IgM Fc and J chain mutations that affect the serum half-life of IgM

Abstract

The present disclosure provides an IgM antibody or an IgM-like antibody, wherein the IgM antibody or the IgM-like antibody comprises a variant J chain and / or a variant IgM heavy chain constant region that can confer an increased serum half-life to the antibody.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 637,186, filed on March 1, 2018, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] The content of the sequence listing submitted electronically in the ASCII text file (name "09789 - 012WO1 - Sequence - Listing; size: 98,048 bytes; and creation date: February 28, 2019") submitted herewith is incorporated herein by reference in its entirety. Background of the Invention

[0005] Multimerizable antibodies and antibody - like molecules, such as IgA and IgM antibodies, have emerged as promising candidates in, for example, the fields of immuno - oncology and infectious diseases, allowing for improved specificity, improved affinity, and the ability to bind multiple binding targets. See, e.g., U.S. Patent Nos. 9,951,134 and 9,938,347, and PCT Publication Nos. WO 2016 / 141303, WO 2016 / 154593, WO 2016 / 168758, WO 2017 / 059387, WO 2017 059380, WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, and WO 2018 / 017761, the contents of which are incorporated herein by reference in their entireties.

[0006] However, the pharmacokinetics (PK) and pharmacodynamics (PD) of multivalent antibodies are complex and depend on the structure of the monoclonal antibody and the physiological system to which it targets. In addition, different antibody classes are typically processed within a subject by different cells and physiological systems. For example, the IgG antibody class has the longest serum half - life of 20 days, while the half - lives of IgM and IgA antibodies are only about 5 to 8 days. Brekke, O.H. and I. Sandlie, Nature Reviews Drug Discovery 2:52 - 62 (2003).

[0007] Antibody molecules in vivo can bind to various receptors on different cells in the blood or different tissues and organs. Binding to these receptors can affect the bioavailability and biodistribution of therapeutic antibodies, as well as their ability to reach the target of interest. For example, antibodies of the IgG isotype are known to have a longer half-life in vivo due to binding to the recycling Fc receptor (FcRn). However, IgA and IgM isotype antibodies do not bind this key recycling receptor. IgM isotype antibodies are known to bind to the Fcμ receptor (FcμR), the Fcα / μ receptor (FcαμR), and the polymeric Ig receptor (pIgR). IgM antibodies with and without the J chain bind to FcμR (Kubagawa, H., et al., Curr. Top. Microbiol. Immunol. 408:25-45 (2017)), but J chain residues contribute at least to the binding of IgM antibodies to FcαμR (Ghumra, A., et al., Eur. J. Immunol. 39:1147-1156 (2009)), and IgM that binds pIgR is J chain-dependent (Braathen R., et al. J. Immunol 178:1589-1597 (2007)). PIgR is responsible for transporting IgM and IgA to the intestinal lumen, salivary glands, and lacrimal glands (see, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002)). FcαμR is responsible for the uptake of antibodies bound to foreign substances by B cells and phagocytes (Akula, S. and Hellman, L., Curr. Top. Microbiol. Immunol. 408:1-23 (2017)). FcμR is important for B cell development (Nguyen, T., et al., Nature Immunol. 18:321-333 (2017)). We hypothesize that the interaction of IgM with one or more of these receptors may affect PK / PD.

[0008] The J chain is an acidic 15-kDa polypeptide that associates with pentameric IgM and dimeric IgA via disulfide bonds involving the penultimate cysteine residue in an 18-amino acid secretory tailpiece (tp) at the C-terminus of the IgM μ or IgA α heavy chain. The precursor human J chain amino acid sequence is represented as SEQ ID NO:1, and the mature human J chain amino acid sequence is represented as SEQ ID NO:2. Assembly of the IgM binding unit into a pentameric structure is thought to involve the Cμ4 and tailpiece domains of the IgM constant region. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002).

[0009] Despite progress in designing multimeric antibodies, there remains a need to be able to manipulate the pharmacokinetic and pharmacodynamic properties of these molecules. SUMMARY OF THE INVENTION

[0010] The present disclosure provides an IgM antibody or IgM-like antibody having an enhanced serum half-life, wherein the antibody comprises five bivalent antibody binding units or variants or fragments thereof and a variant J chain or a functional fragment thereof. Each binding unit of the provided antibody 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 thereof. In certain aspects, the variant J chain or a functional fragment thereof comprises one or more single amino acid substitutions, deletions or insertions relative to a reference J chain that is identical to the variant J chain except for one or more single amino acid substitutions, deletions or insertions. The provided variant J chain can affect the serum half-life of the provided IgM antibody or IgM-like antibody. For example, in certain aspects, the IgM antibody or IgM-like antibody exhibits an increased serum half-life upon administration to a test animal relative to a reference IgM antibody or IgM-like antibody that is identical except for one or more single amino acid substitutions, deletions or insertions in the variant J chain and that is administered to the same animal species in the same manner. In certain aspects, the variant J chain or a functional fragment thereof comprises one, two, three or four single amino acid substitutions, deletions or insertions relative to the reference J chain.

[0011] In some aspects, the variant J chain or a functional fragment thereof comprises an amino acid substitution at an amino acid position corresponding to amino acid Y102 of wild-type human J chain (SEQ ID NO:2). For example, the amino acid corresponding to Y102 of SEQ ID NO:2 can be substituted with alanine (A), serine (S), or arginine (R). In some aspects, the amino acid corresponding to Y102 of SEQ ID NO:2 is substituted with alanine (A). In some aspects, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO:3. In some aspects, the amino acid corresponding to Y102 of SEQ ID NO:2 is substituted with serine (S). In some aspects, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO:4. In some aspects, the amino acid corresponding to Y102 of SEQ ID NO:2 is substituted with arginine (R). In some aspects, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO:5. In some aspects, the variant J chain or a functional fragment thereof comprises an amino acid substitution at an amino acid position corresponding to amino acid T103 of wild-type human J chain (SEQ ID NO:2). For example, the amino acid corresponding to T103 of SEQ ID NO:2 can be substituted with alanine (A). In some aspects, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO:6. In some aspects, the variant J chain or a functional fragment thereof comprises an amino acid substitution at an amino acid position corresponding to amino acid N49 or amino acid S51 of human J chain (SEQ ID NO:2), where S51 is not substituted with threonine (T), or where the J chain comprises amino acid substitutions at amino acid positions corresponding to amino acid N49 and S51 of human J chain (SEQ ID NO:2). For example, the position corresponding to N49 of SEQ ID NO:2 can be substituted with alanine (A), glycine (G), threonine (T), serine (S), or aspartic acid (D). In some aspects, the position corresponding to N49 of SEQ ID NO:2 is substituted with alanine (A). In some aspects, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO:7. In another embodiment, the position corresponding to S51 of SEQ ID NO:2 can be substituted with alanine (A) or glycine (G), for example, the position corresponding to S51 of SEQ ID NO:2 is substituted with alanine (A). In some aspects, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO:8.

[0012] In some aspects, an IgM antibody or IgM-like antibody comprising a variant J chain, IgM heavy chain constant region, or multimeric fragment thereof as provided above may have a variant IgM heavy chain constant region that comprises one or more single amino acid substitutions, deletions, or insertions relative to a reference IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for one or more single amino acid substitutions, deletions, or insertions. According to these aspects, the variant IgM heavy chain constant region may affect the serum half-life of the IgM antibody or IgM-like antibody such that the IgM antibody or IgM-like antibody exhibits a further increased serum half-life upon administration to a subject animal relative to a reference IgM antibody or IgM-like antibody that is identical except for one or more single amino acid substitutions, deletions, or insertions in the IgM heavy chain constant region and that is administered in the same manner to the same animal species. In some aspects, the further increase in serum half-life is cumulative.

[0013] The present disclosure also provides an IgM antibody or IgM-like antibody having an enhanced serum half-life, wherein the IgM antibody or IgM-like antibody comprises five or six bivalent antibody binding units or variants or fragments thereof, and wherein each binding unit comprises two variant IgM heavy chain constant regions or multimeric fragments thereof, each of which is associated with an antigen binding domain or subunit thereof. According to these aspects, each variant IgM heavy chain constant region or multimeric fragment thereof comprises one or more single amino acid substitutions, deletions, or insertions relative to a reference IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for one or more single amino acid substitutions, deletions, or insertions. Additionally, according to these aspects, the variant IgM heavy chain constant region may affect the serum half-life of the IgM antibody or IgM-like antibody. In some aspects, the IgM antibody or IgM-like antibody exhibits an increased serum half-life upon administration to a subject animal relative to a reference IgM antibody or IgM-like antibody that is identical except for one or more single amino acid substitutions, deletions, or insertions in the IgM heavy chain constant region and that is administered in the same manner to the same animal species. In some aspects, the variant IgM heavy chain constant region comprises one, two, three, or four single amino acid substitutions, deletions, or insertions relative to the reference IgM heavy chain constant region.

[0014] In some aspects, the variant IgM heavy chain constant region includes an amino acid substitution at the amino acid position corresponding to amino acid R344 of the wild-type human IgM constant region SEQ ID NO:12. For example, the amino acid corresponding to R344 of SEQ ID NO:12 can be replaced with alanine (A). In some aspects, the variant IgM heavy chain constant region is a variant human IgM heavy chain constant region and includes the amino acid sequence SEQ ID NO:31. In some aspects, the variant IgM heavy chain constant region includes an amino acid substitution at the amino acid position corresponding to amino acid E345 of the wild-type human IgM constant region SEQ ID NO:12. For example, the amino acid corresponding to E345 of SEQ ID NO:12 can be replaced with alanine (A). In some aspects, the variant IgM heavy chain constant region is a variant human IgM heavy chain constant region and includes the amino acid sequence SEQ ID NO:32. In some aspects, the variant IgM heavy chain constant region includes an amino acid substitution at the amino acid position corresponding to amino acid S401 of the wild-type human IgM constant region SEQ ID NO:12. For example, the amino acid corresponding to S401 of SEQ ID NO:12 can be replaced with alanine (A). In some aspects, the variant IgM heavy chain constant region is a variant human IgM heavy chain constant region and includes the amino acid sequence SEQ ID NO:13. In some aspects, the variant IgM heavy chain constant region includes an amino acid substitution at the amino acid position corresponding to amino acid E402 of the wild-type human IgM constant region SEQ ID NO:12. For example, the amino acid corresponding to E402 of SEQ ID NO:12 can be replaced with alanine (A). In some aspects, the variant IgM heavy chain constant region is a variant human IgM heavy chain constant region and includes the amino acid sequence SEQ IDNO:14. In some aspects, the variant IgM heavy chain constant region includes an amino acid substitution at the amino acid position corresponding to amino acid E403 of the wild-type human IgM constant region SEQ ID NO:12. For example, the amino acid corresponding to E403 of SEQ ID NO:12 can be replaced with alanine (A). In some aspects, the variant IgM heavy chain constant region is a variant human IgM heavy chain constant region and includes the amino acid sequence SEQ ID NO:34.

[0015] In the IgM antibodies or IgM-like antibodies provided herein, the increased serum half-life can include an increased alpha half-life (t 1 / 2 α), an increased beta half-life (t 1 / 2 β), or an increased t 1 / 2 α and an increased t 1 / 2β. Similarly, relative to a reference antibody, the IgM antibodies or IgM-like antibodies provided herein may further exhibit increased plasma peak concentration (Cmax), increased area under the curve (AUC), improved clearance time, or any combination thereof.

[0016] In some aspects, the IgM heavy chain constant region or a multimeric fragment or variant thereof of the IgM antibodies or IgM-like antibodies provided herein each comprises a Cμ4 domain and an IgM tailpiece (tp) domain, and may further comprise a Cμ3 domain, a Cμ2 domain, a Cμ1 domain, or any combination thereof.

[0017] In some aspects, the antigen-binding domain of the IgM antibodies or IgM-like antibodies provided herein may be a single-chain Fv (ScFv) fragment or a single-domain variable region (VHH). In some aspects, the antigen-binding domain subunit of the IgM antibodies or IgM-like antibodies provided herein may be a heavy chain variable region (VH).

[0018] In some aspects, each binding unit of the IgM antibodies or IgM-like antibodies provided herein may further comprise two light chain constant regions or fragments or variants thereof, each of which associates with the antigen-binding domain or a subunit thereof. For example, the antigen-binding domain may be an scFv fragment or the antigen-binding domain subunit may be a VL.

[0019] In some aspects, the J chain or a functional fragment or variant thereof of an IgM antibody or IgM-like antibody provided herein may further comprise one or more heterologous polypeptides directly or indirectly fused to the J chain or a functional fragment or variant thereof. In some aspects, the one or more heterologous polypeptides may be fused to the J chain or a fragment thereof via a peptide linker. Exemplary linkers may comprise at least 5 amino acids but no more than 25 amino acids and may consist of: GGGGS (SEQ ID NO:25), GGGGSGGGGS (SEQ ID NO:26), GGGGSGGGGSGGGGS (SEQ ID NO:27), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:28), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:29). In some aspects, the one or more heterologous polypeptides may be fused to the N-terminus of the J chain or a fragment or variant thereof, the C-terminus of the J chain or a fragment or variant thereof, or both the N-terminus and C-terminus of the J chain or a fragment or variant thereof. When two or more heterologous polypeptides are fused to the J chain, the heterologous polypeptides may be the same or different. In some aspects, at least one heterologous polypeptide may be a binding domain, for example, an antibody or an antigen-binding fragment thereof, for example, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment, or any combination thereof. In some aspects, the antigen-binding fragment is an scFv fragment. In some aspects, at least one heterologous polypeptide may specifically bind CD3ε. In some aspects, the J chain may be a variant (V15J) of the modified J chain of SEQ ID NO:9, for example, the amino acid sequence SEQ ID NO:10 (V15J-Y102A), the amino acid sequence SEQ ID NO:23 (V15J-T103A), or the amino acid sequence SEQ ID NO:24 (V15J-N49A).

[0020] The present disclosure also provides a composition comprising an IgM antibody or IgM-like antibody provided herein, and a pharmaceutically acceptable carrier.

[0021] The present disclosure also provides a J chain or a functional fragment thereof, the J chain or the functional fragment thereof comprising one or more single amino acid substitutions, deletions or insertions relative to a reference J chain that is identical except for one or more single amino acid substitutions, deletions or insertions, wherein the variant J chain can affect the serum half-life of an IgM antibody or an IgM-like antibody comprising the variant J chain. In certain aspects, the variant J chain comprises the amino acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:23, SEQ ID NO:24, or any combination thereof.

[0022] The present disclosure also provides an isolated polynucleotide, the isolated polynucleotide comprising a nucleic acid encoding a subunit polypeptide of an IgM or IgM-like antibody provided herein, wherein the subunit polypeptide comprises: (a) an IgM or IgM-like heavy chain constant region or a multimeric fragment thereof, (b) an antibody light chain, or (c) a J chain, a modified J chain, or a functional fragment or variant thereof, or (d) any combination thereof. In certain aspects, the subunit polypeptide comprises an IgM or IgM-like heavy chain constant region or a multimeric fragment thereof. For example, the subunit polypeptide can comprise the amino acid sequence SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:34. In certain aspects, the subunit polypeptide comprises an antibody light chain. In certain aspects, the subunit polypeptide can comprise a J chain, a modified J chain, or any functional fragment or variant thereof provided herein. For example, the subunit polypeptide can comprise the amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:23, or SEQ ID NO:24. The provided polynucleotide can comprise one, two, three, or more nucleic acid sequences encoding two, three, or more subunit polypeptides. An expression vector comprising the provided polynucleotide is also provided. A host cell comprising the provided polynucleotide or the provided expression vector is also provided.

[0023] The present disclosure also provides a method for identifying a variant J chain that can increase the serum half-life of pentameric IgM antibodies or pentameric IgM-like antibodies comprising the variant J chain. The method includes testing, in a test animal, pentameric IgM antibodies or pentameric IgM-like antibodies comprising the variant J chain or a fragment thereof that have an increased serum half-life relative to a reference pentameric IgM antibody or pentameric IgM-like antibody, wherein the variant J chain or a fragment thereof comprises defined amino acid insertions, deletions, or substitutions, and wherein the reference pentameric IgM antibody or pentameric IgM-like antibody comprises a J chain or a fragment thereof that is identical to the variant J chain except for the defined amino acid insertions, deletions, or substitutions; and recovering those variant J chains or fragments thereof that confer an increased serum half-life to the pentameric IgM antibody or pentameric IgM-like antibody relative to the reference pentameric IgM antibody or pentameric IgM-like antibody.

[0024] The present disclosure also provides a method for identifying a variant J chain that can increase the serum half-life of pentameric IgM antibodies or pentameric IgM-like antibodies comprising the variant J chain. The method includes testing the level of binding of pentameric IgM antibodies or pentameric IgM-like antibodies comprising the variant J chain or a fragment thereof to the Fc alpha-mu receptor (FcαμR), polymeric Ig receptor (pIgR), or both FcαμR and pIgR, wherein the variant J chain or a fragment thereof comprises defined amino acid insertions, deletions, or substitutions; and recovering those variant J chains or fragments thereof that confer a reduced FcαμR binding ability, reduced pIgR binding ability, or reduced FcαμR and pIgR binding ability to the pentameric IgM antibody or pentameric IgM-like antibody relative to a reference pentameric IgM antibody or pentameric IgM-like antibody comprising a J chain or a fragment thereof that is identical to the variant J chain except for the defined amino acid insertions, deletions, or substitutions. The method may further include testing, in a test animal, pentameric IgM antibodies or pentameric IgM-like antibodies comprising the recovered variant J chain or fragment thereof that have an increased serum half-life relative to a reference pentameric IgM antibody or pentameric IgM-like antibody comprising a J chain or a fragment thereof that is identical to the recovered variant J chain or fragment thereof except for the defined amino acid insertions, deletions, or substitutions.

[0025] The present disclosure also provides a method for identifying variant IgM heavy chain constant regions that can increase the serum half-life of IgM antibodies or IgM-like antibodies comprising the variant IgM heavy chain constant region. The method includes testing, in a test animal, IgM antibodies or IgM-like antibodies comprising a variant IgM heavy chain constant region that have an increased serum half-life relative to a reference pentameric IgM antibody or pentameric IgM-like antibody, wherein the variant IgM heavy chain constant region comprises defined amino acid insertions, deletions, or substitutions, and wherein the reference IgM antibody or IgM-like antibody comprises an IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for the defined amino acid insertions, deletions, or substitutions; and recovering those IgM antibodies or IgM-like antibodies comprising the variant IgM heavy chain constant region that, relative to the reference IgM antibody or IgM-like antibody, have an increased serum half-life conferred by the variant IgM heavy chain constant region.

[0026] The present disclosure also provides a method for identifying variant IgM heavy chain constant regions that can increase the serum half-life of IgM antibodies or IgM-like antibodies comprising the variant IgM heavy chain constant region. The method includes testing the level of binding of IgM antibodies or IgM-like antibodies comprising a variant IgM heavy chain constant region to the Fc alpha-mu receptor (FcαμR), Fc mu receptor (FcμR), polymeric Ig receptor (pIgR), any combination of the two receptors, or all three receptors, wherein the variant IgM heavy chain constant region comprises defined amino acid insertions, deletions, or substitutions; wherein the reference IgM antibody or IgM-like antibody comprises an IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for the defined amino acid insertions, deletions, or substitutions; and recovering those IgM antibodies or IgM-like antibodies comprising the variant IgM heavy chain constant region that, relative to a reference IgM antibody or IgM-like antibody comprising an IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for the defined amino acid insertions, deletions, or substitutions, have a reduced ability to bind FcαμR, a reduced ability to bind FcμR, a reduced ability to bind pIgR, a reduced ability to bind any two of the receptors, or a reduced ability to bind all three receptors. This method may also include testing, in a test animal, the recovered IgM antibodies or IgM-like antibodies comprising a variant IgM heavy chain constant region that have an increased serum half-life relative to a reference IgM antibody or IgM-like antibody comprising an IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for the defined amino acid insertions, deletions, or substitutions. Brief Description of the Drawings

[0028] Figures 1A to 1BShows the alignment of the amino acid sequence of the human IgM heavy chain constant region (SEQ ID NO:12) with those in mouse (GenBank:CAC20701.1, SEQ ID NO:16), cynomolgus monkey (GenBank:EHH62210.1, SEQ ID NO:30), rhesus monkey (GenBank:AAD02420.1, SEQ ID NO:17), chimpanzee (GenBank:PNI10622.1, SEQ ID NO:18) and Sumatran orangutan (GenBank:PNJ04968.1, SEQ ID NO:19). The amino acids corresponding to amino acids R345, E346, S401, E402 and E403 of SEQ ID NO:12 are boxed.

