Highly sialized polymer-binding molecules

Abstract

The present disclosure provides monoclonal populations of highly sialylated multimeric binding molecules, which include IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules, and which have a higher level of sialic acid content than that found in normal serum IgM. Methods for producing such monoclonal populations of highly sialylated multimeric binding molecules are also provided. TIFF2023509476000018.tif76140

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the rights to U.S. Provisional Patent Application No. 62 / 957,745, filed on 6 January 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing, which was filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 5, 2021, is named 028WO1-Sequence-Listing and is 92,335 bytes in size. [Background technology]

[0003] background Multimerizable antibodies and antibody-like molecules, such as IgA and IgM antibodies, are emerging as promising drug candidates in fields such as immuno-oncology and infectious diseases, enabling improved specificity, enhanced binding affinity, and the ability to bind to multiple binding targets. For example, see U.S. Patent No. 9,951,134 (Patent Document 1), No. 9,938,347 (Patent Document 2), No. 10,351,631 (Patent Document 3), No. 10,400,038 (Patent Document 4), No. 10,570,191 (Patent Document 5), No. 10,604,559 (Patent Document 6), No. 10,618,978 (Patent Document 7), No. 10,689,449 (Patent Document 8), and No. 10,787,520 (Patent Document 9), U.S. Patent Application Publication No. 2019-0330. See Patent Document No. 374 (Patent Document 10), Patent Document No. 2019-0330360 (Patent Document 11), Patent Document No. 2019-0338040 (Patent Document 12), Patent Document No. 2019-0338041 (Patent Document 13), Patent Document No. 2019-0185570 (Patent Document 14), and Patent Document No. 2019-0002566 (Patent Document 15), Patent Document No. 2020-0239572 (Patent Document 16), as well as PCT Publications WO2018 / 187702 (Patent Document 17) and WO2019 / 165340 (Patent Document 18). The contents of these are incorporated herein by reference in their entirety.

[0004] The pharmacokinetics (PK) and pharmacodynamics (PD) of multivalent antibodies are complex and depend on both the translated and posttranslational structures of monoclonal antibodies, as well as the physiological systems they target. Furthermore, different antibody classes are typically processed within a subject via different cells and physiological systems. For example, the serum half-life of the IgG antibody class is 20 days, while the half-lives of IgM and IgA antibodies are only about 5-8 days (Brekke, OH., and I. Sandlie, Nature Reviews Drug Discovery 2:52-62 (2003) (Non-patent Literature 1)).

[0005] One of the key determinants of the pharmacokinetic properties (PK) of antibodies or other biotherapeutic agents is their level and type of glycosylation (Higel, F. et al., Eur. J. Pharm. Biopharm. 139:123-131 (2019) (Non-Patent Literature 2)). The sugar moieties and their derivatives covalently bound to specific residues of an antibody determine how they are recognized by receptors such as the asialoglycoprotein (ASGP) receptor, and in turn, how quickly they are removed from the systemic circulation. Each IgM heavy chain constant region has five asparagine-(N-) linked glycosylation sites, and the J chain has one N- linked glycosylation site. Therefore, the J chain of an IgM pentamer can contain up to 51 glycan moieties, resulting in a complex glycosylation profile (Hennicke, J., et al., Anal. Biochem. 539:162-166 (2017) (Non-Patent Literature 3)). The complexity of glycans can make it difficult to manufacture uniformly glycosylated materials.

