Multimeric bispecific anti-CD123 binding molecules and their uses

Abstract

The present disclosure provides a multivalent bispecific anti-CD123 binding molecule comprising a modified J chain specifically binding to an immune effector cell. A polynucleotide encoding the binding molecule or its subunit and a vector and a host cell comprising the polynucleotide are also provided. The present disclosure also provides a method for producing and / or using a multivalent bispecific anti-CD123 binding molecule comprising a modified J chain specifically binding to an immune effector cell.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 888,475, filed on August 17, 2019, and U.S. Provisional Patent Application Serial No. 62 / 888,702, filed on August 19, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on August 13, 2020, is named 027WO1-Sequence-Listing, and is 204,738 bytes in size. Background Art

[0005] Acute myeloid leukemia (AML) is the leading cause of leukemia death in the United States, with more than 20,000 new patients each year and a 5-year survival rate of less than 30%, with survival rates falling to 10% for patients over 60 years of age (National Cancer Institute Surveillance, Epidemiology and End-Result Program (SEER) data; Oran and Weisdorf 2012, Haematologica 97(12)1916). Over the past 40 years, there have been few advances in the treatment of AML patients, and current treatment options primarily include intensive chemotherapy and stem cell transplantation (Luppi et al. 2018, Cancers 10, 429). Several approaches have been used to target cell surface molecules on AML cells to direct T cells to engage and kill AML cells. One such surface molecule is CD123 (also known as IL-3 receptor alpha chain or IL-3Rα), which is expressed on leukemic cells in more than 90% of AML patients, as well as on leukemic stem cells, which are the cell types that often lead to disease relapse after treatment (Kovtun et al. 2018, Blood Advances 2(8)848; Xie et al. 2017, Blood Cancer Journal 7, e567). In addition, CD123 is highly expressed in patients with gene mutations such as FLT3 that are associated with a very poor prognosis (Xie et al. 2017, Blood Cancer Journal 7, e567). The amino acid sequences of two human isoforms of CD123 are presented as SEQ ID NO: 28 (isoform 1, mature protein: approximately amino acids 23 to 378 of SEQ ID NO: 28) and SEQ ID NO: 29 (isoform 2, mature protein: approximately amino acids 23 to 300 of SEQ ID NO: 29), the cynomolgus macaque CD123 amino acid sequence is presented as SEQ ID NO: 30 (approximately 87% identical to human isoform 1; mature protein: approximately amino acids 23 to 378 of SEQ ID NO: 30), and the mouse CD123 amino acid sequence is presented as SEQ ID NO: 31 (approximately 30% identical to human isoform 1; mature protein: approximately amino acids 17 to 396 of SEQ ID NO: 31).

[0006] CD123 is a clinically validated target for several hematologic malignancies, as evidenced by the FDA approval of a recombinant IL-3 cytokine conjugated to diphtheria toxin for the treatment of blastic plasmacytoid dendritic cell neoplasms (Pemmaraju et al. 2019, NEJM 380:1628). This and other CD123-targeting agents are being tested in preclinical and clinical trials. Xencor (XmAb14045 – IgG-based), Macrogenics (flotetuzumab-DART), and Jansen (JNJ-63709178-duobody) have conducted early phase 1 clinical studies using CD123xCD3 bispecific antibodies. Although early signs of clinical efficacy have been reported in some of these patients, severe cytokine release syndrome and some patient deaths have also been observed with this type of bispecific drug (Ravandi et al. 2018 Blood 132:763; Jacobs et al. 2018, Blood 132:2738; Uy et al. 2018, Blood 132:764). Cytokine release syndrome (or CRS) is characterized by fever, hypotension, coagulation abnormalities, and capillary leakage, which can be life-threatening, and such findings are also associated with other T cell engagement approaches, including CAR-T and BiTE (Teachley et al. 2016, Cancer Discovery 6(6)664; Hay et al. 2017, Blood 130(21)2295). These adverse safety events associated with cytokine release often constitute the dose-limiting toxicity of IgG-based CD3-engaging bispecific antibodies and represent a challenge to the safe, efficient, and tolerable administration of such agents and, due to the resulting dosing limitations, may represent a challenge to the ability to optimize the efficacy of these therapeutics.

[0007] Multimerizable antibodies and antibody-like molecules, such as IgA and IgM antibodies, have become promising drug candidates in, for example, immuno-oncology and infectious disease fields, allowing improved specificity, improved avidity, and the ability to combine multiple binding targets. See, for example, U.S. Patent Nos. 9,951,134, 9,938,347, and 10,618,978, U.S. Patent Application Publication Nos. US2019-0100597, US2019-0185570, and PCT Publication Nos. WO 2016 / 154593, WO 2016 / 168758, WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761, and WO 2019 / 169314, the contents of which are incorporated herein by reference in their entirety.

[0008] There remains a need to target CD123-expressing AML cells and induce T cell-mediated killing of these cells while minimizing CRS. We have evaluated whether targeting CD123 using CD3 bispecific IgM technology will not only effectively target CD123-expressing AML tumor cells for T cell-mediated cytotoxicity, but also generate responses with a favorable safety profile against the sometimes severe cytokine release syndrome seen in patients treated with IgG-based CD123 x CD3 bispecific antibodies. Furthermore, the high avidity binding of IgM antibodies may allow CD123 x CD3 bispecific IgMs to target tumor cells expressing CD123 at relatively low cell surface levels compared to IgG-based bispecific antibodies. Summary of the Invention

[0009] The present disclosure provides a multimer, bispecific or multispecific binding molecule, which includes two or five bivalent binding units and modified J chains, wherein modified J chains include wild-type J chains or their functional fragments or variants and the antigen-binding domains associated with J chains of specific binding immune effector cells.Each binding unit includes two antibody heavy chains, and each heavy chain includes IgA, IgA-like, IgM or IgM-like heavy chain constant regions or their polymeric fragments and at least includes a heavy chain variable region (VH) portion of the antigen-binding domains associated with the binding unit, wherein at least three of the antigen-binding domains associated with the binding unit specifically bind to CD123, and wherein the binding molecule can induce the killing of cells expressing CD123 of immune effector cell dependence.

[0010] In certain embodiments, the modified J chain includes a variant J chain or a fragment thereof, and the variant J chain or its fragment includes one or more single amino acid substitutions, deletions or insertions that can affect the serum half-life of the binding molecule relative to the wild-type J chain, so that relative to the reference binding molecule that is identical except for the one or more single amino acid substitutions, deletions or insertions in the J chain and is administered to the same animal species in the same manner, the binding molecule exhibits an increased serum half-life when administered to the animal. In certain embodiments, the modified J chain includes amino acid substitutions at the amino acid position corresponding to the amino acid Y102 of mature wild-type human J chain (SEQ ID NO: 2). In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be substituted by alanine (A), serine (S) or arginine (R). In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be substituted by alanine (A). In certain embodiments, the J chain is a variant human J chain and includes the amino acid sequence SEQ ID NO: 3 ("J*").

[0011] In certain embodiments, the antigen binding domain associated with the J chain comprises an antibody single-chain Fv (scFv) fragment fused or chemically conjugated to the J chain or a fragment or variant thereof. For example, the scFv fragment can be fused to the J chain via a peptide linker. In certain embodiments, the scFv fragment can be fused to the N-terminus of the J chain or a fragment or variant thereof, to the C-terminus of the J chain or a fragment or variant thereof, or the scFv fragment can be fused to both the N-terminus and the C-terminus of the J chain or a fragment or variant thereof.

[0012] In certain embodiments, the immune effector cell is a T cell or a NK cell. In those embodiments where the immune effector cell is a T cell, the scFv fragment can specifically bind to CD3ε in certain embodiments. In certain embodiments, the T cell is a CD8+ cytotoxic T cell.

[0013] In certain embodiments, the scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, wherein (a) VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, having 0, 1, or 2 amino acid substitutions, and VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, having 0, 1, or 2 amino acid substitutions; and (b) VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 132, and SEQ ID NO: 133, respectively, having 0, 1, or 2 amino acid substitutions. NO: 135, and VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NO: 138, SEQ ID NO: 140, and SEQ ID NO: 142, respectively, having 0, 1, or 2 amino acid substitutions; (c) VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NO: 130, SEQ ID NO: 132, and SEQ ID NO: 135, respectively, having 0, 1, or 2 amino acid substitutions, and VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NO: 138, SEQ ID NO: 140, and SEQ ID NO: 143, respectively, having 0, 1, or 2 amino acid substitutions; (d) VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NO: 131, SEQ ID NO: 133, and SEQ ID NO: 134, respectively, having 0, 1, or 2 amino acid substitutions. NO: 136, and VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 141, and SEQ ID NO: 144, respectively, having 0, 1, or 2 amino acid substitutions; (e) VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequence of SEQ ID NO: 131, SEQ ID NO: 134, and SEQ ID NO: 136, respectively, having 0, 1, or 2 amino acid substitutions, and VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 141, and SEQ ID NO: 145, respectively, having 0, 1, or 2 amino acid substitutions;or (f) VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NO: 131, SEQ ID NO: 134, and SEQ ID NO: 137, respectively, having 0, 1, or 2 amino acid substitutions, and VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NO: 139, SEQ ID NO: 141, and SEQ ID NO: 146, respectively, having 0, 1, or 2 amino acid substitutions;

[0014] In certain embodiments, the scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, respectively, having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or having one, two, or three amino acid substitutions in one or more of the VHCDRs, and wherein the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, respectively, having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or having one, two, or three amino acid substitutions in one or more of the VLCDRs, In certain embodiments, the scFv fragment comprises the VH and VL amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 119 and SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122, SEQ ID NO: 123 and SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126, or SEQ ID NO: 127 and SEQ ID NO: 128, respectively.

[0015] In certain embodiments, the scFv fragment comprises the VH amino acid sequence of SEQ ID NO: 4 and the VL amino acid sequence of SEQ ID NO: 8. In other embodiments, the scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL comprise the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In certain embodiments, the modified J chain comprises amino acids 20 to 420 of SEQ ID NO: 12, amino acids 20 to 412 of SEQ ID NO: 15, or amino acids 23 to 415 of SEQ ID NO: 16.

[0016] In certain embodiments, the immune effector cell is a NK cell, and wherein the scFv fragment specifically binds to CD16.

[0017] In certain embodiments, the modified J chain may further include an immunostimulatory agent ("ISA") fused or chemically conjugated to the J chain or a fragment or variant thereof. In certain embodiments, the ISA includes a cytokine or a receptor-binding fragment or variant thereof. In certain embodiments, the ISA includes: (a) an interleukin-15 (IL-15) protein or a receptor-binding fragment or variant thereof ("I"); and (b) an interleukin-15 receptor-alpha (IL-15Rα) fragment ("R") that is capable of associating with I, including a sushi domain or a variant thereof; wherein at least one of I and R is associated with the J chain or a fragment or variant thereof as a fusion protein, and wherein I and R can associate to function as an ISA. In certain embodiments, the ISA can be fused to the J chain via a peptide linker.

[0018] In certain embodiments, each binding unit of the provided binding molecules further comprises two light chains, each light chain comprising a kappa or lambda light chain constant region and at least a light chain variable region (VL) portion of the antigen binding domain that associates with the binding unit.

[0019] In certain embodiments, the binding molecules provided include at least four, at least five, at least six, at least seven, at least eight, at least nine or ten antigen binding domains associated with the binding unit that specifically bind to CD123. In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten antigen binding domains associated with the binding unit are combined with the same CD123 epitope. In certain embodiments, the antigen binding domains associated with the binding unit of the binding molecules provided are all identical.

[0020] In certain embodiments, the antigen binding domain associated with the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise six immunoglobulin complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise: CDRs of an antibody having VH and VL amino acid sequences comprising or contained in SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71 SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: NO:91, SEQ ID NO:92 and SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ IDNO:101, SEQ ID NO:102 and SEQ ID NO:103, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116, or SEQ ID NO:117 and SEQ ID NO:118; or the CDRs of an antibody comprising or contained in the VH and VL amino acid sequences, respectively, except for one or two amino acid substitutions in one or more of the CDRs: SEQ ID NO:32 and SEQ ID NO:33, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51. SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97 NO:86 and SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO:102 and SEQ ID NO:103, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116, or SEQ ID NO:117 and SEQ ID NO:118.

[0021] In certain embodiments, the antigen binding domains of the associating linking unit comprise antibody VH and VL, wherein VH and VL comprise amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to mature VH and VL amino acid sequences comprising or contained in SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75 SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101, SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 118 and SEQ ID NO: 119, SEQ ID NO: 120 and SEQ ID NO: NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO:102 and SEQ ID NO:103, SEQ ID NO:107 and SEQ ID NO:108.NO:108, SEQ ID NO:109 and SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116, or SEQ ID NO:117 and SEQ ID NO:118.

[0022] In certain embodiments, the binding molecule provided is a dimeric binding molecule comprising two bivalent binding units, wherein each binding unit includes two antibody heavy chains, and each antibody heavy chain includes an IgA or IgA-like heavy chain constant region or a multimeric fragment thereof. In certain embodiments, the dimeric binding molecule provided also includes secretory component or a fragment or variant thereof. In certain embodiments, the IgA or IgA-like heavy chain constant region or its multimeric fragment each includes a Cα3 and a tail piece (tp) domain, and may also include a Cα1 domain, a Cα2 domain, an IgA hinge region or any combination thereof. In certain embodiments, the IgA or IgA-like heavy chain constant region is a human IgA or IgA-like constant region and may include the amino acid sequence SEQ ID NO: 24, SEQ ID NO: 25 or any multimeric variant or fragment thereof. In certain embodiments, each binding unit comprises two IgA or IgA-like heavy chains and two immunoglobulin light chains, each heavy chain comprising a VH located amino-terminal to an IgA constant region or fragment thereof, and each light chain comprising a VL located amino-terminal to an immunoglobulin light chain constant region.

[0023] In certain embodiments, the binding molecule provided is a pentamer binding molecule including five bivalent binding units, wherein each binding unit includes two IgM or IgM sample heavy chain constant regions or its polymeric fragment. In certain embodiments, IgM or IgM sample heavy chain constant regions or its polymeric fragment each include Cμ4 domains and tail piece (tp) domains or its fragment or variant, and may also include Cμ1 domains, Cμ2 domains, Cμ3 domains or any combination thereof. In certain embodiments, IgM or IgM sample heavy chain constant regions are human IgM constant regions and may include amino acid sequence SEQ ID NO:22, SEQ ID NO:23 or its polymeric variant or fragment. In certain embodiments, each binding unit includes two IgM heavy chains and two immunoglobulin light chains, each heavy chain includes the VH located at the amino terminus of IgM constant region or its fragment, and each light chain includes the VL located at the amino terminus of immunoglobulin light chain constant region.

[0024] In certain embodiments, the binding unit comprises a variant human IgM constant region, wherein the multimeric binding molecule has reduced CDC activity relative to a multimeric binding molecule comprising an IgM heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, or a multimeric variant or fragment thereof. In certain embodiments, each IgM heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23 variant, wherein the variant comprises an amino acid substitution at position P311 of SEQ ID NO: 22 or SEQ ID NO: 23, an amino acid substitution at position P313 of SEQ ID NO: 22 or SEQ ID NO: 23, or an amino acid substitution at both positions P311 and P313 of SEQ ID NO: 22 or SEQ ID NO: 23.

[0025] In certain embodiments, the binding unit comprises a variant human IgM constant region having one or more single amino acid substitutions, deletions or insertions relative to a reference IgM heavy chain constant region that is identical except for one or more single amino acid substitutions, deletions or insertions, wherein the binding molecule exhibits increased serum half-life when administered to the subject animal relative to a multimeric binding molecule comprising a reference IgM heavy chain constant region and administered in the same manner to the same animal species. In certain embodiments, the variant IgM heavy chain constant region comprises an amino acid substitution at one or more amino acid positions corresponding to amino acid E345A, S401A, E402A or E403A of the wild-type human IgM constant region SEQ ID NO: 22 or SEQ ID NO: 23.

[0026] The disclosure also provides compositions, such as pharmaceutical compositions, comprising the provided binding molecules.

[0027] The present disclosure also provides a polynucleotide comprising a nucleic acid sequence encoding a polypeptide subunit of a provided binding molecule.

[0028] In certain embodiments, the polypeptide subunit comprises an IgM or IgM-like heavy chain constant region and comprises at least the antibody VH portion of the antigen binding domain of the binding molecule that is associated with the binding unit. In certain embodiments, the polypeptide subunit comprises a human IgM constant region or fragment thereof fused to the C-terminus of a VH comprising: (a) a HCDR1, HCDR2, and HCDR3 region comprising: the CDRs contained in a VH amino acid sequence comprising or comprising SEQ ID NO: 32, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100 NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ IDNO:115 or SEQ ID NO:117;SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124 NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115 or SEQ ID NO:117;or (b) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a mature VH amino acid sequence comprising or contained in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115 or SEQ ID NO:117. ;

[0029] In certain embodiments, the polypeptide subunit comprises the light chain constant region and the antibody VL portion of the antigen binding domain of the multimeric binding molecule. In certain embodiments, the polypeptide subunit comprises a human kappa or lambda light chain constant region, or a fragment thereof, fused to the C-terminus of a VL comprising: (a) LCDR1, LCDR2, and LCDR3 regions comprising: the CDRs contained in a VL amino acid sequence comprising or consisting of SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 90 NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 or SEQ ID NO:118;or the CDRs contained in the following VL amino acid sequences, except for one or two single amino acid substitutions in one or more of the LCDRs: SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100 NO:101, SEQ ID NO:103, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 or SEQ ID NO:118;or (b) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the mature VL amino acid sequence of or contained in SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 or SEQ ID NO:118. ;

[0030] In certain embodiments, the polypeptide subunit comprises a modified J chain, wherein the modified J chain comprises a wild-type J chain or a functional fragment or variant thereof and an antigen binding domain associated with the J chain that specifically binds to an immune effector cell. In certain embodiments, the modified J chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids 20 to 420 of SEQ ID NO: 12, amino acids 20 to 412 of SEQ ID NO: 15, or amino acids 23 to 415 of SEQ ID NO: 16.

[0031] The present disclosure also provides a composition comprising two or more of the aforementioned polynucleotides. The polynucleotides can be located on two or more separate vectors, or on a single vector. Such a vector or vectors are also provided.

[0032] The present disclosure also provides a host cell comprising one or more provided polynucleotides or a provided vector or vectors, wherein the host cell can express the provided binding molecule or its subunit. The present disclosure also provides a method for producing the provided binding molecule, wherein the method comprises culturing the host cell and then recovering the binding molecule.

[0033] The present disclosure also provides a method for treating cancer or other malignant tumors, wherein the method includes administering an effective amount of provided binding molecules to a subject in need of cancer treatment, wherein binding molecules can induce immune effector cell-mediated cancer cell killing. In certain embodiments, cancer or malignant tumor is hematological cancer or malignant tumor, for example, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia (B cell ALL), classical Hodgkin lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), systemic mastocytosis or plasmacytoid dendritic cell leukemia. In certain embodiments, the antigen binding domain associated with J chain binds CD3ε, and binding molecules induce T cell-mediated malignant cell killing. In certain embodiments, relative to corresponding anti-CD123 anti-CD3 bispecific antibodies based on IgG, treatment causes cytokine release to decrease. In certain embodiments, the subject to be treated is a human subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figures 1A-1D Expression, correct assembly, and purification by size exclusion chromatography (SEC) of anti-CD123xCD3 IgM#1 (heavy chain: amino acids 20 to 592 of SEQ ID NO: 35, light chain: amino acids 21 to 240 of SEQ ID NO: 36, modified J chain: amino acids 20 to 420 of SEQ ID NO: 12) and anti-CD123xCD3 IgM#2 (heavy chain: amino acids 20 to 589 of SEQ ID NO: 40, light chain: amino acids 21 to 234 of SEQ ID NO: 41, modified J chain: amino acids 20 to 420 of SEQ ID NO: 12) are shown. Figure 1A : non-reducing gel; Figure 1B : reducing gel; Figure 1C : Size exclusion chromatogram showing purification of anti-CD123xCD3 IgM#1; Figure 1D : Size exclusion chromatogram showing the purification of anti-CD123xCD3 IgM#2.

[0035] Figures 2A-2BShown are the expression, correct assembly and purification by size exclusion chromatography (SEC) of anti-CD123xCD3 IgG#1 (first heavy chain: SEQ ID NO: 104, light chain: SEQ ID NO: 105, second heavy chain: SEQ ID NO: 6).

[0036] Figure 3 Anti-CD123xCD3 IgM#1 (triangle) and anti-CD123xCD3 IgM#2 (inverted triangle) are shown to bind to CD123 in an ELISA assay. Also shown are CD123 binding of monospecific IgG formats anti-CD123 IgG#1 (asterisk, heavy chain: amino acids 20 to 469 of SEQ ID NO:34, light chain: amino acids 21 to 240 of SEQ ID NO:36) and anti-CD123 IgG#2 (star, heavy chain: amino acids 20 to 464 of SEQ ID NO:39, light chain: amino acids 21 to 234 of SEQ ID NO:41).

