggtggagat 360 caaacgtacg 370 <210> 78 <211> 370 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 78 gatgttctgg attcctgcta ccagcagtga tgttgtgatg actcagtctc cactctccct 60 gcctgtcacc cttggacagc ctgcctccat ctcctgcaga tctagtcaga gccttttaca 120 cagtagtgga aacacctatt tagaatggtt tctgcagagg ccaggccaat ctccaaggag 180 gctaatttat aaaatttcca cccgattttc tggggtccca gacagattct ctggcagtgg 240 gtcaggcact gatttcacac tgaaaatcag cagggtggag gctgaggatg ttggggttta 300 ttactgcttt caaggttcac atgttcccta cacctttgga ggagggacca aggtggagat 360 caaacgtacg 370 <210> 79 <211> 370 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 79 gatgttctgg attcctgcta ccagcagtga tgttgtgatg actcagtctc cactctccct 60 gcctgtcacc cttggacagc ctgcctccat ctcctgcaga tctagtcaga gccttttaca 120 cagtagtgga aacacctatt tagaatggtt tcagcagagg ccaggccaat ctccaaagag 180 gctaatttat aaaatttcca cccgattttc tggggtccca gacagattct ctggcagtgg 240 gtcaggcact gatttcacac tgaaaatcag cagggtggag gctgaggatg ttggggttta 300 ttactgcttt caaggttcac atgttcccta cacctttgga ggagggacca aggtggagat 360 caaacgtacg 370 <210> 80 <211> 370 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 80 gatgttctgg attcctgcta ccagcagtga tgttgtgatg actcagtctc cactctccct 60 gcctgtcacc cttggacagc ctgcctccat ctcctgcaga tctagtcaga gccttttaca 120 cagtagtgga aacacctatt tagaatggtt tcagcagagg ccaggccaat ctccaaggcc 180 cctaatttat aaaatttcca cccgattttc tggggtccca gacagattct ctggcagtgg 240 gtcaggcact gatttcacac tgaaaatcag cagggtggag gctgaggatg ttggggttta 300 ttactgcttt caaggttcac atgttcccta cacctttgga ggagggacca aggtggagat 360 caaacgtacg 370 <210> 81 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 81 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcctttta cacagtagtg gaaacaccta tttagaatgg 120 tacctgcaga ggccaggcca atctccaaag cccctaattt ataaaatttc cacccgattt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaaggttc acatgttccc 300 tacacctttg gaggagggac caaggtggag atcaaacgt 339 <210> 82 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 82 gatgttgtga tgactcagtc tccactctcc ctgcctgtca cccttggaca gcctgcctcc 60 atctcctgca gatctagtca gagcctttta cacagtagtg gaaacaccta tttagaatgg 120 taccagcaga ggccaggcca atctccaagg ccctaattt taaaatttc cacccgattt 180 tctggggtcc cagacagatt ctctggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgct ttcaggttc acatgttccc 300 tacacctttg gaggagggac caggtggag atcaacgt 339 <210> 83 <211> 755 <212> PRT <213> Homo sapiens <400> 83 Met Ala Arg Lys Leu Ser Val Ile Leu Ile Leu Thr Phe Ala Leu Ser 1 5 10 15 Val Thr Asn Pro Leu His Glu Leu Lys Ala Ala Ala Phe Pro Gln Thr 20 25 30 Thr Glu Lys Ile Pro Asn Trp Glu Ser Gly Ile Asn Val Asp Leu 35 40 45 Only Three Three Arg Tyrs Have Three Arg Tyr 50 55 60 Gly