[0006] Despite advances in the design of multimeric antibodies, the need to be able to manipulate the physical, pharmacokinetic, and pharmacodynamic properties of these molecules remains. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 9,951,134 [Patent Document 2] U.S. Patent No. 9,938,347 [Patent Document 3] U.S. Patent No. 10,351,631 [Patent Document 4] U.S. Patent No. 10,400,038 [Patent Document 5] U.S. Patent No. 10,570,191 [Patent Document 6] U.S. Patent No. 10,604,559 [Patent Document 7] U.S. Patent No. 10,618,978 [Patent Document 8] U.S. Patent No. 10,689,449 [Patent Document 9] U.S. Patent No. 10,787,520 [Patent Document 10] U.S. Patent Application Publication No. 2019-0330374 [Patent Document 11] U.S. Patent Application Publication No. 2019-0330360 [Patent Document 12] U.S. Patent Application Publication No. 2019-0338040 [Patent Document 13] U.S. Patent Application Publication No. 2019-0338041 [Patent Document 14] U.S. Patent Application Publication No. 2019-0185570 [Patent Document 15] U.S. Patent Application Publication No. 2019-0002566 [Patent Document 16] U.S. Patent Application Publication No. 2020-0239572 [Patent Document 17] WO2018 / 187702 [Patent Document 18] WO2019 / 165340 [Non-patent literature]

[0008] [Non-Patent Document 1] Brekke, OH., and I. Sandlie, Nature Reviews Drug Discovery 2:52-62 (2003) [Non-Patent Document 2] Higel,F. et al.Eur. J. Pharm. Biopharm. 139:123-131 (2019) [Non-Patent Document 3] Hennicke, J., et al., Anal. Biochem. 539:162-166 (2017) [Overview of the project]

[0009] overview Provided herein is a monoclonal population of polymer-binding molecules, each binding molecule comprising 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain specifically binding to a target, each IgM heavy chain constant region comprising at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, each N-linked glycosylation motif comprising the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine, and at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region being occupied by a complex glycan, and the monoclonal population of binding molecules comprising at least 35 moles of sialic acid per mole of binding molecule.

[0010] In some embodiments, the monoclonal population of binding molecules contains at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 124, at least 130, at least 140, or at least 146 moles of sialic acid per mole of binding molecules. In some embodiments, the monoclonal population of binding molecules contains at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 moles of sialic acid per mole of binding molecules. In some embodiments, the monoclonal population of binding molecules contains about 40 to about 70, about 40 to about 60, about 40 to about 55, about 40 to about 50, about 50 to about 70, or about 60 to about 70 moles of sialic acid per mole of binding molecules.

[0011] In some embodiments, the IgM heavy chain constant region is the human IgM heavy chain constant region or a variant thereof, comprising five N-linked glycosylated motifs N-X1-S / T starting from amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04). In some embodiments, motifs N1, N2, and N3 are occupied by complex glycans.

[0012] In some embodiments, monoclonal populations of binding molecules are generated by cell line modification, in vitro glycotechnology, or any combination thereof.

[0013] In some embodiments, cell line modification involves transfecting a cell line that produces a monoclonal population of the binding molecule with a gene encoding sialyltransferase, thereby generating a modified cell line that overexpresses sialyltransferase. In some embodiments, the sialyltransferase includes human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3). In some embodiments, cell line modification further includes transfecting a cell line that produces a monoclonal population of the binding molecule with a gene encoding galactosyltransferase, thereby generating a modified cell line that overexpresses galactosyltransferase. In some embodiments, the galactosyltransferase includes human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).

[0014] In some embodiments, in vitro glycotechnology involves contacting a monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate. In some embodiments, the sialyltransferase comprises a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3). In some embodiments, the soluble variant of ST6GAL1 comprises amino acids x through 406 of SEQ ID NO: 3, where x is an integer from 27 to 120. In some embodiments, the soluble variant of ST6GAL1 includes amino acids 120-406, 115-406, 110-406, 109-406, 105-406, 100-406, 95-406, 90-406, 89-406, 88-406, 87-406, 86-406, 85-406, 84-406, 83-406, 82-406, 81-406, 80-406, 75-406, 70-406, 65-406, 60-406, 55-406, 50-406, 45-406, 40-406, 35-406, 30-406, or 27-406 of SEQ ID NO: 3. In some embodiments, the sialic acid substrate includes cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA).