[0037] Figure 4A Anti-CD123xCD3 IgM#1 (triangles) and anti-CD123xCD3 IgM#2 (inverted triangles) are shown to bind to CD3ε in an ELISA assay, but the monospecific IgG anti-CD123 construct does not. Figure 4B Binding of anti-CD123xCD3 IgM#1 (triangles) and anti-CD123xCD3 IgG#1 (open circles) to CD3ε in an ELISA assay was compared.

[0038] Figures 5A-5D Shown are the binding of IgM and IgG bispecific antibodies to CD123 at different protein concentrations as measured by ELISA. Figure 5A : 3 μg / ml CD123, Figure 5B : 1 μg / ml CD123; Figure 5C : 0.33 μg / ml CD123; and Figure 5D : 0.11μg / ml CD123.

[0039] Figure 6 Shown is the quantification of CD123 expressed on the surface of various AML cell lines.

[0040] Figure 7Figure 2 shows the binding of anti-CD123xCD3 IgM#1 to three different AML cell lines, Kg-1a, MOLM-13, and MV4-11, and the Burkitt's lymphoma cell line, Namalwa (CD123 negative), by flow cytometry. Top row: control anti-CD123 antibody 7G3; bottom row: anti-CD123xCD3 IgM#1, anti-CD123xCD3 IgM#3, and anti-CD123xCD3 IgM#4.

[0041] Figures 8A-8C The AML cell line THP-1 ( Figure 8A ) and MV4-11( Figure 8B ) T cell-dependent killing of Namalwa cells, in which Namalwa cells that did not express CD123 were not killed ( Figure 8C ).

[0042] Figure 9 It is shown that anti-CD123xCD3 IgM#1 enhances the CD25 activation marker on CD8+ T cells but not on CD4+ T cells in the TDCC assay of MV4-11 cells.

[0043] Figure 10A and 10B Comparison of anti-CD123xCD3 IgM#1 (triangles) and anti-CD123xCD3 IgG#1 (open circles) in pan-TDCC assays of MV4-11 cells (panel A) and THP-1 cells (panel B) after 96 hours. Open circles: anti-CD123xCD3 IgG#1, filled triangles: anti-CD123xCD3 IgM#1.

[0044] Figures 11A-11D Comparison of cytokine release between anti-CD123xCD3 IgG#1 and anti-CD123xCD3 IgM#1 in a TDCC assay on MV4-11 cells is shown. Panel A: Interferon gamma (IFNγ) release; Panel B: Interleukin-6 (IL-6) release; Panel C: TNFα release; Panel D: Interleukin-10 (IL10) release.

[0045] Figures 12A-12D Comparison of cytokine release between anti-CD123xCD3 IgG#1 and anti-CD123xCD3 IgM#1 in a TDCC assay on THP-1 cells is shown. Panel A: Interferon gamma (IFNγ) release; Panel B: Interleukin-6 (IL-6) release; Panel C: TNFα release; Panel D: Interleukin-10 (IL10) release.

[0046] Figure 13 Shown is the binding of IgM bispecific antibodies to CD123 at different protein concentrations as measured by ELISA.

[0047] Figure 14 Binding of the IgM bispecific antibody to MV4-11 cells expressing CD123 is shown.

[0048] Figures 15A-15B Figure 2 shows the expression of CD123 in the AML cell line THP-1 ( Figure 15A ) and PL21( Figure 15B ) of T cell-dependent killing.

[0049] Figures 16A-16B Figure 2 shows the expression of CD123 in the AML cell line THP-1 ( Figure 16A ) and PL21( Figure 16B ) of T cell-dependent killing.

[0050] Figure 17 Shown is T cell-dependent killing of the CD123-expressing AML cell line MV4-11 in the presence of anti-CD123xCD3 IgM antibodies with various CD3 binding domains and J chain.

[0051] Figures 18A-18F It shows that when T cells are CD8+T( Figures 18A-18C ) cells or CD4+T( Figures 18D-18F ) cells, the tumor survival ( Figure 18A 、 18D ), T cell proliferation ( Figure 18B 、 18E ) and T cell activation ( Figure 18C 、 18F ).

[0052] Figures 19A-19E Shown is a comparison of cytokine release between anti-CD123xCD3 IgG and anti-CD123xCD3 IgM antibodies in a TDCC assay for the following cytokines: Interferon gamma (IFNγ) ( Figure 19A ); tumor necrosis factor α (TNFα) ( Figure 19B ); interleukin-6 (IL-6) ( Figure 19C ); interleukin-10 (IL-10) ( Figure 19D ); interleukin-2 (IL-2) ( Figure 19E ). DETAILED DESCRIPTION

[0053] definition

[0054] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "binding molecule" should be understood to refer to one or more binding molecules. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0055] Furthermore, when used herein, "and / or" is considered to be a specific disclosure of each of the two specified features or components in the presence or absence of the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0056] Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press provide those of skill with a general dictionary of many of the terms used in this disclosure.

[0057] Units, prefixes, and symbols are all expressed in a form acceptable to the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations of the various embodiments or embodiments of the present disclosure, which can be obtained by reference to the entire specification. Therefore, the terms that will be defined below are more fully defined by reference to the entire specification.

[0058] As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not relate to the specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acids," and any other terms used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide may be derived from a biological source or produced by recombinant technology, but need not be translated from a specified nucleic acid sequence. The polypeptide may be produced in any manner, including by chemical synthesis.

[0059] The size of a polypeptide as disclosed herein can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. A polypeptide can have a defined three-dimensional structure, although it does not necessarily have such a structure. A polypeptide with a defined three-dimensional structure is referred to as folded, and a polypeptide that does not have a defined three-dimensional structure but can adopt many different conformations is referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein through an oxygen-containing or nitrogen-containing side chain of an amino acid (e.g., serine or asparagine).

[0060] An "isolated" polypeptide, or fragment, variant, or derivative thereof, is intended to be a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. As disclosed herein, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides that are separated, fractionated, or partially or substantially purified by any suitable technique.

[0061] As used herein, the term "non-naturally occurring polypeptide" or any grammatical variation thereof is a proviso to a definition that specifically excludes, but only excludes, those forms of the polypeptide that are or could be determined or interpreted by a judge or administrative or judicial body to be "naturally occurring."

[0062] Other polypeptides disclosed herein are fragments, derivatives, analogs or variants of the above-mentioned polypeptides and any combination thereof. The terms "fragment," "variant," "derivative," and "analog" disclosed herein include any polypeptide that retains at least some of the properties (e.g., specific binding antigen) of the corresponding natural antibody or polypeptide. In addition to the specific antibody fragments discussed elsewhere herein, polypeptide fragments include, for example, proteolytic fragments and deletion fragments. For example, variants of polypeptides include fragments as described above, and include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants may be non-naturally occurring. Mutagenesis techniques known in the art can be used to produce non-naturally occurring variants. Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been altered to exhibit additional features not found on the original polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to as "polypeptide analogs" herein. As used herein, a "derivative" of a polypeptide may also refer to a subject polypeptide having one or more amino acids that have been chemically derivatized by the reaction of a functional side group. "Derivatives" also include those peptides containing one or more derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0063] A "conservative amino acid substitution" is a substitution in which one amino acid is replaced by another amino acid with a similar side chain. Families of amino acids with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of polypeptides and antibodies of the present disclosure do not eliminate binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the antibody binds. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0064] The term "polynucleotide" is intended to encompass single nucleic acids as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0065] "Isolated" nucleic acid or polynucleotide means any form of nucleic acid or polynucleotide separated from its natural environment. For example, gel-purified polynucleotides or recombinant polynucleotides encoding polypeptides contained in a vector will be considered to be "isolated". In addition, polynucleotide segments (e.g., PCR products) that have been engineered to have restriction enzyme sites for cloning are considered to be "isolated". Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or purified (partially or substantially) polynucleotides in non-natural solutions such as buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, wherein the transcripts are not transcripts found in nature. Isolated polynucleotides or nucleic acids also include such molecules produced by synthetic means. In addition, polynucleotides or nucleic acids can be regulatory elements or can include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0066] As used herein, the term "non-naturally occurring polynucleotide" or any grammatical variation thereof is a proviso to a definition that specifically excludes, but only excludes, those forms of nucleic acids or polynucleotides that are or could be determined or interpreted by a judge or administrative or judicial body to be "naturally occurring."

[0067] As used herein, "coding region" is a part of a nucleic acid that is composed of codons that are translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are not translated into amino acids, they can be considered as part of a coding region, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, etc. are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, for example, on a single vector, or in a separate polynucleotide construct, for example, on a separate (different) vector. In addition, any vector can contain a single coding region, or can include two or more coding regions, for example, a single vector can encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, respectively. In addition, a vector, polynucleotide or nucleic acid can include a heterologous coding region that is fused or not fused to another coding region. Heterologous coding regions include but are not limited to those that encode special elements or motifs (such as secretory signal peptides or heterologous functional domains).

[0068] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, the polynucleotide comprising a nucleic acid that generally encodes a polypeptide may include a promoter and / or other transcription or translation control elements that are operably associated with one or more coding regions. Operable association is the association of the coding region of a gene product (e.g., a polypeptide) with one or more regulatory sequences, thereby placing the expression of the gene product under the influence or control of the one or more regulatory sequences. If inducing promoter function results in the transcription of the mRNA encoding the desired gene product, and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to guide the expression of the gene product or the ability of the DNA template to be transcribed, then the two DNA fragments (such as a polypeptide coding region and the promoter associated therewith) are "operably associated". Therefore, if the promoter can achieve transcription of the nucleic acid encoding the polypeptide, then the promoter region will be operably associated with that nucleic acid. A promoter can be a cell-specific promoter that guides substantial transcription of DNA in a predetermined cell. In addition to the promoter, other transcription control elements such as enhancers, operators, repressors, and transcription termination signals can be operably associated with the polynucleotide to guide cell-specific transcription.

[0069] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, associated with intron-A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit beta globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0070] Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also known as CITE sequences).

[0071] In other embodiments, the polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0072] Polynucleotides and nucleic acid coding regions can be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides as disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the export of the growing protein chain across the rough endoplasmic reticulum is initiated. One of ordinary skill in the art will appreciate that polypeptides secreted by vertebrate cells may have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a natural signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) or a functional derivative of the sequence that retains the ability to direct the secretion of a polypeptide to which it is operably associated is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0073] As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to a binding target (e.g., an epitope or antigenic determinant). As also described herein, a binding molecule may comprise one or more "antigen binding domains" as described herein. Non-limiting examples of binding molecules are antibodies or antibody-like molecules as described in detail herein that retain antigen-specific binding. In certain embodiments, a "binding molecule" comprises an antibody or antibody-like molecule as described in detail herein.

[0074] As used herein, the term "binding domain" or "antigen binding domain" (used interchangeably) refers to a region of a binding molecule (e.g., an antibody or antibody-like molecule) that is necessary and sufficient for specific binding to a binding target (e.g., an epitope). For example, "Fv", such as the heavy chain variable region and light chain variable region of an antibody, as two separate polypeptide subunits or as a single chain, is considered a "binding domain". Other antigen binding domains include, but are not limited to, the heavy chain variable region (VHH) of an antibody derived from a species of Camelidae or the six immunoglobulin complementary determining regions (CDRs) expressed in a scaffold (e.g., a fibronectin scaffold). "Binding molecules" or "antibodies" as described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more "antigen binding domains". As used herein, an "antigen binding domain associated with a binding unit" refers to an antigen binding domain that is a part of an antibody heavy chain and / or an antibody light chain. The term "antigen binding domain associated with a J chain" refers to an antigen binding domain associated with a modified J chain as described herein, e.g., a scFv fused to a wild-type human J chain or a functional fragment or variant thereof.

[0075] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody (or a fragment, variant or derivative thereof as disclosed herein) includes at least the variable domains of a heavy chain (for Camelidae species) or at least the variable domains of a heavy chain and a light chain. The basic immunoglobulin structure in vertebrate systems is relatively well understood. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988). Unless otherwise indicated, the term "antibody" encompasses anything from a small antigen-binding fragment of an antibody to a full-sized antibody, for example, an IgG antibody comprising 2 complete heavy chains and 2 complete light chains; an IgA antibody comprising 4 complete heavy chains and 4 complete light chains and optionally comprising a J chain and / or secretory component; or an IgM antibody comprising 10 or 12 complete heavy chains and 10 or 12 complete light chains and optionally comprising a J chain or a functional fragment thereof.

[0076] The term "immunoglobulin" includes a wide variety of polypeptides that can be distinguished biochemically. It will be understood by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1 to γ4 or α1 to α2) among them. The nature of this chain determines the "isotype" of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (subtypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., are well characterized and are known to confer functional specificity. In view of the present disclosure, modified versions of each of these immunoglobulins are readily discernible to those skilled in the art and are therefore within the scope of the present disclosure.

[0077] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain category can be combined with a kappa or lambda light chain. Typically, the light chain and the heavy chain are covalently bonded to each other, and when, for example, immunoglobulins are expressed by hybridomas, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence is from the N-terminus at the fork end of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of some antibodies (e.g., IgG antibodies) includes two heavy chain subunits and two light chain subunits, which are covalently linked by disulfide bonds to form a "Y" structure, also referred to herein as an "H2L2" structure, or a "binding unit."

[0078] The term "binding unit" is used herein to refer to a binding molecule, for example, an antibody, an antibody-like molecule, an antigen-binding fragment thereof, or a polymeric fragment thereof, corresponding to a portion of a standard "H2L2" immunoglobulin structure (e.g., two heavy chains or fragments thereof and two light chains or fragments thereof). In certain embodiments, a binding unit may correspond to two heavy chains (e.g., in camelid antibodies). In certain embodiments, for example, when the binding molecule is a bivalent IgG antibody or an antigen-binding fragment thereof, the terms "binding molecule" and "binding unit" are equivalent. In other embodiments, for example, when the binding molecule is a multimer (e.g., a dimeric IgA antibody or an IgA-like antibody, a pentamer IgM antibody or an IgM-like antibody or a hexameric IgM antibody or an IgM-like antibody), the binding molecule comprises two or more "binding units." In the case of an IgA dimer, there are 2 binding units, or in the case of an IgM pentamer or hexamer, there are 5 or 6 binding units, respectively. The binding unit need not include the heavy chain and light chain of a full-length antibody, but will generally be bivalent, i.e., will include two "antigen binding domains" as defined above. As used herein, some binding molecules provided in the present disclosure are "dimers" and include two bivalent binding units comprising an IgA constant region or a multimeric fragment thereof. Some binding molecules provided in the present disclosure are "pentamers" or "hexamers" and include five or six bivalent binding units comprising an IgM constant region or a multimeric fragment thereof. A binding molecule (e.g., an antibody or antibody-like molecule) comprising two or more (e.g., two, five, or six) binding units is referred to herein as a "multimer."

[0079] As used herein, the term "J chain" refers to the J chain of the native sequence of an IgM or IgA antibody of any animal species, any functional fragment thereof, derivative thereof, and / or variant thereof, including the mature human J chain, the amino acid sequence of which is presented as SEQ ID NO: 2. Various J chain variants and modified J chain derivatives are disclosed herein. As will be appreciated by one of ordinary skill in the art, "functional fragments" or "functional variants" include those fragments and variants that are capable of associating with an IgM heavy chain constant region to form a pentameric IgM antibody (or alternatively, capable of associating with an IgA heavy chain constant region to form a dimeric IgA antibody).

[0080] The term "modified J chain" is used herein to refer to a derivative of a native sequence J chain polypeptide, comprising a heterologous portion, such as a heterologous polypeptide, for example, an external binding domain introduced into a native sequence. Introduction can be achieved by any means, including direct or indirect fusion of a heterologous polypeptide or other portion, or by attaching using a peptide linker or a chemical linker. The term "modified human J chain" encompasses, but is not limited to, native sequence human J chains or their functional fragments or functional variants of amino acid sequences modified by introducing a heterologous portion (e.g., a heterologous polypeptide, for example, an external binding domain) comprising SEQ ID NO: 2. In certain embodiments, the heterologous portion does not interfere with IgM being efficiently polymerized into pentamers and does not interfere with the combination of such polymers with targets. Exemplary modified J chains are found in, for example, U.S. Patent Nos. 9,951,134 and 10,618,978, U.S. Patent Application Publication No. US-2019-0185570, each of which is incorporated herein by reference in its entirety.

[0081] As used herein, the terms "IgM-derived binding molecules," "IgM-like antibodies," "IgM-like binding units," or "IgM-like heavy chain constant regions" refer to variant antibody-derived binding molecules, antibodies, binding units, or heavy chain constant regions that still retain the structural portion of the IgM heavy chain, which is necessary for conferring the ability to form multimers (i.e., hexamers) or to associate with the J chain to form pentamers. An IgM-like antibody or IgM-derived binding molecule typically includes at least the Cμ4 and tail piece (tp) domains of the IgM constant region, but may include heavy chain constant region domains of other antibody isotypes (e.g., IgG) from the same species or different species. An IgM-like antibody or IgM-derived binding molecule can also be an antibody fragment in which one or more constant regions are deleted, as long as the IgM-like antibody is able to form hexamer and / or pentamer. Thus, an IgM-like antibody or IgM-derived binding molecule can be, for example, a hybrid IgM / IgG antibody, or can be a "multimeric fragment" of an IgM antibody.

[0082] As used herein, the terms "IgA-derived binding molecules", "IgA-like antibodies", "IgA-like binding units" or "IgA-like heavy chain constant regions" refer to variant antibody-derived binding molecules, antibodies, binding units or heavy chain constant regions that still retain the structural portion of the IgA heavy chain, which is necessary to confer the ability to associate with the J chain to form multimers (i.e., dimers). An IgA-like antibody or IgA-derived binding molecule typically includes at least the Cα3 and tail piece (tp) domains of the IgA constant region, but may include heavy chain constant region domains of other antibody isotypes (e.g., IgG) from the same species or different species. An IgA-like antibody or IgA-derived binding molecule can also be an antibody fragment in which one or more constant regions are deleted, as long as the IgA-like antibody is able to associate with the J chain to form a dimer. Thus, an IgA-like antibody or IgA-derived binding molecule can be, for example, a hybrid IgA / IgG antibody, or can be a "multimeric fragment" of an IgA antibody.

[0083] The terms "valence", "bivalent", "multivalent" and grammatical equivalents refer to the number of antigen binding domains in a given binding molecule (e.g., an antibody or antibody-like molecule) or in a given binding unit. Thus, with reference to a given binding molecule (e.g., an IgM antibody, an IgM-like antibody or a multimeric fragment thereof), the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two antigen binding domains, four antigen binding domains and six antigen binding domains, respectively. Where each binding unit is bivalent, a typical IgM antibody or IgM-like antibody or IgM-derived binding molecule may have 10 or 12 valences. Bivalent or multivalent binding molecules, e.g., antibodies or antibody-like molecules, may be monospecific, i.e., all antigen binding domains are identical, or may be bispecific or multispecific, e.g., wherein two or more antigen binding domains are different, e.g., in conjunction with different epitopes on the same antigen, or in conjunction with completely different antigens.

[0084] The term "epitope" includes any molecular determinant capable of specific binding to an antigen binding domain of an antibody or antibody-like molecule. In certain embodiments, an epitope may include a chemically active surface cluster of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have three-dimensional structural characteristics and or specific charge characteristics. An epitope is a region of a target that is bound by an antigen binding domain of an antibody.

[0085] The term "target" is used in the broadest sense to include substances that can be bound by a binding molecule (e.g., an antibody or antibody-like molecule). A target can be, for example, a polypeptide, nucleic acid, carbohydrate, lipid, or other molecule. In addition, a "target" can be, for example, a cell, organ, or organism that contains an epitope that can be bound by a binding molecule (e.g., an antibody or antibody-like molecule).

[0086] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. The variable regions of the light chain (VL) and the heavy chain (VH) both determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and the heavy chain (e.g., CH1, CH2, CH3, or CH4) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains increases as they are further away from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 (or CH4 in the case of IgM) and CL domains actually comprise the carboxyl termini of the heavy and light chains, respectively.

[0087] A "full-length IgM antibody heavy chain" is a polypeptide that includes, in the N-terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cμ1), an antibody heavy chain constant domain 2 (CM2 or Cμ2), an antibody heavy chain constant domain 3 (CM3 or Cμ3) and an antibody heavy chain constant domain 4 (CM4 or Cμ4) that may include a tail piece.

[0088] As indicated above, one or more variable regions allow a binding molecule (e.g., an antibody or antibody-like molecule) to selectively recognize and specifically bind to an epitope on an antigen. That is, a subset of the VL domain and the VH domain or complementary determining regions (CDRs) of a binding molecule (e.g., an antibody or antibody-like molecule) combines to form an antigen-binding domain. More specifically, an antigen-binding domain can be defined by three CDRs on each VH and VL chain. Certain antibodies form larger structures. For example, IgA can form a molecule comprising two H2L2 binding units and a J chain covalently linked by a disulfide bond, which can also associate with secretory component, and IgM can form a pentamer or hexamer molecule comprising five or six H2L2 binding units and optionally a J chain covalently linked by a disulfide bond.