[0029] Figure 2 Is a table summarizing the binding of human and mouse immunoglobulin receptors to anti-CD20 / anti-CD3 bispecific IgM measured by ELISA, where the anti-CD20 / anti-CD3 bispecific IgM contains various mutations in the J chain neutralization and / or IgM heavy chain constant region. "WT" represents the anti-CD20 1.5.3 antibody with an IgM constant region and a V15J region as described in PCT publication number WO 2016 / 141303 before the introduction of alanine substitutions. The percentages indicate the degree of receptor binding relative to the "WT" starting antibody. The percentages indicate the receptor binding level relative to an IgM pentamer designated as 100%, which contains a wild-type IgM heavy chain constant region ("IgM", SEQ ID NO:12) and a modified J chain ("VJ" or "V15J", SEQ ID NO:9) that is fused to an scFv that binds CD3 at its N-terminus.

[0030] Figures 3A to 3E Is a graph showing the binding of various anti-CD20 / anti-CD3 bispecific IgMs with amino acid substitution mutations at the position corresponding to amino acid Y102 of SEQ ID NO:2 to pIgR. Each graph compares a single mutant with the "wild-type" anti-CD20 1.5.3 IgM antibody 1.5.3VJ (solid square). Figure 3A : Y102A mutation (open square); Figure 3B : Y102F mutation (solid triangle); Figure 3C : Y102T mutation (open triangle); Figure 3D : Y102S mutation (open diamond); Figure 3E : Y102R mutation (solid diamond).

[0031] Figure 4This is a table summarizing the pharmacokinetic data of anti-CD3 monospecific IgM and anti-CD20 / anti-CD3 bispecific IGM in mice containing various mutations in the J chain neutralization and / or the constant region of the IgM heavy chain, compared to the control group. "WT IgM" refers to the anti-CD20 1.5.3 antibody with an IgM constant region as described in PCT Publication No. WO 2016 / 141303 before the introduction of alanine substitutions. "WT J" and "WTVJ" and wild-type J chain ("J") or anti-CD3-modified J chain "VJ" region as described in PCT Publication No. WO 2016 / 141303 before the introduction of alanine substitutions. "VJH" refers to the "VJ" anti-CD3-modified J chain that also contains human serum albumin fused to the C-terminus (represented herein as SEQ ID NO:11). "A" is the serum concentration of the antibody measured at t 1 / 2 alpha; "B" is the serum concentration of the antibody measured at t 1 / 2 beta; "t 1 / 2 alpha" is the alpha half-life (in hours); "t 1 / 2 beta" is the beta half-life measured in hours, "C0" is the serum antibody concentration measured at zero time in μg / ml; "AUC 0-inf " is the area under the curve from zero time to infinity measured in μg / ml*h; and "MRT" is the average residence time of the antibody in serum measured in hours.

[0032] Figure 5 This is a curve showing the effect of the J chain Y102A mutation alone or in combination with the IgM heavy chain S401A or E401A mutation on the total serum half-life of an anti-CD20 / anti-CD3 IgM bispecific antibody compared to an IgG antibody (153IgG) containing the same anti-CD20 VH and VL binding regions. 153IgG: open circles; IgM 1.5.3V15J: solid squares; IgM 1.5.3V15J-Y102A: open squares; IgM 1.5.3S401A / V15J-Y102A: open triangles; IgM 1.5.3E402A / V15J-Y102A: solid inverted triangles.

[0033] Figure 6 This is a table comparing the pharmacokinetic parameters of IgM 1.5.3V15J and IgM 1.5.3V15J-N49A. Detailed Description

[0034] Definitions

[0035] It should be noted that the term "a" or "an" entity refers to one or more of such entities; for example, "binding molecule" is understood to represent one or more binding molecules. Thus, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein.

[0036] In addition, as used herein, "and / or" shall be taken to specifically disclose each of two particular features or components, and to disclose or not disclose the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" is intended to include: "both A and B"; "A or B"; "A" (alone); and "B" (alone). Similarly, the term "and / or" as used in a phrase 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).

[0037] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; 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 a general dictionary of many of the terms used in this disclosure to one of ordinary skill.

[0038] Units, prefixes, and symbols are expressed in their Système International de Unites (SI) acceptable form. Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in the amino to carboxy direction. The headings provided herein do not limit the various aspects or facets of the disclosure, which may be obtained by reference to the entire specification. Thus, the terms defined directly below are more fully defined by reference to the entire specification.

[0039] As used herein, the term "polypeptide" is intended to encompass both a single "polypeptide" and plural "polypeptides", and refers to a molecule composed 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 refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term 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" may be used in place of or interchangeably with any of these terms. The term "polypeptide" also refers to post-expression modification products of polypeptides, 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. Polypeptides may be derived from biological sources or produced by recombinant techniques, but need not be translated from a designated nucleic acid sequence. They may be produced in any manner, including by chemical synthesis.

[0040] The polypeptides disclosed herein may have a size of 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, 1,000 or more, or 2,000 or more amino acids. A polypeptide may have a defined three-dimensional structure, although it need not have such a structure. A polypeptide having a defined three-dimensional structure is called folded, and a polypeptide that does not have a defined three-dimensional structure but can adopt a large number of different conformations is called unfolded. As used herein, the term glycoprotein refers to a protein conjugated to at least one carbohydrate moiety, the at least one carbohydrate moiety being attached to the protein through an oxygen- or nitrogen-containing side chain of an amino acid (e.g., serine or asparagine).

[0041] An "isolated" polypeptide or fragment, variant, or derivative thereof means a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide may be removed from its native or natural environment. Recombinant-produced polypeptides and proteins expressed in host cells are considered to be isolated as disclosed herein, as are native or recombinant polypeptides that are separated, fractionated, or partially or substantially purified by any suitable technique.

[0042] As used herein, the term "non-naturally occurring polypeptide" or any of its grammatical variants is a conditional definition that specifically excludes but only excludes those forms of polypeptides that are determined or interpreted by a judge or administrative or judicial body to be "naturally occurring".

[0043] Other polypeptides disclosed herein are fragments, derivatives, analogs or variants of the above polypeptides and any combination thereof. As used herein, the terms "fragment", "variant", "derivative" and "analog" include any polypeptide that retains at least some properties (e.g., specific binding to an antigen) of the corresponding native antibody or polypeptide. In addition to the specific antibody fragments discussed elsewhere herein, fragments of a polypeptide include, for example, proteolytic fragments and deletion fragments. For example, variants of a polypeptide include fragments as described above and also polypeptides having an altered amino acid sequence due to amino acid substitutions, deletions or insertions. In certain aspects, the variants may be non-naturally occurring. Non-naturally occurring variants can be generated by using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions or additions. A derivative is a polypeptide that has been altered to exhibit additional features not found on the original polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a polypeptide may also refer to a test polypeptide having one or more amino acids chemically derivatized by 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 may replace proline; 5-hydroxylysine may replace lysine; 3-methylhistidine may replace histidine; homoserine may replace serine; and ornithine may replace lysine.

[0044] A "conservative amino acid substitution" is the replacement of one amino acid with another amino acid having a similar side chain. Families of amino acids having 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), nonpolar 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 tyrosine with phenylalanine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the antibody binds. Methods for identifying nucleotide and amino acid conservative substitutions 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)).

[0045] The term "polynucleotide" is intended to encompass both single nucleic acids and multiple nucleic acids and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). Polynucleotides can contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more nucleic acid fragments present in a polynucleotide, e.g., DNA or RNA fragments.

[0046] An "isolated" nucleic acid or polynucleotide means any form of nucleic acid or polynucleotide that is separated from its natural environment. For example, a gel-purified polynucleotide or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered "isolated". In addition, a polynucleotide fragment that has been engineered to have restriction enzyme cleavage sites for cloning (e.g., a PCR product) is considered "isolated". Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in a non-natural solution such as a buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not a transcript that can be found in nature. Isolated polynucleotides or nucleic acids also include such molecules prepared synthetically. In addition, a polynucleotide or nucleic acid can be a regulatory element or can include a regulatory element, such as a promoter, ribosome binding site, or transcription terminator.

[0047] As used herein, the term "non-naturally occurring polynucleotide" or any of its grammatical variants is a conditional definition that specifically excludes but only excludes those forms of nucleic acids or polynucleotides that are determined or interpreted by a judge or administrative or judicial body to be "naturally occurring".

[0048] As used herein, a "coding region" is a portion of a nucleic acid that consists 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 part of the coding region, but any flanking sequences (e.g., promoter, ribosome binding site, transcription terminator, intron, etc.) are not part of the coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. In addition, any vector can contain a single coding region, or can contain two or more coding regions, e.g., 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 regions that encode specific elements or motifs, such as a secretion signal peptide or a heterologous functional domain.

[0049] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide generally may include a promoter and / or other transcriptional or translational control elements 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 such that the expression of the gene product is placed under the influence or control of the one or more regulatory sequences. Two DNA fragments, such as a polypeptide coding region and a promoter associated therewith, are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or with the ability of the DNA template to be transcribed. Thus, a promoter region will be operably associated with the nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA in a predetermined cell. In addition to the promoter, other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription.

[0050] A variety of transcriptional control regions are known to those of ordinary skill in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer fragments from cytomegalovirus (immediate early promoter, associated with intron-A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters that can be induced by interferon or interleukin).

[0051] Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (notably internal ribosome entry sites or IRESs, also known as CITE sequences).

[0052] In other embodiments, the polynucleotide may be RNA, for example in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0053] Polynucleotides and nucleic acid coding regions can be associated with other coding regions encoding a secretory or signal peptide that directs the secretion of a polypeptide encoded by a polynucleotide 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 growing protein chain begins to be exported through the rough endoplasmic reticulum. Those of ordinary skill in the art will recognize that a polypeptide secreted by a vertebrate cell can have a signal peptide fused to the N-terminus of the polypeptide, and the signal peptide can be cleaved from the intact or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide is used, e.g., an immunoglobulin heavy or light chain signal peptide, or a functional derivative of such sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or murine β-glucuronidase.

[0054] As used herein, the term "binding molecule" is meant in the broadest sense to refer to a molecule that specifically binds to a receptor (e.g., an epitope or antigenic determinant). As further described herein, a binding molecule can comprise one of the multiple "antigen-binding domains" 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 aspects, a "binding molecule" comprises an antibody or antibody-like molecule as described in detail herein.

[0055] As used herein, the terms "binding domain" or "antigen-binding domain" (used interchangeably) refer to the region of a binding molecule (e.g., an antibody or antibody-like molecule) that is necessary and sufficient for specific binding to an epitope. For example, an "Fv," e.g., the variable heavy and variable light chains of an antibody, either 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, variable heavy chains (VHHs) of antibodies derived from camelid species, or six immunoglobulin complementarity-determining regions (CDRs) expressed in a fibronectin scaffold. A "binding molecule" or "antibody" as described herein can comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more "antigen-binding domains."

[0056] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof disclosed herein) includes at least the variable domain of the heavy chain (in camelid species) or at least the variable domains of the heavy and light chains. The basic immunoglobulin structure in the vertebrate system is relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988). Unless otherwise specified, the term "antibody" encompasses anything from small antigen-binding fragments of an antibody to a full-length antibody, e.g., an IgG antibody comprising two full-length heavy chains and two full-length light chains, an IgA antibody comprising four full-length heavy chains and four full-length light chains and optionally comprising a J chain and / or a secretory component, or an IgM antibody comprising ten or twelve full-length heavy chains and ten or twelve full-length light chains and optionally comprising a J chain or a functional fragment thereof.

[0057] The term "immunoglobulin" encompasses a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), some of which have subclasses (e.g., γ1 to γ4 or α1 to α2). The nature of this chain determines the "isotype" of the antibody to be IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., have been well characterized and are known to have functional specificities. Given the present disclosure, modified forms of each of these immunoglobulins will be readily discernible to those skilled in the art and are thus within the scope of the present disclosure.

[0058] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can associate with a κ or λ light chain. Generally, the light and heavy chains are covalently bonded to each other, and when an immunoglobulin is expressed, e.g., by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In a heavy chain, the amino acid sequence proceeds 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 certain antibodies (e.g., IgG antibodies) includes two heavy chain subunits and two light chain subunits covalently linked by disulfide bonds to form a "Y" structure (also referred to herein as an "H2L2" structure) or a "binding unit."

[0059] As used herein, the term "binding unit" refers to a portion of a binding molecule such as an antibody, antibody-like molecule, antigen-binding fragment thereof, or multimeric fragment thereof, the portion corresponding to the standard "H2L2" immunoglobulin structure, i.e., two heavy chains or fragments thereof and two light chains or fragments thereof. In some aspects, e.g., where the binding molecule is a bivalent IgG antibody or antigen-binding fragment thereof, the terms "binding molecule" and "binding unit" are equivalent. In other aspects, e.g., where the binding molecule is a multimer, e.g., a dimeric IgA antibody or IgA-like antibody, a pentameric IgM antibody or IgM-like antibody, or a hexameric IgM antibody or IgM-like antibody, the binding molecule comprises two or more "binding units". Two binding units in the case of an IgA dimer, or five or six binding units in the case of an IgM pentamer or hexamer, respectively. The binding unit need not include the heavy and light chains of a full-length antibody but will typically be bivalent, i.e., will include two "antigen-binding domains" as defined above. As used herein, certain binding molecules provided in the present disclosure are "dimers" and include two bivalent binding units comprising an IgA constant region or multimeric fragment thereof. Certain 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 multimeric fragment thereof. A binding molecule, such as 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".

[0060] As used herein, an "IgM-like antibody" refers to a variant antibody that still retains the ability to form a hexamer or associate with a J chain to form a pentamer. An IgM-like antibody typically includes at least the Cμ4-tp domain of the IgM constant region but may include heavy chain constant region domains from other antibody isotypes (e.g., IgG) from the same or different species. An IgM-like antibody may likewise be an antibody fragment in which one or more constant regions are deleted, provided that the IgM-like antibody is capable of forming a hexamer and / or pentamer. Thus, an IgM-like antibody may be a hybrid IgM / IgG antibody or may be a multimeric fragment of an IgM antibody.

[0061] 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 respect 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" denote the presence of two, four and six antigen-binding domains, respectively. In the case where each binding unit is bivalent, a typical IgM antibody or IgM-like antibody can have a valence of 10 or 12. A bivalent or multivalent binding molecule, e.g., an antibody or antibody-like molecule, can be monospecific, i.e., all antigen-binding domains are identical, or can be bispecific or multispecific, e.g., where two or more antigen-binding domains are different, e.g., binding to different epitopes on the same antigen or binding to completely different antigens.

[0062] The term "epitope" includes any molecular determinant capable of specifically binding to the antigen-binding domain of an antibody or antibody-like molecule. In some aspects, an epitope can include the chemically reactive surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in some aspects, can have three-dimensional structural features and / or charge features. An epitope is the target region bound by the antigen-binding domain of an antibody.

[0063] 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). The target can be, for example, a polypeptide, nucleic acid, carbohydrate, lipid or other molecule. Additionally, a "target" can be, for example, a cell, organ or organism that contains an epitope to which a binding molecule (e.g., an antibody or antibody-like molecule) can bind.

[0064] The light and heavy chains are divided into regions that are structurally and functionally homologous. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of the variable light chain (VL) and variable heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (e.g., CH1, CH2, CH3 or CH4) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they get further 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 contain the carboxyl termini of the heavy and light chains, respectively.

[0065] "Full-length IgM antibody heavy chain" is a polypeptide that, in the N-terminal to C-terminal direction, comprises 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 comprise a tailpiece.

[0066] As noted 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, subsets of the VL domain and VH domain or complementarity-determining regions (CDRs) of a binding molecule (e.g., an antibody or antibody-like molecule) combine to form an antigen-binding domain. More specifically, the 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 that comprises two H2L2 binding units covalently linked by a disulfide bond and a J chain, and the molecule can also associate with a secretory component, and IgM can form a pentameric or hexameric molecule that comprises five or six H2L2 binding units covalently linked by disulfide bonds and optionally a J chain.

[0067] The six "complementarity-determining regions" or "CDRs" present in an antibody antigen-binding domain are short, non-contiguous amino acid sequences that, when the antibody assumes its three-dimensional conformation in an aqueous environment, are specifically positioned to form the antigen-binding domain. The remaining amino acids in the antigen-binding domain are referred to as "framework" regions, which exhibit less inter-molecular variability. The framework regions predominantly 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 the positioning of the CDRs in the correct orientation through inter-chain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunologically reactive antigen. This complementary surface facilitates the 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, as they are defined in a variety of different ways (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entirety).

[0068] Where two or more definitions exist for terms used and / or recognized within the art, the definition of a term used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementary determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described, for example, by Kabat et al., U.S. Dept. 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. The Kabat and Chothia definitions include an overlap or subset of amino acids when compared to each other. However, unless otherwise specified, reference to the CDR of an antibody or its variant using either definition (or other definitions known to one of ordinary skill in the art) is intended to fall within the scope of the term as defined and used herein. The appropriate amino acids encompassing the CDR defined by each of the above-cited references are listed in Table 1 below for comparison. The exact number of amino acids encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, one of ordinary skill in the art can routinely determine which amino acids comprise a particular CDR.

[0069] Table 1 CDR Definitions *

[0070] 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

[0071] * The numbering of all CDR definitions in Table 1 is according to the numbering convention proposed by Kabat et al. (see below).

[0072] For example, the IMGT information system (imgt_dot_cines_dot_fr / )( / V-Quest) can also be used to analyze antibody variable domains to identify variable region segments, including CDRs. (See, e.g., Brochet et al., Nucl. Acids Res., 36:W503-508, 2008).

[0073] Kabat et al. also defined a numbering system applicable to the sequences of variable domains of any antibody. A person of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain 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. Dept. 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 use consecutive numbering.

[0074] 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. Dept. of Health and Human Services (1991). The IgM constant region can be numbered sequentially (i.e., amino acid #1 starts from the first amino acid of the constant region), or numbered using the Kabat numbering scheme. The following lists the comparison of the sequential numbering (represented herein as SEQ ID NO:12) and the numbering by the Kabat system of the human IgM constant region:

[0075] Sequential (SEQ ID NO:12) / KABAT Numbering Key for IgM Heavy Chain

[0076]

[0077] Binding molecules, e.g., antibodies, antibody-like molecules, antigen-binding fragments thereof, variants or derivatives, 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 in, e.g., U.S. Patent No. 5,892,019.

[0078] "Specific binding" generally refers to a binding molecule (e.g., an antibody or a fragment, variant, or derivative thereof) that binds to an epitope through its antigen binding domain, and binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, when a binding molecule (e.g., an antibody or antibody-like molecule) more easily binds to an epitope through its antigen binding domain than a random, unrelated epitope binding, it can be judged as "specifically binding" to the epitope. The term "specificity" is used herein to define the relative affinity of a binding molecule to a certain epitope. For example, it can be considered that for a given epitope, binding molecule "A" has a higher specificity than binding molecule "B", or binding molecule "A" can be judged to have a higher specificity for binding to epitope "C" than for binding to a related epitope "D".