[0015] In some embodiments, the mass ratio of the binding molecule to the sialic acid substrate is about 1:4 to about 40:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is about 80:1 to about 5000:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is about 500:1. In some embodiments, the mass ratio of the binding molecule to the sialic acid substrate to the sialyltransferase is about 500:62.5:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is about 2000:1. In some embodiments, the mass ratio of the binding molecule to the sialic acid substrate to the sialyltransferase is about 2000:500:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is about 80:1. In some embodiments, the mass ratio of the binding molecule to the sialic acid substrate to the sialyltransferase is about 80:500:1.

[0016] In some embodiments, contact of the monoclonal aggregate of the binding molecule with soluble sialyltransferase and a sialic acid substrate includes contact for at least 30 minutes. In some embodiments, contact includes contact for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, contact of the monoclonal aggregate of the binding molecule with soluble sialyltransferase and a sialic acid substrate occurs at a temperature of about 2°C to about 40°C. In some embodiments, contact occurs at a temperature of 15°C to about 37°C, 15°C to about 30°C, or 15°C to about 25°C.

[0017] In some embodiments, in vitro glycotechnology further comprises contacting a monoclonal population of binding molecules with a galactosyltransferase and a galactose substrate. In some embodiments, the galactosyltransferase comprises a soluble variant of human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4). In some embodiments, the soluble variant of B4GALT4 comprises amino acids x through 344 of SEQ ID NO: 4, where x is an integer from 39 to 120. In some embodiments, the soluble variant of B4GALT4 includes amino acids 120-344, 115-344, 110-344, 105-344, 100-344, 95-344, 90-344, 85-344, 80-344, 75-344, 70-344, 65-344, 60-344, 55-344, 50-344, 45-344, 40-344, or 39-344 of SEQ ID NO: 4. In some embodiments, the galactose substrate includes uridine diphosphate-α-D-galactose (UDP-Gal). In some embodiments, contact with galactosyltransferase and the galactose substrate occurs before or simultaneously with contact with soluble sialyltransferase and the sialic acid substrate.

[0018] In some embodiments, each binding molecule is multispecific, and two or more binding domains bound to the IgM heavy chain constant region of each binding molecule specifically bind to different targets. In some embodiments, the binding domains bound to the IgM heavy chain constant region of each binding molecule specifically bind to the same target. In some embodiments, the binding domains bound to the IgM heavy chain constant region of each binding molecule are identical.

[0019] In some embodiments, the binding domain is an antigen-binding domain derived from the antibody. In some embodiments, each binding molecule is a pentameric or hexamer IgM antibody containing five or six bivalent IgM binding units, each binding unit containing two IgM heavy chains, each containing a VH located at the amino-terminus of the variant IgM constant region, and two immunoglobulin light chains, each containing a light chain variable domain (VL) located at the amino-terminus of the immunoglobulin light chain constant region, where the VH and VL bind to form an antigen-binding domain that specifically binds to the target. In some embodiments, each antigen-binding domain of each binding molecule binds to the same target. In some embodiments, each antigen-binding domain of each binding molecule is identical.

[0020] In some embodiments, the target is a target epitope, a target antigen, a target cell, a target organ, or a target virus.

[0021] In some embodiments, each binding molecule is a pentamer and further comprises a J chain or a functional fragment or a functional variant thereof. In some embodiments, the J chain is a mature human J chain comprising the amino acid sequence of SEQ ID NO: 6, or a functional fragment or a functional variant thereof. In some embodiments, the J chain comprises an N-linked glycosylated motif N-X1-S / T (motif N6) starting from the amino acid position corresponding to amino acid 49 of SEQ ID NO: 6.

[0022] In some embodiments, the J chain is a functional variant J chain that includes one or more single amino acid substitutions, deletions, or insertions compared 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, and the monoclonal population of binding molecules is identical to the variant J chain except for one or more single amino acid substitutions, deletions, or insertions, and exhibits an increased serum half-life upon administration to the target animal compared to binding molecules derived from reference IgM administered using the same method to the same animal species. In some embodiments, the variant J chain or its functional fragment includes one, two, three, or four single amino acid substitutions, deletions, or insertions compared to the reference J chain. In some embodiments, the variant J chain or its functional fragment includes an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J chain of SEQ ID NO: 6.