Glu Asn Asn Ser Leu Ser Val Glu Gly Phe Arg Lys Leu Leu Gln 65 70 75 80 Asn To Gly To Asp To Lys To Lys Arg To His To His To Asp His 85 90 95 Asp His His Ser Asp His Glu His His Ser Asp His Glu Arg His Ser 100 105 110 Asp His Glu His His Ser Glu His Glu His His Ser Asp His Asp His 115 120 125 His Ser His His Asn His Ala Ala Ser Gly Lys Asn Lys Arg Lys Ala 130 135 140 Leu Cys Pro Asp His Asp Ser Asp Ser Ser Gly Lys Asp Pro Arg Asn 145 150 155 160 Ser Gln Gly Lys Gly Ala His Arg Pro Glu His Ala Ser Gly Arg Arg 165 170 175 Asn Val Lys Asp Ser Val Ser Ala Ser Glu Val Thr Ser Thr Val Tyr 180 185 190 Asn Thr Val Ser Glu Gly Thr His Phe Leu Glu Thr Ile Glu Thr Pro 195 200 205 Arg Pro Gly Lys Leu Phe Pro Lys Asp Val Ser Ser Ser Thr Pro Pro 210 215 220 Ser Val Thr Ser Lys Ser Arg Val Ser Arg Leu Ala Gly Arg Lys Thr 225 230 235 240 Asn Glu Ser Val Ser Glu Pro Arg Lys Gly Phe Met Tyr Ser Arg Asn 245 250 255 Thr Asn Glu Asn Pro Gln Glu Cys Phe Asn Ala Ser Lys Leu Leu Thr 260 265 270 Ser His Gly Met Gly Ile Gln Val Pro Leu Asn Ala Thr Glu Phe Asn 275 280 285 Tyr Leu Cys Pro Ala Ile Ile Asn Gln Ile Asp Ala Arg Ser Cys Leu 290 295 300 Ile His Thr Ser Glu Lys Lys Ala Glu Ile Pro Pro Lys Thr Tyr Ser 305 310 315 320 Leu Gln Ile Ala Trp Val Gly Gly Phe Ile Ala Ile Ser Ile Ile Ser 325 330 335 Phe Leu Ser Leu Leu Gly Val Ile Leu Val Pro Leu Met Asn Arg Val 340 345 350 Phe Phe Lys Phe Leu Leu Ser Phe Leu Val Ala Leu Ala Val Gly Thr 355 360 365 Leu Ser Gly Asp Ala Phe Leu His Leu Leu Pro His Ser His Ala Ser 370 375 380 His His His Ser His Ser His Glu Glu Pro Ala Met Glu Met Lys Arg 385 390 395 400 Gly Pro Leu Phe Ser His Leu Ser Ser Gln Asn Ile Glu Glu Ser Ala 405 410 415 Tyr Phe Asp Ser Thr Trp Lys Gly Leu Thr Ala Leu Gly Gly Leu Tyr 420 425 430 Phe Met Phe Leu Val Glu His Val Leu Thr Leu Ile Lys Gln Phe Lys 435 440 445 Asp Lys Lys Lys Lys Asn Gln Lys Lys Pro Glu Asn Asp Asp Asp Val 450 455 460 Glu Ile Lys Lys Gln Leu Ser Lys Tyr Glu Ser Gln Leu Ser Thr Asn 465 470 475 480 Glu Glu Lys Val Asp Thr Asp Asp Arg Thr Glu Gly Tyr Leu Arg Ala 485 490 495 Asp Ser Gln Glu Pro Ser His Phe Asp Ser Gln Gln Pro Ala Val Leu 500 505 510 Glu Glu Glu Glu Val Met Ile Ala His Ala His Pro Gln Glu Val Tyr 515 520 525 Asn Glu Tyr Val Pro Arg Gly Cys Lys Asn Lys Cys His Ser His Phe 530 535 540 His Asp Thr Leu Gly Gln Ser Asp Asp Leu Ile His His His His Asp 545 550 555 560 Tyr His His Ile Leu His His His His His Gln Asn His His Pro His 565 570 575 Ser His Ser Gln Arg Tyr Ser Arg Glu Glu Leu Lys Asp Ala Gly Val 580 585 590 Ala Thr Leu Ala Trp Met Val Ile Met Gly Asp Gly Leu His Asn Phe 595 600 605 Ser Asp Gly Leu Ala Ile Gly Ala Ala Phe Thr Glu Gly Leu Ser Ser 610 615 