[0079] The binding molecules (e.g., antibodies or fragments, variants, or derivatives thereof) disclosed herein can be judged to have a binding affinity of less than or equal to 5×10 -2 Second -1 , 10 -2 Second -1 , 5X 10 -3 Second -1 , 10 -3 Second -1 , 5X 10 -4 Second -1 , 10 -4 Second -1 , 5X 10 -5 Second -1 or 10 -5 Second -1 , 5X 10 -6 Second -1 , 10 -6 Second -1 , 5X 10 -7 Second -1 or 10 -7 Second -1 The dissociation rate (k(off)) of the protein binds to the target antigen.

[0080] The binding molecules (e.g., antibodies or antigen-binding fragments, variants or derivatives) disclosed herein can be determined to bind to the target antigen with an association rate (k(on)) greater than or equal to 10 3 M -1 seconds -1 、5 X 10 3 M -1 seconds -1 、10 4 M -1 seconds -1 、5 X 10 4 M -1 seconds -1 、10 5 M -1 seconds -1 、5 X 10 5 M -1 seconds -1 、10 6 M -1 seconds -1 、or 5 X 10 6 M -1 seconds -1 or 10 7 M -1 seconds -1 and bind to the target antigen.

[0081] If a binding molecule (e.g., an antibody or a fragment, variant or derivative thereof) preferentially binds to an epitope such that it blocks to some extent the binding of a reference antibody or antigen-binding fragment to a given epitope, it can be determined to competitively inhibit the binding of the reference antibody or antigen-binding antibody to that epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. The binding molecule can be determined to competitively inhibit the binding of the 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%.

[0082] As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope to one or more antigen-binding domains of, for example, an immunoglobulin molecule. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988), pp. 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, e.g., Harlow, pp. 29-34. Avidity is related not only to the affinity of the individual antigen-binding domains in the population for a particular epitope, but also to the valency of the immunoglobulin and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen having a highly repetitive epitope structure such as a polymer will be a high-avidity interaction. The interaction between a bivalent monoclonal antibody and a receptor present at high density on the cell surface will also have high avidity.

[0083] Binding molecules (e.g., antibodies or fragments, variants, or derivatives thereof) as disclosed herein may also be described or specified according to 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) specific for one antigen to react with a second antigen; a measure of the relatedness 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. Cross-reactive epitopes typically contain many of the same complementary structural features as the inducing epitope and, in some cases, may actually be more suitable than the original epitope.

[0084] Binding molecules (e.g., antibodies or fragments, variants, or derivatives thereof) may also be described or specified according to their binding affinity for an antigen. For example, a binding molecule may have a binding affinity of not greater than 5x10 -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, 5x10-11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M or 10 -15 Dissociation constant of M or K D Binds to the antigen.

[0085] "Antigen-binding antibody fragments" that include single-chain antibodies or other antigen-binding domains can exist alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domains, J chain, or secretory component. Also included are antigen-binding fragments that can include any combination of one or more variable regions with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domains, J chain, or secretory component. Binding molecules, e.g., antibodies or antigen-binding fragments thereof, can be from any animal source, including birds and mammals. Antibodies can be antibodies of human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In another embodiment, the variable region can be of chondricthoid origin (e.g., from sharks). As used herein, "human" antibodies include antibodies having the amino acid sequences of human immunoglobulins and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and can in some cases express endogenous immunoglobulins and in some cases not, as described hereinbelow and, for example, as described in U.S. Patent No. 5,939,598 to Kucherlapati et al. According to aspects of the present disclosure, IgM or IgM-like antibodies provided herein can include antigen-binding fragments of the antibody, e.g., scFv fragments, provided that the IgM or IgM-like antibody is capable of forming multimers, e.g., hexamers or pentamers.

[0086] As used herein, the term "heavy chain subunit" includes an amino acid sequence derived from an immunoglobulin heavy chain. A binding molecule comprising a heavy chain subunit (e.g., an antibody or antibody-like molecule) can include at least one of the following: a VH domain, a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof. For example, a binding molecule (e.g., an antibody, antibody-like molecule, or fragment, variant, or derivative thereof) can include, but is not limited to: in addition to the VH domain, a CH1 domain; a CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In certain aspects, a binding molecule (e.g., an antibody, antibody-like molecule, or fragment, variant, or derivative thereof) can include, in addition to the VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, and a J chain. In addition, a binding molecule (e.g., an antibody or antibody-like molecule) for use in the present disclosure can lack certain constant region portions, e.g., all or part of the CH2 domain. Those of ordinary skill in the art will understand that these domains (e.g., heavy chain subunits) can be modified such that they differ in amino acid sequence from the original immunoglobulin molecule. According to aspects of the present disclosure, the IgM or IgM-like antibodies provided herein comprise a sufficient portion of the IgM heavy chain constant region to allow the IgM or IgM-like antibody to form a multimer, e.g., a hexamer or pentamer.

[0087] As used herein, the term "light chain subunit" includes an amino acid sequence derived from an immunoglobulin light chain. The light chain subunit includes at least VL and can also include a CL (e.g., Cκ or Cλ) domain.

[0088] A binding molecule (e.g., an antibody, antibody-like molecule, an antigen-binding fragment, variant, or derivative thereof, or a multimeric fragment thereof) can be described or specified according to one or more epitopes or portions of an antigen that it recognizes or specifically binds to. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant". A target antigen can comprise a single epitope or at least two epitopes and can include any number of epitopes, depending on the size, conformation, and type of the antigen.

[0089] As previously described, the subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is at the amino terminus of the hinge region of a typical IgG heavy chain molecule.

[0090] As used herein, the term "CH2 domain" includes the portion of the heavy chain molecule that, for example, extends from approximately amino acid 244 to amino acid 360 of an IgG antibody using a conventional numbering scheme (amino acids 244 to 360, Kabat numbering system; and amino acids 231 to 340, EU numbering system; see Kabat EA et al., op. cit). The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 amino acids. Certain immunoglobulin classes, e.g., IgM, also include a CH4 region.

[0091] 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.

[0092] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0093] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained from or derived from a first species and the constant region (which may be full-length, 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 will be human.

[0094] The term "multispecific antibody" or "bispecific antibody" refers to an antibody or antibody-like molecule that has antigen-binding domains directed against two or more different epitopes within a single antibody molecule. In addition to the standard antibody structure, other binding molecules can be constructed with two binding specificities. The epitopes bound by a bispecific or multispecific antibody can be simultaneous or sequential. Tricomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed recombinantly. ( and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). Bispecific antibodies can also be diabodies.

[0095] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in the heavy chain and / or the light chain is altered by at least partial replacement of one or more amino acids in the CDR or framework region. In some aspects, entire CDRs from an antibody of known specificity can be transplanted into the framework region of a heterologous antibody. Although the substituted CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework region is derived, the CDRs can also be derived from antibodies of different classes, e.g., antibodies from different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity are transplanted into a human heavy or light chain framework region is referred to herein as a "humanized antibody". In some aspects, not all of the CDRs are replaced with the complete CDRs from the donor variable region, but the antigen-binding ability of the donor can still be transferred to the receptor variable domain. Functional engineered or humanized antibodies can be obtained by performing routine experiments or by trial and error, as explained, for example, in U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, which are entirely within the capabilities of those skilled in the art.

[0096] As used herein, the term "engineered" 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, or some combination of these techniques.

[0097] As used herein, the terms "linked", "fused", or other grammatical equivalents are used interchangeably. These terms refer to the joining together of two or more elements or components by any means, including chemical conjugation or recombinant means. "In-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous, longer ORF in such a way as to maintain the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more fragments corresponding to polypeptides encoded by the original ORFs (the fragments of which are not normally joined together in nature). Although the reading frame is thus continuous throughout the fusion fragment, the fragments can be physically or spatially separated, e.g., by an in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused in-frame but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, provided that the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0098] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in the polypeptide in the direction from the amino terminus to the carboxyl terminus, where amino acids that are adjacent to each other in the sequence are contiguous in the primary structure of the polypeptide. The portion of the polypeptide at the other end, the "amino terminus" or "N-terminus", is the portion that occurs earlier in the sequential polypeptide chain. Similarly, the portion of the polypeptide at the other end, the "carboxyl terminus" or "C-terminus", is the portion that occurs 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.

[0099] 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 within a cell, including but not limited to gene knockdown as well as transient and stable expression. It includes but is not limited to the transcription of a gene into RNA, e.g., messenger RNA (mRNA), and the translation of such mRNA into one or more polypeptides. If the end product desired is a biochemical substance, expression includes creating that 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 can be a polypeptide translated from the transcript. The gene products 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.).

[0100] Terms such as "treat" or "alleviate" refer to therapeutic measures that cure, slow down, relieve the symptoms of an existing diagnosed pathological disorder or condition and / or halt or slow the progression of an existing diagnosed pathological disorder or condition. Terms such as "prevent", "avoid", "deter", etc. refer to preventive measures or prophylactic measures that prevent the development of an undiagnosed target pathological disorder or condition. Thus, "those in need of treatment" can include those who already have a disorder; those who are predisposed to a disorder; and those in whom a disorder will be prevented.

[0101] As used herein, the term "serum half-life" or "plasma half-life" refers to the time (e.g., in minutes, hours or days) required after administration to reduce the serum or plasma concentration of a drug (e.g., a binding molecule as described herein, such as an antibody, antibody-like molecule or fragment thereof) by 50%. Two half-lives can be described: the alpha half-life, α half-life or t 1 / 2 α, which is the rate of decline in plasma concentration due to the redistribution of the drug from the central compartment (e.g., blood in the case of intravenous delivery) to peripheral compartments (e.g., tissues or organs), and the beta half-life, β half-life or t 1 / 2 β, which is the rate of decline due to excretion or metabolism processes.

[0102] As used herein, the term "area under the plasma drug concentration-time curve" or "AUC" reflects the actual exposure of a human to a drug after administration of a given dose of the drug and is expressed in mg*h / L. This area under the curve is measured from time 0 (t0) to infinity (∞) and depends on the rate of drug clearance from the body and the dose administered.

[0103] As used herein, the term "mean residence time" or "MRT" refers to the average length of time a drug remains in the body.

[0104] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject in need of diagnosis, prognosis, or treatment, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0105] As used herein, phrases such as "subjects who will benefit from treatment" and "animals in need of treatment" refer to a subset of subjects from all intended subjects who will benefit from administration of a given therapeutic agent, e.g., a binding molecule such as an antibody that comprises one or more antigen-binding domains. Such binding molecules, e.g., antibodies, can be used, for example, in diagnostic procedures and / or for the treatment or prevention of diseases.

[0106] IgM or IgM-like antibody

[0107] IgM is the first immunoglobulin produced by B cells in response to antigen stimulation and is naturally present in serum at approximately 1.5 mg / ml, with a half-life of approximately 5 days. IgM is a pentameric or hexameric molecule and thus consists of five or six binding units. An IgM binding unit typically consists of two light chains and two heavy chains. While the IgG heavy chain constant region contains three heavy chain constant regions (CH1, CH2, and CH3), the heavy (μ) constant region of IgM also contains a fourth constant region (CH4) and includes a C-terminal "tail piece". The human IgM constant region typically contains the amino acid sequence SEQ ID NO:12 (identical to, for example, GenBank accession numbers pir||S37768, CAA47708.1, and CAA47714.1). The human Cμ1 region ranges from approximately amino acid 5 to approximately amino acid 102 of SEQ ID NO:12; the human Cμ2 region ranges from approximately amino acid 114 to approximately amino acid 205 of SEQ ID NO:12, the human Cμ3 region ranges from approximately amino acid 224 to approximately amino acid 319 of SEQ ID NO:12, the Cμ4 region ranges from approximately amino acid 329 to approximately amino acid 430 of SEQ ID NO:12, and the tail piece ranges from approximately amino acid 431 to approximately amino acid 453 of SEQ ID NO:12. SEQ ID NO:12 is shown below.

[0108] SEQ ID NO:12:

[0109] GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0110] There are other forms of human IgM constant regions with minor sequence variations, including but not limited to GenBank accession numbers P01871.4, CAB37838.1, and pir||MHHU. Amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO:12 described and claimed elsewhere in the present disclosure can also be incorporated into alternative human IgM sequences, as well as into IgM constant region amino acid sequences of other species, such as those shown in FIG. 1.

[0111] Each IgM heavy chain constant region can associate with an antigen-binding domain (e.g., scFv or VHH) or a subunit of an antigen-binding domain (e.g., VH region).

[0112] Five IgM binding units can form a complex with another small polypeptide chain (J chain) to form a pentameric IgM antibody or IgM-like antibody. The precursor form of the human J chain, SEQ ID NO:1, is shown below. The signal peptide (underlined) extends from amino acid 1 of SEQ ID NO:1 to approximately amino acid 22, and the mature human J chain extends from approximately amino acid 23 of SEQ ID NO:1 to amino acid 159. The mature human J chain includes the amino acid sequence SEQ ID NO:2.

[0113] SEQ ID NO:1:

[0114] MKNHLLFWGVLAVFIKAVHVKA QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0115] SEQ ID NO:2 is shown below.

[0116] SEQ ID NO:2:

[0117] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0118] In the absence of the J chain, IgM antibodies or IgM-like antibodies typically assemble into hexamers containing up to twelve antigen-binding domains. In the presence of the J chain, IgM antibodies or IgM-like antibodies typically assemble into pentamers containing up to ten antigen-binding domains, or more antigen-binding domains if the J chain is a modified J chain that includes a heterologous polypeptide containing additional antigen-binding domains. The assembly of five or six IgM binding units into pentameric or hexameric IgM antibodies or IgM-like antibodies is thought to involve Cμ4 and the tailpiece domain. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Thus, the pentameric or hexameric IgM antibodies provided in the present disclosure typically include at least the Cμ4 and / or tailpiece domains (collectively referred to herein as Cμ4-tp). Thus, the "polymeric fragment" of the IgM heavy chain constant region includes at least the Cμ4-tp domain. The IgM heavy chain constant region may also include the Cμ3 domain or a fragment thereof, the Cμ2 domain or a fragment thereof, the Cμ1 domain or a fragment thereof, and / or other IgM heavy chain domains. In certain aspects, a binding molecule (e.g., an IgM antibody or IgM-like antibody provided herein) may include the complete IgM heavy (μ) chain constant domain, e.g., SEQ ID NO:12, or a variant, derivative, or analogue thereof.

[0119] In certain aspects, 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 a polymeric fragment or variant thereof, each of which is associated with an antigen-binding domain or a subunit thereof. In certain aspects, the two IgM heavy chain constant regions are human heavy chain constant regions.

[0120] When the IgM or IgM-like antibody provided herein is pentameric, the IgM or IgM-like antibody typically further comprises a J chain or a functional fragment or variant thereof. In certain aspects, the J chain is a modified J chain or a variant thereof that further comprises one or more heterologous moieties attached thereto, as described elsewhere herein. In certain aspects, as discussed elsewhere herein, the J chain may be mutated to affect (e.g., enhance) the serum half-life of the IgM or IgM-like antibody provided herein.

[0121] The IgM heavy chain constant region may include one or more Cμ1 domains or fragments or variants thereof, Cμ2 domains or fragments or variants thereof, Cμ3 domains or fragments or variants thereof, and / or Cμ4 domains or fragments or variants thereof, provided that the constant region can perform the required functions in an IgM or IgM-like antibody, for example, associate with a second IgM constant region to form an antigen-binding domain, and / or associate with other binding units (and the J chain in the case of a pentamer) to form a hexamer or pentamer. In some aspects, two IgM heavy chain constant regions or fragments or variants thereof within a single binding unit each comprise a Cμ4 domain or fragment or variant thereof, a tailpiece (tp) or fragment or variant thereof, or a combination of a Cμ4 domain and a TP or fragment or variant thereof. In some aspects, two IgM heavy chain constant regions or fragments or variants thereof within 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.

[0122] Modified J chain

[0123] In some aspects, the J chain of the pentameric IgM or IgM-like antibodies provided herein can be modified, for example, by introducing a heterologous moiety or two or more heterologous moieties (e.g., polypeptides), without interfering with the ability of the IgM or IgM-like antibody to assemble and bind to one or more of its binding targets. See U.S. Patent No. 9,951,134, PCT Publication No. WO 2017 / 059387, and PCT Publication No. WO 2017 / 059380, each of which is incorporated herein by reference in its entirety. Accordingly, the IgM or IgM-like antibodies provided herein, including the multispecific IgM or IgM-like antibodies described elsewhere herein, can comprise a modified J chain or a functional fragment or variant thereof, the modified J chain or a functional fragment or variant thereof comprising a heterologous moiety introduced into the J chain or a fragment or variant thereof, e.g., a heterologous polypeptide. In some aspects, the heterologous moiety can be a peptide or polypeptide sequence that is in-frame fused or chemically conjugated to the J chain or a fragment or variant thereof, wherein the heterologous polypeptide is directly or indirectly fused to the variant J chain or a functional fragment thereof. In some aspects, the heterologous polypeptide is fused to the J chain or a functional fragment thereof via a peptide linker (e.g., a peptide linker consisting of at least 5 amino acids but not more than 25 amino acids). In some aspects, the peptide linker comprises GGGGS (SEQ ID NO:25), GGGGSGGGGS (SEQ ID NO:26), GGGGSGGGGSGGGGS (SEQ ID NO:27), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:28), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:29). In some aspects, the heterologous moiety can be a chemical moiety conjugated to the J chain. The heterologous moiety to be attached to the J chain can include, but is not limited to, a binding moiety (e.g., an antibody or an antigen-binding fragment thereof, e.g., a single-chain Fv (ScFv) molecule), a stabilizing peptide that can increase the half-life of the IgM or IgM-like antibody, or a chemical moiety such as a polymer or a cytotoxin.

[0124] In some embodiments, the modified J chain can comprise an antigen-binding domain, which can include, but is not limited to, a polypeptide (including a small peptide) capable of specifically binding to a target antigen. In certain aspects, the antigen-binding domain associated with the modified J chain can be an antibody or an antigen-binding fragment thereof, as described elsewhere herein. In certain aspects, the antigen-binding domain can be, for example, a scFv antigen-binding domain or a single-chain antigen-binding domain derived from camelid or chondrichthyan antibodies. The antigen-binding domain can be introduced into the J chain at any position that allows the antigen-binding domain 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. Insertion positions include, but are not limited to, at or near the C-terminus, at or near the N-terminus, or an internal position accessible based on the three-dimensional structure of the J chain. In certain aspects, the antigen-binding domain can be introduced into the mature human J chain of SEQ ID NO:2 between cysteine residues 92 and 101. In another aspect, the antigen-binding domain can be introduced into the human J chain of SEQ ID NO:2 at or near a glycosylation site. In another aspect, the antigen-binding domain 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 acids from the N-terminus.