[0089] The six "complementarity determining regions" or "CDRs" present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domain are called "framework" regions, which exhibit less intermolecular variability. The framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Thus, the framework regions serve to form a scaffold that provides for positioning the CDRs in the correct orientation through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on the target antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify the amino acids that make up the CDRs and framework regions, respectively, since they have been defined in a variety of ways (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are herein incorporated by reference in their entireties).

[0090] In the case where there are two or more definitions of terms used and / or recognized in the art, unless explicitly stated otherwise, the definition of the terms used herein is intended to include all such meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable region of both heavy and light chain polypeptides. These specific regions have been described, for example, by Kabat et al., US Patent. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987), which are incorporated herein by reference. Kabat and Chothia define overlapping or subsets of amino acids when compared to each other. However, unless otherwise indicated, the application of any definition (or other definitions known to those of ordinary skill in the art) of the CDR of an antibody or its variants is intended to fall within the scope of the terms defined and used herein. The appropriate amino acids encompassing the defined CDRs in each reference cited above are listed in Table 1 below for comparison. The exact number of amino acids that encompass a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which amino acids constitute a particular CDR.

[0091] Table 1 CDR definitions *

[0092] Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

[0093] * The numbering of all CDR definitions in Table 1 is according to the numbering convention proposed by Kabat et al. (see below).

[0094] For example, you can also use the IMGT information system (imgt_dot_cines_dot_fr / )( The antibody variable domains were analyzed using PCR / V-Quest to identify variable region segments, including CDRs (see, e.g., Brochet et al., Nucl. Acids Res., 36: W503-508, 2008).

[0095] Kabat et al. also defined a numbering system applicable to the variable and constant region sequences of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). However, unless explicitly stated to use the Kabat numbering system, all amino acid sequences in this disclosure are numbered consecutively.

[0096] The Kabat numbering system for human IgM constant domains can be found in Kabat et al. "Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, β-2 Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, α-2 Macroglobulins, and Other Related Proteins," U. S. Pat. of Health and Human Services (1991). The IgM constant regions can be numbered sequentially (i.e., amino acid #1 starts with the first amino acid of the constant region) or by using the Kabat numbering scheme. Listed below are two alleles of the human IgM constant region, numbered sequentially (presented herein as SEQ ID NO: 22 (allele IGHM*03) and SEQ ID NO: 23 (allele IGHM*04)) and a comparison of the numbering by the Kabat system. Underlined amino acid residues are not considered in the Kabat system (hereinafter double-underlined " X " can be serine (S) (SEQ ID NO: 22) or glycine (G) (SEQ ID NO: 23)):

[0097] Sequence of IgM heavy chain (SEQ ID NO: 22 or SEQ ID NO: 23) / KABAT numbering legend

[0098]

[0099] Binding molecules (e.g., antibodies, antibody-like molecules), antigen-binding fragments, variants or derivatives thereof, and / or polymeric fragments thereof include, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments comprising VL or VH domains, fragments produced by Fab expression libraries. scFv molecules are known in the art and are described, e.g., in U.S. Patent No. 5,892,019.

[0100] "Specific binding" generally means that a binding molecule (e.g., an antibody or a fragment, variant, or derivative thereof) binds to an epitope through its antigen binding domain, and that the binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it is more likely to bind to an epitope through its antigen binding domain than a binding molecule (e.g., an antibody or antibody-like molecule) will bind to a random, unrelated epitope. The term "specificity" is used herein to describe the relative affinity with which a binding molecule binds to an epitope. For example, it can be said that binding molecule "A" has a higher specificity for a given epitope than binding molecule "B", or it can be said that binding molecule "A" binds to epitope "C" with a higher specificity than for a related epitope "D".

[0101] It can be said that the binding molecules disclosed herein (e.g., antibodies or fragments, variants or derivatives thereof) have a specific activity of less than or equal to 5×10 -2 sec -1 , 10 -2 sec -1 , 5X 10 -3 sec -1 , 10 -3 sec -1 , 5X 10 -4 sec -1 , 10 -4 sec -1 , 5X 10 -5 sec -1 or 10 -5 sec -1 , 5X10 -6 sec -1 , 10 -6 sec-1 , 5X 10 -7 sec -1 or 10 -7 sec -1 The dissociation rate (k(off)) of the protein binds to the target antigen.

[0102] It can be said that the binding molecules (eg, antibodies or antigen-binding fragments, variants, or derivatives) disclosed herein have a molecular weight greater than or equal to 10 3 M -1 sec -1 , 5X 10 3 M -1 sec -1 , 10 4 M -1 sec -1 , 5X 10 4 M -1 sec -1 , 10 5 M -1 sec -1 , 5X 10 5 M -1 sec -1 , 10 6 M -1 sec -1 , or 5X 10 6 M -1 sec -1 or 10 7 M -1 sec -1 The binding rate (k(on)) of the target antigen is determined.

[0103] If a binding molecule (e.g., an antibody or fragment, variant, or derivative thereof) preferentially binds to a given epitope to such an extent that it blocks, to some extent, the binding of a reference antibody or antigen-binding fragment to that epitope, then the binding molecule is said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to that epitope. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. A binding molecule can be said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0104] As used herein, the term "affinity" refers to a measure of the strength of binding of a single epitope to, for example, one or more antigen-binding domains of an immunoglobulin molecule. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988), pages 27 to 28. As used herein, the term "avidity" refers to the overall stability of the complex between an antigen-binding domain group and an antigen. See, for example, Harlow, pages 29 to 34. Avidity is related to the affinity of a single antigen-binding domain in the group to a specific epitope, and is related to the price of the immunoglobulin and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitope structure (such as a polymer) will be a high-affinity interaction. The interaction between a bivalent monoclonal antibody and a receptor present at high density on the cell surface will also have high avidity.

[0105] Binding molecules (e.g., antibodies or fragments, variants or derivatives thereof) as disclosed herein may also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule (e.g., an antibody or fragment, variant or derivative thereof) that is specific for one antigen to react with a second antigen; a measure of the correlation between two different antigenic substances. Thus, a binding molecule is cross-reactive if it binds to an epitope other than the epitope that induced its formation. A cross-reactive epitope typically contains many of the same complementary structural features as the induced epitope, and in some cases, may actually be more suitable than the original epitope.

[0106] A binding molecule (e.g., an antibody or fragment, variant, or derivative thereof) may also be described or specified in terms of its binding affinity to an antigen. For example, a binding molecule may have a binding affinity of no greater than 5 x 10 -2 M, 10 -2 M, 5x10 -3 M, 10 -3 M, 5x10 -4 M, 10 -4 M, 5x10 -5 M, 10 -5 M, 5x10 -6 M, 10 -6 M, 5x10 -7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11M, 10 -11 M, 5x10 -12 M, 10 -12 M, 5x10 -13 M, 10 -13 M, 5x10 -14 M, 10 -14 M, 5x10 -15 M or 10 -15 The dissociation constant or K of M D Binds to antigen.

[0107] "Antigen-binding antibody fragments" including single-chain antibodies or other antigen-binding domains may exist alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3 or CH4 domain, J chain or secretory component. Also included are antigen-binding fragments that may include any combination of one or more of the hinge region, CH1, CH2, CH3 or CH4 domain, J chain or secretory component and one or more variable regions. The binding molecule (e.g., an antibody or antigen-binding fragment thereof) may be from any animal source, including birds and mammals. The antibody may be, for example, an antibody of human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In another embodiment, the variable region may be of condricthoid origin (e.g., from shark). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from a transgenic animal for one or more human immunoglobulins, which transgenic animal may in some cases express endogenous immunoglobulins and in some cases not express them, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al. According to embodiments of the present disclosure, an IgM or IgM-like antibody or an IgM-derived binding molecule as provided herein may include an antigen-binding fragment (e.g., scFv fragment) of an antibody, as long as the IgM or IgM-like antibody is able to form multimers, such as hexamers or pentamers.

[0108] As used herein, the term "heavy chain subunit" includes an amino acid sequence derived from an immunoglobulin heavy chain, and the binding molecule (e.g., antibody or antibody-like molecule) comprising a heavy chain subunit may include at least one of the following: VH domain, CH1 domain, hinge (e.g., upper, middle and / or lower hinge region) domain, CH2 domain, CH3 domain, CH4 domain, tail piece (tp) or its variant or fragment. For example, in addition to the VH domain, the binding molecule (e.g., antibody, antibody-like molecule or its fragment, variant or derivative) may also include, but is not limited to, any combination of the CH1 domain, hinge, CH2 domain, CH3 domain, CH4 domain or tail piece (tp) of one or more antibody isotypes and / or species. In certain embodiments, the binding molecule (e.g., antibody, antibody-like molecule) or its fragment, variant or derivative may also include, in addition to the VH domain, a CH3 domain and a CH4-tp domain; or a CH3 domain, a CH4-tp domain and a J chain. In addition, the binding molecules (e.g., antibodies or antibody-like molecules) used in the present disclosure may lack certain constant region portions, such as all or part of the CH2 domain. These domains (e.g., heavy chain subunits) may be modified so that they differ from the original immunoglobulin molecules in amino acid sequence. According to embodiments of the present disclosure, as provided herein, the IgM or IgM-like antibodies include a sufficient portion of the IgM heavy chain constant region to allow the IgM or IgM-like antibodies to form multimers (e.g., hexamers or pentamers), e.g., the IgM heavy chain constant region includes a "multimeric fragment" of the IgM heavy chain constant region.

[0109] As used herein, the term "light chain subunit" includes an amino acid sequence derived from an immunoglobulin light chain. A light chain subunit includes at least a VL and may further include a CL (eg, CK or Cλ) domain.

[0110] Binding molecules (e.g., antibodies, antibody-like molecules, antigen-binding fragments, variants or derivatives thereof, or multimeric fragments thereof) can be described or specified based on one or more epitopes or portions of an antigen that they recognize or specifically bind. The portion of the target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant." A target antigen may comprise a single epitope or at least two epitopes, and may comprise any number of epitopes, depending on the size, conformation, and type of the antigen.

[0111] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain in IgG, IgA, and IgD heavy chains. This hinge region contains approximately 25 amino acids and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0112] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a sulfhydryl group that can form a disulfide bond with or bridge to a second sulfhydryl group.

[0113] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, partial, or modified) is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0114] The term "multispecific antibody" or "bispecific antibody" refers to an antibody or antibody-like molecule that has antigen-binding domains for two or more different epitopes within a single antibody molecule. In addition to the typical antibody structure, other binding molecules can be constructed with two binding specificities.

[0115] As used herein, the term "engineered antibody" refers to an antibody wherein the variable domains in heavy chain or light chain or both are replaced by at least part of one or more amino acids in CDR or framework region and changed. In certain embodiments, the whole CDR from a known specific antibody can be transplanted into the framework region of a heterologous antibody. Although the substituted CDR can be derived from the same class or even subclass of antibody derived from the framework region, CDR can also be derived from antibodies of different classes, for example, antibodies derived from different species. The engineered antibodies wherein one or more "donor" CDRs from a non-human antibody of known specificity are transplanted into human heavy chain or light chain framework region are referred to herein as "humanized antibodies." In certain embodiments, not all CDRs are replaced by the complete CDR from the donor variable region, but the antigen binding capacity of the donor can still be transferred to the receptor variable domains. Given the explanations set forth in, for example, US Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, one of ordinary skill in the art will be well able to obtain functionally engineered or humanized antibodies by performing routine experimentation.

[0116] As used herein, the term "engineering" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, nucleic acids or glycans, or some combination of these techniques).

[0117] As used herein, the terms "connected," "fused," or "fusion," or other grammatical equivalents, are used interchangeably. These terms refer to joining two or more elements or components together by whatever means, including chemical conjugation or recombinant means. "In-frame fusion" refers to joining two or more polynucleotide ORFs to form a continuous, longer ORF in a manner that maintains the translational reading frame of the original open reading frame (ORF). Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to polypeptides encoded by the original ORFs that would not normally be so connected in nature. Although the reading frame is thus continuous throughout the fused segment, these segments can be physically or spatially separated by, for example, in-frame linker sequences. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused in-frame, but can be separated by polynucleotides encoding at least one immunoglobulin framework region or another CDR region, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0118] In the context of polypeptides, a "linear sequence" or "sequence" is the sequential sequence of amino acids in a polypeptide, running from the amino terminus to the carboxyl terminus, wherein adjacent amino acids in the sequence are contiguous in the primary structure of the polypeptide. The portion of a polypeptide that is at the "amino terminus" or "C-terminus" of another portion of a polypeptide is that portion that appears earlier in the sequential polypeptide chain. Similarly, the portion of a polypeptide that is at the "carboxyl terminus" or "C-terminus" of another portion of a polypeptide is that portion that appears later in the sequential polypeptide chain. For example, in a typical antibody, the variable domain is at the "N-terminus" of the constant region, and the constant region is at the "C-terminus" of the variable domain.

[0119] As used herein, the term "expression" refers to the process by which a gene produces a biochemical substance, such as a polypeptide. The process includes any manifestation of the functional presence of a gene in a cell, including but not limited to gene knockout and transient expression and stable expression. It includes but is not limited to transcribing a gene into RNA, for example, messenger RNA (mRNA), and translating this mRNA into one or more polypeptides. If the final desired product is a biochemical substance, expression includes producing the biochemical substance and any precursors. The expression of a gene produces a "gene product." As used herein, a gene product can be a nucleic acid (e.g., messenger RNA produced by gene transcription) or a polypeptide translated from a transcript. Gene products as described herein also include nucleic acids with post-transcriptional modifications (e.g., polyadenylation), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, etc.).

[0120] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to curing, alleviating, lessening the symptoms of, and / or halting or slowing the progression of, an existing diagnosed disease, pathological condition, or disorder. Terms such as "prevent," "prevention," "avoid," "deterrence," and the like refer to prophylactic or preventative measures that prevent the development of an undiagnosed target disease, pathological condition, or disorder. Thus, a "subject in need of treatment" can include subjects already suffering from a disorder; those susceptible to the disorder; and those for whom the disorder is to be prevented.

[0121] As used herein, the term "serum half-life" or "plasma half-life" refers to the time (e.g., in minutes, hours, or days) it takes for the serum or plasma concentration of a protein or drug (e.g., a binding molecule as described herein, such as an antibody, antibody-like molecule, or fragment thereof) to decrease by 50% after administration. Two half-lives can be described: alpha half-life, α half-life, or t half-life. 1 / 2 α, which is the rate of decrease in plasma concentration due to the process of redistribution of the drug from the central compartment (e.g., blood in the case of intravenous delivery) to the peripheral compartment (e.g., tissues or organs); and beta half-life, β half-life, or t 1 / 2 β, which is the rate of decline due to excretion or metabolic processes.

[0122] As used herein, the term "area under the plasma drug concentration-time curve" or "AUC" reflects the actual exposure of the human body to a drug after administration of a dose of the drug and is expressed in mg*h / L. The area under this curve is measured from time 0 (t0) to infinity (∞) and depends on the rate of clearance of the drug from the body and the dose administered.

[0123] As used herein, the term "mean residence time" or "MRT" refers to the average length of time a drug remains in the body.

[0124] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0125] As used herein, phrases such as "a subject who will benefit from treatment" and "an animal in need of treatment" refer to a subset of subjects from among all expected subjects who will benefit from the administration of a given therapeutic agent (e.g., a binding molecule such as an antibody or antibody-like molecule comprising one or more antigen binding domains). Such binding molecules (e.g., antibodies or antibody-like molecules) can be used, for example, in diagnostic procedures and / or in the treatment or prevention of disease.

[0126] IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules

[0127] IgM is the first immunoglobulin produced by B cell response antigen stimulation, and is naturally present in serum with about 1.5mg / ml, and its half-life is about 5 days.IgM is a pentamer or hexamer molecule, and therefore includes five or six binding units.IgM binding units generally include two light chains and two heavy chains.Although the IgG heavy chain constant region contains three heavy chain constant domains (CH1, CH2 and CH3), the heavy chain (μ) constant region of IgM contains the fourth constant domain (CH4) in addition and includes C-terminal " tail piece " (tp).Although there are some human alleles, human IgM constant region generally includes amino acid sequence SEQ ID NO:22 (IMGT allele IGHM*03, identical with, for example, GenBank accession number pir||S37768) or SEQ ID NO:23 (IMGT allele IGHM*04, identical with, for example, GenBank accession number sp|P01871.4). The range of the human Cμ1 region is about amino acid 5 to about amino acid 102 of SEQ ID NO:22 or SEQ ID NO:23; the range of the human Cμ2 region is about amino acid 114 to about amino acid 205 of SEQ ID NO:22 or SEQ ID NO:23; the range of the human Cμ3 region is about amino acid 224 to about amino acid 319 of SEQ ID NO:22 or SEQ ID NO:23; the range of the Cμ4 region is about amino acid 329 to about amino acid 430 of SEQ ID NO:22 or SEQ ID NO:23; and the range of the tail piece is about amino acid 431 to about amino acid 453 of SEQ ID NO:22 or SEQ ID NO:23.

[0128] There are other forms of human IgM constant regions with minor sequence variations, including but not limited to GenBank Accession Nos. CAB37838.1 and pir||MHHU. Amino acid substitutions, insertions and / or deletions at positions corresponding to SEQ ID NO: 22 or SEQ ID NO: 23 described and claimed elsewhere in this disclosure can also be incorporated into alternative human IgM sequences, as well as into IgM constant region amino acid sequences of other species.

[0129] Each IgM heavy chain constant region can be associated with an antigen binding domain (eg, scFv or VHH) or a subunit of an antigen binding domain (eg, a VH region).

[0130] The five IgM binding units can form a complex with another small polypeptide chain (J chain) or a functional fragment, variant, or derivative thereof to form a pentameric IgM antibody or IgM-like antibody. The precursor form of the human J chain is presented in SEQ ID NO: 1. The signal peptide (underlined) extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 1, and the mature human J chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO: 1. The mature human J chain has the amino acid sequence SEQ ID NO: 2.

[0131] Exemplary variants and modified J chains are provided elsewhere herein. In the absence of J chains, IgM antibodies or IgM-like antibodies are generally assembled into hexamers, and hexamers include six binding units and up to 12 antigen-binding domains associated with binding units. In the case of J chains, IgM antibodies or IgM-like antibodies are generally assembled into pentamers, and pentamers include five binding units and up to ten antigen-binding domains associated with binding units, or if J chains are modified J chains comprising one or more heterologous polypeptides (heterologous polypeptides can be, for example, other, antigen-binding domains associated with J chains), pentamers include more antigen-binding domains. Five or six IgM binding units are assembled into pentamers or hexamer IgM antibodies or IgM-like antibodies and are considered to be related to the interaction between C μ 4 and tail piece domains. See, for example, Braathen, R., et al., J.Biol.Chem.277:42755-42762 (2002). Therefore, the constant region of the pentamer or hexamer IgM antibody or antibody-like molecule provided in the present disclosure generally includes at least Cμ4 and / or tail piece domain (also collectively referred to as Cμ4-tp herein). Therefore, the "multimeric fragment" of the IgM heavy chain constant region includes at least the Cμ4-tp domain. The IgM heavy chain constant region may additionally include a Cμ3 domain or a fragment thereof, a Cμ2 domain or a fragment thereof, a Cμ1 domain or a fragment thereof. In certain embodiments, the binding molecule as provided herein (e.g., IgM antibody or IgM-like antibody) may include a complete IgM heavy (μ) chain constant domain (e.g., SEQ ID NO: 22 or SEQ ID NO: 23) or a variant, derivative or analog thereof (e.g., as provided herein).

[0132] In certain embodiments, the present disclosure provides a pentameric IgM or IgM-like antibody comprising five bivalent binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimeric fragments or variants thereof, each of which is associated with an antigen binding domain or a subunit of an antigen binding domain. In certain embodiments, the two IgM heavy chain constant regions are human heavy chain constant regions.

[0133] In the case where the IgM or IgM-like antibodies provided herein are pentamers, IgM or IgM-like antibodies generally also include a J chain or its functional fragment or variant. As provided herein, in some embodiments, the J chain is a modified J chain, which includes specific binding to immune effector cells (e.g., CD8+ cytotoxic T cells or NK cells), an antigen binding domain associated with the J chain. In certain embodiments, the modified J chain may also include one or more heterologous portions attached thereto, such as immunostimulants. In certain embodiments, the J chain may be mutated to affect (e.g., enhance) the serum half-life of the IgM or IgM-like antibodies provided herein, as discussed elsewhere herein. In certain embodiments, the J chain may be mutated to affect glycosylation, as discussed elsewhere in the present disclosure.

[0134] In some embodiments, the multimeric binding molecule is a hexamer and comprises six bivalent binding units or variants or fragments thereof. In some embodiments, the multimeric binding molecule is a hexamer and comprises six bivalent binding units or variants or fragments thereof, and wherein each binding unit comprises two IgM heavy chain constant regions or multimeric fragments or variants thereof.