620 Gly Leu Ser Thr Ser Val Ala Val Phe Cys His Glu Leu Pro His Glu 625 630 635 640 Leu Gly Asp Phe Ala Val Leu Leu Lys Ala Gly Met Thr Val Lys Gln 645 650 655 Ala Val Leu Tyr Asn Ala Leu Ser Ala Met Leu Ala Tyr Leu Gly Met 660 665 670 Ala Thr Gly Ile Phe Ile Gly His Tyr Ala Glu Asn Val Ser Met Trp 675 680 685 Ile Phe Ala Leu Thr Ala Gly Leu Phe Met Tyr Val Ala Leu Val Asp 690 695 700 Met Val Pro Glu Met Leu His Asn Asp Ala Ser Asp His Gly Cys Ser 705 710 715 720 Arg Trp Gly Tyr Phe Phe Leu Gln Asn Ala Gly Met Leu Leu Gly Phe 725 730 735 Gly Ile Met Leu Leu Ile Ser Ile Phe Glu His Lys Ile Val Phe Arg 740 745 750 Ile Asn Phe 755 <210> 84 <211> 749 <212> PRT <213> Homo sapiens <400> 84 Met Ala Arg Lys Leu Ser Val Ile Leu Ile Leu Thr Phe Ala Leu Ser 1 5 10 15 Val Thr Asn Pro Leu His Glu Leu Lys Ala Ala Ala Phe Pro Gln Thr 20 25 30 Thr Glu Lys Ile Ser Pro Asn Trp Glu Ser Gly Ile Asn Val Asp Leu 35 40 45 Ala Ile Ser Thr Arg Gln Tyr His Leu Gln Gln Leu Phe Tyr Arg Tyr 50 55 60 Gly Glu Asn Asn Ser Leu Ser Val Glu Gly Phe Arg Lys Leu Leu Gln 65 70 75 80 Asn Ile Gly Ile Asp Lys Ile Lys Arg Ile His Ile His His Asp His 85 90 95 Asp His His Ser Asp His Glu His His Ser Asp His Glu Arg His Ser 100 105 110 Asp His Glu His His Ser Asp His Glu His His Ser Asp His Asn His 115 120 125 Ala Ala Ser Gly Lys Asn Lys Arg Lys Ala Leu Cys Pro Asp His Asp 130 135 140 Ser Asp Ser Ser Gly Lys Asp Pro Arg Asn Ser Gln Gly Lys Gly Ala 145 150 155 160 His Arg Pro Glu His Ala Ser Gly Arg Arg Asn Val Lys Asp Ser Val 165 170 175 Ser Ala Ser Glu Val Thr Ser Thr Val Tyr Asn Thr Val Ser Glu Gly 180 185 190 Thr His Phe Leu Glu Thr Ile Glu Thr Pro Arg Pro Gly Lys Leu Phe 195 200 205 Pro Lys Asp Val Ser Ser Ser Thr Pro Pro Ser Val Thr Ser Lys Ser 210 215 220 Arg Val Ser Arg Leu Ala Gly Arg Lys Thr Asn Glu Ser Val Ser Glu 225 230 235 240 Pro Arg Lys Gly Phe Met Tyr Ser Arg Asn Thr Asn Glu Asn Pro Gln 245 250 255 Glu Cys Phe Asn Ala Ser Lys Leu Leu Thr Ser His Gly Met Gly Ile 260 265 270 Gln Val Pro Leu Asn Ala Thr Glu Phe Asn Tyr Leu Cys Pro Ala Ile 275 280 285 Ile Asn Gln Ile Asp Ala Arg Ser Cys Leu Ile His Thr Ser Glu Lys 290 295 300 Lys Ala Glu Ile Pro Pro Lys Thr Tyr Ser Leu Gln Ile Ala Trp Val 305 310 315 320 Gly Gly Phe Ile Ala Ile Ser Ile Ile Ser Phe Leu Ser Leu Leu Gly 325 330 335 Val Ile Leu Val Pro Leu Met Asn Arg Val Phe Phe Lys Phe Leu Leu 340 345 350 Ser Phe Leu Val Ala Leu Ala Val Gly Thr Leu Ser Gly Asp Ala Phe 355 360 365 Leo His Leo Leo Pro His Ser His Ala Ser His His His Ser His Ser 370 375 380 His Glu Glu Pro Ala Met Glu Met Lys Arg Gly Pro Leu Phe Ser His 385 390 395 400 Leu Ser Ser Gln Asn Ile Glu Glu Ser Ala Tyr Phe Asp Ser Thr Trp 405 410 415 