[0125] Pentameric IgM or IgM-like antibodies with J chain mutations that alter serum half-life

[0126] The present disclosure provides an IgM antibody or a multimeric fragment thereof having an enhanced serum half-life, e.g., a pentameric IgM-like antibody or a multimeric fragment thereof. In certain aspects, the IgM or IgM-like antibody provided herein comprises five bivalent antibody binding units or variants or multimeric fragments thereof and a variant of the J chain or a functional fragment thereof. A "functional fragment" of the J chain refers to a J chain fragment (or a fragment of a variant J chain or a modified J chain provided herein) that is still capable of associating with five IgM binding units to form a pentamer or with two IgA binding units to form a dimer. Each binding unit of the provided IgM antibody or IgM-like antibody comprises two IgM heavy chain constant regions or fragments or variants thereof (wherein the fragment or variant is capable of multimerization), wherein each constant region is associated with an antigen-binding domain or a subunit thereof. As provided herein, a "variant J chain or a functional fragment thereof" may include one or more single amino acid substitutions, deletions, or insertions relative to a reference J chain or a functional fragment thereof, wherein the reference J chain is identical to the variant J chain except for one or more single amino acid substitutions, deletions, or insertions. In certain aspects, the reference J chain is a wild-type J chain. The variant J chain or a functional fragment thereof provided herein may affect (e.g., enhance) the serum half-life of an IgM antibody or an IgM-like antibody comprising the variant J chain or a functional fragment thereof. The variant J chain or a functional fragment thereof provided herein may also affect (e.g., inhibit) the ability of an IgM antibody or an IgM-like antibody comprising the variant J chain or a functional fragment thereof to bind to a cognate receptor (e.g., the Fcαμ receptor (FcαμR) or the polymeric Ig receptor (pIgR)). The term "one or more single amino acid substitutions, insertions, and deletions" means that each amino acid of the amino acid sequence of the variant J chain or a functional fragment thereof may be individually substituted, deleted, or may have a single amino acid inserted adjacent thereto relative to the reference J chain, provided that the variant J chain or a functional fragment thereof must still be capable of functioning to assemble with an IgM heavy chain or an IgM-like heavy chain and an antibody light chain to form an IgM pentamer or an IgM-like pentamer.

[0127] In some aspects, variant J chains or functional fragments thereof provided herein can have a single amino acid substitution, insertion, or deletion, a combination of two single amino acid substitutions, insertions, or deletions (e.g., two single amino acid substitutions or one single amino acid substitution and one single amino acid insertion or deletion), a combination of three single amino acid substitutions, insertions, or deletions, a combination of four single amino acid substitutions, insertions, or deletions, or more, wherein one, two, three, four, or more single amino acid substitutions, insertions, or deletions can affect (e.g., enhance) the serum half-life of an IgM antibody or IgM-like antibody comprising the variant J chain or a functional fragment thereof. Thus, relative to a reference IgM or IgM-like antibody that is otherwise identical except for one or more single amino acid substitutions, deletions, or insertions in the variant J chain or a functional fragment thereof, the provided IgM or IgM-like antibody can exhibit an increased serum half-life when administered to a test animal (e.g., a mouse model), wherein the provided antibody and the reference antibody are administered to the same animal species in the same manner.

[0128] In some aspects, relative to a reference antibody, the serum half-life of the provided IgM or IgM-like antibody comprising a variant J chain, e.g., an alpha half-life (t 1 / 2 alpha), a beta half-life (t 1 / 2 beta), t 1 / 2 alpha and t 1 / 2 beta, or a total half-life can be increased by at least 0.1-fold, at least 0.5-fold, at least 1-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more. In some aspects, the increase in serum half-life approaches the increase in serum half-life of an IgG antibody comprising the same antigen-binding domain.

[0129] In some aspects, relative to a reference antibody, an IgM antibody or IgM-like antibody comprising a variant J chain provided herein also exhibits other improved pharmacokinetic parameters, e.g., an increased plasma peak concentration (C max ), an increased area under the curve from T0 to infinity (AUC), e.g., an improved clearance time, an increased mean residence time (MRT), or any combination thereof. In some aspects, relative to a reference IgM or IgM-like antibody that is otherwise identical except for one or more single amino acid substitutions, deletions, or insertions in the variant J chain or a functional fragment thereof, the AUC can be increased by at least 0.1-fold, at least 0.5-fold, at least 1-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more, wherein the provided antibody and the reference antibody are administered to the same animal species in the same manner.

[0130] In some aspects, the J chain of the IgM antibody or IgM-like antibody provided herein contains an amino acid substitution at the amino acid position corresponding to amino acid Y102 (SEQ ID NO:2) of the mature wild-type human J chain. "The amino acid corresponding to amino acid Y102 of the mature wild-type human J chain" refers to the amino acid in the J chain sequence of any species that is homologous to Y102 in the human J chain. 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. See U.S. Patent No. 9,951,134 Figure 4 , which is incorporated herein by reference. As shown in the examples, certain mutations at the position corresponding to Y102 of SEQ ID NO:2 can inhibit the binding of certain immunoglobulin receptors, such as human or murine Fcαμ receptor, murine Fcμ receptor, and / or human or murine polymeric Ig receptor (pIg receptor) to the IgM pentamer containing the mutant J chain. See, for example, Figure 2 . In some aspects, the amino acid corresponding to Y102 of SEQ ID NO:2 can be replaced by any amino acid. In some aspects, the amino acid corresponding to Y102 of SEQ ID NO:2 can be replaced by alanine (A), serine (S), or arginine (R). In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO:2 can be replaced by alanine. In a particular aspect, the J chain or its functional fragment or variant is a variant human J chain and contains the amino acid sequence SEQ ID NO:3. SEQ ID NO:3 is shown below.

[0131] SEQ ID NO:3:

[0132] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCATYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0133] In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO:2 can be replaced by serine. In a particular aspect, the J chain or its functional fragment or variant is a variant human J chain and contains the amino acid sequence SEQ ID NO:4. SEQ ID NO:4 is shown below.

[0134] SEQ ID NO:4

[0135] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCSTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0136] In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO:2 can be replaced with arginine. In a particular aspect, the J chain or a functional fragment or variant thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO:5. SEQ ID NO:5 is shown below.

[0137] SEQ ID NO:5

[0138] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCRTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0139] In certain aspects, the J chain of the IgM antibody or IgM-like antibody provided herein comprises an amino acid substitution at the amino acid position corresponding to amino acid T103 of the wild-type mature human J chain (SEQ ID NO:2). "The amino acid corresponding to amino acid T103 of the wild-type mature human J chain" refers to the amino acid in the J chain sequence of any species that is homologous to T103 in the human J chain. The position corresponding to T103 in SEQ ID NO:2 is conserved in the J chain amino acid sequences of at least 37 other species. See Figure 4 U.S. Patent No. 9,951,134, which is incorporated herein by reference. As shown in the Examples, certain mutations at the position corresponding to T103 in SEQ ID NO:2 can inhibit the binding of the human Fcαμ receptor to the IgM pentamer comprising the mutant J chain. See Figure 2 In certain aspects, the amino acid corresponding to T103 of SEQ ID NO:2 can be replaced with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO:2 can be replaced with alanine. In a particular aspect, the J chain or a functional fragment or variant thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO:6. SEQ ID NO:6 is shown below.

[0140] SEQ ID NO:6:

[0141] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYAYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0142] In some aspects, the variant J chain or a functional fragment thereof of the IgM antibody or IgM-like antibody provided herein comprises an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of the mature wild-type human J chain (SEQ ID NO:2), provided that S51 is not substituted with threonine (T), or wherein the J chain comprises amino acid substitutions at the amino acid positions corresponding to amino acid N49 and S51 of the wild-type mature human J chain (SEQ ID NO:2). Similarly, "the amino acid corresponding to amino acid N49 of SEQ ID NO:2 of the wild-type mature human J chain or the amino acid corresponding to S51 of SEQ ID NO:2" refers to the amino acid in the J chain sequence of any species that is homologous to N49 and / or S51 in the human J chain. The positions corresponding to N49 and S51 in SEQ ID NO:2 are conserved in the J chain amino acid sequences of at least 43 other species. See Figure 4 of U.S. Patent No. 9,951,134, which is incorporated herein by reference. The amino acids corresponding to N49 and S51 of SEQ ID NO:2 and the amino acid corresponding to I50 of SEQ ID NO:2 contain an N-linked glycosylation motif in the J chain. Thus, mutations at N49 and / or S51 (except for the single threonine substitution at S51) can prevent glycosylation of this motif. In some aspects, the asparagine at the position corresponding to N49 of SEQ ID NO:2 can be substituted with any amino acid. In some aspects, the asparagine at the position corresponding to N49 of SEQ ID NO:2 can be substituted with alanine (A), glycine (G), threonine (T), serine (S), or aspartic acid (D). In a particular aspect, the position corresponding to N49 of SEQ ID NO:2 can be substituted with alanine (A). In a particular aspect, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO:7. SEQ ID NO:7 is shown below.

[0143] SEQ ID NO:7:

[0144] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0145] In some aspects, the serine at the position corresponding to S51 of SEQ ID NO:2 can be replaced with any amino acid other than threonine. In some aspects, the serine at the position corresponding to S51 of SEQ ID NO:2 can be replaced with alanine (A) or glycine (G). In a particular aspect, the position corresponding to S51 of SEQ ID NO:2 can be replaced with alanine (A). In a particular aspect, the variant J chain or its functional fragment is a variant human J chain and comprises the amino acid sequence SEQ ID NO:8. SEQ ID NO:8 is shown below.

[0146] SEQ ID NO:8:

[0147] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENIADPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0148] In some aspects, the provided variant J chain or functional fragment thereof of an IgM antibody or IgM-like antibody provided herein is a modified J chain, for example, as provided in U.S. Patent No. 9,951,134. In some aspects, the modified J chain further comprises a heterologous polypeptide, wherein the heterologous polypeptide is directly or indirectly fused to the variant J chain or a functional fragment thereof. In some aspects, the heterologous polypeptide is fused to the variant J chain or a functional fragment thereof via a peptide linker (e.g., a peptide linker consisting of at least 5 but no more than 25 amino acids). In some aspects, the peptide linker comprises GGGGS (SEQ ID NO:25), GGGGSGGGGS (SEQ ID NO:26), GGGGSGGGGSGGGGS (SEQ ID NO:27), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:28), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:29). The heterologous polypeptide can be fused to the N-terminus of the variant J chain or a functional fragment thereof, the C-terminus of the variant J chain or a functional fragment thereof, or the heterologous polypeptide can be fused to both the N-terminus and the C-terminus of the variant J chain or a functional fragment thereof. In some aspects, the heterologous polypeptide comprises an antigen-binding domain. In some aspects, the binding domain of the heterologous polypeptide is an antibody or an antigen-binding fragment thereof, for example, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment, or any combination thereof. In some aspects, the heterologous polypeptide can specifically bind CD3ε. For example, a modified variant J chain or a functional fragment thereof that can increase the serum half-life of the provided IgM or IgM-like antibody is a variant of the modified J chain "V15J" (SEQ ID NO:9) and can comprise, for example, the amino acid sequence SEQ ID NO:10 (V15J-Y102A), or SEQ ID NO:23 (V15J-T103A), or SEQ ID NO:24 (V15J-N49A). In some aspects, the modified J chain can comprise a heterologous polypeptide fused to the N-terminus of the J chain, for example, an scFv antibody fragment, that specifically binds CD3ε, and can further comprise another heterologous polypeptide that affects the serum half-life of an IgM antibody that comprises the modified J chain fused to the C-terminus of the J chain, the other heterologous polypeptide being, for example, human serum albumin. In a particular aspect, the modified J chain comprises an amino acid sequence SEQ ID NO:11 (VJH) or a variant thereof having one or more amino acid substitutions, insertions, or deletions at positions corresponding to Y102 or T103 of SEQ ID NO:2, and the substitutions, insertions, or deletions can affect the serum half-life of a pentameric IgM antibody or IgM-like antibody comprising the J chain.

[0149] In some aspects, the IgM or IgM-like antibodies provided herein comprise a variant J chain provided herein or a functional fragment thereof, the variant J chain or functional fragment thereof comprising the amino acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:23, SEQ ID NO:24, or any combination thereof.

[0150] In some aspects, an IgM or IgM-like antibody comprising a variant J chain provided herein further comprises a variant IgM heavy chain constant region comprising one or more single amino acid substitutions, deletions, or insertions relative to a reference IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for one or more single amino acid substitutions, deletions, or insertions, wherein the variant IgM heavy chain constant region can also affect the serum half-life of the provided IgM antibody or IgM-like antibody. In some aspects, such an IgM antibody or IgM-like antibody can exhibit a further increased serum half-life upon administration to an animal relative to a reference IgM antibody or IgM-like antibody that is identical except for one or more single amino acid substitutions, deletions, or insertions in the IgM heavy chain constant region and that is administered to the same animal species in the same manner. According to this aspect, the reference IgM antibody or IgM-like antibody can be an IgM antibody or IgM-like antibody comprising a variant J chain provided herein, e.g., a variant J chain comprising the amino acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:23, SEQ ID NO:24, or any combination thereof. In some aspects, the increased serum half-life can be additive, or greater than additive, or less than additive. Exemplary variant IgM heavy chain constant regions are provided elsewhere herein and include, but are not limited to, variant human IgM heavy chain constant regions comprising the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:34.

[0151] In some aspects, relative to a reference antibody comprising only a variant J chain or only a variant IgM constant region, the serum half-life of an IgM or IgM-like antibody comprising a variant J chain provided herein and a variant IgM heavy chain constant region provided herein comprises an increase in serum half-life, i.e., the cumulative increase in the individual serum half-life is greater than additive or less than additive.

[0152] J Chain Variants Affecting the Serum Half-Life of IgM

[0153] The present disclosure provides a isolated variant J chain or a functional fragment thereof, which, as part of a pentameric IgM antibody or a pentameric IgM-like antibody, can increase the serum half-life of the antibody. The provided variant J chain or its functional fragment can be of any species and contains one or more single amino acid substitutions, deletions or insertions relative to a reference J chain that is identical to the variant J chain except for one or more single amino acid substitutions, insertions or deletions. The term "one or more single amino acid substitutions, insertions and deletions" means that each amino acid of the amino acid sequence of the variant J chain can be individually substituted, deleted or can have a single amino acid inserted adjacent to it, provided that the variant J chain or its functional fragment must still be able to function to assemble with an IgM heavy chain or an IgM-like heavy chain and an antibody light chain to form an IgM pentamer or an IgM-like pentamer. In certain aspects, the variant J chain or its functional fragment provided herein can have a single amino acid substitution, insertion or deletion, a combination of two single amino acid substitutions, insertions or deletions (e.g., two single amino acid substitutions or one single amino acid substitution and one single amino acid insertion or deletion), a combination of three single amino acid substitutions, insertions or deletions, a combination of four single amino acid substitutions, insertions or deletions, or more, wherein one, two, three, four or more single amino acid substitutions, insertions or deletions can individually or jointly affect the serum half-life of an IgM antibody or an IgM-like antibody comprising the variant J chain or its functional fragment. In certain aspects, one or more single amino acid substitutions, insertions or deletions inhibit the interaction of an IgM antibody or an IgM-like antibody comprising the variant J chain, e.g., binding to a receptor, e.g., FcαμR or pIgR.

[0154] In certain aspects, relative to a reference antibody, the serum half-life (e.g., α half-life, β half-life or total half-life) of an IgM or IgM-like antibody comprising the provided variant J chain or its functional fragment provided herein can be increased by at least 0.1-fold, at least 0.5-fold, at least 1-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more. In certain aspects, the increase in serum half-life is close to the increase in serum half-life of an IgG antibody comprising the same antigen-binding domain.

[0155] In certain aspects, relative to a reference antibody, an IgM antibody or an IgM-like antibody comprising the provided variant J chain also exhibits other improved pharmacokinetic parameters, e.g., increased plasma peak concentration (C max) Increased area under the T0 to ∞ curve (AUC), improved clearance time, increased mean residence time (MRT), or any combination thereof. In some aspects, the AUC can be increased by at least 0.1-fold, at least 0.5-fold, at least 1-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more relative to a reference IgM or IgM-like antibody that is otherwise identical except for one or more single amino acid substitutions, deletions, or insertions in the variant J chain or a functional fragment thereof, wherein the provided antibody and the reference antibody are administered to the same animal species in the same manner.

[0156] In some aspects, the isolated variant J chain or a functional fragment thereof provided herein comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO:2). "Amino acid corresponding to amino acid Y102 of the mature wild-type human J chain" refers to an amino acid in the J chain sequence of any species that is homologous to Y102 in the human J chain. 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. See U.S. Patent No. 9,951,134 Figure 4 , which is incorporated herein by reference. As described in the following examples, certain amino acid substitutions at the position corresponding to Y102 of SEQ ID NO:2 inhibit the binding of the variant J chain to the Fc alpha-mu (Fcαμ) receptor and the polymeric Ig receptor (pIg receptor or pIgR). In some aspects, the amino acid corresponding to Y102 of SEQ ID NO:2 can be substituted with any amino acid. In some aspects, the amino acid corresponding to Y102 of SEQ ID NO:2 can be substituted with alanine (A), serine (S), or arginine (R). In a particular aspect, Y102 of SEQ ID NO:2 can be substituted with alanine. In a particular aspect, the variant J chain or a functional fragment thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO:3. In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO:2 can be substituted with serine. In a particular aspect, the variant J chain or a functional fragment thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO:4. In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO:2 can be substituted with arginine. In a particular aspect, the variant J chain or a functional fragment thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO:5.

[0157] In some aspects, the isolated variant J chain or functional fragment thereof provided herein comprises an amino acid substitution at the amino acid position corresponding to amino acid T103 of the mature wild-type human J chain (SEQ ID NO:2). "The amino acid corresponding to amino acid T103 of the mature wild-type human J chain" refers to the amino acid in the J chain sequence of any species that is homologous to T103 in the human J chain. The position corresponding to T103 in SEQ ID NO:2 is conserved in the J chain amino acid sequences of at least 37 other species. See Figure 4 , which is incorporated herein by reference. As described in the following examples, the amino acid substitution at the position corresponding to T103 of SEQ ID NO:2 inhibits the binding of the variant J chain to the immunoglobulin Fcαμ receptor. In some aspects, the amino acid corresponding to T103 of SEQ ID NO:2 can be substituted with any amino acid. In some aspects, the amino acid corresponding to T103 of SEQ ID NO:2 can be substituted with alanine (A). In a particular aspect, the variant J chain or functional fragment thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO:6.

[0158] In some aspects, the present disclosure provides an isolated variant J chain or functional fragment thereof, the isolated variant J chain or functional fragment thereof comprising an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of the mature human J chain (SEQ ID NO:2), provided that S51 is not substituted with threonine (T), or wherein the J chain comprises amino acid substitutions at the amino acid positions corresponding to amino acid N49 and S51 of the mature wild-type human J chain (SEQ ID NO:2). Again, "the amino acid corresponding to amino acid N49 of SEQ ID NO:2 of the wild-type human J chain or S51 of SEQ ID NO:2" refers to the amino acid in the J chain sequence of any species that is homologous to N49 and / or S51 in the human J chain. The positions corresponding to N49 and S51 in SEQ ID NO:2 are conserved in the J chain amino acid sequences of at least 43 other species. See Figure 4, which is incorporated herein by reference. The amino acids at N49 and S51 corresponding to SEQ ID NO:2 and the amino acid at I50 corresponding to SEQ ID NO:2 contain an N-linked glycosylation motif in the J chain, and mutations at N49 and / or S51 (except for the threonine substitution at S51) can thus prevent possible glycosylation at this motif. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO:2 can be replaced by any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO:2 can be replaced by alanine (A), glycine (G), threonine (T), serine (S), or aspartic acid (D). In a particular aspect, the position corresponding to N49 of SEQ ID NO:2 can be replaced by alanine (A). In a particular aspect, the variant J chain or its functional fragment is a variant human J chain and contains the amino acid sequence SEQ ID NO:7. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO:2 can be replaced by any amino acid (except threonine). In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO:2 can be replaced by alanine (A) or glycine (G). In a particular aspect, the position corresponding to S51 of SEQ ID NO:2 can be replaced by alanine (A). In a particular aspect, the variant J chain or its functional fragment is a variant human J chain and contains the amino acid sequence SEQ ID NO:8.

[0159] In certain aspects, the variant J chain or its functional fragment provided herein contains the amino acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:23, SEQ ID NO:24, or any combination thereof.