[0023] In some embodiments, the amino acid corresponding to Y102 in SEQ ID NO: 6 is substituted with alanine (A). In some embodiments, the J chain contains the amino acid sequence of SEQ ID NO: 7.

[0024] In some embodiments, the J chain or its fragment or variant is a modified J chain further comprising a heterologous moiety, the heterologous moiety fused or conjugated to the J chain or its fragment or variant. In some embodiments, the heterologous moiety is a polypeptide fused to the J chain or its fragment or variant. In some embodiments, the heterologous polypeptide is fused to the J chain or its fragment or variant via a peptide linker. In some embodiments, the peptide linker comprises at least 5 but no more than 25 amino acids. In some embodiments, the peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 43).

[0025] In some embodiments, a heterogeneous polypeptide is fused to the N-terminus of the J chain or its fragment or variant, or to the C-terminus of the J chain or its fragment or variant. In some embodiments, the same or different heterogeneous moieties are fused to both the N-terminus and the C-terminus of the J chain or its fragment or variant.

[0026] In some embodiments, the heterologous polypeptide includes a binding domain. In some embodiments, the antigen-binding domain of the heterologous polypeptide is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the heterologous scFv fragment binds to CD3ε. In some embodiments, the modified J chain includes amino acids 20-420 (VJH) of SEQ ID NO: 36 (V15J), SEQ ID NO: 37 (V15J*), SEQ ID NO: 38 (SJ*), SEQ ID NO: 31 (A-55-J*), SEQ ID NO: 32 (A-56-J*), SEQ ID NO: 33 (A-57-J*), SEQ ID NO: 34, amino acids 20-420 (VJ*H) of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO: 6 or 7 fused via a peptide linker to the scFv of anti-CD3ε, which includes the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, including SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0027] Pharmaceutical compositions comprising a monoclonal population of the binding molecules disclosed herein and pharmaceutically acceptable excipients are also provided herein.

[0028] Recombinant host cells that generate monoclonal populations of the binding molecules disclosed herein are also provided herein.

[0029] A method for generating a monoclonal population of the binding molecules disclosed herein is also provided herein, comprising culturing the host cells disclosed herein and recovering the population of binding molecules.

[0030] A method for generating a monoclonal population of highly sialylated multimer-binding molecules, comprising: providing a cell line expressing the monoclonal population of binding molecules; culturing the cell line; and recovering the monoclonal population of binding molecules, wherein each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, and each IgM heavy chain constant region comprising at least 3, at least 4, or at least 5 asparagine (N)-linked glycosylation motifs, and N-linked glycosylation The glycosylation motif comprises the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine, and on average, at least one, at least two, or at least three N-linked glycosylation motifs on each IgM heavy chain constant region in the population are occupied by complex glycans, and cell lines, culture conditions, harvesting processes, or combinations thereof are optimized to enrich complex glycans containing at least one, two, three, or four sialic acid-terminal monosaccharides per glycan, methods are also provided herein.

[0031] A method for generating a monoclonal population of highly sialylated multimer-binding molecules, comprising: providing a cell line expressing the monoclonal population of binding molecules; culturing the cell line; and recovering the monoclonal population of binding molecules, wherein each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, and each IgM heavy chain constant region comprising at least 3, at least 4, or at least 5 asparagine (N)-linked glycosylation motifs, and N-linked glycosylation The sylation motif comprises the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine, and on average, at least one, at least two, or at least three N-linked glycosylation motifs on each IgM heavy chain constant region in the population are occupied by complex glycans, and cell lines, harvesting processes, or combinations thereof are optimized to enrich complex glycans containing at least one, two, three, or four sialic acid-terminal monosaccharides per glycan, methods are also provided herein.