Lys Gly Leu Thr Ala Leu Gly Gly Leu Tyr Phe Met Phe Leu Val Glu 420 425 430 His Val Leu Thr Leu Ile Lys Gln Phe Lys Asp Lys Lys Lys Lys Asn 435 440 445 Gln Lys Lys Pro Glu Asn Asp Asp Asp Val Glu Ile Lys Lys Gln Leu 450 455 460 Ser Lys Tyr Glu Ser Gln Leu Ser Thr Asn Glu Glu Lys Val Asp Thr 465 470 475 480 Asp Asp Arg Thr Glu Gly Tyr Leu Arg Ala Asp Ser Gln Glu Pro Ser 485 490 495 His Phe Asp Ser Gln Gln Pro Ala Val Leu Glu Glu Glu Glu Val Met 500 505 510 Ile Ala His Ala His Pro Gln Glu Val Tyr Asn Glu Tyr Val Pro Arg 515 520 525 Gly Cys Lys Asn Lys Cys His Ser His Phe His Asp Thr Leu Gly Gln 530 535 540 Ser Asp Asp Leu Ile His His His His Asp Tyr His His Ile Leu His 545 550 555 560 His His His His Gln Asn His His Pro His Ser His Ser Gln Arg Tyr 565 570 575 Ser Arg Glu Glu Leu Lys Asp Ala Gly Val Ala Thr Leu Ala Trp Met 580 585 590 Val Ile Met Gly Asp Gly Leu His Asn Phe Ser Asp Gly Leu Ala Ile 595 600 605 Gly Ala Ala Phe Thr Glu Gly Leu Ser Ser Gly Leu Ser Thr Ser Val 610 615 620 Ala Val Phe Cys His Glu Leu Pro His Glu Leu Gly Asp Phe Ala Val 625 630 635 640 Leu Leu Lys Ala Gly Met Thr Val Lys Gln Ala Val Leu Tyr Asn Ala 645 650 655 Leu Ser Ala Met Leu Ala Tyr Leu Gly Met Ala Thr Gly Ile Phe Ile 660 665 670 Gly His Tyr Ala Glu Asn Val Ser Met Trp Ile Phe Ala Leu Thr Ala 675 680 685 Gly Leu Phe Met Tyr Val Ala Leu Val Asp Met Val Pro Glu Met Leu 690 695 700 His Asn Asp Ala Ser Asp His Gly Cys Ser Arg Trp Gly Tyr Phe Phe 705 710 715 720 Leu Gln Asn Ala Gly Met Leu Leu Gly Phe Gly Ile Met Leu Leu Ile 725 730 735 Ser Ile Phe Glu His Lys Ile Val Phe Arg Ile Asn Phe 740 745 <210> 85 <211> 741 <212> PRT <213> Cynomolgus sp. <400> 85 Met Ala Arg Lys Leu Ser Val Ile Leu Ile Leu Thr Phe Thr Leu Ser 1 5 10 15 Val Thr Asn Pro Leu His Glu Leu Lys Ser Ala Ala Ala Phe Pro Gln 20 25 30 Thr Thr Glu Lys Ile Ser Pro Asn Trp Glu Ser Gly Ile Asn Val Asp 35 40 45 Leu Ala Ile Thr Thr Arg Gln Tyr His Leu Gln Gln Leu Phe Tyr Arg 50 55 60 Tyr Gly Glu Asn Asn Ser Leu Ser Val Glu Gly Phe Arg Lys Leu Leu 65 70 75 80 Gln Asn Ile Gly Ile Asp Lys Ile Lys Arg Ile His Ile His His Asp 85 90 95 His Asp His His Ser Asp His Glu His His Ser Asp His Glu His His 100 105 110 Ser Asp His Glu His His Ser His Arg Asn His Ala Ala Ser Gly Lys 115 120 125 Asn Lys Arg Lys Ala Leu Cys Pro Glu His Asp Ser Asp Ser Ser Gly 130 135 140 Lys Asp Pro Arg Asn Ser Gln Gly Lys Gly Ala His Arg Pro Glu His 145 150 155 160 Ala Asn Gly Arg Arg Asn Val Lys Asp Ser Val Ser Thr Ser Glu Val 165 170 175 Thr Ser Thr Val Tyr Asn Thr Val Ser Glu Gly Thr His Phe Leu Glu 180 185 190 Thr Ile Glu Thr Pro Lys Leu Phe Pro Lys Asp Val Ser Ser Ser Thr 195 200 205 Pro Pro Ser Val Thr Glu Lys Ser Leu Val