[0160] Variant IgM constant regions conferring increased serum half-life

[0161] The present disclosure also provides an IgM antibody or IgM-like antibody having an enhanced serum half-life, wherein the IgM antibody or IgM-like antibody comprises five or six bivalent antibody binding units or variants or fragments thereof, wherein each binding unit comprises two variant IgM heavy chain constant regions or multimeric fragments thereof, each of which is associated with an antigen-binding domain or subunit thereof. The provided variant IgM heavy chain constant regions or multimeric fragments thereof each comprise one or more single amino acid substitutions, deletions or insertions relative to a reference IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for one or more single amino acid substitutions, deletions or insertions. In certain aspects, the variant IgM heavy chain constant regions may alone or in combination with variant J chains, variant modified J chains or functional fragments thereof provided elsewhere in the present disclosure affect (e.g., increase) the serum half-life of the provided IgM antibody or IgM-like antibody. A "reference IgM heavy chain constant region" refers to an IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for one or more single amino acid substitutions, deletions or insertions. In certain aspects, when expressed as part of a pentameric or hexameric IgM or IgM-like antibody provided herein, the variant IgM constant region or multimeric fragment thereof confers an increased serum half-life to the IgM or IgM-like antibody upon administration to a test animal relative to a reference IgM antibody or IgM-like antibody administered to the same animal in the same manner, wherein the reference IgM antibody or IgM-like antibody is identical except for one or more single amino acid substitutions, deletions or insertions in the IgM heavy chain constant region and is administered to the same animal species in the same manner. In certain aspects, relative to the reference IgM constant region, the variant IgM constant region comprises, for example, one or more amino acid substitutions, insertions or deletions in the Cμ4 domain. Assays for measuring serum half-life are well known to those of ordinary skill in the art, and exemplary assays are described herein and, for example, in U.S. Patent Application Publication No. US-2018-0265596-A1, which is incorporated herein by reference in its entirety.

[0162] In certain aspects, the variant IgM heavy chain constant regions or multimeric fragments thereof provided herein that confer an increased serum half-life to an IgM antibody or IgM-like antibody may also increase the serum half-life of the provided pentameric IgM antibody or IgM-like antibody having a variant J chain (such as the variant J chain provided elsewhere herein) that also increases the serum half-life of the IgM antibody or IgM-like antibody. In certain aspects, the increase in serum half-life is additive. In certain aspects, the increase in serum half-life is greater than or less than additive.

[0163] As provided herein, a variant IgM constant region or a multimeric fragment thereof can include one or more single amino acid substitutions, deletions, or insertions that affect the serum half-life of an IgM antibody or IgM-like antibody comprising the variant IgM constant region or fragment. The term "one or more single amino acid substitutions, insertions, and deletions" means that each amino acid of the IgM constant region or a multimeric fragment thereof can be individually substituted, deleted, or can have a single amino acid inserted adjacent thereto, provided that the variant IgM constant region or a multimeric fragment thereof must still be able to function within an IgM or IgM-like binding molecule (e.g., an antibody) to form an IgM pentamer or hexamer or an IgM-like pentamer or hexamer. In certain aspects, the variant IgM constant region or a multimeric fragment thereof provided herein can have a single amino acid substitution, insertion, or deletion, a combination of two single amino acid substitutions, insertions, and / or deletions (e.g., two single amino acid substitutions or one single amino acid substitution and one single amino acid insertion or deletion), a combination of three single amino acid substitutions, insertions, and / or deletions, a combination of four single amino acid substitutions, insertions, and / or deletions, or more, wherein one, two, three, four, or more single amino acid substitutions, insertions, and / or deletions can affect the serum half-life of an IgM antibody or IgM-like antibody comprising the variant IgM constant region or a multimeric fragment thereof. Thus, relative to a reference IgM antibody or IgM-like antibody that is identical except for one or more single amino acid substitutions, deletions, and / or insertions in the variant IgM constant region or a multimeric fragment thereof, the provided IgM antibody or IgM-like antibody exhibits an increased serum half-life when administered to an animal, wherein the provided antibody and the reference antibody are administered to the same animal species in the same manner.

[0164] In certain aspects, relative to a reference antibody, the serum half-life (e.g., α half-life, β half-life, or total half-life) of an IgM or IgM-like antibody can be increased by at least 0.1-fold, at least 0.5-fold, at least 1-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more. In certain aspects, the increase in serum half-life approaches the increase in serum half-life of an IgG antibody comprising the same antigen-binding domain. In certain aspects, the variant IgM constant region or a multimeric fragment thereof can be combined with other IgM antibody or IgM-like antibody modifications to further increase the serum half-life. For example, the variant IgM constant region or a multimeric fragment thereof provided herein can be combined with a J chain comprising amino acid substitutions, deletions, and / or insertions described elsewhere herein to provide an additive or greater than additive, e.g., synergistic, increase in serum half-life.

[0165] In certain aspects, the IgM antibodies or IgM-like antibodies comprising the variant IgM constant regions or multimeric fragments thereof provided herein also exhibit other improved pharmacokinetic parameters relative to a reference antibody, for example, an increased peak plasma concentration (C max ), increased T0 to ∞ area under the curve (AUC), improved clearance time, increased mean residence time (MRT), or any combination thereof.

[0166] In some aspects, the variant IgM constant region of the IgM antibody or IgM-like antibody provided herein or its multimeric fragment comprises an amino acid replacement at the amino acid position corresponding to the amino acid S401 of the wild-type human IgM constant region (SEQ ID NO: 12), which then corresponds to the amino acid S524 according to the Kabat numbering system. "The amino acid corresponding to the amino acid S401 of the wild-type human IgM constant region" refers to the amino acid in the IgM constant region sequence of any species homologous to S401 in the human IgM constant region. The position corresponding to S401 in SEQ ID NO: 12 is conserved in the IgM constant region amino acid sequences of some species (e.g., non-human primates), but is not conserved in other species (e.g., mice). Referring to Fig. 1. In some aspects, the S401 of SEQ ID NO: 12 can be replaced by any amino acid. In some aspects, the S401 of SEQ ID NO: 12 can be replaced by alanine (A). In a specific aspect, the variant IgM constant region is a variant human IgM constant region comprising an S401A mutation, represented herein as SEQ ID NO: 13. SEQ ID NO: 13 is shown below, wherein the S401A mutation is represented in bold underline:

[0167] SEQ ID NO:13

[0168] GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTV A EEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0169] In some aspects, the variant IgM constant region or multimeric fragment thereof of the IgM antibody or IgM-like antibody provided herein contains an amino acid substitution at the amino acid position corresponding to amino acid E402 of the wild-type human IgM constant region (SEQ ID NO:12), which in turn corresponds to amino acid E525 according to the Kabat numbering system. "The amino acid corresponding to amino acid E402 of the wild-type human IgM constant region" refers to the amino acid in the IgM constant region sequence of any species that is homologous to E402 in the human IgM constant region. The position corresponding to E402 in SEQ ID NO:12 is conserved in the IgM constant region amino acid sequences of some species (e.g., non-human primates and mice). See Figure 1. In some aspects, E402 of SEQ ID NO:12 can be substituted with any amino acid. In some aspects, E402 of SEQ ID NO:12 can be substituted with alanine (A). In a particular aspect, the variant IgM constant region is a variant human IgM constant region containing the E402A mutation, designated herein as SEQ ID NO:14. SEQ ID NO:14 is shown below, with the E402A mutation shown in bold underline:

[0170] SEQ ID NO:14:

[0171]

[0172] In some aspects, the variant IgM constant regions or multimeric fragments thereof of the IgM antibodies or IgM-like antibodies provided herein contain an amino acid substitution at the amino acid position corresponding to amino acid E403 of the wild-type human IgM constant region (SEQ ID NO:12), which in turn corresponds to amino acid E526 according to the Kabat numbering system. "The amino acid corresponding to amino acid E403 of the wild-type human IgM constant region" refers to the amino acid in the IgM constant region sequence of any species that is homologous to E403 in the human IgM constant region. The position corresponding to E403 in SEQ ID NO:12 is conserved in the IgM constant region amino acid sequences of some species (e.g., non-human primates and mice). See Figure 1. In some aspects, E403 of SEQ ID NO:12 can be substituted with any amino acid. In some aspects, E403 of SEQ ID NO:12 can be substituted with alanine (A). In a particular aspect, the variant IgM constant region is a variant human IgM constant region containing the E403A mutation, designated herein as SEQ ID NO:34. SEQ ID NO:34 is shown below, with the E403A mutation in bold and underlined:

[0173] SEQ ID NO:34:

[0174] NTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTA

[0175] In some aspects, the variant IgM constant regions or multimeric fragments thereof of the IgM antibodies or IgM-like antibodies provided herein contain an amino acid substitution at the amino acid position corresponding to amino acid R344 of the wild-type human IgM constant region (SEQ ID NO:12), which in turn corresponds to amino acid R467 according to the Kabat numbering system. "The amino acid corresponding to amino acid R344 of the wild-type human IgM constant region" refers to the amino acid in the IgM constant region sequence of any species that is homologous to R344 in the human IgM constant region. The position corresponding to R344 in SEQ ID NO:12 is conserved in the IgM constant region amino acid sequences of some species (e.g., non-human primates and mice). See Figure 1. In some aspects, R344 of SEQ ID NO:12 can be substituted with any amino acid. In some aspects, R344 of SEQ ID NO:12 can be substituted with alanine (A). In a particular aspect, the variant IgM constant region is a variant human IgM constant region containing the R344A mutation, designated herein as SEQ ID NO:31. SEQ ID NO:31 is shown below, with the R344A mutation in bold and underlined:

[0176] SEQ ID NO:31

[0177]

[0178] In some aspects, the variant IgM constant region or multimeric fragment thereof of the IgM antibody or IgM-like antibody provided herein contains an amino acid substitution at the amino acid position corresponding to amino acid E345 of the wild-type human IgM constant region (SEQ ID NO:12), which in turn corresponds to amino acid E468 according to the Kabat numbering system. "The amino acid corresponding to amino acid E345 of the wild-type human IgM constant region" refers to the amino acid in the IgM constant region sequence of any species that is homologous to E345 in the human IgM constant region. The position corresponding to E345 in SEQ ID NO:12 is conserved in the IgM constant region amino acid sequences of some species (e.g., non-human primates and mice). See Figure 1. In some aspects, E345 of SEQ ID NO:12 can be substituted with any amino acid. In some aspects, E345 of SEQ ID NO:12 can be substituted with alanine (A). In a particular aspect, the variant IgM constant region is a variant human IgM constant region containing the E345A mutation, designated herein as SEQ ID NO:32. SEQ ID NO:32 is shown below, with the E345A mutation shown in bold underlined:

[0179] SEQ ID NO:32

[0180]

[0181] Variant human IgM constant region with reduced CDC activity

[0182] In some aspects, when expressed as part of an IgM antibody or IgM-like antibody provided herein, a variant human IgM constant region may additionally exhibit reduced complement-dependent cytotoxicity (CDC) activity against cells in the presence of complement, relative to a reference IgM antibody or IgM-like antibody having the same corresponding reference human IgM constant region except for mutations that confer reduced CDC activity. These CDC mutations can be combined with any of the mutations provided herein that confer increased serum half-life. "Corresponding reference human IgM constant region" refers to a human IgM constant region or a portion thereof, e.g., the Cμ3 domain, that is identical to the variant IgM constant region except for one or more modifications in the constant region that affect CDC activity. In some aspects, relative to the wild-type human IgM constant region described, for example, in PCT application number PCT / US2018 / 026474, which is incorporated herein by reference in its entirety, the variant human IgM constant region comprises, for example, one or more amino acid substitutions in the Cμ3 domain. Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described, for example, in PCT application number PCT / US2018 / 026474.

[0183] In some aspects, the variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO:12. In other aspects, the variant human IgM constant region provided herein contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO:12. In other aspects, the variant human IgM constant region provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO:12 and position P313 of SEQ ID NO:12. The variant IgM constant region at amino acid position P311 of SEQ ID NO:12 can be substituted, for example, with alanine (P311A), serine (P311S), or glycine (P311G). The variant IgM constant region at amino acid position P313 of SEQ ID NO:12 can be substituted, for example, with alanine (P313A), serine (P313S), or glycine (P313G). The variant IgM constant region at amino acid positions P311 and P313 of SEQ ID NO:12 can be substituted with alanine (P311A) and serine (P313S) (SEQ ID NO:15), respectively, or any combination of alanine, serine, and / or glycine. SEQ ID NO:15 is shown below, where the P311A and P313S mutations are shown in bold underlined:

[0184] SEQ ID NO:15

[0185]

[0186] In one aspect, relative to a binding molecule comprising a corresponding wild-type IgM constant region, the IgM antibodies or IgM-like antibodies provided herein that comprise a variant human IgM constant region comprising an amino acid substitution at P311 and / or P313 (e.g., P311A, P311S, P311G, P313A, P313S, and / or P313G or any combination thereof) have a maximum CDC obtained in a dose-response assay that is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0187] Polynucleotides, vectors, and host cells

[0188] The present disclosure also provides polynucleotides, e.g., isolated, recombinant, and / or non-naturally occurring polynucleotides, that comprise a nucleic acid sequence encoding a polypeptide subunit of an IgM or IgM-like antibody provided herein. A "polypeptide subunit" refers to a part of a binding molecule, binding unit, IgM antibody, IgM-like antibody, or antigen-binding domain that can be translated independently. Examples include, but are not limited to, antibody variable domains, e.g., VH or VL, J chain, 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.

[0189] In certain aspects, the polypeptide subunit can comprise an IgM heavy chain constant region or an IgM-like heavy chain constant region or a multimeric fragment thereof, which can be fused to an antigen-binding domain or a subunit thereof, e.g., fused to the VH portion of an antigen-binding domain, all as provided herein. In certain aspects, the polynucleotide can encode a polypeptide subunit that comprises a human IgM heavy chain constant region, a human IgM-like heavy chain constant region, or a multimeric fragment thereof, e.g., SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:34, any of which can be fused to an antigen-binding domain or a subunit thereof, e.g., the C-terminus of VH.

[0190] In certain aspects, the polypeptide subunit can comprise the VL portion of an antibody of an antigen-binding domain as described elsewhere herein. In certain aspects, the polypeptide subunit can comprise an antibody light chain constant region (e.g., a human antibody light chain constant region) or a fragment thereof, which can be fused to the C-terminus of VL.

[0191] In some aspects, as provided herein, the polypeptide subunit can comprise a J chain, a modified J chain, or any functional fragment or variant thereof. In some aspects, the polypeptide subunit can comprise a human J chain or a functional fragment or variant thereof, including a modified J chain. In some aspects, the J chain can comprise the amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:23, or SEQ ID NO:24.

[0192] In some aspects, the polynucleotides provided herein (e.g., expression vectors such as plasmids) can comprise a nucleic acid sequence encoding one polypeptide subunit (e.g., an IgM heavy chain or IgM-like heavy chain, a light chain, or a J chain), or can comprise two or more or all three nucleic acid sequences encoding two or more polypeptide subunits of an IgM antibody or IgM-like antibody provided herein. Alternatively, the nucleic acid sequences encoding the three polypeptide subunits can be on a separate polynucleotide, e.g., a separate expression vector. The present disclosure provides such single or multiple expression vectors. The present disclosure also provides a host cell or host cells encoding one or more of the polynucleotides or expression vectors provided.

[0193] Thus, to form an antigen-binding domain, a nucleic acid sequence encoding the variable region of an antibody can be inserted into an expression vector template for an IgM or IgM-like structure, particularly those nucleic acid sequences encoding the variant IgM heavy chain constant regions provided herein, such as SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:34, and a polynucleotide encoding the J chain provided herein or a functional fragment or variant thereof can also be combined, e.g., encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:23, or SEQ ID NO:24, more specifically a variant J chain comprising the amino acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:23, or SEQ ID NO:24, thereby generating an IgM antibody or IgM-like antibody having five or six binding units and having an increased serum half-life relative to an IgM antibody or IgM-like antibody that is otherwise structurally identical except for one or more single amino acid substitutions, insertions, or deletions in the IgM heavy chain constant region and / or the J chain described elsewhere herein. In short, nucleic acid sequences encoding the variable domain sequences of the heavy and light chains can be synthesized or amplified from existing molecules and inserted into one or more vectors in the appropriate orientation and in-frame such that upon expression, the vectors will produce the desired full-length heavy or light chain. Vectors useful for these purposes are known in the art. Such vectors may also contain enhancers and other sequences required to effect the expression of the desired chain. Multiple vectors or a single vector can be used, and a variant J chain or a functional fragment thereof can also be encoded. This vector or these vectors can be transfected into a host cell, and then the heavy and / or light chain and the variant J chain or a functional fragment thereof are expressed, and the IgM or IgM-like antibody is assembled and purified. After expression, the chains form a fully functional polymeric IgM or IgM-like antibody with an enhanced serum half-life. The fully assembled polymeric IgM or IgM-like antibody can then be purified by standard methods. If desired, the expression and purification processes can be carried out on a commercial scale.

[0194] The present disclosure also provides a composition comprising two or more polynucleotides, wherein the two or more polynucleotides together can encode an IgM or IgM-like antibody having an increased serum half-life as described above. In certain aspects, the composition can include a polynucleotide encoding a wild-type or variant IgM or IgM-like heavy chain or a multimeric fragment thereof, e.g., a wild-type or variant human IgM heavy chain comprising the constant region amino acid sequences SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:34 as described above, wherein the IgM or IgM-like heavy chain further comprises an antigen-binding domain or a subunit thereof, e.g., a VH domain. The composition can further include a polynucleotide encoding a light chain or a fragment thereof, e.g., a human kappa or lambda light chain comprising at least the VL of the antigen-binding domain. The provided polynucleotide composition can further include a polynucleotide encoding a J chain, e.g., a variant J chain or a functional fragment thereof having at least one single amino acid substitution, insertion, or deletion that can enhance the serum half-life of the IgM or IgM-like antibody, e.g., a variant and / or modified J chain comprising the amino acid sequences SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:23, or SEQ ID NO:24. In certain aspects, the polynucleotides constituting the compositions provided herein can be located on two, three, or more separate vectors, e.g., expression vectors. Such vectors are provided by the present disclosure. In certain aspects, two or more polynucleotides constituting the compositions provided herein can be located on a single vector, e.g., an expression vector. Such a vector is provided by the present disclosure.

[0195] 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 an IgM or IgM-like antibody or any subunit thereof provided herein, a polynucleotide composition provided herein, or a vector or two, three, or more vectors encoding an IgM or IgM-like antibody or any subunit thereof provided herein.

[0196] In a related aspect, the present disclosure provides a method of preparing an IgM or IgM-like antibody having an increased serum half-life as provided by the present disclosure, wherein the method comprises culturing a host cell provided herein and recovering the IgM or IgM-like antibody.

[0197] Method for identifying variant J chains and / or variant IgM heavy chain constant regions that can confer an increased serum half-life

[0198] The present disclosure also provides various methods for identifying variant antibody subunits that can increase the serum half-life of antibodies (e.g., IgM antibodies or IgM-like antibodies) containing the subunit. These methods utilize standard methods of molecular biology and site-directed mutagenesis with which those of ordinary skill in the art will be familiar. Exemplary methods are provided in the Examples section.