[0135] The IgM heavy chain constant region may include one or more of the following: Cμ1 domain or its fragment or variant, Cμ2 domain or its fragment or variant, Cμ3 domain or its fragment or variant, Cμ4 domain or its fragment or variant and / or tail piece (tp) or its fragment or variant, provided that the constant region can play a desired function in IgM or IgM-like antibodies, for example, by associating with the second IgM constant region to form a binding unit with 1, 2 or more antigen-binding domains, and / or by associating with other binding units (and J chain in the case of pentamers) to form a hexamer or pentamer. In certain embodiments, the two IgM heavy chain constant regions or their fragments or variants in a single binding unit each include: Cμ4 domain or its fragment or variant; tail piece (tp) or its fragment or variant; or a combination of Cμ4 domain and tp or its fragment or variant. In certain embodiments, the two IgM heavy chain constant regions, or fragments or variants thereof, in a single binding unit each further comprise: a Cμ3 domain, or fragment or variant thereof; a Cμ2 domain, or fragment or variant thereof; a Cμ1 domain, or fragment or variant thereof; or any combination thereof.

[0136] In some embodiments, the binding unit of an IgM or IgM-like antibody comprises two light chains. In some embodiments, the binding unit of an IgM or IgM-like antibody comprises two light chain fragments. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments, each binding unit comprises two immunoglobulin light chains, each light chain comprising a VL located amino-terminal to an immunoglobulin light chain constant region.

[0137] IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules with enhanced serum half-life

[0138] The polymer bispecific binding molecules derived from some IgM provided herein can be modified to have enhanced serum half-life. The exemplary IgM heavy chain constant region mutations for the serum half-life of the binding molecules derived from IgM can be enhanced and disclosed in PCT Publication No. WO 2019 / 169314A1, which are incorporated herein by reference in their entirety. For example, the variant IgM heavy chain constant region of the binding molecules derived from IgM as provided herein can include amino acid substitutions at the amino acid positions corresponding to amino acid S401, E402, E403, R344 and / or E345 of wild-type human IgM constant region (for example, SEQ ID NO: 22 or SEQ ID NO: 23). "Amino acids corresponding to amino acids S401, E402, E403, R344, and / or E345 of a wild-type human IgM constant region" refers to amino acids in the sequence of an IgM constant region of any species that are homologous to S401, E402, E403, R344, and / or E345 in a human IgM constant region. In certain embodiments, the amino acids corresponding to S401, E402, E403, R344, and / or E345 of SEQ ID NO: 22 or SEQ ID NO: 23 may be substituted with any amino acid (e.g., alanine).

[0139] IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules with reduced CDC activity

[0140] With respect to the reference IgM antibody or IgM-like antibody with corresponding reference human IgM constant region, except the mutation that gives the CDC activity of reduction, it is possible to engineer the multimeric binding molecules derived from some IgM as provided herein to show the complement dependent cytotoxicity (CDC) activity of the reduction to cells in the presence of complement. These CDC mutations can be combined with any mutation that blocks N-linked glycosylation and / or gives increased serum half-life as provided herein." Corresponding reference human IgM constant region" means human IgM constant region or a portion thereof (e.g., C μ 3 domains), which is identical to variant IgM constant region except for one or more modifications of the CDC activity of the influence in the constant region. In certain embodiments, variant human IgM constant region includes one or more amino acid substitutions (e.g., in C μ 3 domains) relative to wild-type human IgM constant region (e.g., described in PCT Publication No. WO / 2018 / 187702, which is incorporated herein by reference in its entirety). Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described, for example, in PCT Publication No. WO / 2018 / 187702.

[0141] In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises amino acid substitutions corresponding to the wild-type human IgM constant region at positions L310, P311, P313, and / or K315 of SEQ ID NO: 22 (human IgM constant region allele IGHM*03) or SEQ ID NO: 23 (human IgM constant region allele IGHM*04). In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises amino acid substitutions corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, the variant IgM constant region as provided herein contains amino acid substitutions corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, variant IgM constant region as provided herein contains and SEQ ID NO:22 or SEQ ID NO:23 P311 and / or SEQ ID NO:22 or SEQ ID NO:23 wild-type human IgM constant region at P313 position corresponding to the combination of replacement.These proline residues can be independently replaced by any amino acid (for example, alanine, serine or glycine).In certain embodiments, the variant human IgM constant region giving the CDC activity of reduction includes and SEQ ID NO:22 or SEQ ID NO:23 K315 position corresponding to the amino acid replacement of the wild-type human IgM constant region.Lysine residue can be independently replaced by any amino acid (for example, alanine, serine, glycine or aspartic acid).In certain embodiments, the variant human IgM constant region giving the CDC activity of reduction includes and SEQ ID NO:22 or SEQ ID NO:23 K315 position corresponding to the amino acid replacement carried out with aspartic acid of the wild-type human IgM constant region. In certain embodiments, the variant human IgM constant region that gives reduced CDC activity includes and SEQ ID NO:22 or SEQ ID NO:23 L310 position corresponding to the wild-type human IgM constant region amino acid replacement.Lysine residues can be independently replaced by any amino acid (e.g., alanine, serine, glycine or aspartic acid).In certain embodiments, the variant human IgM constant region that gives reduced CDC activity includes and SEQ ID NO:22 or SEQ ID NO:23 L310 position corresponding to the wild-type human IgM constant region amino acid replacement with aspartic acid.

[0142] Human and certain non-human primate IgM constant regions typically include five (5) naturally occurring asparagine (N) linked glycosylation motifs or sites. As used herein, an "N-linked glycosylation motif" comprises or consists of the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). Glycans are attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd Edition). Oxford University Press, USA. N-linked glycosylation motifs appear in the human IgM heavy chain constant region of SEQ ID NO: 22 or SEQ ID NO: 23, starting at positions 46 ("N1"), 209 ("N2"), 272 ("N3"), 279 ("N4"), and 440 ("N5"). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five motifs are conserved in mouse IgM heavy chain constant regions. Thus, in some embodiments, the IgM heavy chain constant region of a multimeric binding molecule as provided herein comprises five N-linked glycosylation motifs: N1, N2, N3, N4, and N5. In some embodiments, at least three N-linked glycosylation motifs (e.g., N1, N2, and N3) on each IgM heavy chain constant region are occupied by complex glycans.

[0143] In certain embodiments, at least one, at least two, at least three or at least four N-X1-S / T motifs may include amino acid insertions, deletions or substitutions that prevent glycosylation at the motif. In certain embodiments, the multimeric binding molecules derived from IgM may include amino acid insertions, deletions or substitutions at any combination of two or more, three or more or all four of the N1 motif, N2 motif, N3 motif, N5 motif or N1, N2, N3 or N5 motifs, wherein amino acid insertions, deletions or substitutions prevent glycosylation at the motif. In some embodiments, relative to the wild-type human IgM constant region, the IgM constant region is included in SEQ ID NO: 22 (human IgM constant region allele IGHM*03) or SEQ ID NO: 23 (human IgM constant region allele IGHM*04) at 46, 209, 272 or 440 positions. See, e.g., U.S. Provisional Application No. 62 / 891,263, which is incorporated herein by reference in its entirety.

[0144] IgA antibodies, IgA-like antibodies, and IgA-derived binding molecules

[0145] IgA plays a key role in mucosal immunity and comprises approximately 15% of the total immunoglobulins produced. IgA can be monomeric or multimeric, primarily forming dimeric molecules, but can also assemble into trimers, tetramers, and / or pentamers. See, e.g., de Sousa-Pereira, P. and JM Woof, Antibodies 8:57 (2019).

[0146] In some embodiments, the multimeric binding molecule is a dimer and comprises two bivalent binding units or variants or fragments thereof. In some embodiments, the multimeric binding molecule is a dimer, comprises two bivalent binding units or variants or fragments thereof, and further comprises a J chain or functional fragment or variant thereof as described herein. In some embodiments, the multimeric binding molecule is a dimer, comprises two bivalent binding units or variants or fragments thereof, and further comprises a J chain or functional fragment or variant thereof as described herein, wherein each binding unit comprises two IgA heavy chain constant regions or multimeric fragments or variants thereof.

[0147] In some embodiments, the multimeric binding molecule is a tetramer and comprises four bivalent binding units or variants or fragments thereof. In some embodiments, the multimeric binding molecule is a tetramer, comprises four bivalent binding units or variants or fragments thereof, and further comprises a J chain or functional fragment or variant thereof as described herein. In some embodiments, the multimeric binding molecule is a tetramer, comprises four bivalent binding units or variants or fragments thereof, and further comprises a J chain or functional fragment or variant thereof as described herein, wherein each binding unit comprises two IgA heavy chain constant regions or multimeric fragments or variants thereof.

[0148] In certain embodiments, the multimeric binding molecules provided by the present disclosure are dimeric binding molecules, which include an IgA heavy chain constant region or a multimeric fragment thereof, and each of the IgA heavy chain constant region or its multimeric fragment is associated with an antigen binding domain, for a total of four antigen binding domains. As provided herein, IgA antibodies, IgA-derived binding molecules or IgA-like antibodies include two binding units and a J chain, such as a modified J chain, which includes an scFv antibody fragment bound to CD3 or an IL-15 and / or IL-15 receptor-αsushi domain fused to a modified J chain, as described elsewhere herein. Each binding unit provided includes two IgA heavy chain constant regions or their multimeric fragments or variants. In certain embodiments, at least three or all four antigen binding domains of the multimeric binding molecule bind to the same target antigen. In certain embodiments, at least three or all four binding polypeptides of the multimeric binding molecule are identical.

[0149] The bivalent IgA-derived binding unit comprises two IgA heavy chain constant regions, and the dimeric IgA-derived binding molecule comprises two binding units. IgA contains the following heavy chain constant domains: Cα1 (or alternatively CA1 or CH1), a hinge region, Cα2 (or alternatively CA2 or CH2) and Cα3 (or alternatively CA3 or CH3), and a C-terminal "tail piece." Human IgA has two subtypes: IgA1 and IgA2. The human IgA1 constant region generally comprises the amino acid sequence SEQ ID NO: 24. The human Cα1 domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 24; the human IgA1 hinge region extends from about amino acid 102 to about amino acid 124 of SEQ ID NO: 24, the human Cα2 domain extends from about amino acid 125 to about amino acid 219 of SEQ ID NO: 24, the human Cα3 domain extends from about amino acid 228 to about amino acid 330 of SEQ ID NO: 24, and the tail extends from about amino acid 331 to about amino acid 352 of SEQ ID NO: 24. The human IgA2 constant region generally includes the amino acid sequence of SEQ ID NO: 25. The human Cα1 domain extends from approximately amino acid 6 to approximately amino acid 98 of SEQ ID NO:25; the human IgA2 hinge region extends from approximately amino acid 102 to approximately amino acid 111 of SEQ ID NO:25, the human Cα2 domain extends from approximately amino acid 113 to approximately amino acid 206 of SEQ ID NO:25, the human Cα3 domain extends from approximately amino acid 215 to approximately amino acid 317 of SEQ ID NO:25, and the tail extends from approximately amino acid 318 to approximately amino acid 340 of SEQ ID NO:25.

[0150] Two IgA binding units can form a complex with two additional polypeptide chains: J chain (e.g., SEQ ID NO: 2) and secretory component (precursor, SEQ ID NO: 26, mature, SEQ ID NO: 27) to form a bivalent secretory IgA (sIgA) derived binding molecule as provided herein. The assembly of two IgA binding units into dimeric IgA derived binding molecules is believed to involve Cα3 and tail piece domains. See, e.g., Braathen, R., et al., J.Biol.Chem.277:42755-42762 (2002). Thus, the multimeric dimeric IgA derived binding molecules provided in the present disclosure typically include an IgA constant region comprising at least Cα3 and tail piece domains. Four IgA binding units can also form a tetrameric complex with the J chain. sIgA antibodies can also form higher order multimers, such as tetramers.

[0151] The IgA heavy chain constant region may further include a Cα2 domain or a fragment thereof, an IgA hinge region or a fragment thereof, a Cα1 domain or a fragment thereof and / or other IgA (or other immunoglobulins, such as IgG) heavy chain domains including, for example, an IgG hinge region. In certain embodiments, a binding molecule as provided herein may include a complete IgA heavy (α) chain constant domain (e.g., SEQ ID NO: 24 or SEQ ID NO: 25) or a variant, derivative or analog thereof. In some embodiments, the IgA heavy chain constant region or its multimeric fragment is a human IgA constant region.

[0152] In certain embodiments, each binding unit of the multimeric binding molecule as provided herein includes two IgA heavy chain constant regions or multimeric fragments or variants thereof, each IgA heavy chain constant region or multimeric fragments or variants thereof includes at least an IgA C α 3 domain and an IgA tail domain. In certain embodiments, the IgA heavy chain constant region can also each include an IgA C α 2 domain located at the N-terminus of the IgA C α 3 domain and the IgA tail domain. For example, the IgA heavy chain constant region can include amino acids 125 to 353 of SEQ ID NO: 24 or amino acids 113 to 340 of SEQ ID NO: 25. In certain embodiments, the IgA heavy chain constant region can also each include an IgA or IgG hinge region located at the N-terminus of the IgA C α 2 domain. For example, the IgA heavy chain constant region can include amino acids 102 to 353 of SEQ ID NO: 24 or amino acids 102 to 340 of SEQ ID NO: 25. In certain embodiments, the IgA heavy chain constant regions may each further include an IgA Cα1 domain located N-terminal to the IgA hinge region.

[0153] In some embodiments, each binding unit of an IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprises two light chains. In some embodiments, each binding unit of an IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprises two light chain fragments. In some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain. In some embodiments, the light chain is a chimeric kappa-lambda light chain. In some embodiments, each binding unit comprises two immunoglobulin light chains, each light chain comprising a VL located amino-terminal to an immunoglobulin light chain constant region.

[0154] Modified J chain and / or variant J chain

[0155] In certain embodiments, the multimeric binding molecules provided herein include J chain or its functional fragment or variant. In certain embodiments, the multimeric binding molecules provided herein are pentamers and include J chain or its functional fragment or variant. In certain embodiments, the multimeric binding molecules provided herein are dimeric IgA molecules or pentamer IgM molecules and include J chain or its functional fragment or variant. In some embodiments, the multimeric binding molecules may include naturally occurring J chain sequences, such as mature human J chain sequences (e.g., SEQ ID NO: 2). In some embodiments, the multimeric binding molecules may include naturally occurring J chain or a functional fragment of a variant J chain.

[0156] In certain embodiments, the J chain of pentamer IgM or IgM-like antibody or dimer IgA or IgA-like antibody as provided herein can be, for example, modified by introducing a heterologous part or two or more heterologous parts (for example, polypeptide), without interfering with the ability of IgM or IgM-like antibody or IgA or IgA-like antibody to assemble and combine its binding target.Referring to U.S. Patent number 9,951,134 and 10,618,978 and U.S. Patent Application Publication No. US-2019-0185570, the document is each incorporated herein by reference in its entirety.Therefore, as provided herein IgM or IgM-like antibody or IgA or IgA-like antibody, including bispecific or multispecific IgM or IgM-like antibody or IgA or IgA-like antibody as described elsewhere herein, modified J chain or its functional fragment or variant can be included, modified J chain or its functional fragment or variant also include heterologous part, such as heterologous polypeptide, it is introduced into J chain or its fragment or variant. In certain embodiments, the heterologous portion can be a peptide or polypeptide fused or chemically conjugated to a J chain or its fragment or variant frame. For example, a heterologous polypeptide can be fused to a J chain or its functional fragment or variant. In certain embodiments, the heterologous polypeptide is fused to a J chain or its functional fragment or variant via a linker (e.g., a peptide linker consisting of at least 5 amino acids but generally no more than 25 amino acids). In certain embodiments, the peptide linker is composed of: GGGGS (SEQ ID NO: 17), GGGGSGGGGS (SEQ ID NO: 18), GGGGSGGGGSGGGGS (SEQ ID NO: 19), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 21). In certain embodiments, the heterologous portion can be a chemical portion conjugated to a J chain. The heterologous moiety to be attached to the J chain can include, but is not limited to, a binding moiety, such as an antibody or antigen-binding fragment thereof, such as a single-chain Fv (scFv) molecule; a stabilizing peptide that can increase the half-life of an IgM or IgM-like antibody; or a chemical moiety such as a polymer or cytotoxin. In some embodiments, the heterologous moiety comprises a stabilizing peptide that can increase the half-life of a binding molecule (e.g., human serum albumin (HSA) or an HSA-binding molecule).

[0157] In some embodiments, the modified J chain includes an antigen-binding domain associated with the J chain, such as a polypeptide capable of specifically binding to a target antigen. In certain embodiments, the antigen-binding domain associated with the J chain can be an antibody or an antigen-binding fragment thereof, as described elsewhere herein. In certain embodiments, the antigen-binding domain associated with the J chain can be a single-chain Fv (scFv) antigen-binding domain or a single-chain antigen-binding domain derived from, for example, a camelid or cartilaginous fish antibody. The antigen-binding domain associated with the J chain can be introduced into the J chain at any position that allows the antigen-binding domain associated with the J chain to bind to its binding target without interfering with the function of the J chain or the function of the associated IgM or IgA antibody. The insertion position includes but is not limited to an internal position accessible to the three-dimensional structure of the C-terminus, the N-terminus, or the J-chain. In certain embodiments, the antigen-binding domain associated with the J chain can be introduced into the mature human J chain of SEQ ID NO: 2 between cysteine ​​residues 92 and 101 of SEQ ID NO: 2. In another embodiment, the antigen binding domain associated with the J chain can be introduced into the human J chain of SEQ ID NO: 2 at or near the glycosylation site. In another embodiment, the antigen binding domain associated with the J chain can be introduced into the human J chain of SEQ ID NO: 2 within about 10 amino acid residues from the C-terminus or within about 10 amino acid residues from the N-terminus. As described elsewhere herein, the present disclosure provides a multimeric bispecific binding molecule comprising a modified J chain, wherein the modified J chain comprises an antigen binding domain associated with the J chain that specifically binds to an immune effector cell (e.g., a T cell, such as a CD4+ T cell or a CD8+ cytotoxic T cell or a NK cell).

[0158] In some embodiments, the modified J chain may also include an immunostimulatory agent (ISA), such as a cytokine (e.g., interleukin-2 (IL-2) or interleukin-15 (IL-15) or a receptor-binding fragment or variant thereof), which in certain embodiments can associate with a portion of its receptor (e.g., the sushi domain of IL-15 receptor-α) by binding or covalent attachment. Such ISAs are described in detail in co-pending U.S. Provisional Application No. 62 / 887,458, which is incorporated herein by reference in its entirety.

[0159] In certain embodiments, the J chain of the IgM antibody, IgM-like antibody, IgA antibody, IgA-like antibody or IgM or IgA-derived binding molecule as provided herein is a variant J chain, which comprises one or more amino acid substitutions that can change the serum half-life of the IgM antibody, IgM-like antibody, IgA antibody, IgA-like antibody or IgM or IgA-derived binding molecule, for example, provided herein. For example, relative to a binding molecule derived from a reference IgM that is identical and administered to the same animal species in the same manner except for one or more single amino acid substitutions, deletions or insertions in the variant J chain, some amino acid substitutions, deletions or insertions can cause the IgM-derived binding molecule to show an increased serum half-life when administered to a test animal. In certain embodiments, the variant J chain can comprise 1, 2, 3 or 4 single amino acid substitutions, deletions or insertions relative to the reference J chain.

[0160] In some embodiments, the multimeric binding molecule may include a variant J chain sequence, such as a variant sequence described herein with reduced glycosylation or reduced binding to one or more polymer Ig receptors (e.g., pIgR, Fcα-μ receptor (FcαμR) or Fcμ receptor (FcμR)). See, for example, PCT Publication No. WO 2019 / 169314, which is incorporated herein by reference in its entirety. In certain embodiments, the variant J chain may include an amino acid substitution at an amino acid position corresponding to amino acid Y102 of a mature wild-type human J chain (SEQ ID NO: 2). "Amino acid corresponding to amino acid Y102 of a mature wild-type human J chain" means an amino acid in a J chain sequence of any species homologous to Y102 in a human J chain. See PCT Publication No. WO 2019 / 169314, which is incorporated herein by reference in its entirety. The position corresponding to Y102 in SEQ ID NO: 2 is conserved in the J chain amino acid sequences of at least 43 other species. Referring to Fig. 4 of U.S. Patent number 9,951,134, described document is incorporated herein by reference.With SEQ ID NO:2 Y102 corresponding position certain mutation can suppress certain immunoglobulin receptor (for example, people or mouse Fc α μ receptor, mouse Fc μ receptor and / or people or mouse polymer Ig receptor (pIg receptor)) and comprise the IgM pentamer of mutation J chain and combine.Compared to except replacement all identical and in the same manner to the corresponding antibody of identical species administration, antibody-like molecule or binding molecule, comprising and SEQ ID NO:2 Y102 corresponding amino acid place IgM antibody, IgM-like molecule and IgM derived binding molecule have improved serum half-life when animal is administered.In certain embodiments, with SEQ ID NO:2 Y102 corresponding amino acid can be replaced by any amino acid.In certain embodiments, with SEQ ID NO:2 Y102 corresponding amino acid can be replaced by alanine (A), serine (S) or arginine (R). In a specific embodiment, the amino acid corresponding to Y102 of SEQ ID NO: 2 may be substituted with alanine. In a specific embodiment, the J chain or a functional fragment or variant thereof is a variant human J chain and comprises the amino acid sequence of SEQ ID NO: 3 (referred to herein as "J*" J chain).