[0032] In some embodiments, cell lines, culture conditions, recovery processes, or combinations thereof are optimized to yield a monoclonal population of binding molecules containing at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 124, at least 130, at least 140, or at least 146 moles of sialic acid per mole of binding molecules. In some embodiments, cell lines, recovery processes, or combinations thereof are optimized to yield a monoclonal population of binding molecules containing at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or at least 60 moles of sialic acid per mole of binding molecules. In some embodiments, the cell line, recovery process, or combination thereof is optimized to yield a monoclonal population of binding molecules containing at least 30, at least 35, at least 40, at least 45, at least 50, or at least 60 moles of sialic acid per mole of binding molecule. In some embodiments, the cell line, recovery process, or combination thereof is optimized to yield a monoclonal population of binding molecules containing about 40–70, about 40–60, about 40–55, about 40–50, about 50–70, or about 60–70 moles of sialic acid per mole of binding molecule.

[0033] In some embodiments, the IgM heavy chain constant region is derived from the human IgM heavy chain constant region, which includes five N-linked glycosylated motifs N-X1-S / T that start at amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04). In some embodiments, on average one, two, or all three of motifs N1, N2, and N3 in the cluster of binding molecules are occupied by complex glycans.

[0034] In some embodiments, the provided cell line is modified to overexpress sialyltransferase. In some embodiments, the sialyltransferase includes human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).

[0035] In some embodiments, the recovery process includes subjecting a monoclonal population of the binding molecule to in vitro glycotechnology. In some embodiments, the in vitro glycotechnology includes contacting the monoclonal population of the binding molecule with a soluble sialyltransferase and a sialic acid substrate. In some embodiments, the sialyltransferase includes a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3). In some embodiments, the soluble variant of ST6GAL1 includes amino acids x through 406 of SEQ ID NO: 3, where x is an integer from 27 to 120. In some embodiments, the soluble variant of ST6GAL1 includes amino acids 120-406, 115-406, 110-406, 109-406, 105-406, 100-406, 95-406, 90-406, 89-406, 88-406, 87-406, 86-406, 85-406, 84-406, 83-406, 82-406, 81-406, 80-406, 75-406, 70-406, 65-406, 60-406, 55-406, 50-406, 45-406, 40-406, 35-406, 30-406, or 27-406 of SEQ ID NO: 3. In some embodiments, the sialic acid substrate includes cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA).

[0036] In some embodiments, the mass ratio of the binding molecule to the sialic acid substrate is about 1:4 to about 40:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is about 80:1 to about 10000:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is about 500:1. In some embodiments, the mass ratio of the binding molecule to the sialic acid substrate to the sialyltransferase is about 500:62.5:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is about 2000:1. In some embodiments, the mass ratio of the binding molecule to the sialic acid substrate to the sialyltransferase is about 2000:500:1. In some embodiments, the molar ratio of the binding molecule to the sialyltransferase is about 80:1. In some embodiments, the molar ratio of the binding molecule to the sialic acid substrate to the sialyltransferase is about 80:500:1.

[0037] In some embodiments, contact of the monoclonal aggregate of the binding molecule with soluble sialyltransferase and a sialic acid substrate includes contact for at least 30 minutes. In some embodiments, contact includes contact for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, contact of the monoclonal aggregate of the binding molecule with soluble sialyltransferase and a sialic acid substrate occurs at a temperature of about 2°C to about 40°C. In some embodiments, contact occurs at a temperature of 15°C to about 37°C, 15°C to about 30°C, or 15°C to about 25°C.