[0199] In certain aspects, the present disclosure provides a method for identifying a variant J chain that can increase the serum half-life of pentameric IgM antibodies, pentameric IgM-like antibodies, dimeric IgA antibodies, and / or dimeric IgA-like antibodies containing the variant J chain. The method includes testing for an increase in the serum half-life in a test animal of a pentameric IgM antibody or pentameric IgM-like antibody assembled to include the variant J chain or a fragment thereof, or alternatively a dimeric IgA antibody or dimeric IgA-like antibody, relative to a reference antibody, where the reference antibody is identical to the test antibody except for the variant J chain. According to these aspects, a series of variant J chains or fragments thereof containing defined amino acid insertions, deletions, or substitutions are constructed and tested relative to a reference J chain (e.g., J chain, modified J chain, and / or functional fragment thereof) that is identical to the variant J chain except for the defined amino acid insertions, deletions, or substitutions. In certain aspects, the variant J chain can include one, two, three, four, or more defined single amino acid substitutions, insertions, or deletions, as described elsewhere herein. In certain aspects, limited regions of the J chain are targeted for mutagenesis, e.g., those regions necessary or contributing to binding to certain immunoglobulin receptors (e.g., Fc alpha-mu receptor (FcαμR), polymeric Ig receptor (pIgR), Fc-mu receptor (FcμR), two or more receptors, or all three receptors), as described in more detail in Examples 1 and 2. In certain aspects, the full length of the J chain can be subjected to mutagenesis, e.g., by substituting the amino acid at each position of the J chain such as SEQ ID NO:2 with another amino acid (e.g., alanine, serine, or threonine). The mutated J chain can then be co-expressed with, e.g., a reference IgM heavy chain and an antibody light chain, and the resulting pentameric IgM antibody is analyzed for proper assembly, antigen binding, and any other desired functions. The increase in the serum half-life of the resulting IgM antibody or IgM-like antibody in a test animal (e.g., the mouse model described in the Examples) is then evaluated. Those J chains that confer an increase in the serum half-life of the corresponding IgM antibody or IgM-like antibody are then recovered and further characterized as needed. This method can also be used to identify variant J chains that can confer an increase in the serum half-life of dimeric IgA antibodies or IgA-like antibodies containing the variant J chain using a similar method.

[0200] In some aspects, the present disclosure provides a method for identifying variant J chains that can increase the serum half-life of pentameric IgM antibodies, pentameric IgM-like antibodies, dimeric IgA antibodies, and / or dimeric IgA-like antibodies that comprise the variant J chain. The method includes testing for reduced binding of pentameric IgM antibodies or pentameric IgM-like antibodies assembled to include a variant J chain or a fragment thereof, or alternatively dimeric IgA antibodies or dimeric IgA-like antibodies, to certain immunoglobulin receptors (e.g., Fc alpha-mu receptor (FcαμR), polymeric Ig receptor (pIgR), Fc-mu receptor (FcμR), two or more of the receptors, or all three receptors). Regions of the J chain to be subjected to mutagenesis can be targeted by determining, for example, those regions necessary or contributory to receptor binding as described in Example 1. Alternatively, amino acids along the full length of the J chain can be mutagenized and the resulting antibodies comprising the variant J chain can be tested for receptor binding. Reduced receptor binding can be measured by any suitable assay, e.g., by the ELISA assay described in Example 2. Variant J chains or fragments thereof are constructed to include amino acid insertions, deletions, or substitutions as defined above and elsewhere herein. Those variant J chains that confer reduced antibody binding to one or more receptors can then be recovered and further characterized as needed. The method can further include testing pentameric IgM antibodies or pentameric IgM-like antibodies, or dimeric IgA antibodies or dimeric IgA-like antibodies that comprise the recovered variant J chain or fragment thereof, for increased serum half-life in a test animal (e.g., in the mouse model described in the Examples) relative to a reference antibody that is identical except for the amino acid substitutions, deletions, or insertions defined in the variant J chain or fragment thereof. This method can also be used to identify variant J chains that can confer increased serum half-life to dimeric IgA antibodies or IgA-like antibodies that comprise a variant J chain using a similar method.

[0201] The present disclosure also provides a method for identifying a variant IgM heavy chain constant region (or variant IgA heavy chain constant region) that increases the serum half-life of an IgM antibody or IgM-like antibody (or IgA antibody or IgA-like antibody) comprising the variant IgM (or IgA) heavy chain constant region. The method for IgM comprises testing, in a test animal, an IgM antibody or IgM-like antibody comprising a variant IgM heavy chain constant region for an increased serum half-life relative to a reference IgM antibody or IgM-like antibody, wherein the variant IgM heavy chain constant region comprises defined amino acid insertions, deletions, or substitutions, and wherein the reference IgM antibody or IgM-like antibody comprises an IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for the defined amino acid insertions, deletions, or substitutions. The method further comprises recovering those IgM antibodies or IgM-like antibodies comprising the variant IgM heavy chain constant region that, relative to the reference IgM antibody or IgM-like antibody, have an increased serum half-life conferred by the variant IgM heavy chain constant region. Any number of variant IgM or IgM-like heavy chain constant regions comprising, for example, one, two, three, four, or more defined single amino acid substitutions, insertions, or deletions can be tested. The regions of the IgM or IgM-like heavy chain constant region to be subjected to mutagenesis can be determined empirically, for example, by identifying those regions in, for example, the Cμ4 domain that are essential or contributory to binding to various immunoglobulins such as the Fc alpha-mu receptor (FcαμR), Fc mu receptor (FcμR), polymeric Ig receptor (pIgR), any combination of the two receptors, or all three receptors as described in the examples. Alternatively, the entire length of the selected Cμ domain or the entire IgM heavy chain constant region can be subjected to mutagenesis, for example, by substituting the amino acid at each position with a different amino acid (e.g., alanine, serine, or threonine). The variant IgM heavy chain constant region is then assembled with other antibody subunits (e.g., an antibody light chain and an optional J chain), and proper assembly and antigen binding are tested. The increase in serum half-life of the test antibody in a test animal (e.g., the mouse model described in the examples) can then be evaluated. This method can also be used to identify variant IgA or IgA-like heavy chain constant regions that can confer an increased serum half-life to a dimeric IgA antibody or IgA-like antibody comprising a variant constant region using a similar method.

[0202] The present disclosure also provides a method for identifying a variant IgM heavy chain constant region (or alternatively a variant IgA heavy chain constant region) that increases the serum half-life of an IgM antibody or IgM-like antibody (or IgA antibody or IgA-like antibody) comprising the variant heavy chain constant region. The method for IgM comprises testing an IgM antibody or IgM-like antibody comprising a variant IgM heavy chain constant region for reduced binding to the Fc alpha-mu receptor (FcαμR), Fc mu receptor (FcμR), polymeric Ig receptor (pIgR), any combination of two receptors, or all three receptors, relative to a reference IgM antibody or IgM-like antibody, wherein the variant IgM heavy chain constant region comprises defined amino acid insertions, deletions, or substitutions, and wherein the reference IgM antibody or IgM-like antibody comprises an IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for the defined amino acid insertions, deletions, or substitutions. The method further comprises recovering those IgM antibodies or IgM-like antibodies comprising the variant IgM heavy chain constant region that, relative to the reference IgM antibody or IgM-like antibody, have a reduced ability to bind FcαμR, a reduced ability to bind FcμR, a reduced ability to bind pIgR, a reduced ability to bind any two receptors, or a reduced ability to bind all three receptors. Any number of variant IgM or IgM-like heavy chain constant regions comprising, for example, one, two, three, four, or more defined single amino acid substitutions, insertions, or deletions can be tested. The region of the IgM or IgM-like heavy chain constant region to be subjected to mutagenesis can be determined empirically, for example, by identifying those regions, such as in the Cμ4 domain, that are essential or contribute to binding to various immunoglobulins, such as the Fc alpha-mu receptor (FcαμR), Fc mu receptor (FcμR), polymeric Ig receptor (pIgR), any combination of two receptors, or all three receptors, as described in the examples. Alternatively, the entire length of the selected Cμ domain or the entire IgM heavy chain constant region can be subjected to mutagenesis, for example, by substituting the amino acid at each position with a different amino acid (e.g., alanine, serine, or threonine). The variant IgM heavy chain constant region is then assembled with other antibody subunits (e.g., an antibody light chain and optionally a J chain), and proper assembly and antigen binding are tested. The variant IgM heavy chain constant region is then assembled with other antibody subunits (e.g., an antibody light chain and optionally a J chain), and proper assembly and antigen binding are tested. Reduced receptor binding can be measured by any suitable assay, for example, by the ELISA assay described in Example 2. Then, an increase in serum half-life can be evaluated in a test animal (e.g., the mouse model described in the examples) of a test antibody comprising the recovered variant IgM or IgM-like heavy chain constant region.This method can also be used to identify variant IgA or IgA-like heavy chain constant regions that can confer an increased serum half-life to dimeric IgA antibodies or IgA-like antibodies comprising variant constant regions using a similar method.

[0203] Method of use

[0204] The present disclosure also provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an IgM or IgM-like antibody provided herein. "Therapeutically effective dose or amount" or "effective amount" means an amount of the IgM or IgM-like antibody which, when administered, produces a positive immunotherapeutic response relative to the treatment of the subject.

[0205] The effective dose of the composition for treating cancer varies depending on many different factors, including the mode of administration, the target site, the physiological state of the subject, whether the subject is human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Generally, the subject is human, but non-human mammals, including transgenic mammals, can also be treated. Conventional methods known to those of skill in the art can be used to titrate the therapeutic dose to optimize safety and efficacy.

[0206] The subject to be treated can be any animal in need of treatment, e.g., a mammal, and in certain aspects, the subject is a human subject.

[0207] In its simplest form, the formulation to be administered to the subject is an IgM or IgM-like antibody provided herein or a multimeric antigen-binding fragment thereof, administered in a conventional dosage form, which can be combined with a pharmaceutical excipient, carrier or diluent described elsewhere herein.

[0208] The compositions of the present disclosure can be administered by any suitable method, e.g., parenterally, intracerebroventricularly, orally, by aerosol inhalation, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0209] Pharmaceutical compositions and methods of administration

[0210] In view of the present disclosure, methods for preparing and administering the IgM or IgM-like antibodies provided herein to a subject in need thereof are well known or readily determinable by those skilled in the art. The route of administration can be, for example, intratumoral, oral, parenteral, by inhalation, or topical. As used herein, the term "parenteral" includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Although these forms of administration are considered suitable, another example of a form of administration is an injectable solution, particularly a solution for intratumoral, intravenous, or intraarterial injection or infusion. Suitable pharmaceutical compositions can contain buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), optional stabilizers (e.g., human albumin), and the like.

[0211] As discussed herein, the IgM or IgM-like antibodies provided herein can be administered in a pharmaceutically effective amount to treat a subject in need thereof. In this regard, it should be understood that the disclosed IgM or IgM-like antibodies can be formulated to facilitate administration and promote the stability of the active agent. Thus, the pharmaceutical compositions can contain a pharmaceutically acceptable non-toxic sterile carrier, such as physiological saline, non-toxic buffers, preservatives, and the like. A pharmaceutically effective amount of the IgM or IgM-like antibodies provided herein refers to an amount sufficient to achieve effective binding to the target and obtain a therapeutic benefit. Suitable formulations are described in Remington's Pharmaceutical Sciences, for example, the 21st edition (Lippincott Williams & Wilkins) (2005).

[0212] Certain pharmaceutical compositions 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 spray or inhalation. Such compositions are prepared as solutions in physiological saline with benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents.

[0213] The amount of IgM or IgM-like receptor that can be combined with a carrier substance to produce a single dosage form will vary depending on, for example, the subject being treated and the particular mode of administration. The compositions can be administered in single doses, multiple doses, or over a defined period of infusion. The dosage regimen can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0214] Within the scope of the present disclosure, the IgM or IgM-like antibodies provided herein can be administered to a subject in need of treatment in an amount sufficient to produce a therapeutic effect. The IgM or IgM-like antibodies provided herein can be administered to a subject in conventional dosage forms, which are prepared by combining the antibodies or their multivalent antigen-binding fragments, variants or derivatives of the present disclosure with conventional pharmaceutically acceptable carriers or diluents according to known techniques. The form and characteristics of the pharmaceutically acceptable carrier or diluent can be determined by the amount of the active ingredient combined therewith, the route of administration and other well-known variables.

[0215] The present disclosure also provides the use of the IgM or IgM-like antibodies provided herein in the manufacture of a medicament for treating, preventing or controlling cancer.

[0216] Unless otherwise indicated, the present disclosure employs conventional techniques in the fields of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art. Such techniques are well described in the literature. See, for example, 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); D.N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2nd ed. (IRL Press), vols. 1-4; Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press); Mullis et al., U.S. Patent 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 ed. (Wiley-Interscience); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); S.C. Makrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science); Methods in Enzymology, vols. 151-155 (Academic Press, Inc., N.Y.); 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).

[0217] The general principles of antibody engineering are presented, for example, in Strohl, W.R. and L.M. Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). The general principles of protein engineering are presented, for example, in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press). The general principles of immunology are presented, for example, in Abbas and Lichtman (2017) Cellular and Molecular Immunology 9th Edition (Elsevier). In addition, standard immunological methods known in the art can be followed, for example, the methods 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, 2nd Edition (Cold Spring Harbor Press); and Ossipow and Fischer, eds. (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press).

[0218] All of the references cited above and all references cited herein are hereby incorporated by reference in their entirety.

[0219] The following examples are provided for illustration and not limitation.

[0220] Examples

[0221] We hypothesized that interactions between IgM and one or more of its Ig receptors, the Fcμ receptor (FcμR), the Fcα / μ receptor (FcαμR), and / or the polymeric Ig receptor (pIgR) contribute to the specific pharmacokinetics and pharmacodynamics of IgM antibodies observed in vivo. Accordingly, we set out to identify specific regions of IgM associated with binding to these receptors and to ask whether alterations (e.g., amino acid substitutions) in these regions could modulate, e.g., inhibit, receptor binding to an IgM antibody containing the alteration. We then tested whether some of these alterations affected the in vivo plasma utilization of an IgM antibody containing the alteration.

[0222] Example 1: Screening of J chain and Fcμ peptide arrays for receptor binding

[0223] To identify regions on the IgM constant region and J chain that bind FcμR, FcαμR, and / or pIgR, we used a peptide array-based technology (PEPperPRINT; WWW_dot_pepperprint_dot_com). A complete set of peptides based on the amino acid sequence of the human J chain (SEQ ID NO:2) and the Cμ3 and Cμ4 domains of human IgM (amino acids approximately 224 to approximately 430 of SEQ ID NO:12) were synthesized and immobilized on a solid support as follows. The sequences of IgM μ3, μ4, and J chain were extended at the N- and C-termini with neutral GSGSGSG linkers (SEQ ID NO:33) to avoid truncated peptides. The extended protein sequences were translated into 9- and 13-amino acid peptides with peptide-peptide overlaps of 8 and 12 amino acids, respectively. After peptide synthesis, all peptides were cyclized via a thioether bond between the thiol group of the C-terminal cysteine side chain and the appropriately modified N-terminus. Together with control peptides, the resulting IgM peptide microarray contained 844 different peptides, printed in duplicate (1,688 peptide spots). Restricted peptides with one residue increment were probed with 1, 10, or 100 μg / mL recombinant human pIgR (amino acids 19 to 638 of SEQ ID NO:20, available from R&D Systems), human FcμR (amino acids 18 to 251 of SEQ ID NO:21, available from R&D Systems), and human FcαμR (amino acids 17 to 450 of SEQ ID NO:22, available from R&D Systems), each having a 6X HIS tag in incubation buffer (PBS, pH 7.4 with 0.005% Tween 20 and 10% Rockland blocking buffer (MB-070)), followed by staining with a secondary antibody, mouse anti-6x-His epitope tag DyLight680, and a control antibody, and reading at a scan intensity of 7 / 7 (red / green).

[0224] Quantification of spot intensity and peptide annotation was performed on 16-bit grayscale tiff files at scan intensity 7 / 7, which showed a higher dynamic range than 24-bit color tiff files; microarray image analysis was performed with an analyzer. The software algorithm decomposes the fluorescence intensity of each spot into raw signal, foreground signal, and background signal (see "Raw Data" tab), and calculates the average median foreground intensity and the point-to-point deviation of spot replicates. Based on the average median foreground intensity, an intensity map is generated.

[0225] The average spot intensity measured was plotted against the protein sequence from the N-terminus of the IgM μ3 chain to the C-terminus of the IgM J chain for the target protein to visualize the overall spot intensity and signal-to-noise ratio. The intensity map was correlated with the peptide and intensity map as well as the visual inspection of the microarray scan to identify the major interactions of the target protein.

[0226] Using the raw data, the key regions of the J chain and the IgM HC constant region that bind to the receptor in the PEPperPRINT platform were identified.

[0227] Human FcαμR binds to the following underlined regions of the mature J chain SEQ ID NO:2:

[0228]

[0229] Thus, the regions of the mature human J chain that contribute to human FcαμR binding may include, for example, amino acids within amino acids 1 to 10 of SEQ ID NO:2 and / or amino acids overlapping with said amino acids and / or amino acids including, consisting of, or consisting essentially of said amino acids, amino acids within amino acids 87 to 105 of SEQ ID NO:2 and / or amino acids overlapping with said amino acids and / or amino acids including, consisting of, or consisting essentially of said amino acids, and / or amino acids within amino acids 125 to 137 of SEQ ID NO:2 and / or amino acids overlapping with said amino acids and / or amino acids including, consisting of, or consisting essentially of said amino acids. As would be understood by one of ordinary skill in the art, pentameric IgM antibodies or IgM-like antibodies containing variant J chains can be tested for, for example, inhibition of FcαμR binding as described in Example 2 and / or enhancement of serum half-life as described in Example 3, said variant J chains having mutations within, overlapping with, and / or corresponding to these regions (e.g., in the J chain amino acid sequence from another species), e.g., amino acid insertions, deletions, and / or substitutions.

[0230] Human pIgR binds to the following regions of the mature J chain SEQ ID NO:2:

[0231]

[0232] Thus, regions of the mature human J chain that contribute to human pIgR binding can include, for example, amino acids within amino acids 12 to 24 of SEQ ID NO:2 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, and / or amino acids within amino acids 93 to 105 of SEQ ID NO:2 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids. As would be understood by one of ordinary skill in the art, pentameric IgM antibodies or IgM-like antibodies containing variant J chains can be tested for, for example, inhibition of pIgR binding as described in Example 2 and / or enhancement of serum half-life as described in Example 3, wherein the variant J chain has mutations within, overlapping with, and / or corresponding to these regions (e.g., in the J chain amino acid sequence from another species), such as amino acid insertions, deletions, and / or substitutions.

[0233] Human FcμR binds to the following of mature J chain SEQ ID NO:2 regions:

[0234]

[0235] Thus, regions of the mature human J chain that contribute to human FcμR binding can include, for example, amino acids within amino acids 1 to 4 of SEQ ID NO:2 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, amino acids within amino acids 87 to 105 of SEQ ID NO:2 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, and / or amino acids within amino acids 125 to 137 of SEQ ID NO:2 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids. As would be understood by one of ordinary skill in the art, pentameric IgM antibodies or IgM-like antibodies containing variant J chains can be tested for, for example, inhibition of FcμR binding as described in Example 2 and / or enhancement of serum half-life as described in Example 3, wherein the variant J chain has mutations within, overlapping with, and / or corresponding to these regions (e.g., in the J chain amino acid sequence from another species), such as amino acid insertions, deletions, and / or substitutions.

[0236] Human FcαμR binds to the following regions of Cμ3 and Cμ4 of human IgM constant region SEQ ID NO:12 underlined (numbered SEQ ID NO:12 / the provided Kabat):

[0237] Order of IgM heavy chain (SEQ ID NO:12) / Kabat numbering key

[0238]

[0239] Thus, the regions of the human IgM constant region that contribute to human FcαμR binding may include, for example, the amino acids within amino acids 241 to 253 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, the amino acids within amino acids 282 to 294 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, the amino acids within amino acids 342 to 354 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, and / or the amino acids within amino acids 393 to 415 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids. As would be understood by one of ordinary skill in the art, IgM antibodies or IgM-like antibodies containing an IgM heavy chain constant region can be tested for, for example, inhibition of FcαμR binding as described in Example 2 and / or enhancement of serum half-life as described in Example 3, wherein the IgM heavy chain constant region has mutations within, overlapping with, and / or corresponding to these regions (e.g., in the amino acid sequence of an IgM heavy chain constant region from another species such as those shown in FIG. 1), such as amino acid insertions, deletions, and / or substitutions.