[0161] Wild-type J chain generally includes an N-linked glycosylation site.In certain embodiments, the variant J chain of the multimeric binding molecule as provided herein or its functional fragment include asparagine (N) link glycosylation motif N-X1-S / T (for example, starting with the amino acid position corresponding to amino acid 49 (N6 motif) of mature human J chain (SEQ ID NO:2) or J* (SEQ ID NO:3)), wherein N is asparagine, X1 is any amino acid except proline, and S / T is serine or threonine, and wherein mutation prevents the glycosylation at this motif. As shown in PCT Publication No. WO 2019 / 169314, preventing the glycosylated mutation at this site can result in relative to the reference multimeric binding molecule that is identical and administered to the same animal species in the same manner except for one or more mutations that prevent the glycosylation in the variant J chain, as provided herein, showing increased serum half-life when administered to the test animal.

[0162] For example, in certain embodiments, the variant J chain or its functional fragment of the binding molecule comprising a J chain as provided herein may include an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO:2 or SEQ ID NO:3, provided that the amino acid corresponding to S51 is not replaced by threonine (T), or wherein the variant J chain includes an amino acid substitution at the amino acid position corresponding to amino acid N49 and S51 of SEQ ID NO:2 or SEQ ID NO:3. In certain embodiments, the position corresponding to N49 of SEQ ID NO:2 or SEQ ID NO:3 is replaced by any amino acid (e.g., alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D)). In specific embodiments, the position corresponding to N49 of SEQ ID NO:2 or SEQ ID NO:3 may be replaced by alanine (A). In another specific embodiment, the position corresponding to N49 of SEQ ID NO:2 or SEQ ID NO:3 may be replaced by aspartic acid (D). In some embodiments, the position corresponding to S51 of SEQ ID NO: 2 or SEQ ID NO: 3 is substituted with alanine (A) or glycine (G). In some embodiments, the position corresponding to S51 of SEQ ID NO: 2 or SEQ ID NO: 3 is substituted with alanine (A).

[0163] Multimeric bispecific or multispecific anti-CD123 binding molecules with modified J chains that bind to immune effector cells.

[0164] The present disclosure provides a polymer, bispecific or multispecific binding molecule for treating cancer (e.g., hematological cancer, such as acute myeloid leukemia (AML)), wherein the binding molecule is bispecific and targets CD123 (IL-3R α) on cancer cells with high avidity, while also targeting immune effector cells (e.g., CD4+ or CD8+ T cells or NK cells) through a single antigen binding domain, thereby contributing to effector cell-mediated cancer cell killing while minimizing excessive release of cytokines. In certain aspects, the multimeric, bispecific anti-CD123 binding molecule is an anti-CD123 x anti-CD3 binding molecule.

[0165] Therefore, the present disclosure provides a kind of multimer, bispecific or multispecific binding molecule, it includes two IgA or IgA sample bivalent binding units or five IgM or IgM sample bivalent binding units and modified J chain, wherein modified J chain at least includes wild-type J chain or its functional fragment or variant and the antigen-binding domains associated with J chain of specific binding immune effector cell.Each binding unit includes two antibody heavy chains, and each heavy chain includes IgA, IgA sample, IgM or IgM sample heavy chain constant region or its polymeric fragment (as described elsewhere herein) and at least includes the heavy chain variable region (VH) part of the antigen-binding domains associated with binding unit.At least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all ten specific binding CD123 in the antigen-binding domains associated with binding unit.As provided herein, binding molecules can induce immune effector cell dependent cells (for example, cancer cells expressing CD123) to kill.

[0166] In certain embodiments, the modified J chains of the binding molecules provided herein include variants of wild-type J chains or fragments thereof, wherein relative to the wild-type J chain, the variant includes one or more single amino acid substitutions, deletions or insertions that can affect the serum half-life of the binding molecule; and wherein relative to a reference binding molecule that is identical except for one or more single amino acid substitutions, deletions or insertions in the J chain and is administered to the same animal species in the same manner, the binding molecule exhibits increased serum half-life when administered to the animal. For example, in certain embodiments, the J chain is a variant human J chain comprising the amino acid sequence SEQ ID NO: 3 ("J*").

[0167] In certain embodiments, the antigen-binding domains associated with J chain of the binding molecule provided include antibodies or their fragments. In certain embodiments, the antibody fragment is a single-chain Fv (scFv) fragment. scFv can be fused or chemically conjugated with J chain or its fragment or variant (e.g., J*). In certain embodiments, the scFv fragment is fused with J chain by a peptide linker (e.g., SEQ ID NO: 17 to 21). As noted elsewhere in the disclosure, the scFv fragment can be fused with J chain or its fragment or variant in any way, as long as the function of the J chain (i.e., assembled with IgM, IgM-like, IgA or IgA-like binding units to form dimers or pentamers) is not affected. For example, the scFv fragment can be fused with the N-terminal of J chain or its fragment or variant, fused with the C-terminal of J chain or its fragment or variant, or fused with both the N-terminal and C-terminal of J chain or its fragment or variant.

[0168] The immune effector cell combined by the antigen binding domains of modified J chain can be any immune effector cell that gives beneficial effect (for example, mediating the CD123+ cancer cell killing based on cell) when the cancer cell of CD123 targeting is associated.In certain embodiments, immune effector cell can be but is not limited to:T cell (for example, CD4+T cell, CD8+T cell, NKT cell or γ δ T cell), B cell, plasma cell, macrophage, dendritic cell or natural killer (NK) cell.In certain embodiments, immune effector cell is T cell, such as CD4+ or CD8+T cell.In certain embodiments, immune effector cell is CD8+ cytotoxic T cell.In certain embodiments, immune effector cell is NK cell.

[0169] In the case where the immune effector cell is a T cell (e.g., CD8+ T cell), the scFv fragment associated with the J chain can specifically bind to the T cell surface antigen CD3 (e.g., CD3ε). In certain embodiments, the anti-CD3εscFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises a VH complementary determining region VHCDR1, VHCDR2, and VHCDR3, which respectively comprise an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or an amino acid sequence having one, two, or three amino acids substituted in one or more of VHCDR: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and wherein VL comprises a VL complementary determining region VLCDR1, VLCDR2, and VLCDR3, which respectively comprise an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, or an amino acid sequence having one, two, or three amino acids substituted in one or more of VLCDR: 9, SEQ ID NO: 10, and SEQ ID NO: 11. In certain embodiments, the scFv fragment comprises the VH amino acid sequence of SEQ ID NO: 4 and the VL amino acid sequence of SEQ ID NO: 8. In other embodiments, the anti-CD3ε scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL comprise the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In specific embodiments, the modified J chain comprises an amino acid sequence comprising amino acids 20 to 420 of SEQ ID NO: 12, amino acids 20 to 412 of SEQ ID NO: 15, or amino acids 23 to 415 of SEQ ID NO: 16.

[0170] In certain other embodiments, the immune effector cell is a NK cell, and the scFv fragment can specifically bind to CD16 or CD56.

[0171] As provided herein, the modified J chain of the multimeric bispecific anti-CD123 binding molecules (for example, anti-CD123 x anti-CD3 binding molecules) can be further modified to include the other heterologous part attached to the J chain.Exemplary parts are described in, for example, U.S. Patent No. 9,951,134 and U.S. Patent Application Publication No. US2019-0185570 and U.S. Patent No. 10,618,978 and U.S. Provisional Application No. 62 / 887,458, and the documents are all incorporated herein by reference as a whole. In certain embodiments, as provided herein, the modified J chain of the multimeric bispecific anti-CD123 binding molecules (for example, anti-CD123x anti-CD3 binding molecules) can also include an immunostimulatory agent (" ISA ") fused to J chain or its fragment or variant or chemically conjugated.For example, ISA may include a cytokine or its receptor binding fragment or variant. In certain embodiments, an ISA associated with a J chain may include: (a) an interleukin-15 (IL-15) protein or a receptor-binding fragment or variant thereof ("I"); and (b) an interleukin-15 receptor-α (IL-15Rα) fragment ("R") capable of associating with I, comprising a sushi domain or a variant thereof; wherein at least one of I and R, or both I and R, are associated with the J chain or a fragment or variant thereof as a fusion protein, and wherein I and R can associate to function as an ISA. In certain embodiments, the ISA can be fused to the J chain via a peptide linker.

[0172] Antigen binding domain associated with anti-CD123 binding unit

[0173] In addition to two heavy chains, each binding unit of an anti-CD123 bispecific multimeric binding molecule (e.g., an anti-CD123 x anti-CD3 binding molecule) as provided herein can further comprise two light chains, wherein each light chain comprises a kappa or lambda light chain constant region (e.g., a human kappa or lambda light chain constant region) and comprises at least a light chain variable region (VL) portion of the antigen binding domain that associates with the binding unit.

[0174] In certain embodiments, the multimeric binding molecule provided is multispecific (e.g., bispecific, trispecific or tetraspecific), wherein two or more binding domains associated with the heavy chain constant region of the binding molecule are specifically bound to different targets. In certain embodiments, the binding domains of the multimeric binding molecule are all specifically bound to CD123. In certain embodiments, the binding domains of the multimeric binding molecule are identical. In such cases, for example, if the binding domains with different specificities are a part of a modified J chain as described elsewhere herein, the multimeric binding molecule can still be bispecific. In certain embodiments, the binding domain is an antibody-derived antigen-binding domain, for example, the VH subunit of the scFv associated with the heavy chain constant region or the antibody binding domain associated with the heavy chain constant region.

[0175] In addition, as provided herein, anti-CD123 bispecific multimeric binding molecules (e.g., anti-CD123 x anti-CD3 binding molecules) may include at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten antigen binding domains associated with a binding unit that specifically bind to CD123. In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all ten antigen binding domains associated with a binding unit are combined with the same CD123 epitope. In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all ten antigen binding domains associated with a binding unit are identical. In certain embodiments, the antigen binding domains associated with a binding unit are all identical.

[0176] In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten antigen binding domains associated with a binding unit of a provided binding molecule comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the six immunoglobulin complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise: the CDR amino acid sequences of an antibody comprising the VH and VL amino acid sequences, the VH and VL amino acid sequences comprising or contained in SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 SEQ ID NO: 78 and SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101, SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ IDNO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, or SEQ ID NO:102 and SEQ ID NO:103; or CDRs of an antibody comprising VH and VL amino acid sequences comprising or contained in the following, respectively, except for one or two amino acid substitutions in one or more of the CDRs: SEQ ID NO:32 and SEQ ID NO:33, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71 SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ IDNO: 101, SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 107 and SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116, or SEQ ID NO: 117 and SEQ ID NO: 118. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise CDR amino acid sequences of an antibody comprising VH and VL amino acid sequences comprising SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 113 and SEQ ID NO: 114, or SEQ ID NO: 111 and SEQ ID NO: 112, respectively, with 0, 1, or 2 amino acid substitutions. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise CDR amino acid sequences of an antibody comprising VH and VL amino acid sequences comprising SEQ ID NO: 113 and SEQ ID NO: 114, respectively, with 0, 1, or 2 amino acid substitutions (such as 0 amino acid substitutions).

[0177] In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten antigen binding domains associated with a binding unit of a provided binding molecule comprise an antibody VH and VL, wherein the VH and VL comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the mature VH and VL amino acid sequences, respectively, comprising or contained in SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71 SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101, SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115 NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ IDNO:102 and SEQ IDNO:103, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116, or SEQ ID NO:117 and SEQ ID NO:118.

[0178] In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten antigen binding domains associated with a binding unit of a provided binding molecule comprise antibody VH and VL, wherein VH and VL comprise amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the mature VH and VL amino acid sequences comprising SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 113 and SEQ ID NO: 114, or SEQ ID NO: 111 and SEQ ID NO: 112, respectively. In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten antigen binding domains associated with a binding unit of a provided binding molecule comprise antibody VH and VL, wherein VH and VL comprise amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical (such as 100% identical) to the mature VH and VL amino acid sequences comprising SEQ ID NO: 113 and SEQ ID NO: 114, respectively.

[0179] In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten antigen binding domains associated with the binding units of the provided binding molecules comprise antibody VH and VL, wherein VH and VL comprise the amino acid sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively. In certain embodiments, the provided binding molecules are IgM antibodies and each binding unit comprises two IgM heavy chains and two kappa light chains, the heavy chain comprising amino acids 20 to 592 of SEQ ID NO: 35 and the light chain comprising amino acids 21 to 240 of SEQ ID NO: 36.

[0180] In certain embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten antigen binding domains associated with the binding units of the provided binding molecules comprise antibody VH and VL, wherein VH and VL comprise the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively. In certain embodiments, the provided binding molecules are IgM antibodies and each binding unit comprises two IgM heavy chains and two kappa light chains, the heavy chain comprising amino acids 20 to 589 of SEQ ID NO: 40 and the light chain comprising amino acids 21 to 234 of SEQ ID NO: 41.

[0181] Polynucleotides, vectors and host cells

[0182] The present disclosure also provides a polynucleotide (e.g., isolated, recombinant and / or non-naturally occurring polynucleotide), which includes a nucleic acid sequence encoding a polypeptide subunit of an anti-CD123 multimeric bispecific binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) as provided herein." Polypeptide subunit" means a part that can be independently translated in a binding molecule, a binding unit, an IgM antibody, an IgM-like antibody, an IgA antibody or an IgA-like antibody, a J chain, a modified J chain or an antigen-binding domain. Examples include, but are not limited to: antibody variable domains, e.g., VH or VL; J chain, including a modified J chain as provided herein; secretory component; single-chain Fv; antibody heavy chain; antibody light chain; antibody heavy chain constant region; antibody light chain constant region; and / or any fragment, variant or derivative thereof.

[0183] In certain embodiments, the polypeptide subunit may include an IgM heavy chain constant region or an IgM-like heavy chain constant region or a multimeric fragment thereof, or an IgA heavy chain constant region or an IgA-like heavy chain constant region or a multimeric fragment thereof, which can be fused to an antigen binding domain or a subunit thereof, for example, to the VH portion of an antigen binding domain or the VL portion of an antigen binding domain, each of which is as provided herein. In certain embodiments, the polynucleotide may encode a polypeptide subunit comprising a human IgM heavy chain constant region, a human IgM-like heavy chain constant region, a human IgA heavy chain constant region, a human IgA-like heavy chain constant region, or a multimeric fragment thereof (e.g., SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25), any of which can be fused to an antigen binding domain or a subunit thereof (e.g., the C-terminus of a VH).

[0184] In certain embodiments, a VH may comprise HCDR1, HCDR2, and HCDR3 regions comprising a CDR amino acid sequence contained within a VH amino acid sequence comprising or contained within SEQ ID NO: 32, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100 NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102; SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115 or SEQ ID NO:117; or a CDR amino acid sequence contained in a VH amino acid sequence comprising or contained in,SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120 NO: 102, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. In certain embodiments,The VH may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a mature VH amino acid sequence comprising or contained in SEQ ID NO: 32, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94 NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115 or SEQ ID NO:117. ,

[0185] In certain embodiments, the polypeptide subunit may include an antibody VL portion of an antigen binding domain as described elsewhere herein. In certain embodiments, the polypeptide subunit may include an antibody light chain constant region (e.g., a human antibody light chain constant region) or a fragment thereof, which may be fused to the C-terminus of the VL.

[0186] In certain embodiments, the VL may include LCDR1, LCDR2, and LCDR3 regions, and the LCDR1, LCDR2, and LCDR3 regions include: a CDR amino acid sequence contained in a VL amino acid sequence comprising or contained in SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 or SEQ ID NO:118; or a CDR amino acid sequence contained in a VL amino acid sequence comprising or contained in,SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120 NO: 103, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, or SEQ ID NO: 118. In certain embodiments,The VH may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a mature VL amino acid sequence comprising or contained in SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 or SEQ ID NO:118. ,

[0187] In certain embodiments, the polypeptide subunit can be a modified J chain as described elsewhere herein. For example, the polypeptide subunit can include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids 20 to 420 of SEQ ID NO: 12, amino acids 20 to 412 of SEQ ID NO: 15, or amino acids 23 to 415 of SEQ ID NO: 16.

[0188] In certain embodiments, the disclosure provides a kind of compositions, it comprises two, three or more polynucleotides as provided herein, wherein polynucleotides can encode together as provided herein polymer bispecific anti-CD123 binding molecules (for example, anti-CD123x anti-CD3 binding molecules).In certain embodiments, polynucleotides can be located on a separate carrier.In certain embodiments, two or more in the polynucleotides can be located on the same carrier.Such carrier is provided by the disclosure equally.

[0189] In certain embodiments, the polynucleotides as provided herein are located on an expression vector such as a plasmid, and may include a nucleic acid sequence encoding a polypeptide subunit (e.g., IgM heavy chain or IgM-like heavy chain, IgA heavy chain or IgA-like heavy chain, light chain or J chain (e.g., modified J chain)), or may include two or more nucleic acid sequences encoding two or more or all three polypeptide subunits of a binding molecule as provided herein. Alternatively, the nucleic acid sequence encoding the three polypeptide subunits may be on a separate polynucleotide (e.g., a separate expression vector). The present disclosure provides such a single or multiple expression vectors. The present disclosure also provides one or more host cells encoding the polynucleotides or expression vectors provided.

[0190] The present disclosure also provides a host cell (e.g., a prokaryotic or eukaryotic host cell) comprising: a polynucleotide, or two or more polynucleotides encoding a multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) or any subunit thereof as provided herein; a polynucleotide composition as provided herein; or a vector, or two, three or more vectors that collectively encode a binding molecule as provided herein or any subunit thereof.

[0191] In a related embodiment, the present disclosure provides a method of producing a multimeric binding molecule as provided herein, wherein the method comprises culturing a host cell as provided herein and recovering the multimeric binding molecule.

[0192] How to use

[0193] The present disclosure also provides a method for treating a disease or disorder (e.g., cancer or other malignancies, e.g., hematological cancers or malignancies) of a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) as provided herein. "Therapeutically effective dose or amount" or "effective amount" means the amount of a binding molecule that produces a positive response (e.g., tumor cell killing) in a subject when administered.

[0194] In certain embodiments, the cancer to be treated can be any cancer in which malignant cells express or overexpress CD123. For example, cancer can be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia (B cell ALL), classical Hodgkin lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), systemic mastocytosis or plasmacytoid dendritic cell leukemia.

[0195] The effective dosage of a composition for treating cancer varies depending on many different factors, including the route of administration, the target site, the physiological state of the subject, whether the subject is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the subject is human, but non-human mammals, including transgenic mammals, can also be treated. Conventional methods known to those skilled in the art can be used to titrate the therapeutic dose to optimize safety and efficacy.

[0196] The subject to be treated can be any animal (eg, a mammal) in need of treatment, and in certain embodiments, the subject is a human subject.

[0197] In its simplest form, the formulation to be administered to a subject is a multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) or multimeric antigen-binding fragment thereof as provided herein, administered in a conventional dosage form, which can be combined with a pharmaceutical excipient, carrier, or diluent as described elsewhere herein.

[0198] The compositions of the present disclosure can be administered by any suitable method, for example, parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0199] Pharmaceutical compositions and methods of administration

[0200] According to the present disclosure, those skilled in the art are well aware of or easily determine the preparation of multimeric bispecific anti-CD123 binding molecules (e.g., anti-CD123 x anti-CD3 binding molecules) as provided herein and the method of administering them to a subject in need thereof. The route of administration can be, for example, intratumoral, oral, parenteral, by inhalation or local. The term "parenteral" as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Although these forms of administration are envisioned as suitable forms, another example of form of administration will be a solution for injection, especially for intratumoral, intravenous or intraarterial injection or infusion. Suitable pharmaceutical compositions can include buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbate), optionally stabilizers (e.g., human albumin), etc.

[0201] As discussed herein, the multimeric bispecific anti-CD123 binding molecules (e.g., anti-CD123 x anti-CD3 binding molecules) as provided herein can be administered in a pharmaceutically effective amount for the treatment of subjects in need thereof. In this regard, it will be appreciated that the disclosed multimeric bispecific anti-CD123 binding molecules (e.g., anti-CD123 x anti-CD3 binding molecules) can be formulated to facilitate administration and promote the stability of the active agent. Therefore, the pharmaceutical composition may include a pharmaceutically acceptable non-toxic sterile carrier (such as normal saline), a non-toxic buffer, a preservative, etc. The pharmaceutically effective amount of the multimeric binding molecules comprising ISA as provided herein means an amount sufficient to achieve effective binding to the target and achieve therapeutic benefit. Suitable formulations are described in Remington's Pharmaceutical Sciences, e.g., the 21st edition (Lippincott Williams & Wilkins) (2005).

[0202] Certain pharmaceutical compositions provided herein can be administered orally in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions, or solutions. Certain pharmaceutical compositions can also be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, adsorption promoters to enhance bioavailability, and / or other conventional stabilizers or dispersants.

[0203] The amount of the multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) that can be combined with a carrier material to produce a single dosage form will vary depending on, for example, the subject being treated and the specific mode of administration. The composition can be administered as a single dose, multiple doses, or in the form of an infusion over an established time period. The dosage regimen can also be adjusted to provide the optimal desired response (e.g., a therapeutic response or a preventive response).