[0038] In some embodiments, in vitro glycotechnology further comprises contacting a monoclonal population of binding molecules with a galactosyltransferase and a galactose substrate. In some embodiments, the galactosyltransferase comprises a soluble variant of human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4). In some embodiments, the galactose substrate comprises uridine diphosphate-α-D-galactose (UDP-Gal). In some embodiments, contact with the galactosyltransferase and galactose substrate occurs before or simultaneously with contact with a soluble sialyltransferase and a sialic acid substrate. [Invention 1001] A monoclonal population of multimer-binding molecules, each binding molecule comprising 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least 1, at least 2, at least 3, at least 4, or at least 5 asparagine (N)-linked glycosylation motifs, wherein the N-linked glycosylation motifs are of the amino acid sequence NX 1 -Includes S / T, where N is asparagine and X 1 The monoclonal population of the polymer-binding molecule is any amino acid other than proline, and S / T is serine or threonine, and at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region are occupied by a complex glycan, and the monoclonal population of the binding molecule contains at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 moles of sialic acid per mole of the binding molecule. [Invention 1002] A monoclonal collection of the binding molecules of Invention 1001, each containing approximately 40-70 moles, 40-60 moles, 40-55 moles, 40-50 moles, 50-70 moles, and 60-70 moles of sialic acid per mole of binding molecule. [Invention 1003] The IgM heavy chain constant region consists of five N-linked glycosylated motifs NX, which start at amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04). 1 A monoclonal population of the binding molecules of the present invention 1001, which include the human IgM heavy chain constant region or its variants, including -S / T. [Invention 1004] A monoclonal population of the binding molecules of the present invention 1001, generated by cell line modification, in vitro glycotechnology, or any combination thereof. [Invention 1005] The monoclonal population of the binding molecule according to the present invention 1004, wherein the cell line modification includes transfecting a cell line that generates a monoclonal population of the binding molecule with a gene encoding sialyltransferase, thereby generating a modified cell line that overexpresses the sialyltransferase. [Invention 1006] The monoclonal group of binding molecules of the present invention 1004, wherein in vitro glycosylation involves contacting the monoclonal group of the binding molecules with a soluble sialyltransferase and a sialic acid substrate. [Invention 1007] A monoclonal population of the binding molecules of the present invention 1006, wherein the sialyltransferase comprises a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3), and / or the sialic acid substrate comprises cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA). [Invention 1008] A monoclonal collection of the binding molecule of Invention 1006, wherein the mass ratio of the binding molecule to the sialic acid substrate is approximately 1:4 to approximately 40:1, and / or the mass ratio of the binding molecule to the sialyltransferase is approximately 80:1 to approximately 5000:1. [Invention 1009] The monoclonal group of the binding molecule according to Invention 1006, wherein contact of the monoclonal group of the binding molecule with the soluble sialyltransferase and the sialic acid substrate includes contact at a temperature of about 2°C to about 40°C for at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours. [Invention 1010] A monoclonal population of binding molecules according to the present invention 1001, wherein each binding molecule is a pentameric or hexamer IgM antibody containing five or six bivalent IgM binding units, and each binding unit contains two IgM heavy chains each containing a VH located at the amino-terminal end of the variant IgM constant region, and two immunoglobulin light chains each containing a light chain variable domain (VL) located at the amino-terminal end of the immunoglobulin light chain constant region, and the VH and VL bind to form an antigen-binding domain that specifically binds to the target. [Invention 1011] A monoclonal collection of binding molecules according to the present invention 1010, wherein each binding molecule is a pentamer and further comprises a J chain or a functional fragment thereof or a functional variant thereof. [Invention 1012] A monoclonal population of the binding molecule of the present invention 1011, wherein the J chain is a mature human J chain containing the amino acid sequence of SEQ ID NO: 6, or a functional fragment thereof, or a functional variant thereof. [Invention 1013] A monoclonal population of the binding molecules of the present invention 1011, wherein the variant J chain or its functional fragment contains an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J chain of SEQ ID NO: 6. [Invention 