[0240] Human pIgR binds to the following regions of Cμ3 and Cμ4 of human IgM constant region SEQ ID NO:12 (numbered SEQ ID NO:12 / the provided Kabat):

[0241]

[0242] Thus, regions of the human IgM constant region that contribute to human pIgR binding can include, for example, amino acids within amino acids 232 to 244 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, amino acids within amino acids 287 to 304 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, and / or amino acids within amino acids 397 to 413 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids. As would be understood by one of ordinary skill in the art, IgM antibodies or IgM-like antibodies comprising an IgM heavy chain constant region can be tested for, e.g., inhibition of pIgR binding as described in Example 2 and / or enhancement of serum half-life as described in Example 3, wherein the IgM heavy chain constant region has mutations within, overlapping with, and / or corresponding to these regions (e.g., in the amino acid sequence of an IgM heavy chain constant region from another species such as those shown in FIG. 1), e.g., amino acid insertions, deletions, and / or substitutions.

[0243] Human FcμR binds to the following of Cμ3 and Cμ4 of the human IgM constant region SEQ ID NO:12 regions (numbered SEQ ID NO:12 / Kabat provided):

[0244]

[0245] Thus, regions of the human IgM constant region that contribute to human FcμR binding can include, for example, amino acids within amino acids 282 to 305 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids, and / or amino acids within amino acids 393 to 417 of SEQ ID NO:12 and / or amino acids that overlap with said amino acids and / or amino acids that include, consist of, or consist essentially of said amino acids. As would be understood by one of ordinary skill in the art, IgM antibodies or IgM-like antibodies comprising an IgM heavy chain constant region can be tested for, e.g., inhibition of FcμR binding as described in Example 2 and / or enhancement of serum half-life as described in Example 3, wherein the IgM heavy chain constant region has mutations within, overlapping with, and / or corresponding to these regions (e.g., in the amino acid sequence of an IgM heavy chain constant region from another species such as those shown in FIG. 1), e.g., amino acid insertions, deletions, and / or substitutions.

[0246] Example 2: Construction of alanine-scanned IgM J chain and Fc mutants and screening for receptor binding inhibition by ELISA

[0247] Screening for receptor binding inhibition

[0248] Alanine-scanning mutagenesis was performed on the J chain and IgM constant regions of the monospecific anti-CD20 IgM antibody 1.5.3 (with the mature human J chain of SEQ ID NO:2) as a monospecific pentamer and the bispecific anti-CD20 IgM antibody 1.5.3IgM (with the "V15J" J chain, SEQ ID NO:9) as a bispecific pentamer, both as described in PCT Publication No. WO 2016 / 141303, to determine whether certain regions identified by PEPperPRINT analysis (Example 1) could be modified to reduce or inhibit the binding of FcαμR, FcμR, and / or pIgR. The mutants tested included J chain alanine substitutions (Y102A and T103A) at positions corresponding to Y102 and T103 of the human mature J chain of SEQ ID NO:2. The modified "V15J" J chain containing the Y102A mutation is designated SEQ ID NO:10, and the modified "V15J" J chain containing the T103A mutation is designated SEQ ID NO:23. SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:23 are shown below.

[0249] SEQ ID NO:9:

[0250] QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0251] SEQ ID NO:10:

[0252] QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCATYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0253] SEQ ID NO:23:

[0254] QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYAYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0255] The mutants tested on the human IgM constant region included alanine mutations (R344A, SEQ ID NO: 31, E345A, SEQ ID NO: 32, S401A, SEQ ID NO: 13, E402A, SEQ ID NO: 14, and E403A, SEQ ID NO: 34) at positions corresponding to R344, E345, S401, and E402 of SEQ ID NO: 12. Two variant IgM constant region mutations SEQ ID NO: 13 and SEQ ID NO: 14 were tested for their effect on the in vivo half-life of IgM containing the mutations, using both the "wild-type" V15J J chain (SEQ ID NO: 9), and the V15J J chain containing the Y102A mutation (SEQ ID NO: 10), see Example 3).

[0256] DNA fragments encoding the heavy, light, and J chains of the selected mutants were synthesized by a commercial vendor. The DNA constructs were transformed into competent bacteria and plated on LB plates with multiple selective antibiotics. Several bacterial colonies were picked and DNA preparation was performed by standard molecular biology techniques. The constructs encoding the heavy and light chains were verified by sequencing. The plasmid constructs encoding the heavy, light, and J chains were co-transfected into HEK293 / Expi293 / CHO cells according to standard methods, and cells expressing the CD20 IgM antibody were selected. The antibody present in the cell supernatant was recovered using Capture Select IgM (Catalog 2890.05, BAC, Thermo Fisher) according to the manufacturer's protocol. The antibody was evaluated on SDS polyacrylamide gel electrophoresis under non-reducing conditions to show the assembly as described previously, e.g., the assembly in PCT Publication No. WO 2016 / 141303. The expression and assembly of each mutant were verified, and the bispecific construct was verified to retain its ability to promote T cell-mediated cytotoxicity.

[0257] Receptor binding with certain IgM mutants and corresponding IgM and IgG controls was measured by ELISA as follows. White opaque 96-well polystyrene ELISA plates were coated with 1 μg / mL of polymeric immunoglobulin receptor (mouse PIGR: R&D systems, Cat#2800-PG; human PIGR: Cat#2717-PG), human Fc alpha / mu receptor (R&D systems, Cat#9278-FC-050), or human Fc mu receptor / protein (Sino Biological, Cat#13556-H02H or R&D systems, Cat#9494-MU-050) in 100 μL of PBS overnight at 4 °C. Then, the plates were washed 5 times with 0.05% PBS-Tween and blocked with 2% BSA-PBS for 2 hours at room temperature. After blocking and washing 5 times, serial dilutions of IgM or IgG protein in 100 μL of 2% BSA-PBS were added to the wells and incubated for 2 hours at room temperature. Then, the plates were washed 5 times and incubated with HRP-conjugated mouse anti-human κ (Southern Biotech, Cat#9230-05. Diluted 1:6000 in 2% BSA-PBS) for 30 minutes. After the secondary incubation, the plates were washed 10 times, and then 100 μL of SuperSignal chemiluminescent substrate (Thermo Fisher, Cat#37070) was added to each well. The luminescence signal was measured, and the data were plotted and analyzed using GraphPad Prism with a 4-parameter logistic model.

[0258] The binding of various mutant IgMs to human and mouse receptors is summarized in Figure 2 . The T103A mutation in the J chain inhibits human FcαμR binding but not human pIgR or human FcμR binding (data not shown).

[0259] The effect of bispecific anti-CD20 / anti-CD3 antibodies with additional amino acid substitutions at the amino acid position corresponding to Y102 of SEQ ID NO:2 in the V15J-modified J chain on binding to the pIgR receptor was also evaluated. Tyrosine at position 102 was replaced with aspartic acid (Y102D), phenylalanine (Y102F), arginine (Y102R), serine (Y102S), and threonine (Y102T). The mutations were incorporated into the anti-CD20 / anti-CD3 bispecific antibody as described above. Except for IgM containing the Y102D mutation, which exhibited reduced expression, the mutant IgMs were expressed and assembled as expected. The results of binding to pIgR are shown in Figures 3A to 3EShown in. The Y102F and Y102T mutations allow binding to pIgR, while like the Y102A mutation, the Y102S and Y102R mutations disrupt pIgR binding.

[0260] Example 3: Pharmacokinetic Analysis of Anti-CD20 Antibodies Containing Selected Mutations Affecting Receptor Binding

[0261] This example demonstrates that certain amino acid substitutions in the J chain or IgM constant region of pentameric IgM monospecific or bispecific antibodies can increase the serum half-life of those antibodies. The J chain and IgM constant region of the monospecific anti-CD20 IgM antibody 1.5.3 (with the mature J chain of SEQ ID NO:2) as a monospecific pentamer and the bispecific anti-CD20 IgM antibody 1.5.3IgM (with the "V15J" J chain, SEQ ID NO:9) as a bispecific pentamer, both as described in PCT Publication No. WO 2016 / 141303, were subjected to alanine scanning mutagenesis, and the changes in pharmacokinetic parameters were tested in mice. The mutants tested included J chain alanine substitutions (Y102A and T103A) at positions corresponding to Y102 and T103 of the human mature J chain of SEQ ID NO:2. The modified "V15J" J chain containing the Y102A mutation is designated SEQ ID NO:10.

[0262] The pharmacokinetic parameters of various IgM antibodies were measured in an in vivo mouse model as follows. Each antibody (100 μg) was injected into Balb / c mice by intravenous infusion. At approximately zero time and each time point, 500 μL of blood was collected by terminal cardiac puncture, with 3 mice at each time point, and a total of 8 or 15 time points for each antibody. The serum concentration of each antibody in the blood at each time point was measured using a standard ELISA assay. Quality metrics were verified on all ELISAs, and PK parameters, including T 1 / 2-阿尔法 、T 1 / 2-贝塔 and the area under the concentration curve from zero time to infinity (AUC 0-∞ , measured in μg / ml*hr), were derived using standard curve fitting techniques (Win Non Lin, Phoenix Software). The PK results, including alpha and beta half-lives and AUC, are shown in Figure 4 . The results indicate that mutations corresponding to Y102 in the mature human J chain SEQ ID NO:2 and mutations corresponding to S401 and E401 in the human IgM constant region can improve the PK parameters of the resulting IgM antibodies either alone or in combination.

[0263] Figure 5Shows a comparison of the serum concentrations over time of certain bispecific mutant IgM antibodies with IgG antibodies containing the same VH and VL regions. As these results show, the serum half-life of IgM having a single Y102A mutation in the J chain or having the Y102A mutation plus additional mutations in the IgM heavy chain is close to that of the comparable IgG antibody.

[0264] Example 4: Prolonged half-life of IgM antibody molecules containing the N49A variant human J chain.

[0265] This example demonstrates that another single amino acid substitution in the human J chain can increase the serum half-life of IgM antibodies containing the variant J chain. The previously described modified J chain (“V15J,” SEQ ID NO:9, see U.S. Patent No. 9,951,134) containing the mature human J chain and a heterologous antigen-binding domain that binds to CD3 fused to its N-terminus via a 15-amino acid linker was mutated using standard techniques to introduce an alanine substitution at the position corresponding to N49 of the wild-type mature human J chain (SEQ ID NO:2) to produce the modified J chain “V15J-N49A” (SEQ ID NO:24). SEQ ID NO:24 is shown below.

[0266] SEQ ID NO:24:

[0267] QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

[0268] This J-chain construct was used to generate exemplary pentameric IgM antibodies, 1.5.3V15J and 1.5.3V15J-N49A, that bind to CD20. The CD20 antigen-binding domain and methods for preparing the IgM antibodies are described in PCT Publication No. WO / 2016 / 141303. The correct assembly of the antibodies, antigen binding, and the ability of the CD3 binder to activate T cells were tested.

[0269] Pharmacokinetic (PK) studies were conducted in Balb / c mice as described in Example 3 to evaluate the clearance rate of the IgM antibodies. Quality metrics were verified on all ELISAs, and PK parameters, including T 1 / 2-阿尔法 、T 1 / 2-贝塔 and the area under the concentration curve from zero time to infinity (AUC 0-∞ , measured in μg / ml*hr), were derived using standard curve-fitting techniques (Win Non Lin, Phoenix Software).

[0270] The results are shown in Figure 6 As shown, IgM 1.5.3V15J-N49A showed an almost 50% increase in T 1 / 2-阿尔法 and a 1.6-fold increase in the area under the concentration curve.