[0204] Consistent with the scope of the present disclosure, a multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) as provided herein can be administered to a subject in need of treatment in an amount sufficient to produce a therapeutic effect. A multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) as provided herein can be administered to a subject in conventional dosage forms, which are prepared by combining an antibody of the present disclosure or its multimeric antigen-binding fragment, variant or derivative with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. The form and characteristics of a pharmaceutically acceptable carrier or diluent can be determined by the amount of the active ingredient combined therewith, route of administration and other well-known variables.

[0205] The present disclosure also provides the use of a multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) as provided herein in the manufacture of a medicament for treating, preventing, or controlling cancer or other malignancies. The present disclosure also provides a multimeric bispecific anti-CD123 binding molecule (e.g., anti-CD123 x anti-CD3 binding molecule) as provided herein for use in treating, preventing, or controlling cancer.

[0206] Unless otherwise indicated, the present disclosure employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are fully explained in the literature. See, e.g., Green and Sambrook, eds. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DN Glover and BD Hames, eds. (1995) DNA Cloning, 2nd ed. (IRL Press), Vols. 1-4; Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press); Mullis et al., U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1985) Nucleic Acid Hybridization (IRL Press); Hames and Higgins, eds. (1984) Transcription And Translation (IRL Press); Freshney (2016) Culture Of Animal Cells, 7th ed. (Wiley-Blackwell); Woodward, J., Immobilized Cells and Enzymes (IRL Press) (1985); Perbal (1988) A Practical Guide To Molecular Cloning; 2nd Edition (Wiley-Interscience); Miller and Calos (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); SCMakrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science); Methods in Enzymology, Volume 151-155 (Academic Press, Inc., NY); Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Weir and Blackwell, eds.; and Ausubel et al. (1995) Current Protocols in Molecular Biology (John Wiley and Sons).

[0207] The general principles of antibody engineering are set forth in, for example, Strohl, WR and LM Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). The general principles of protein engineering are set forth in, for example, Park and Cochran (eds.) (2009), Protein Engineering and Design (CDC Press). The general principles of immunology are set forth in, for example, Abbas and Lichtman (2017) Cellular and Molecular Immunology, 9th edition (Elsevier). In addition, standard immunological methods known in the art, such as those in Current Protocols in Immunology (Wiley Online Library); Wild, D. (2013), The Immunoassay Handbook 4th Edition (Elsevier Science); Greenfield, ed. (2013), Antibodies, a Laboratory Manual, 2d Edition (Cold Spring Harbor Press); and Ossipow and Fischer, eds. (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press), can be followed.

[0208] All references cited above and all references cited herein are incorporated by reference in their entirety.

[0209] The following examples are offered by way of illustration and not limitation.

[0210] Example

[0211] Example 1: Antibody Generation and Purification

[0212] Anti-CD123 X CD3 IgM #1 and #2 and anti-CD123 IgG #1 and #2

[0213] As exemplary constructs, the VH and VL regions of four anti-CD123 antibodies were incorporated into IgM (with SJ* chains, SEQ ID NO:12 amino acids 20 to 420, forming bispecific IgM antibodies) and IgG forms according to standard cloning procedures. Anti-CD123#1 constructs include VH and VL amino acid sequences SEQ ID NO:32 and SEQ ID NO:33, respectively, and anti-CD123#2 constructs include VH and VL amino acid sequences SEQ ID NO:37 and SEQ ID NO:38, respectively, and anti-CD123#3 constructs include VH and VL amino acid sequences SEQ ID NO:102 and SEQ ID NO:103, respectively, and anti-CD123#4 constructs include VH and VL amino acid sequences SEQ ID NO:107 and SEQ ID NO:108, respectively. These antibody constructs were expressed and purified as described below. IgM bispecific antibodies (plus modified J chain-SJ*) were resolved on reducing and non-reducing gels as follows. Figure 1A An exemplary non-reducing gel resolving high molecular weight IgM is shown, and Figure 1B An exemplary reducing gel showing IgM heavy and light chains is shown. For non-reducing gels, samples were mixed with NuPage LDS sample buffer (Life Technologies#NP0007) and loaded onto NativePage Novex 3 to 12% Bis-Tris gels (Life Technologies#BN1003). Gel electrophoresis was performed using Novex Tris-Acetate SDS running buffer (Life Technologies#LA0041), and the gel was stained with Colloidal Blue Stain (Life Technologies#LC6025). For reducing gels, samples were mixed with sample buffer and NuPage reducing agent (Life Technologies#NP0004) and heated to 80°C for 10 minutes, and loaded onto NuPage Novex 4 to 12% Bis-Tris gels (Life Technologies#NP0322). Gel electrophoresis was performed using NuPage MESSDS running buffer (Life Technologies#NP0002), and the gel was stained with Colloidal Blue.

[0214] Additional anti-CD123 x CD3 and anti-CD123 IgG constructs

[0215] Anti-CD123 x anti-CD3 BiTE constructs are described in PCT application publication number WO 2017 / 210443A1. Construct includes: a first heavy chain comprising an anti-CD123 VH sequence (VH=SEQ ID NO:32; heavy chain=SEQ ID NO:104); a light chain comprising an anti-CD123 VL sequence (VL=SEQ ID NO:33, light chain=SEQ ID NO:105); and a second heavy chain comprising an anti-CD3 scFv (heavy chain=SEQ ID NO:106) fused to an IgG heavy chain constant region. This construct is synthesized, expressed, and purified by commercial suppliers (Creative Biolabs and ATUM) and is herein named anti-CD123xCD3 IgG#1. The purified protein was purified by reduction and non-reduction gel ( Figure 2A ) distinguish, through Figure 2B Proteins were resolved by size exclusion chromatography as shown in .

[0216] Protein expression and purification

[0217] Transfection. DNA for the heavy chain, light chain, and modified J chain (SJ*) (for IgM pentamer constructs) are transfected into, for example, CHO cells or Expi 293 cells. DNA for the expression vector is mixed with a polyethylamine (PEI) reagent (ExpiFectamine TM 293 transfection kit) was mixed and then added to the cells. PEI transfection using CHO-S or 293expi cells was performed according to established techniques (see "Biotechnology and Bioengineering, Vol. 87, 553-545").

[0218] IgG expression products were expressed and purified by commercial suppliers.

[0219] According to the manufacturer's recommendations, use, for example, Capto Core 400 (GE life science) and POROS TM 50HQ strong anion exchange resin (Thermo Fisher) was used to purify the IgM expression product. For the IgM expression product, the protein peak was resolved by size exclusion chromatography, such as Figure 1C and Figure 1D shown.

[0220] Example 2: Antibody specificity measured by ELISA

[0221] Anti-CD123xCD3 IgM#1 and anti-CD123xCD3 IgM#2 specificity to human CD123 and CD3ε, and control anti-CD123 IgG#1 and anti-CD123 IgG#2 specificity to CD123 and bispecific anti-CD123 x CD3 IgG#1 specificity to CD3ε were measured as follows in ELISA assays. At 4°C, 96-well white polystyrene ELISA plates (Pierce 15042) were coated overnight with 100 μL 0.5 μg / mL of recombinant human CD123 protein (Sino Biological 10518-H08H-50) or recombinant human CD3ε protein (Acro Biosystems, CDE-H5256-100) per well. The plate was then washed 5 times with 0.05% PBS-Tween and blocked with 2% BSA-PBS. After blocking, the CD123 IgM or IgG serial dilutions, standards and controls of 100 μL are added to the wells and incubated at room temperature for 2 hours.Then, the plate is washed 10 times and the plate is incubated for 30 minutes with mouse anti-human κ (Southern Biotech, 9230-05, 1:6000 diluted in 2% BSA-PBS) conjugated to HRP. After 10 final washes using 0.05% PBS-Tween, SuperSignal chemiluminescent substrate (ThermoFisher, 37070) is used to read the plate. Luminescent data are collected on EnVision plate reader (Perkin-Elmer) and analyzed using GraphPadPrism with a 4-parameter logistic model. Figure 3 Binding of IgM bispecific antibody to CD123 is shown in FIG, and Figures 4A-4B Binding of IgM and IgG bispecific antibodies to CD3ε is shown in .

[0222] To compare the binding of IgM and IgG bispecific antibodies to CD123 at different protein concentrations, 384-well white plates were coated with 25 μl of different CD123 protein concentrations (3 μg / ml, 1 μg / ml, 0.33 μg / ml, and 0.11 μg / ml) at 37°C for one hour. The plates were washed and blocked for 15 minutes using blocking buffer Starting Block T20 (Thermo, 37539). Serial dilutions of 25 μl CD123 X CD3 IgM or IgG#1 were added to the plates and incubated at 37°C for 30 minutes, washed 10 times, and bound IgM or IgG (Abcam, ab202549) was detected using a secondary antibody conjugated to HRP, anti-human κEPR5367-8. The results were shown in Table 1. Figures 5A-5D The IgM antibody showed excellent binding at all concentrations.

[0223] Example 3: Binding to AML cell lines

[0224] AML cell lines - CD123 surface quantification

[0225] AML cell lines were purchased from ATCC or DSMZ (MV4-11, THP-1, Namalwa, KG-1a, Molm-13, JM-1, REH, K562, HL-60, and Oci-Ly9) and cultured in appropriate culture media according to the vendor's recommendations. Commercial anti-CD123 antibodies conjugated to PE (Biolegend, Clone 6H6, 306006) and Quantum TM R-PE MESF beads (Bangs Laboratories, 827) were used to quantify CD123 surface expression. Figure 6 MV4-11, Molm-3, Thp-1, KG-1a, and JM-1 cells expressed detectable levels of CD123.

[0226] AML cell line IgM and IgG binding assays

[0227] To evaluate the ability of IgG and IgM antibodies to bind to CD123 on AML cells expressing CD123 protein, a binding assay was performed as follows. Cells were washed with FACS staining buffer (BD Pharmigen catalog number 554656) and pre-incubated with Fc Block (BD, 564220) for 10 minutes at room temperature. At 4°C, 1x10 5 Each cell was stained with 1 μg anti-CD123 antibody, 1 μg / mL IgG isotype control (Jackson ImmunoResearch #009-000-003) or 1 μg / mL IgM isotype control (Jackson ImmunoResearch #009-000-012) for 30 minutes. The cells were washed twice and then stained with 5 μg / mL anti-human κ-AF488 secondary antibody (Biolegend #316512) at 4°C for 30 minutes. The cells were washed twice, resuspended in FACS staining buffer, and acquired by flow cytometry. The results were shown in Figure 2. Figure 7 Shown in.

[0228] Example 4: T cell-directed killing of AML cells

[0229] To demonstrate that the bispecific CD123 x CD3 IgM binding molecule can kill target cells in the presence of human T cells, a co-culture experiment was performed. 5x10 β-cells were co-cultured in a 96-well round-bottom tissue culture plate in the presence of serial dilutions of anti-CD123xCD3 IgM#1 in a total volume of 100 μL per well of AIM-V medium supplemented with 3% heat-inactivated fetal bovine serum (FBS). 3 Tumor cells MV4-11, THP-1, and Namalwa (all expressing firefly luciferase) were co-cultured with T cells at different effector-target ratios (E:T). After incubation for 72 or 96 hours at 37°C in a 5% CO2 incubator, 50 μl of supernatant was removed and frozen at -80°C for later cytokine release analysis. 50 μl of luciferase substrate (e.g., ONE-GloEX luciferase assay system, Promega) was added to the wells. The plate was shaken briefly to mix the reagents, and the luciferase luminescence signal was measured on an EnVision plate reader (Perkin-Elmer). The data was then analyzed with GraphPad Prism to determine the EC 50 . Figures 8A-8C Representative dose-response curves are shown in . THV-1 cells (EC50: 11.12 pM) and MV4-11 cells (EC50: 13.63 pM) expressing detectable levels of CD123 were effectively killed, whereas Namalwa cells that did not express CD123 were not killed.

[0230] Example 5: Anti-CD123xCD3 IgM#1 activates CD8+ T cells but not CD4+ T cells

[0231] The ability of anti-CD123 x CD3 IgM #1 to enhance T cell activation was evaluated as follows. Human pan T cells were isolated from PBMC using the MACS pan T cell isolation kit according to the manufacturer's instructions. T cells were then labeled with cell tracer purple dye (Thermo, C34557). 10x10 cells were plated per well in the presence of 2.5 μg / ml anti-CD123 x CD3 IgM #1 or 1 μg / ml anti-CD3 monoclonal antibody (SP34ebioscience, Thermo 16-0037-85). 3 MV4-11 cells and 40x10 3Individual Pan T cells were co-cultured for 72 hours. Cells were stained for FACS analysis using the following staining kit: anti-CD8 BV510, anti-CD25 APC, and anti-CD4 BV785 from Biolegend, and fixable viability dye LIVE-OR-DYE 750 / 788 (Biotium, 32008). Figure 9 Anti-CD123xCD3IgM#1 enhanced the CD25 activation marker on CD8+ T cells, but not on CD4+ T cells.

[0232] Example 6: Cytokine Release

[0233] Supernatants from the T cell-directed cytotoxicity assay performed in Example 4 were collected at time points where 0%, 20%, 50% and 95% of cells were killed. Figure 10A and 10B Comparison of anti-CD123xCD3 IgM#1 (triangles) and anti-CD123xCD3 IgG#1 (open circles) in pan-TDCC assays of MV4-11 cells (panel A) and THP-1 cells (panel B) at the indicated points on the curves. Open circles: anti-CD123xCD3 IgG#1, filled triangles: anti-CD123xCD3 IgM#1. Samples were collected at the indicated killing levels.

[0234] Supernatants from the T cell-directed cytotoxicity assay performed in Example 4 were collected as indicated and assayed for a cytokine panel including IFNγ, IL-4, TNF, IL-10, and IL-6 in the supernatants using the V-PLEX Proinflammatory Panel (human) (MSD, K15049D-2) according to the manufacturer's protocol. The results were then analyzed using GraphPad Prism. Figures 11A-11D The results for MV4-11 cells on day 4 are shown, and Figures 12A-12D Results are shown for THP cells at day 4. Even with 95% cell killing, the IgM construct resulted in minimal cytokine release, whereas the IgG construct resulted in high levels of cytokine release.

[0235] Example 7: Additional Antibody Generation and Purification

[0236] Additional exemplary antibodies, as indicated in Table 2, were generated and purified as described in Example 1. The antibodies assembled into pentamers with the J chain (data not shown).

[0237] Table 2: Antibodies generated

[0238]

[0239]

[0240] Example 8: Antibody specificity measured by ELISA

[0241] Binding of an additional subset of anti-CD123xCD3 IgM antibodies to CD123 at varying protein concentrations was measured in an ELISA assay as described in Example 2. Figure 13 The data were then analyzed using GraphPad Prism to determine the EC 50 , and the results are shown in Table 3.

[0242] Table 3: Antibody Binding IC 50

[0243] Antibody <![CDATA[EC 50 (pM)]]> IGM#FbJ*-H1 529.7 IGM#BbJ*-H1 298.0 IGM#CbJ*-H1 354.4 IGM#AbJ*-H1 198.0

[0244] Example 9: MV4-11-IgM and IgG binding assay

[0245] To evaluate the ability of a subset of additional anti-CD123xCD3 IgM antibodies to bind to CD123 on MV4-11 cells expressing CD123 protein, binding assays were performed on various concentrations of the antibodies by the method described in Example 3 under the heading "AML cell line IgM and IgG binding assays". Figure 14 Shown in.

[0246] Example 10: T cell-directed AML cell killing—different CD123 binding domains

[0247] To demonstrate that bispecific CD123 x CD3 IgM binding molecules comprising different CD123 binding domains can kill target cells in the presence of human T cells, a co-culture experiment was performed using four exemplary anti-CD123 x CD3 IgM binding molecules (IGM#FbJ*-H1, IGM#BbJ*-H1, IGM#CbJ*-H1, and IGM#AbJ*-H1), each comprising a different CD123 binding domain. 5x10 T cells were co-cultured in the presence of serial dilutions of the antibodies in a 96-well round-bottom tissue culture plate in a total volume of 100 μL per well in AIM-V medium supplemented with 3% heat-inactivated fetal bovine serum (FBS). 3Tumor cells MV4-11, THP-1 and PL-21 (all expressing firefly luciferase) were co-cultured with T cells (strong donor or weak donor T cells) at an effector-target ratio (E:T) of 7:1. After incubation for 72 or 96 hours at 37°C in a 5% CO2 incubator, 50 μl of luciferase substrate (e.g., ONE-Glo EX luciferase assay system, Promega) was added to the wells. The plate was shaken briefly to mix the reagents and the luciferase luminescence signal was measured on an EnVision plate reader (Perkin-Elmer). The data were then analyzed with GraphPad Prism to determine EC 50 . Figure 15A and 15B The results of co-cultures of strong donor T cells and THP1 or PL21 are shown in Table 4, and the EC values ​​of all co-cultures are shown in Table 5. 50 value.

[0248] Table 4: T cell-directed EC killing 50 (pM)

[0249]

[0250] Example 11: T cell-directed killing of AML cells - different modified J chains

[0251] To demonstrate that bispecific CD123 x CD3 IgM binding molecules comprising different CD3 binding domains can kill target cells in the presence of human T cells, a co-culture experiment was performed using four exemplary anti-CD123 x CD3 IgM binding molecules (IGM#AcJ*-H1, IGM#AdJ*-H1, IGM#AeJ*-H1, and IGM#AbJ*-H1), each comprising a different CD123 binding domain. 5x10 50 μL of bispecific CD123 x CD3 IgM binding molecules were co-cultured in the presence of serial dilutions of the antibodies in a 96-well round-bottom tissue culture plate in a total volume of 100 μL per well in AIM-V medium supplemented with 3% heat-inactivated fetal bovine serum (FBS). 3 Tumor cells MV4-11, THP-1 and PL-21 (all expressing firefly luciferase) were co-cultured with T cells (strong donor or weak donor T cells) at an effector-target ratio (E:T) of 7:1. After incubation for 72 or 96 hours at 37°C in a 5% CO2 incubator, 50 μl of luciferase substrate (e.g., ONE-Glo EX luciferase assay system, Promega) was added to the wells. The plate was shaken briefly to mix the reagents and the luciferase luminescence signal was measured on an EnVision plate reader (Perkin-Elmer). The data were then analyzed with GraphPad Prism to determine EC 50 . Figure 16A and16B The results of co-cultures of strong donor T cells and THP1 or PL21 are shown in Table 5, and the EC values ​​of all co-cultures are shown in Table 5. 50 value.

[0252] Table 5: T cell-directed EC killing 50 (pM)

[0253]

[0254] To compare other CD3 binding domains and J* compared to J-HSA, the assay was repeated essentially as described above. MV4-11 cells were co-cultured with strong donor cells at an E:T ratio of 3:1 in the presence of IGM#AbJ*-H1, IGM#Af-JH-H1, IGM#AfJ*-H1, or IGM#AaJ*-H1. Figure 17 Shown in.

[0255] Example 12: CD4+ and CD8+ T cell-guided AML cell killing

[0256] The ability of an additional subset of anti-CD123 x CD3 IgM antibodies to enhance T cell activation was assessed as described in Example 5. Figures 18A-18F Anti-CD123 x CD3 IgM potent tumor mediated CD8+ T cell cytotoxicity and T cell proliferation, but not CD4+ T cell cytotoxicity and T cell proliferation.

[0257] Example 13: Cytokine Release

[0258] To determine the amount of various cytokines released upon exposure to the bispecific CD123 x CD3 IgM binding molecules, co-culture experiments were performed using three exemplary anti-CD123 x CD3 IgM binding molecules (IGM#BbJ*-H1, IGM#AcJ*-H1, or IGM#AbJ*-H1) or anti-CD123xCD3 IgG#1. 5x10 IgG were co-cultured in 96-well round-bottom tissue culture plates in a total volume of 100 μL per well in AIM-V medium supplemented with 3% heat-inactivated fetal bovine serum (FBS) in the presence of 50 pM or 1 nM antibody. 3 MV4-11 tumor cells expressing firefly luciferase were co-cultured with T cells at an effector-to-target ratio (E:T) of 7:1. After incubation at 37°C in a 5% CO2 incubator for 72 or 96 hours, 50 μl of supernatant was removed from the co-culture when 100% of the tumor cells had been killed and frozen at -80°C until analysis.

[0259] The supernatant was assayed for cytokine levels including IFNγ, TNFα, IL-6, IL-10, and IL-2 using the V-PLEX Proinflammatory Panel (Human) (MSD, K15049D-2) according to the manufacturer's protocol. The results were then analyzed using GraphPad Prism. Figures 19A-19E and are shown in Table 6. For all cytokines assayed, IgM antibodies resulted in less cytokine release at both antibody concentrations.