Ser Arg Leu Ala Gly Arg 210 215 220 Lys Thr Asn Glu Ser Met Ser Glu Pro Arg Lys Gly Phe Met Tyr Ser 225 230 235 240 Arg Asn Thr Asn Glu Asn Pro Gln Glu Cys Phe Asn Ala Ser Lys Leu 245 250 255 Leu Thr Ser His Gly Met Gly Ile Gln Val Pro Leu Asn Ala Thr Glu 260 265 270 Phe Asn Tyr Leu Cys Pro Ala Ile Ile Asn Gln Ile Asp Ala Arg Ser 275 280 285 Cys Leu Ile His Thr Ser Glu Lys Lys Ala Glu Ile Pro Pro Lys Thr 290 295 300 Tyr Ser Leu Gln Ile Ala Trp Val Gly Gly Phe Ile Ala Ile Ser Ile 305 310 315 320 Ile Ser Phe Leu Ser Leu Leu Gly Val Ile Leu Val Pro Leu Met Asn 325 330 335 Arg Val Phe Phe Lys Phe Leu Leu Ser Phe Leu Val Ala Leu Ala Val 340 345 350 Gly Thr Leu Ser Gly Asp Ala Phe Leu His Leu Leu Pro His Ser His 355 360 365 Ala Ser His His His Ser His Ser His Glu Glu Pro Ala Met Glu Met 370 375 380 Lys Arg Gly Pro Leu Phe Ser His Leu Ser Ser Gln Asn Ile Glu Glu 385 390 395 400 Ser Ala Tyr Phe Asp Ser Thr Trp Lys Gly Leu Thr Ala Leu Gly Gly 405 410 415 Leu Tyr Phe Met Phe Leu Val Glu His Val Leu Thr Leu Ile Lys Gln 420 425 430 Phe Lys Asp Lys Lys Lys Lys Asn Gln Lys Lys Pro Glu Asn Asp Asp 435 440 445 Asp Val Glu Ile Lys Lys Gln Leu Ser Lys Tyr Glu Ser Gln Leu Ser 450 455 460 Thr Asn Glu Glu Lys Val Asp Thr Asp Asp Arg Thr Glu Gly Tyr Leu 465 470 475 480 Arg Ala Asp Ser Gln Glu Pro Ser His Phe Asp Ser Gln Gln Pro Ala 485 490 495 Ile Leu Glu Glu Glu Glu Val Met Ile Ala His Ala His Pro Gln Glu 500 505 510 Val Tyr Asn Glu Tyr Val Pro Arg Gly Cys Lys Asn Lys Cys His Ser 515 520 525 His Phe His Asp Thr Leu Gly Gln Ser Asp Asp Leu Ile His His His 530 535 540 His Asp Tyr His His Ile Leu His His His His His Gln Asn His His 545 550 555 560 Pro His Ser His Ser Gln Arg Tyr Ser Arg Glu Glu Leu Lys Asp Ala 565 570 575 Gly Ile Ala Thr Leu Ala Trp Met Val Ile Met Gly Asp Gly Leu His 580 585 590 Asn Phe Ser Asp Gly Leu Ala Ile Gly Ala Ala Phe Thr Glu Gly Leu 595 600 605 Ser Ser Gly Leu Ser Thr Ser Val Ala Val Phe Cys His Glu Leu Pro 610 615 620 His Glu Leu Gly Asp Phe Ala Val Leu Leu Lys Ala Gly Met Thr Val 625 630 635 640 Lys Gln Ala Val Leu Tyr Asn Ala Leu Ser Ala Met Leu Ala Tyr Leu 645 650 655 Gly Met Ala Thr Gly Ile Phe Ile Gly His Tyr Ala Glu Asn Val Ser 660 665 670 Met Trp Ile Phe Ala Leu Thr Ala Gly Leu Phe Met Tyr Val Ala Leu 675 680 685 Val Asp Met Val Pro Glu Met Leu His Asn Asp Ala Ser Asp His Gly 690 695 700 Cys Ser Arg Trp Gly Tyr Phe Phe Leu Gln Asn Ala Gly Met Leu Leu 705 710 715 720 Gly Phe Gly Ile Met Leu Leu Ile Ser Ile Phe Glu His Lys Ile Val 725 730 735 Phe Arg Ile Asn Phe 740

Claims

[Claim 1] The invention as hereinbefore described in the description and / or drawings.

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