[0271] The breadth and scope of the present disclosure should not be limited to the above exemplary embodiments, but should be defined solely by the appended claims and their equivalents. Sequence Listing <110> IGM BIOSCIENCES, INC. <120> IgM Fc and J-chain Mutations Affecting IgM Serum Half-Life <130> 09789.012WO1 <140> <141> <150> 62 / 637,186 <151> 2018-03-01 <160> 34 <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 sequence: synthetic Polypeptide <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> 137 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 4 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 Ser 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> 5 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 5 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 Arg 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> 6 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence description: Synthetic Polypeptide <400> 6 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 Ala 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> 7 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 7 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 Ala 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> 8 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence description: Synthetic Polypeptide <400> 8 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 Ala 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> 9 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 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 Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 260 265 270 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 275 280 285 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 290 295 300 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 305 310 315 320 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 325 330 335 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 340 345 350 Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 355 360 365 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 370 375 380 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 385 390 <210> 10 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 10 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr 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 Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 260 265 270 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 275 280 285 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 290 295 300 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 305 310 315 320 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 325 330 335 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 340 345 350 Ala Thr Glu Thr Cys Ala Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 355 360 365 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 370 375 380 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 385 390 <210> 11 <211> 1012 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 11 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 Val 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 Tyr 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 Gly Gly Gly Gly 405 410 415 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ala His Lys Ser 420 425 430 Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala 435 440 445 Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu 450 455 460 Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 465 470 475 480 Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu 485 490 495 Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly 500 505 510 Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys 515 520 525 Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg 530 535 540 Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr 545 550 555 560 Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 565 570 575 Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 580 585 590 Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 595 600 605 Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 610 615 620 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 625 630 635 640 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala 645 650 655 Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys 660 665 670 Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala 675 680 685 Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 690 695 700 Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 705 710 715 720 Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 725 730 735 Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 740 745 750 Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr 755 760 765 Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 770 775 780 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val 785 790 795 800 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 805 810 815 Gln Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln Asn 820 825 830 Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 835 840 845 Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 850 855 860 Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 865 870 875 880 Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val 885 890 895 Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 900 905 910 Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 915 920 925 Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 930 935 940 Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 945 950 955 960 Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 965 970 975 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu 980 985 990 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala 995 1000 1005 Ala Leu Gly Leu 1010 <210> 12 <211> 453 <212> PRT <213> Homo sapiens <400> 12 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> 13 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence description: Synthetic Polypeptide <400> 13 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 Ala 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> 14 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 14 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 Ala 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> 15 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence description: Synthetic Polypeptide <400> 15 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 Ala Ser Ser 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> 16 <211> 455 <212> PRT <213> Mouse (Mus musculus) <400> 16 Ala Ser Gln Ser Phe Pro Asn Val Phe Pro Leu Val Ser Cys Glu Ser 1 5 10 15 Pro Leu Ser Asp Lys Asn Leu Val Ala Met Gly Cys Leu Ala Arg Asp 20 25 30 Phe Leu Pro Ser Thr Ile Ser Phe Thr Trp Asn Tyr Gln Asn Asn Thr 35 40 45 Glu Val Ile Gln Gly Ile Arg Thr Phe Pro Thr Leu Arg Thr Gly Gly 50 55 60 Lys Tyr Leu Ala Thr Ser Gln Val Leu Leu Ser Pro Lys Ser Ile Leu 65 70 75 80 Glu Gly Ser Asp Glu Tyr Leu Val Cys Lys Ile His Tyr Gly Gly Lys 85 90 95 Asn Arg Asp Leu His Val Pro Ile Pro Ala Val Ala Glu Met Asn Pro 100 105 110 Asn Val Asn Val Phe Val Pro Pro Arg Asp Gly Phe Ser Gly Pro Ala 115 120 125 Pro Arg Lys Ser Lys Leu Ile Cys Glu Ala Thr Asn Phe Thr Pro Lys 130 135 140 Pro Ile Thr Val Ser Trp Leu Lys Asp Gly Lys Leu Val Glu Ser Gly 145 150 155 160 Phe Thr Thr Asp Pro Val Thr Ile Glu Asn Lys Gly Ser Thr Pro Gln 165 170 175 Thr Tyr Lys Val Ile Ser Thr Leu Thr Ile Ser Glu Ile Asp Trp Leu 180 185 190 Asn Leu Asn Val Tyr Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe 195 200 205 Leu Lys Asn Val Ser Ser Thr Cys Ala Ala Ser Pro Ser Thr Asp Ile 210 215 220 Leu Asn Phe Thr Ile Pro Pro Ser Phe Ala Asp Ile Phe Leu Ser Lys 225 230 235 240 Ser Ala Asn Leu Thr Cys Leu Val Ser Asn Leu Ala Thr Tyr Glu Thr 245 250 255 Leu Ser Ile Ser Trp Ala Ser Gln Ser Gly Glu Pro Leu Glu Thr Lys 260 265 270 Ile Lys Ile Met Glu Ser His Pro Asn Gly Thr Phe Ser Ala Lys Gly 275 280 285 Val Ala Ser Val Cys Val Glu Asp Trp Asn Asn Arg Lys Glu Phe Val 290 295 300 Cys Thr Val Thr His Arg Asp Leu Pro Ser Pro Gln Lys Lys Phe Ile 305 310 315 320 Ser Lys Pro Asn Glu Val His Lys His Pro Pro Ala Val Tyr Leu Leu 325 330 335 Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Val Thr 340 345 350 Cys Leu Val Lys Gly Phe Ser Pro Ala Asp Ile Ser Val Gln Trp Lys 355 360 365 Gln Arg Gly Gln Leu Leu Pro Gln Glu Lys Tyr Val Thr Ser Ala Pro 370 375 380 Met Pro Glu Pro Gly Ala Pro Gly Phe Tyr Phe Thr His Ser Ile Leu 385 390 395 400 Thr Val Thr Glu Glu Glu Trp Asn Ser Gly Glu Thr Tyr Thr Cys Val 405 410 415 Val Gly His Glu Ala Leu Pro His Leu Val Thr Glu Arg Thr Val Asp 420 425 430 Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Ile Met Ser 435 440 445 Asp Thr Gly Gly Thr Cys Tyr 450 455 <210> 17 <211> 472 <212> PRT <213> Rhesus macaque (Macaca mulatta) <400> 17 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ala Pro Leu Asp Thr Asn Glu Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Phe Lys Asn Asn Ser 35 40 45 Asn Ile Ser Lys Gly Val Trp Gly Phe Pro Ser Val Leu Arg Gly Gly 50 55 60 Lys Tyr Ala Ala Thr Ser Gln Val Leu Leu Ala Ser Lys Asp Val Met 65 70 75 80 Gln Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly 85 90 95 Asn Lys Glu Gln Asn Val Pro Leu Pro Val Leu Ala Glu Arg Pro Pro 100 105 110 Asn Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Val Gly Asn Pro 115 120 125 Arg Glu Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln 130 135 140 Ile Glu Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Ile 145 150 155 160 Thr Thr Asp Arg Val Glu Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr 165 170 175 Phe Lys Val Thr Ser Thr Leu Thr Val Ser Glu Arg Asp Trp Leu Ser 180 185 190 Gln Ser Val Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln 195 200 205 Lys Asn Val Ser Ser Val Cys Gly Pro Asn Pro Asp Thr Ala Ile Arg 210 215 220 Val Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser 225 230 235 240 Thr Lys Leu Thr Cys Leu Val Thr Asp Leu Ala Thr Tyr Asp Ser Val 245 250 255 Thr Ile Thr Trp Thr Arg Gln Asn Gly Glu Ala Leu Lys Thr His Thr 260 265 270 Asn Ile Ser Glu Ser His Pro Asn Gly Thr Phe Ser Ala Val Gly Glu 275 280 285 Ala Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Arg Cys 290 295 300 Thr Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser 305 310 315 320 Arg Pro Lys Gly Val Ala Met His Arg Pro Asp Val Tyr Leu Leu Pro 325 330 335 Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys 340 345 350 Leu Val Thr Gly Phe Ser Pro Ala Asp Ile Phe Val Gln Trp Met Gln 355 360 365 Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met 370 375 380 Pro Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr 385 390 395 400 Val Ser Glu Glu Asp Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val 405 410 415 Ala His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys 420 425 430 Ser Thr Glu Gly Glu Val Ser Ala Asp Glu Glu Gly Phe Glu Asn Leu 435 440 445 Trp Ala Thr Ala Ser Thr Phe Ile Val Leu Phe Leu Leu Ser Leu Phe 450 455 460 Tyr Ser Thr Thr Val Thr Leu Phe 465 470 <210> 18 <211> 488 <212> PRT <213> Chimpanzee (Pan troglodytes) <400> 18 Thr Thr Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Phe 1 5 10 15 Pro Leu Val Ser Cys Glu Asn Ser Pro Ser Asp Thr Ser Ser Val Ala 20 25 30 Val Gly Cys Leu Ala Gln Asp Phe Leu Pro Asp Ser Ile Thr Phe Ser 35 40 45 Trp Lys Tyr Lys Asn Asn Ser Asp Ile Ser Ser Thr Arg Gly Phe Pro 50 55 60 Ser Val Leu Arg Gly Gly Lys Tyr Ala Ala Thr Ser Gln Val Leu Leu 65 70 75 80 Pro Ser Lys Glu Val Met Gln Gly Thr Asp Glu His Val Val Cys Lys 85 90 95 Val Gln His Pro Asn Gly Asn Lys Glu Lys Asn Val Pro Leu Pro Val 100 105 110 Thr Ala Glu Leu Pro Pro Lys Val Ser Ile Phe Val Pro Pro Arg Asp 115 120 125 Gly Phe Phe Gly Asn Pro Arg Ser Ser Lys Leu Ile Cys Gln Ala Thr 130 135 140 Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys 145 150 155 160 Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys 165 170 175 Gln Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys 180 185 190 Glu Ser Asp Trp Leu Ser Gln Ser Val Phe Thr Cys Arg Val Asp His 195 200 205 Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Ser Pro Gly 210 215 220 Glu Ser Arg His Ser His Pro Gly Leu Cys His Pro Pro Ser Phe Ala 225 230 235 240 Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Ala Cys Leu Val Thr Asp 245 250 255 Leu Thr Thr Tyr Asp Ser Leu Thr Ile Ser Trp Thr Arg Gln Asn Gly 260 265 270 Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala 275 280 285 Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn 290 295 300 Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser 305 310 315 320 Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Glu Val Ala Leu His Arg 325 330 335 Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg 340 345 350 Glu Leu Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp 355 360 365 Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys 370 375 380 Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr 385 390 395 400 Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly 405 410 415 Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val 420 425 430 Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Thr His Pro Val Gln 435 440 445 Arg Val Pro Gly His Val Arg His Ser Trp His Leu Leu Leu Thr Leu 450 455 460 Leu Ala Cys Pro Gln Ala Gln Gly Gly Arg Pro Leu Cys Val Cys Ala 465 470 475 480 Cys Lys Leu Thr Val Ser Thr Gly 485 <210> 19 <211> 453 <212> PRT <213> Sumatran orangutan (Pongo abelii) <400> 19 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Leu 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 Thr Gly Ser Lys 50 55 60 Tyr Val 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 Ile Phe Ile Pro Pro Arg Asp Gly Phe Phe Gly Ser 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 Ala Ser Gly Ile 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 Asn 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 Lys 195 200 205 Asn Ala Ser Ser Met Cys Ser Pro Asn Pro Asn 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 Ala Ser Tyr Asp Ser Met 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 Ala 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 Asp 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> 20 <211> 764 <212> PRT <213> Homo sapiens <400> 20 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 Ser 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> 21 <211> 390 <212> PRT <213> Homo sapiens <400> 21 Met Asp Phe Trp Leu Trp Pro Leu Tyr Phe Leu Pro Val Ser Gly Ala 1 5 10 15 Leu Arg Ile Leu Pro Glu Val Lys Val Glu Gly Glu Leu Gly Gly Ser 20 25 30 Val Thr Ile Lys Cys Pro Leu Pro Glu Met His Val Arg Ile Tyr Leu 35 40 45 Cys Arg Glu Met Ala Gly Ser Gly Thr Cys Gly Thr Val Val Ser Thr 50 55 60 Thr Asn Phe Ile Lys Ala Glu Tyr Lys Gly Arg Val Thr Leu Lys Gln 65 70 75 80 Tyr Pro Arg Lys Asn Leu Phe Leu Val Glu Val Thr Gln Leu Thr Glu 85 90 95 Ser Asp Ser Gly Val Tyr Ala Cys Gly Ala Gly Met Asn Thr Asp Arg 100 105 110 Gly Lys Thr Gln Lys Val Thr Leu Asn Val His Ser Glu Tyr Glu Pro 115 120 125 Ser Trp Glu Glu Gln Pro Met Pro Glu Thr Pro Lys Trp Phe His Leu 130 135 140 Pro Tyr Leu Phe Gln Met Pro Ala Tyr Ala Ser Ser Ser Lys Phe Val 145 150 155 160 Thr Arg Val Thr Thr Pro Ala Gln Arg Gly Lys Val Pro Pro Val His 165 170 175 His Ser Ser Pro Thr Thr Gln Ile Thr His Arg Pro Arg Val Ser Arg 180 185 190 Ala Ser Ser Val Ala Gly Asp Lys Pro Arg Thr Phe Leu Pro Ser Thr 195 200 205 Thr Ala Ser Lys Ile Ser Ala Leu Glu Gly Leu Leu Lys Pro Gln Thr 210 215 220 Pro Ser Tyr Asn His His Thr Arg Leu His Arg Gln Arg Ala Leu Asp 225 230 235 240 Tyr Gly Ser Gln Ser Gly Arg Glu Gly Gln Gly Phe His Ile Leu Ile 245 250 255 Pro Thr Ile Leu Gly Leu Phe Leu Leu Ala Leu Leu Gly Leu Val Val 260 265 270 Lys Arg Ala Val Glu Arg Arg Lys Ala Leu Ser Arg Arg Ala Arg Arg 275 280 285 Leu Ala Val Arg Met Arg Ala Leu Glu Ser Ser Gln Arg Pro Arg Gly 290 295 300 Ser Pro Arg Pro Arg Ser Gln Asn Asn Ile Tyr Ser Ala Cys Pro Arg 305 310 315 320 Arg Ala Arg Gly Ala Asp Ala Ala Gly Thr Gly Glu Ala Pro Val Pro 325 330 335 Gly Pro Gly Ala Pro Leu Pro Pro Ala Pro Leu Gln Val Ser Glu Ser 340 345 350 Pro Trp Leu His Ala Pro Ser Leu Lys Thr Ser Cys Glu Tyr Val Ser 355 360 365 Leu Tyr His Gln Pro Ala Ala Met Met Glu Asp Ser Asp Ser Asp Asp 370 375 380 Tyr Ile Asn Val Pro Ala 385 390 <210> 22 <211> 532 <212> PRT <213> Homo sapiens <400> 22 Met Pro Leu Phe Leu Ile Leu Cys Leu Leu Gln Gly Ser Ser Phe Ala 1 5 10 15 Leu Pro Gln Lys Arg Pro His Pro Arg Trp Leu Trp Glu Gly Ser Leu 20 25 30 Pro Ser Arg Thr His Leu Arg Ala Met Gly Thr Leu Arg Pro Ser Ser 35 40 45 Pro Leu Cys Trp Arg Glu Glu Ser Ser Phe Ala Ala Pro Asn Ser Leu 50 55 60 Lys Gly Ser Arg Leu Val Ser Gly Glu Pro Gly Gly Ala Val Thr Ile 65 70 75 80 Gln Cys His Tyr Ala Pro Ser Ser Val Asn Arg His Gln Arg Lys Tyr 85 90 95 Trp Cys Arg Leu Gly Pro Pro Arg Trp Ile Cys Gln Thr Ile Val Ser 100 105 110 Thr Asn Gln Tyr Thr His His Arg Tyr Arg Asp Arg Val Ala Leu Thr 115 120 125 Asp Phe Pro Gln Arg Gly Leu Phe Val Val Arg Leu Ser Gln Leu Ser 130 135 140 Pro Asp Asp Ile Gly Cys Tyr Leu Cys Gly Ile Gly Ser Glu Asn Asn 145 150 155 160 Met Leu Phe Leu Ser Met Asn Leu Thr Ile Ser Ala Gly Pro Ala Ser 165 170 175 Thr Leu Pro Thr Ala Thr Pro Ala Ala Gly Glu Leu Thr Met Arg Ser 180 185 190 Tyr Gly Thr Ala Ser Pro Val Ala Asn Arg Trp Thr Pro Gly Thr Thr 195 200 205 Gln Thr Leu Gly Gln Gly Thr Ala Trp Asp Thr Val Ala Ser Thr Pro 210 215 220 Gly Thr Ser Lys Thr Thr Ala Ser Ala Glu Gly Arg Arg Thr Pro Gly 225 230 235 240 Ala Thr Arg Pro Ala Ala Pro Gly Thr Gly Ser Trp Ala Glu Gly Ser 245 250 255 Val Lys Ala Pro Ala Pro Ile Pro Glu Ser Pro Pro Ser Lys Ser Arg 260 265 270 Ser Met Ser Asn Thr Thr Glu Gly Val Trp Glu Gly Thr Arg Ser Ser 275 280 285 Val Thr Asn Arg Ala Arg Ala Ser Lys Asp Arg Arg Glu Met Thr Thr 290 295 300 Thr Lys Ala Asp Arg Pro Arg Glu Asp Ile Glu Gly Val Arg Ile Ala 305 310 315 320 Leu Asp Ala Ala Lys Lys Val Leu Gly Thr Ile Gly Pro Pro Ala Leu 325 330 335 Val Ser Glu Thr Leu Ala Trp Glu Ile Leu Pro Gln Ala Thr Pro Val 340 345 350 Ser Lys Gln Gln Ser Gln Gly Ser Ile Gly Glu Thr Thr Pro Ala Ala 355 360 365 Gly Met Trp Thr Leu Gly Thr Pro Ala Ala Asp Val Trp Ile Leu Gly 370 375 380 Thr Pro Ala Ala Asp Val Trp Thr Ser Met Glu Ala Ala Ser Gly Glu 385 390 395 400 Gly Ser Ala Ala Gly Asp Leu Asp Ala Ala Thr Gly Asp Arg Gly Pro 405 410 415 Gln Ala Thr Leu Ser Gln Thr Pro Ala Val Gly Pro Trp Gly Pro Pro 420 425 430 Gly Lys Glu Ser Ser Val Lys Arg Thr Phe Pro Glu Asp Glu Ser Ser 435 440 445 Ser Arg Thr Leu Ala Pro Val Ser Thr Met Leu Ala Leu Phe Met Leu 450 455 460 Met Ala Leu Val Leu Leu Gln Arg Lys Leu Trp Arg Arg Arg Thr Ser 465 470 475 480 Gln Glu Ala Glu Arg Val Thr Leu Ile Gln Met Thr His Phe Leu Glu 485 490 495 Val Asn Pro Gln Ala Asp Gln Leu Pro His Val Glu Arg Lys Met Leu 500 505 510 Gln Asp Asp Ser Leu Pro Ala Gly Ala Ser Leu Thr Ala Pro Glu Arg 515 520 525 Asn Pro Gly Pro 530 <210> 23 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 23 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 Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 260 265 270 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 275 280 285 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 290 295 300 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 305 310 315 320 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 325 330 335 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 340 345 350 Ala Thr Glu Thr Cys Tyr Ala Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 355 360 365 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 370 375 380 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 385 390 <210> 24 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <400> 24 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 Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 260 265 270 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 275 280 285 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 290 295 300 Ala Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 305 310 315 320 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 325 330 335 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 340 345 350 Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 355 360 365 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 370 375 380 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 385 390 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Peptide <400> 25 Gly Gly Gly Gly Ser 1 5 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Peptide <400> 26 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 27 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Peptide <400> 27 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 28 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Peptide <400> 28 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> 29 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Peptide <400> 29 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> 30 <211> 487 <212> PRT <213> Cynomolgus monkey (Macaca fascicularis) <400> 30 Phe Trp Gly Gln Gly Ala Leu Val Thr Val Ser Ser Gly Glu Ser Ala 1 5 10 15 Gly Pro Phe Lys Trp Glu Pro Ser Val Ser Ser Pro Asn Ala Pro Leu 20 25 30 Asp Thr Asn Glu Val Ala Val Gly Cys Leu Ala Gln Asp Phe Leu Pro 35 40 45 Asp Ser Ile Thr Phe Ser Trp Lys Phe Lys Asn Asn Ser Asp Ile Ser 50 55 60 Lys Gly Val Trp Gly Phe Pro Ser Val Leu Arg Gly Gly Lys Tyr Ala 65 70 75 80 Ala Thr Ser Gln Val Leu Leu Ala Ser Lys Asp Val Met Gln Gly Thr 85 90 95 Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn Lys Glu 100 105 110 Gln Asn Val Pro Leu Pro Val Val Ala Glu Arg Pro Pro Asn Val Ser 115 120 125 Val Phe Val Pro Pro Arg Asp Gly Phe Val Gly Asn Pro Arg Glu Ser 130 135 140 Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Glu Val 145 150 155 160 Ser Trp Leu Arg Asp Gly Lys Gln Val Gly Ser Gly Ile Thr Thr Asp 165 170 175 Arg Val Glu Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Phe Lys Val 180 185 190 Thr Ser Thr Leu Thr Val Ser Glu Arg Asp Trp Leu Ser Gln Ser Val 195 200 205 Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Lys Asn Val 210 215 220 Ser Ser Val Cys Gly Pro Asn Pro Asp Thr Ala Ile Arg Val Phe Ala 225 230 235 240 Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu 245 250 255 Thr Cys Leu Val Thr Asp Leu Ala Thr Tyr Asp Ser Val Thr Ile Thr 260 265 270 Trp Thr Arg Gln Asn Gly Glu Ala Leu Lys Thr His Thr Asn Ile Ser 275 280 285 Glu Ser His Pro Asn Gly Thr Phe Ser Ala Val Gly Glu Ala Ser Ile 290 295 300 Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Arg Cys Thr Val Thr 305 310 315 320 His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys 325 330 335 Gly Val Ala Met His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg 340 345 350 Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr 355 360 365 Gly Phe Ser Pro Ala Asp Ile Phe Val Gln Trp Met Gln Arg Gly Gln 370 375 380 Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro 385 390 395 400 Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu 405 410 415 Glu Asp Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu 420 425 430 Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly 435 440 445 Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Ile Leu Trp Thr Thr Leu 450 455 460 Ser Thr Phe Val Ala Leu Phe Val Leu Thr Leu Leu Tyr Ser Gly Ile 465 470 475 480 Val Thr Phe Ile Lys Val Arg 485 <210> 31 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 31 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 Ala 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> 32 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence description: Synthetic Polypeptide <400> 32 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 Ala 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> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence description: Synthetic Peptide <400> 33 Gly Ser Gly Ser Gly Ser Gly 1 5 <210> 34 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence description: Synthetic Polypeptide <400> 34 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 Ala 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

Claims

1. An IgM antibody with an enhanced serum half-life, said IgM antibody comprising five bivalent antibody binding units and a variant human J chain, wherein each binding unit comprises two IgM heavy chain constant regions, each of which is associated with an antigen-binding domain or a subunit thereof, wherein the amino acid sequence of said variant human J chain is based on SEQ ID NO: 2: (i) the amino acid Y102 is replaced by alanine (A); or (ii) the amino acid N49 is replaced by alanine (A); and wherein said IgM antibody exhibits an increased serum half-life when administered to a test animal, relative to a reference IgM antibody administered to the same animal species in the same manner.

2. The IgM antibody according to claim 1, wherein the amino acid Y102 of SEQ ID NO: 2 is replaced by alanine (A), and wherein said variant human J chain consists of the amino acid sequence of SEQ ID NO:

3.

3. The IgM antibody according to claim 1, wherein the amino acid N49 of SEQ ID NO: 2 is replaced by alanine (A), and wherein said variant human J chain consists of the amino acid sequence of SEQ ID NO:

7.

4. The IgM antibody according to claim 1, wherein each IgM heavy chain constant region is a human IgM heavy chain constant region consisting of the amino acid sequence of SEQ ID NO:

12.

5. The IgM antibody according to claim 1, wherein each IgM heavy chain constant region is a variant human IgM heavy chain constant region, wherein the variant human IgM heavy chain constant region is based on SEQ ID NO: 12, and the amino acid S401 or E402 of SEQ ID NO: 12 is replaced by alanine (A), and wherein said IgM antibody exhibits a further increased serum half-life when administered to a test animal, relative to a reference IgM antibody administered to the same animal species in the same manner.

6. The IgM antibody according to claim 5, wherein each IgM heavy chain constant region consists of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

7. An IgM antibody with an enhanced serum half-life, said IgM antibody comprising five or six bivalent antibody binding units, wherein each binding unit comprises two variant IgM heavy chain constant regions, each of which is associated with an antigen-binding domain or a subunit thereof, Each of the variant IgM heavy chain constant regions is based on SEQ ID NO: 12, with the amino acid S401 or E402 in SEQ ID NO: 12 replaced by alanine (A); And wherein said IgM antibody exhibits an increased serum half-life upon administration to a test animal relative to a reference IgM antibody administered to the same animal species in the same manner.

8. The IgM antibody according to claim 7, wherein each variant human IgM heavy chain constant region consists of the amino acid sequence SEQ ID NO: 13 or SEQ ID NO:

14.

9. The IgM antibody according to claim 7, which is pentameric and further comprises a variant human J chain consisting of the amino acid sequence SEQ ID NO:

2.

10. The IgM antibody according to claim 7, which is pentameric and further comprises a variant human J chain consisting of the amino acid sequence SEQ ID NO:

3.

11. The IgM antibody according to any one of claims 1 to 10, wherein said increased serum half-life comprises an increased alpha half-life (t 1 / 2 α), an increased beta half-life (t 1 / 2 β), or an increased t 1 / 2 α and an increased t 1 / 2 β.

12. The IgM antibody according to claim 11, wherein relative to said reference antibody, said IgM antibody further exhibits an increased plasma peak concentration (Cmax), an increased area under the curve (AUC), an improved clearance time, or any combination thereof.

13. The IgM antibody according to any one of claims 1 to 10, wherein said antigen-binding domain comprises a single-chain Fv (ScFv) fragment or a single-domain variable region (VHH).

14. The IgM antibody according to any one of claims 1 to 10, wherein said antigen-binding domain subunit comprises a heavy chain variable region (VH).

15. The IgM antibody according to claim 14, wherein each binding unit further comprises two human light chain constant regions, each of which associates with a light chain variable region (VL).

16. The IgM antibody according to any one of claims 1 to 6, 9 or 10, wherein said human J chain or variant human J chain further comprises one or more heterologous polypeptides fused to the human J chain or variant human J chain.

17. The IgM antibody according to claim 16, wherein said one or more heterologous polypeptides are fused to the human J chain or variant human J chain or a fragment thereof via a peptide linker.

18. The IgM antibody according to claim 16, wherein the one or more heterologous polypeptides are fused to the N-terminus of the human J chain or variant human J chain, the C-terminus of the human J chain or variant human J chain, or wherein the heterologous polypeptide is fused to both the N-terminus and C-terminus of the human J chain or variant human J chain, and wherein the heterologous polypeptides may be the same or different.

19. The IgM antibody according to claim 16, wherein at least one heterologous polypeptide comprises a scFv.

20. The IgM antibody according to claim 19, wherein the scFv specifically binds to CD3ε.

21. A composition comprising the IgM antibody according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

22. An isolated polynucleotide comprising a nucleic acid encoding the IgM antibody according to any one of claims 1 to 10.

23. An expression vector comprising the polynucleotide according to claim 22.

24. A host cell comprising the polynucleotide according to claim 22 or the expression vector according to claim 23.

Images