[0260] Table 6: Released cytokine concentrations (pg / mL)

[0261]

[0262] Table 7: Sequences presented in this disclosure

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279] Sequence Listing <110> IGM BIOSCIENCES, INC. <120> Multimeric bispecific anti-CD123 binding molecules and their uses <130> 001-027WO1 <140> <141> <150> 62 / 888,702 <151> 2019-08-19 <150> 62 / 888,475 <151> 2019-08-17 <160> 146 <170> PatentIn version 3.5 <210> 1 <211> 159 <212> PRT <213> Homo sapiens <400> 1 Met Lys Asn His Leu Leu Phe Trp Gly Val Leu Ala Val Phe Ile Lys 1 5 10 15 Ala Val His Val Lys Ala Gln Glu Asp Glu Arg Ile Val Leu Val Asp 20 25 30 Asn Lys Cys Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser 35 40 45 Glu Asp Pro Asn Glu Asp Ile Val Glu Arg Asn Ile Arg Ile Ile Val 50 55 60 Pro Leu Asn Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg 65 70 75 80 Thr Arg Phe Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro 85 90 95 Thr Glu Val Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn 100 105 110 Ile Cys Asp Glu Asp Ser Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg 115 120 125 Asn Lys Cys Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr 130 135 140 Lys Met Val Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp 145 150 155 <210> 2 <211> 137 <212> PRT <213> Homo sapiens <400> 2 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 1 5 10 15 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 20 25 30 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 35 40 45 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 50 55 60 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 65 70 75 80 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 85 90 95 Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 100 105 110 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 115 120 125 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 130 135 <210> 3 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 3 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 1 5 10 15 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 20 25 30 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 35 40 45 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 50 55 60 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 65 70 75 80 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 85 90 95 Ala Thr Glu Thr Cys Ala Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 100 105 110 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 115 120 125 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 130 135 <210> 4 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Thr Tyr Ala Met Asn 1 5 <210> 6 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 6 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 7 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 8 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 8 Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 9 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 10 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 11 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 12 <211> 420 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 12 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95 Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val 115 120 125 Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 145 150 155 160 Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr 165 170 175 Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn 180 185 190 Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu 195 200 205 Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser 210 215 220 Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln 225 230 235 240 Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu 245 250 255 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly 260 265 270 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Glu Asp Glu Arg 275 280 285 Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala Arg Ile Thr Ser Arg 290 295 300 Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp Ile Val Glu Arg Asn 305 310 315 320 Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu Asn Ile Ser Asp Pro 325 330 335 Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His Leu Ser Asp Leu Cys 340 345 350 Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp Asn Gln Ile Val Thr 355 360 365 Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser Ala Thr Glu Thr Cys 370 375 380 Ala Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala Val Val Pro Leu Val 385 390 395 400 Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala Leu Thr Pro Asp Ala 405 410 415 Cys Tyr Pro Asp 420 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <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 Tyr Thr Phe Ile Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 412 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 15 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Ile Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn 65 70 75 80 Gln Lys Leu Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys 180 185 190 Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala 195 200 205 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 210 215 220 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 225 230 235 240 Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys 245 250 255 Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 260 265 270 Gly Gly Ser Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys 275 280 285 Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro 290 295 300 Asn Glu Asp Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn 305 310 315 320 Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe 325 330 335 Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val 340 345 350 Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp 355 360 365 Glu Asp Ser Ala Thr Glu Thr Cys Ala Thr Tyr Asp Arg Asn Lys Cys 370 375 380 Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val 385 390 395 400 Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp 405 410 <210> 16 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 16 Met Lys Asn His Leu Leu Phe Trp Gly Val Leu Ala Val Phe Ile Lys 1 5 10 15 Ala Val His Val Lys Ala Gln Glu Asp Glu Arg Ile Val Leu Val Asp 20 25 30 Asn Lys Cys Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser 35 40 45 Glu Asp Pro Asn Glu Asp Ile Val Glu Arg Asn Ile Arg Ile Ile Val 50 55 60 Pro Leu Asn Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg 65 70 75 80 Thr Arg Phe Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro 85 90 95 Thr Glu Val Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn 100 105 110 Ile Cys Asp Glu Asp Ser Ala Thr Glu Thr Cys Ala Thr Tyr Asp Arg 115 120 125 Asn Lys Cys Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr 130 135 140 Lys Met Val Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 165 170 175 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 180 185 190 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr Thr Met 195 200 205 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Tyr 210 215 220 Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu Lys Asp 225 230 235 240 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr Met Glu 245 250 255 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 260 265 270 Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr 275 280 285 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 290 295 300 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 305 310 315 320 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser 325 330 335 Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 340 345 350 Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser 355 360 365 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 370 375 380 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser 385 390 395 400 Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 405 410 415 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 17 Gly Gly Gly Gly Ser 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 18 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 20 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 20 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> twenty one <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty one Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> twenty two <211> 453 <212> PRT <213> Homo sapiens <400> twenty two Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 23 <211> 453 <212> PRT <213> Homo sapiens <400> 23 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 24 <211> 353 <212> PRT <213> Homo sapiens <400> 24 Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys Ser Thr 1 5 10 15 Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro 100 105 110 Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser 115 120 125 Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn 130 135 140 Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe 145 150 155 160 Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu 165 170 175 Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys 180 185 190 Ala Glu Pro Trp Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr 195 200 205 Pro Glu Ser Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn 210 215 220 Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu 225 230 235 240 Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser 245 250 255 Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro 260 265 270 Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly 275 280 285 Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp 290 295 300 Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu 305 310 315 320 Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro 325 330 335 Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys 340 345 350 Tyr <210> 25 <211> 340 <212> PRT <213> Homo sapiens <400> 25 Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr 1 5 10 15 Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Pro Val Pro Pro Pro Pro Pro Cys Cys His Pro 100 105 110 Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125 ​​​​Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly 145 150 155 160 Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170 175 Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 180 185 190 Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys 195 200 205 Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser 210 215 220 Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg 225 230 235 240 Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255 Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265 270 Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala 275 280 285 Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His 290 295 300 Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala 305 310 315 320 Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp 325 330 335 Gly Thr Cys Tyr 340 <210> 26 <211> 764 <212> PRT <213> Homo sapiens <400> 26 Met Leu Leu Phe Val Leu Thr Cys Leu Leu Ala Val Phe Pro Ala Ile 1 5 10 15 Ser Thr Lys Ser Pro Ile Phe Gly Pro Glu Glu Val Asn Ser Val Glu 20 25 30 Gly Asn Ser Val Ser Ile Thr Cys Tyr Tyr Pro Pro Thr Ser Val Asn 35 40 45 Arg His Thr Arg Lys Tyr Trp Cys Arg Gln Gly Ala Arg Gly Gly Cys 50 55 60 Ile Thr Leu Ile Ser Ser Glu Gly Tyr Val Ser Ser Lys Tyr Ala Gly 65 70 75 80 Arg Ala Asn Leu Thr Asn Phe Pro Glu Asn Gly Thr Phe Val Val Asn 85 90 95 Ile Ala Gln Leu Ser Gln Asp Asp Ser Gly Arg Tyr Lys Cys Gly Leu 100 105 110 Gly Ile Asn Ser Arg Gly Leu Ser Phe Asp Val Ser Leu Glu Val Ser 115 120 125 Gln Gly Pro Gly Leu Leu Asn Asp Thr Lys Val Tyr Thr Val Asp Leu 130 135 140 Gly Arg Thr Val Thr Ile Asn Cys Pro Phe Lys Thr Glu Asn Ala Gln 145 150 155 160 Lys Arg Lys Ser Leu Tyr Lys Gln Ile Gly Leu Tyr Pro Val Leu Val 165 170 175 Ile Asp Ser Ser Gly Tyr Val Asn Pro Asn Tyr Thr Gly Arg Ile Arg 180 185 190 Leu Asp Ile Gln Gly Thr Gly Gln Leu Leu Phe Ser Val Val Ile Asn 195 200 205 Gln Leu Arg Leu Ser Asp Ala Gly Gln Tyr Leu Cys Gln Ala Gly Asp 210 215 220 Asp Ser Asn Ser Asn Lys Lys Asn Ala Asp Leu Gln Val Leu Lys Pro 225 230 235 240 Glu Pro Glu Leu Val Tyr Glu Asp Leu Arg Gly Ser Val Thr Phe His 245 250 255 Cys Ala Leu Gly Pro Glu Val Ala Asn Val Ala Lys Phe Leu Cys Arg 260 265 270 Gln Ser Ser Gly Glu Asn Cys Asp Val Val Val Asn Thr Leu Gly Lys 275 280 285 Arg Ala Pro Ala Phe Glu Gly Arg Ile Leu Leu Asn Pro Gln Asp Lys 290 295 300 Asp Gly Ser Phe Ser Val Val Ile Thr Gly Leu Arg Lys Glu Asp Ala 305 310 315 320 Gly Arg Tyr Leu Cys Gly Ala His Ser Asp Gly Gln Leu Gln Glu Gly 325 330 335 Ser Pro Ile Gln Ala Trp Gln Leu Phe Val Asn Glu Glu Ser Thr Ile 340 345 350 Pro Arg Ser Pro Thr Val Val Lys Gly Val Ala Gly Gly Ser Val Ala 355 360 365 Val Leu Cys Pro Tyr Asn Arg Lys Glu Ser Lys Ser Ile Lys Tyr Trp 370 375 380 Cys Leu Trp Glu Gly Ala Gln Asn Gly Arg Cys Pro Leu Leu Val Asp 385 390 395 400 Ser Glu Gly Trp Val Lys Ala Gln Tyr Glu Gly Arg Leu Ser Leu Leu 405 410 415 Glu Glu Pro Gly Asn Gly Thr Phe Thr Val Ile Leu Asn Gln Leu Thr 420 425 430 Ser Arg Asp Ala Gly Phe Tyr Trp Cys Leu Thr Asn Gly Asp Thr Leu 435 440 445 Trp Arg Thr Thr Val Glu Ile Lys Ile Ile Glu Gly Glu Pro Asn Leu 450 455 460 Lys Val Pro Gly Asn Val Thr Ala Val Leu Gly Glu Thr Leu Lys Val 465 470 475 480 Pro Cys His Phe Pro Cys Lys Phe Ser Ser Tyr Glu Lys Tyr Trp Cys 485 490 495 Lys Trp Asn Asn Thr Gly Cys Gln Ala Leu Pro Ser Gln Asp Glu Gly 500 505 510 Pro Ser Lys Ala Phe Val Asn Cys Asp Glu Asn Ser Arg Leu Val Ser 515 520 525 Leu Thr Leu Asn Leu Val Thr Arg Ala Asp Glu Gly Trp Tyr Trp Cys 530 535 540 Gly Val Lys Gln Gly His Phe Tyr Gly Glu Thr Ala Ala Val Tyr Val 545 550 555 560 Ala Val Glu Glu Arg Lys Ala Ala Gly Ser Arg Asp Val Ser Leu Ala 565 570 575 Lys Ala Asp Ala Ala Pro Asp Glu Lys Val Leu Asp Ser Gly Phe Arg 580 585 590 Glu Ile Glu Asn Lys Ala Ile Gln Asp Pro Arg Leu Phe Ala Glu Glu 595 600 605 Lys Ala Val Ala Asp Thr Arg Asp Gln Ala Asp Gly Ser Arg Ala Ser 610 615 620 Val Asp Ser Gly Ser Ser Glu Glu Gln Gly Gly Ser Ser Arg Ala Leu 625 630 635 640 Val Ser Thr Leu Val Pro Leu Gly Leu Val Leu Ala Val Gly Ala Val 645 650 655 Ala Val Gly Val Ala Arg Ala Arg His Arg Lys Asn Val Asp Arg Val 660 665 670 Ser Ile Arg Ser Tyr Arg Thr Asp Ile Ser Met Ser Asp Phe Glu Asn 675 680 685 Ser Arg Glu Phe Gly Ala Asn Asp Asn Met Gly Ala Ser Ser Ile Thr 690 695 700 Gln Glu Thr Ser Leu Gly Gly Lys Glu Glu Phe Val Ala Thr Thr Glu 705 710 715 720 Ser Thr Thr Glu Thr Lys Glu Pro Lys Lys Ala Lys Arg Ser Ser Lys 725 730 735 Glu Glu Ala Glu Met Ala Tyr Lys Asp Phe Leu Leu Gln Ser Ser Thr 740 745 750 Val Ala Ala Glu Ala Gln Asp Gly Pro Gln Glu Ala 755 760 <210> 27 <211> 585 <212> PRT <213> Homo sapiens <400> 27 Lys Ser Pro Ile Phe Gly Pro Glu Glu Val Asn Ser Val Glu Gly Asn 1 5 10 15 Ser Val Ser Ile Thr Cys Tyr Tyr Pro Pro Thr Ser Val Asn Arg His 20 25 30 Thr Arg Lys Tyr Trp Cys Arg Gln Gly Ala Arg Gly Gly Cys Ile Thr 35 40 45 Leu Ile Ser Ser Glu Gly Tyr Val Ser Ser Lys Tyr Ala Gly Arg Ala 50 55 60 Asn Leu Thr Asn Phe Pro Glu Asn Gly Thr Phe Val Val Asn Ile Ala 65 70 75 80 Gln Leu Ser Gln Asp Asp Ser Gly Arg Tyr Lys Cys Gly Leu Gly Ile 85 90 95 Asn Ser Arg Gly Leu Ser Phe Asp Val Ser Leu Glu Val Ser Gln Gly 100 105 110 Pro Gly Leu Leu Asn Asp Thr Lys Val Tyr Thr Val Asp Leu Gly Arg 115 120 125 Thr Val Thr Ile Asn Cys Pro Phe Lys Thr Glu Asn Ala Gln Lys Arg 130 135 140 Lys Ser Leu Tyr Lys Gln Ile Gly Leu Tyr Pro Val Leu Val Ile Asp 145 150 155 160 Ser Ser Gly Tyr Val Asn Pro Asn Tyr Thr Gly Arg Ile Arg Leu Asp 165 170 175 Ile Gln Gly Thr Gly Gln Leu Leu Phe Ser Val Val Ile Asn Gln Leu 180 185 190 Arg Leu Ser Asp Ala Gly Gln Tyr Leu Cys Gln Ala Gly Asp Asp Ser 195 200 205 Asn Ser Asn Lys Lys Asn Ala Asp Leu Gln Val Leu Lys Pro Glu Pro 210 215 220 Glu Leu Val Tyr Glu Asp Leu Arg Gly Ser Val Thr Phe His Cys Ala 225 230 235 240 Leu Gly Pro Glu Val Ala Asn Val Ala Lys Phe Leu Cys Arg Gln Ser 245 250 255 Ser Gly Glu Asn Cys Asp Val Val Val Asn Thr Leu Gly Lys Arg Ala 260 265 270 Pro Ala Phe Glu Gly Arg Ile Leu Leu Asn Pro Gln Asp Lys Asp Gly 275 280 285 Ser Phe Ser Val Val Ile Thr Gly Leu Arg Lys Glu Asp Ala Gly Arg 290 295 300 Tyr Leu Cys Gly Ala His Ser Asp Gly Gln Leu Gln Glu Gly Ser Pro 305 310 315 320 Ile Gln Ala Trp Gln Leu Phe Val Asn Glu Glu Ser Thr Ile Pro Arg 325 330 335 Ser Pro Thr Val Val Lys Gly Val Ala Gly Gly Ser Val Ala Val Leu 340 345 350 Cys Pro Tyr Asn Arg Lys Glu Ser Lys Ser Ile Lys Tyr Trp Cys Leu 355 360 365 Trp Glu Gly Ala Gln Asn Gly Arg Cys Pro Leu Leu Val Asp Ser Glu 370 375 380 Gly Trp Val Lys Ala Gln Tyr Glu Gly Arg Leu Ser Leu Leu Glu Glu 385 390 395 400 Pro Gly Asn Gly Thr Phe Thr Val Ile Leu Asn Gln Leu Thr Ser Arg 405 410 415 Asp Ala Gly Phe Tyr Trp Cys Leu Thr Asn Gly Asp Thr Leu Trp Arg 420 425 430 Thr Thr Val Glu Ile Lys Ile Ile Glu Gly Glu Pro Asn Leu Lys Val 435 440 445 Pro Gly Asn Val Thr Ala Val Leu Gly Glu Thr Leu Lys Val Pro Cys 450 455 460 His Phe Pro Cys Lys Phe Ser Ser Tyr Glu Lys Tyr Trp Cys Lys Trp 465 470 475 480 Asn Asn Thr Gly Cys Gln Ala Leu Pro Ser Gln Asp Glu Gly Pro Ser 485 490 495 Lys Ala Phe Val Asn Cys Asp Glu Asn Ser Arg Leu Val Ser Leu Thr 500 505 510 Leu Asn Leu Val Thr Arg Ala Asp Glu Gly Trp Tyr Trp Cys Gly Val 515 520 525 Lys Gln Gly His Phe Tyr Gly Glu Thr Ala Ala Val Tyr Val Ala Val 530 535 540 Glu Glu Arg Lys Ala Ala Gly Ser Arg Asp Val Ser Leu Ala Lys Ala 545 550 555 560 Asp Ala Ala Pro Asp Glu Lys Val Leu Asp Ser Gly Phe Arg Glu Ile 565 570 575 Glu Asn Lys Ala Ile Gln Asp Pro Arg 580 585 <210> 28 <211> 378 <212> PRT <213> Homo sapiens <400> 28 Met Val Leu Leu Trp Leu Thr Leu Leu Leu Ile Ala Leu Pro Cys Leu 1 5 10 15 Leu Gln Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met 20 25 30 Lys Ala Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr 35 40 45 Asp Ile Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn 50 55 60 Asn Ser Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn 65 70 75 80 Tyr Thr Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe 85 90 95 Pro Glu Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys 100 105 110 Trp Ile His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro 115 120 125 Gly Ala Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn 130 135 140 Arg Arg Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly 145 150 155 160 Thr Arg Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser Ser Gly 165 170 175 Ser Gln Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Phe Gly 180 185 190 Ile Pro Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu Ile Leu 195 200 205 Thr Pro Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met 210 215 220 His Trp Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu 225 230 235 240 Gln Ile Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val Arg Asp 245 250 255 Arg Thr Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val Gln Ile 260 265 270 Arg Ala Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro 275 280 285 Gln Arg Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg Ala Trp 290 295 300 Arg Thr Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Val Cys 305 310 315 320 Val Phe Val Ile Cys Arg Arg Tyr Leu Val Met Gln Arg Leu Phe Pro 325 330 335 Arg Ile Pro His Met Lys Asp Pro Ile Gly Asp Ser Phe Gln Asn Asp 340 345 350 Lys Leu Val Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu 355 360 365 Val Thr Glu Val Gln Val Val Gln Lys Thr 370 375 <210> 29 <211> 300 <212> PRT <213> Homo sapiens <400> 29 [[ID=3,6]]Met Val Leu Leu Trp Leu Thr Leu Leu Leu Ile Ala Leu Pro Cys Leu 1 5 10 15 Leu Gln Thr Lys Glu Gly Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu 20 25 30 Thr Cys Trp Ile His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val 35 40 45 Gly Pro Gly Ala Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val 50 55 60 Ala Asn Arg Arg Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala 65 70 75 80 Gln Gly Thr Arg Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser 85 90 95 Ser Gly Ser Gln Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala 100 105 110 Phe Gly Ile Pro Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu 115 120 125 Ile Leu Thr Pro Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser 130 135 140 Phe Met His Trp Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr 145 150 155 160 Glu Leu Gln Ile Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val 165 170 175 Arg Asp Arg Thr Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val 180 185 190 Gln Ile Arg Ala Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser 195 200 205 Thr Pro Gln Arg Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg 210 215 220 Ala Trp Arg Thr Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu 225 230 235 240 Val Cys Val Phe Val Ile Cys Arg Arg Tyr Leu Val Met Gln Arg Leu 245 250 255 Phe Pro Arg Ile Pro His Met Lys Asp Pro Ile Gly Asp Ser Phe Gln 260 265 270 Asn Asp Lys Leu Val Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu 275 280 285 Cys Leu Val Thr Glu Val Gln Val Val Gln Lys Thr 290 295 300 <210> 30 <211> 378 <212> PRT <213> Macaca fascicularis (Macaca fascicularis) <400> 30 Met Thr Leu Leu Trp Leu Thr Leu Leu Leu Val Ala Thr Pro Cys Leu 1 5 10 15 Leu Arg Thr Lys Glu Asp Pro Asn Ala Pro Ile Arg Asn Leu Arg Met 20 25 30 Lys Glu Lys Ala Gln Gln Leu Met Trp Asp Leu Asn Arg Asn Val Thr 35 40 45 Asp Val Glu Cys Ile Lys Gly Thr Asp Tyr Ser Met Pro Ala Met Asn 50 55 60 Asn Ser Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn 65 70 75 80 Tyr Thr Val Arg Val Ala Ser Pro Pro Phe Ser Thr Trp Ile Leu Phe 85 90 95 Pro Glu Asn Ser Gly Thr Pro Arg Ala Gly Ala Glu Asn Leu Thr Cys 100 105 110 Trp Val His Asp Val Asp Phe Leu Ser Cys Ser Trp Val Val Gly Pro 115 120 125 Ala Ala Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Asn Pro Asn 130 135 140 Ser His Glu Gln Tyr Arg Cys Leu His Tyr Lys Thr Asp Ala Arg Gly 145 150 155 160 Thr Gln Ile Gly Cys Arg Phe Asp Asp Ile Ala Pro Leu Ser Arg Gly 165 170 175 Ser Gln Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Val Ser 180 185 190 Ile Pro Cys Thr Asp Lys Phe Val Phe Phe Ser Gln Ile Glu Arg Leu 195 200 205 Thr Pro Pro Asn Met Thr Gly Glu Cys Asn Glu Thr His Ser Phe Met 210 215 220 His Trp Lys Met Lys Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu 225 230 235 240 Arg Ile Gln Lys Arg Met Gln Pro Val Arg Thr Glu Gln Val Arg Asp 245 250 255 Thr Thr Ser Phe Gln Leu Pro Asn Pro Gly Thr Tyr Thr Val Gln Ile 260 265 270 Arg Ala Arg Glu Thr Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro 275 280 285 Gln Arg Phe Glu Cys Asp Gln Glu Glu Gly Ala Ser Ser Arg Ala Trp 290 295 300 Arg Thr Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Leu Cys 305 310 315 320 Val Phe Leu Ile Cys Arg Arg Tyr Leu Val Met Gln Arg Leu Phe Pro 325 330 335 Arg Ile Pro His Met Lys Asp Pro Ile Gly Asp Thr Phe Gln Gln Asp 340 345 350 Lys Leu Val Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu 355 360 365 Val Ser Glu Val Gln Val Val Glu Lys Thr 370 375 <210> 31 <211> 396 <212> PRT <213> Mus musculus <400> 31 Met Ala Ala Asn Leu Trp Leu Ile Leu Gly Leu Leu Ala Ser His Ser 1 5 10 15 Ser Asp Leu Ala Ala Val Arg Glu Ala Pro Pro Thr Ala Val Thr Thr 20 25 30 Pro Ile Gln Asn Leu His Ile Asp Pro Ala His Tyr Thr Leu Ser Trp 35 40 45 Asp Pro Ala Pro Gly Ala Asp Ile Thr Thr Gly Ala Phe Cys Arg Lys 50 55 60 Gly Arg Asp Ile Phe Val Trp Ala Asp Pro Gly Leu Ala Arg Cys Ser 65 70 75 80 Phe Gln Ser Leu Ser Leu Cys His Val Thr Asn Phe Thr Val Phe Leu 85 90 95 Gly Lys Asp Arg Ala Val Ala Gly Ser Ile Gln Phe Pro Pro Asp Asp 100 105 110 Asp Gly Asp His Glu Ala Ala Ala Gln Asp Leu Arg Cys Trp Val His 115 120 125 Glu Gly Gln Leu Ser Cys Gln Trp Glu Arg Gly Pro Lys Ala Thr Gly 130 135 140 Asp Val His Tyr Arg Met Phe Trp Arg Asp Val Arg Leu Gly Pro Ala 145 150 155 160 His Asn Arg Glu Cys Pro His Tyr His Ser Leu Asp Val Asn Thr Ala 165 170 175 Gly Pro Ala Pro His Gly Gly His Glu Gly Cys Thr Leu Asp Leu Asp 180 185 190 Thr Val Leu Gly Ser Thr Pro Asn Ser Pro Asp Leu Val Pro Gln Val 195 200 205 Thr Ile Thr Val Asn Gly Ser Gly Arg Ala Gly Pro Val Pro Cys Met 210 215 220 Asp Asn Thr Val Asp Leu Gln Arg Ala Glu Val Leu Ala Pro Pro Thr 225 230 235 240 Leu Thr Val Glu Cys Asn Gly Ser Glu Ala His Ala Arg Trp Val Ala 245 250 255 Arg Asn Arg Phe His His Gly Leu Leu Gly Tyr Thr Leu Gln Val Asn 260 265 270 Gln Ser Ser Arg Ser Glu Pro Gln Glu Tyr Asn Val Ser Ile Pro His 275 280 285 Phe Trp Val Pro Asn Ala Gly Ala Ile Ser Phe Arg Val Lys Ser Arg 290 295 300 Ser Glu Val Tyr Pro Arg Lys Leu Ser Ser Trp Ser Glu Ala Trp Gly 305 310 315 320 Leu Val Cys Pro Pro Glu Val Met Pro Val Lys Thr Ala Leu Val Thr 325 330 335 Ser Val Ala Thr Val Leu Gly Ala Gly Leu Val Ala Ala Gly Leu Leu 340 345 350 Leu Trp Trp Arg Lys Ser Leu Leu Tyr Arg Leu Cys Pro Pro Ile Pro 355 360 365 Arg Leu Arg Leu Pro Leu Ala Gly Glu Met Val Val Trp Glu Pro Ala 370 375 380 Leu Glu Asp Cys Glu Val Thr Pro Val Thr Asp Ala 385 390 395 <210> 32 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 32 Gln Val Gln Leu Gln 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 Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 33 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 33 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 34 <211> 469 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 34 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Met Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro Gly Lys 465 <210> 35 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 35 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Met Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala 130 135 140 Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn Ser Pro Ser Asp Thr 145 150 155 160 Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp Phe Leu Pro Asp Ser 165 170 175 Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser Asp Ile Ser Ser Thr 180 185 190 Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys Tyr Ala Ala Thr Ser 195 200 205 Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln Gly Thr Asp Glu His 210 215 220 Val Val Cys Lys Val Gln His Pro Asn Gly Asn Lys Glu Lys Asn Val 225 230 235 240 Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val 245 250 255 Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile 260 265 270 Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu 275 280 285 Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln 290 295 300 Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr 305 310 315 320 Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln Ser Met Phe Thr Cys 325 330 335 Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met 340 345 350 Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro 355 360 365 Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu 370 375 380 Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg 385 390 395 400 Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His 405 410 415 Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp 420 425 430 Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp 435 440 445 Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala 450 455 460 Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu 465 470 475 480 Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser 485 490 495 Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser 500 505 510 Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro 515 520 525 Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp 530 535 540 Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro 545 550 555 560 Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr 565 570 575 Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 580 585 590 <210> 36 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 36 Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Arg 1 5 10 15 Gly Ala Arg Cys Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Thr Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 37 <211> 115 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 37 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 Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 38 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 38 Glu Val Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 39 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 39 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn 65 70 75 80 Gln Lys Phe Lys Asp Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 225 230 235 240 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 245 250 255 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295 300 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 305 310 315 320 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 370 375 380 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 385 390 395 400 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410 415 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 420 425 430 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 40 <211> 587 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 40 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn 65 70 75 80 Gln Lys Phe Lys Asp Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Thr Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro 130 135 140 Leu Val Ser Cys Glu Asn Ser Pro Ser Asp Thr Ser Ser Val Ala Val 145 150 155 160 Gly Cys Leu Ala Gln Asp Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp 165 170 175 Lys Tyr Lys Asn Asn Ser Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser 180 185 190 Val Leu Arg Gly Gly Lys Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro 195 200 205 Ser Lys Asp Val Met Gln Gly Thr Asp Glu His Val Val Cys Lys Val 210 215 220 Gln His Pro Asn Gly Asn Lys Glu Lys Asn Val Pro Leu Pro Val Ile 225 230 235 240 Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp Gly 245 250 255 Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly 260 265 270 Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys Gln 275 280 285 Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys Glu 290 295 300 Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys Glu 305 310 315 320 Ser Asp Trp Leu Ser Gln Ser Met Phe Thr Cys Arg Val Asp His Arg 325 330 335 Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp Gln 340 345 350 Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile 355 360 365 Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp Leu Thr 370 375 380 Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala 385 390 395 400 Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala Thr Phe 405 410 415 Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly 420 425 430 Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser Pro Leu 435 440 445 Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala Leu His Arg Pro Asp 450 455 460 Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser 465 470 475 480 Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val Phe 485 490 495 Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val 500 505 510 Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala 515 520 525 His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr 530 535 540 Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val Thr Glu 545 550 555 560 Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser 565 570 575 Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 580 585 <210> 41 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 41 Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Arg 1 5 10 15 Gly Ala Arg Cys Glu Val Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser 35 40 45 Ile Ser Lys Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Asn 100 105 110 Lys Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 42 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 42 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Ala Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Thr Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 43 <211> 113 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 43 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Pro Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 44 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 44 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 45 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 45 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 46 <211> 120 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met His Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 47 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 47 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 48 <211> 120 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 49 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 49 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Val Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 50 <211> 120 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: synthetic polypeptide <400> 50 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 51 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 51 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Val Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 52 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 52 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Asp Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Gly Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Glu Gly Phe Tyr Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 53 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 53 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Phe Phe Gly 20 25 30 Ser Thr Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Ile 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 54 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 54 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Asp Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Gly Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Glu Gly Phe Tyr Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 55 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 55 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Phe Phe Gly 20 25 30 Ser Thr Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Ile 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 56 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 56 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 Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 57 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 57 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 58 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 58 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 59 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 59 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 60 <211> 120 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 60 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 61 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 61 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 62 <211> 117 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 62 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Asp Tyr Ser Ile Thr Ser Gly [[ID=二十九]]20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ser Arg Gly Glu Gly Phe Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 63 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 63 Asp Ile Met Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Phe Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Phe Phe Gly 20 25 30 Ser Thr Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala 65 70 75 80 Ile Ser Ser Val Met Pro Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 64 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 64 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met His Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 65 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 65 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 66 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Glu Trp Phe Ser Glu Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 67 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 67 Gln Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln Lys Val 1 5 10 15 Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Asn Asn Tyr Val 20 25 30 Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Asn Asn Arg Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln Thr Gly 65 70 75 80 Asp Glu Ala Asp Tyr Phe Cys Gly Thr Trp Asp Ser Ser Leu Ser Ala 85 90 95 Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 68 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 68 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Ser Asp Ala Leu Asp Ile Trp Gly Arg Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 69 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 69 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Gly Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Pro Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 70 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Asp Tyr Tyr Glu Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 71 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 71 Asn Phe Met Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ala Asp Val Gly Gly Asp 20 25 30 Tyr Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Thr Ile Tyr Asp Val Ser Glu Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Tyr Thr Ser Ser 85 90 95 Gly Thr Trp Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 72 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Thr Arg Gly Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 73 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 73 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 <210> 74 <211> 130 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 74 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 Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Lys Tyr Ser Gln Lys Leu 50 55 60 Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Glu Tyr Asp Phe Trp Ser Gly Tyr Gly Ser Trp Tyr 100 105 110 Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115, 120, 125 Ser Ser 130 <210> 75 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 75 Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 76 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 76 Glu Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Tyr Tyr Asp Ser Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 77 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 77 Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Gln Phe Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Lys Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Ser Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn His Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 78 <211> 120 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 78 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 ...... 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Gln Gln Trp Pro Asp Asp Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 79 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 79 Gln Ala Gly Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ala Cys Ser Gly Ser Ser Ser Ser Asn Ile Gly Thr Tyr 20 25 30 Thr Val Asn Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Tyr Gln Arg Pro Leu Glu Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Gly Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Arg Leu 85 90 95 Asn Gly Phe Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 80 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 80 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Trp Tyr Asp Glu Asn Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Phe Arg Asp Tyr Tyr Phe Asp Val Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 81 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 81 Gln Ser Val Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ile Asp Val Asp Lys Asp 20 25 30 Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Arg Val Pro Lys Leu 35 40 45 Ile Ile Tyr Asp Val Asn Lys Arg Pro Ser Gly Val Pro Asp His Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95 Leu Ser Ser Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 82 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 82 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Asp Tyr Asp Phe Trp Ser Gly Tyr Tyr Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 83 <211> 111 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 83 Leu Pro Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Arg Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Gln Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Gly Ser 85 90 95 Ser Thr Leu Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 84 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 84 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Thr Ile Pro Lys Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Val Val Pro Ala Ala Ile Val Glu Ala Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 85 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 85 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Thr Ile Pro Lys Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Val Val Pro Ala Ala Ile Val Glu Ala Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 86 <211> 126 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: synthetic polypeptide <400> 86 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 Leu Ser Met Tyr 20 25 30 Gly Ile Ser Trp Val Arg His Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Tyr Thr Gly Asp Arg Lys Tyr Ala Gln Arg Phe 50 55 60 Gln Gly Arg Leu Thr Val Thr Thr Asp Thr Ser Thr Ala Thr Ser Tyr 65 70 75 80 Met Glu Leu Thr Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Glu Tyr His Asp Ser Met Ile Gly Tyr Tyr Val Gly Gly 100 105 110 Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 87 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 87 Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Pro Gln Leu Thr Val Leu 100 105 <210> 88 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 88 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Asn Trp Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met 100 105 110 Val Thr Val Ser Ser 115 <210> 89 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 89 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly His 1 5 10 15 Glu Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Ala Ser Glu Ile Pro Asp Arg Phe Phe 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Val Ser Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Asn Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 90 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 90 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Asp Tyr Asp Phe Trp Ser Gly Tyr Tyr Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 91 <211> 111 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 91 Leu Pro Val Leu Thr Gln Ser Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Arg Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Gln Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Gly Ser 85 90 95 Ser Thr Leu Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 92 <211> 124 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 92 Glu 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 Gly 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 Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Glu Asp Tyr Tyr Gly Ser Gly Glu His Tyr Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 93 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 93 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu Asn Trp Tyr His Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Arg Ser Gly Chr Asp Phe Thr Leu Thr Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100 105 <210> 94 <211> 121 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 94 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 Gly 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 Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln 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 Asp Met Asn Ile Leu Ala Thr Val Pro Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 95 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 95 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Ser Val Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 96 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 96 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 Gly 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 Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Ile Ser Thr Val 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 Asp Met Asn Ile Leu Ala Thr Val Pro Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 97 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 97 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Ser Val Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 98 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 98 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 Ile Phe Thr Gly Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln 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 Gly Leu Arg Ser Asp Asp Pro Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Met Asn Ile Leu Ala Thr Val Pro Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 99 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 99 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Ser Val Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 100 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 100 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Ser 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 Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Ile Ser Thr Val 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 Asp Met Asn Ile Leu Ala Thr Val Pro Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 101 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 101 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Ser Val Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 102 <211> 120 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 102 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Ala Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Thr Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 103 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 103 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Pro Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 104 <211> 449 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 104 Gln Val Gln Leu Gln 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 Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asp Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 105 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 105 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 106 <211> 485 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polypeptide <400> 106 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Lys Pro 115 120 125 Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly 130 135 140 Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly 145 150 155 160 Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr 165 170 175 Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ser Pro Arg 180 185 190 Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg 195 200 205 Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly 210 215 220 Ala Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser 225 230 235 240 Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Glu Pro 245 250 255 Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro 260 265 270 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 275 280 285 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 290 295 300 Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 305 310 315 320 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 325 330 335 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 340 345 350 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 355 360 365 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 370 375 380 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln 385 390 395 400 Val Lys Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 405 410 415 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 420 425 430 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 435 440 445 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 450 455 460 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 465 470 475 480 Leu Ser Pro Gly Lys 485 <210> 107 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 107 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu 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 Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 108 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 108 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 109 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 109 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu 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 Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 110 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 110 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 111 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 111 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 112 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 112 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 113 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 113 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Ser Phe Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ser Ala Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu His Ile Asn Asp Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Ser Gly Gly Tyr Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 114 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 114 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 115 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 115 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 116 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 116 Asp Phe Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 117 <211> 121 <212> PRT <213> Artificial Sequence <220> <223>Description of artificial sequence: Synthetic polypeptide <400> 117 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 Ile Phe Thr Ser Ser 20 25 30 Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Lys Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Gl...

Claims

1. A bispecific antibody, which is a multimer and comprises five bivalent binding units and a modified J chain, wherein the modified J chain is a variant J chain, the variant J chain is fused with an antigen binding domain associated with the J chain, the antigen binding domain associated with the J chain is a single-chain Fv (scFv), and the scFv specifically binds to CD3, wherein the variant J chain is a mature wild-type human J chain (SEQ ID NO: 2), but has an alanine (A) amino acid substitution at amino acid Y102 of SEQ ID NO: 2; wherein each binding unit comprises two antibody heavy chains, each heavy chain comprising an IgM or IgM-like heavy chain constant region or a multimeric fragment thereof and comprising at least a heavy chain variable region (VH) portion of an antigen binding domain associated with the binding unit, and each binding unit comprises two light chains, each light chain comprising a kappa or lambda light chain constant region and comprising at least a light chain variable region (VL) of an antigen binding domain associated with the binding unit; wherein each antigen binding domain associated with the binding unit is identical and specifically binds to CD123, and The antibody induces T cell-dependent killing of cells expressing CD123.

2. The antibody of claim 1, which exhibits increased serum half-life when administered to an animal relative to a reference antibody that is identical except for an amino acid substitution at amino acid Y102 of SEQ ID NO: 2 and is administered in the same manner to the same animal species.

3. The antibody of claim 1, wherein the amino acid sequence of the variant J chain is SEQ ID NO:

3.

4. The antibody of claim 1, wherein the scFv is fused to the N-terminus of the variant J chain.

5. The antibody of claim 1, wherein the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, wherein (a) the amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, and the amino acid sequences of VLCDR1, VLCDR2 and VLCDR3 are SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; (b) the amino acid sequences of said VHCDR1, VHCDR2 and VHCDR3 are SEQ ID NO: 130, SEQ ID NO: 132 and SEQ ID NO: 135, respectively, and the amino acid sequences of said VLCDR1, VLCDR2 and VLCDR3 are SEQ ID NO: 138, SEQ ID NO: 140 and SEQ ID NO: 142, respectively; (c) the amino acid sequences of said VHCDR1, VHCDR2 and VHCDR3 are SEQ ID NO:130, SEQ ID NO:132 and SEQ ID NO:135, respectively, and the amino acid sequences of said VLCDR1, VLCDR2 and VLCDR3 are SEQ ID NO:138, SEQ ID NO:140 and SEQ ID NO:143, respectively; (d) the amino acid sequences of said VHCDR1, VHCDR2 and VHCDR3 are SEQ ID NO:131, SEQ ID NO:133 and SEQ ID NO:136, respectively, and the amino acid sequences of said VLCDR1, VLCDR2 and VLCDR3 are SEQ ID NO:139, SEQ ID NO:141 and SEQ ID NO:144, respectively; (e) the amino acid sequences of said VHCDR1, VHCDR2 and VHCDR3 are SEQ ID NO:131, SEQ ID NO:134 and SEQ ID NO:136, respectively, and the amino acid sequences of said VLCDR1, VLCDR2 and VLCDR3 are SEQ ID NO:139, SEQ ID NO:141 and SEQ ID NO:145, respectively; or (f) the amino acid sequences of said VHCDR1, VHCDR2 and VHCDR3 are SEQ ID NO:131, SEQ ID NO:134 and SEQ ID NO:137, respectively, and the amino acid sequences of said VLCDR1, VLCDR2 and VLCDR3 are SEQ ID NO:139, SEQ ID NO:141 and SEQ ID NO:146, respectively.

6. The antibody of claim 5, wherein the scFv comprises VH and VL amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 119 and SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122, SEQ ID NO: 123 and SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126, or SEQ ID NO: 127 and SEQ ID NO: 128, respectively.

7. The antibody of claim 6, wherein the scFv comprises VH and VL amino acid sequences of SEQ ID NO: 119 and SEQ ID NO: 120, respectively.

8. The antibody of claim 1, wherein each antigen binding domain associated with a binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein each binding unit comprises two IgM heavy chains and two immunoglobulin light chains, each IgM heavy chain comprising a copy of VH located at the amino terminus of an IgM constant region, and each immunoglobulin light chain comprising a copy of VL located at the amino terminus of an immunoglobulin light chain constant region.

9. The antibody of claim 8, wherein the VH and VL comprise a total of six immunoglobulin complementary determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are: CDRs of an antibody comprising VH and VL amino acid sequences, wherein the VH and VL amino acid sequences are SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, : 54 and SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO:102 and SEQ ID NO:103 and SEQ ID NO:104.NO:103, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116 or SEQ ID NO:117 and SEQ ID NO:

118.

10. The antibody of claim 9, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of each antigen binding domain associated with the binding unit are: CDRs of an antibody comprising VH and VL amino acid sequences, wherein the VH and VL amino acid sequences are SEQ ID NO: 113 and SEQ ID NO: 114, respectively.

11. The antibody of claim 10, wherein the antigen binding domain scFv associated with the J chain comprises VH and VL amino acid sequences of SEQ ID NO: 119 and SEQ ID NO:

120.

12. The antibody of any one of claims 1 to 11, wherein each IgM or IgM-like heavy chain constant region comprises a Cμ4 domain and a tail piece (tp) domain.

13. The antibody of claim 12, wherein each of the IgM or IgM-like heavy chain constant regions further comprises a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof.

14. The antibody of claim 13, wherein each IgM heavy chain constant region is a human IgM constant region comprising the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:

23.

15. A composition comprising the antibody of any one of claims 1 to 14.

16. A polynucleotide comprising a sequence encoding the antibody of any one of claims 1 to 14. A vector comprising the polynucleotide according to claim 16 .

18. A host cell comprising the polynucleotide of claim 16 or the vector of claim 17, wherein the host cell is capable of expressing the antibody of any one of claims 1 to 14.

19. A method of producing the antibody of any one of claims 1 to 14, comprising culturing the host cell of claim 18 and recovering the antibody.

20. Use of an effective amount of an antibody according to any one of claims 1 to 14 in the preparation of a medicament for treating acute myeloid leukemia (AML) in a subject, wherein the antibody is capable of inducing T cell-mediated killing of AML cells.

21. The use of claim 20, wherein the treatment results in reduced cytokine release relative to an IgG-based anti-CD123 anti-CD3 bispecific antibody.

22. The use of claim 20, wherein the subject is a human.

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