1014] A monoclonal collection of the binding molecules of the present invention 1013, wherein the amino acid corresponding to Y102 in SEQ ID NO: 6 is substituted with alanine (A). [Invention 1015] The J chain is a monoclonal collection of the binding molecule of the present invention 1014, wherein the J chain contains the amino acid sequence of SEQ ID NO: 7. [Invention 1016] A monoclonal collection of binding molecules according to the present invention 1011, wherein the J chain or its fragment or variant is a modified J chain further comprising a heterogeneous portion, and the heterogeneous portion is fused to or conjugated to the J chain or its fragment or variant. [Invention 1017] A monoclonal collection of binding molecules according to the present invention 1016, wherein the heterogeneous portion is a polypeptide fused to the J chain or a fragment or variant thereof. [Invention 1018] A monoclonal collection of binding molecules according to the present invention 1017, wherein the heterogeneous polypeptide is fused to the J chain or a fragment or variant thereof via a peptide linker containing at least 5 but 25 or fewer amino acids. [Invention 1019] A monoclonal collection of binding molecules according to the present invention 1017, wherein the heterogeneous polypeptide is 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 the C-terminus of the J chain or a fragment or variant thereof, and the heterogeneous polypeptide fused to both the N-terminus and the C-terminus may be the same or different. [Invention 1020] A monoclonal collection of the binding molecule of the present invention 1017, wherein the heterogeneous polypeptide contains an scFv fragment. [Invention 1021] A monoclonal collection of the binding molecule of the present invention 1020, wherein the aforementioned heterogeneous scFv fragments bind to CD3ε. [Invention 1022] A pharmaceutical composition comprising a monoclonal group of any of the binding molecules described in invention 1001 to 1021 and a pharmaceutically acceptable excipient. [Invention 1023] Recombinant host cells that generate a monoclonal population of any of the binding molecules described in invention 1001 to 1021. [Invention 1024] A method for generating a monoclonal population of any of the binding molecules of the present invention 1001 to 1021, comprising culturing a host cell of the present invention 1022 and recovering the population of the binding molecules. [Invention 1025] A method for generating a monoclonal population of highly sialylated multimer-binding molecules, comprising: providing a cell line expressing the monoclonal population of the binding molecules; culturing the cell line; and recovering the monoclonal population of the binding molecules, wherein each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least 3, at least 4, or at least 5 asparagine (N)-linked glycosylation motifs, the N-linked glycosylation motifs having an amino acid sequence NX 1 -Includes S / T, where N is asparagine and X 1 The method wherein is any amino acid other than proline, and S / T is serine or threonine, and on average at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region in the population are occupied by complex glycans, and the cell line, harvesting process, or combination thereof is optimized to enrich complex glycans containing at least one, two, three, or four sialic acid-terminal monosaccharides per glycan. [Invention 1026] The method of the present invention 1025, wherein the cell line, the recovery process, or a combination thereof is optimized to yield a monoclonal population of binding molecules containing at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, or at least 65 moles of sialic acid per mole of binding molecule, or about 40 to about 70, about 40 to about 60, about 40 to about 55, about 40 to about 50, about 50 to about 70, or about 60 to about 70 moles of sialic acid per mole of binding molecule. [Invention 1027] The IgM heavy chain constant region consists of five N-linked glycosylated motifs NX, which start at amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04). 1 A method of the present invention 1025, derived from the human IgM heavy chain constant region, including -S / T. [Invention 1028] The method of the present invention 1025, wherein the provided cell line is modified to overexpress sialyltransferase. [Invention 1029] The method of the present invention 1025, wherein the recovery process comprises subjecting the monoclonal population of the binding molecule to in vitro glycosylation, and the in vitro glycosylation comprises contacting the monoclonal population of the binding molecule with a soluble sialyltransferase and a sialic acid substrate. [Invention 1030] The method of the present invention 1029, wherein the sialyltransferase comprises a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3), and / or the sialic acid substrate comprises cytidine monophosphate (CMP)-N-acetylneuraminic acid (CMP-NANA). [Invention 1031] The method of the present invention 1029, wherein the mass ratio of the binding molecule to the sialic acid substrate is about 1:4 to about 40:1, and / or the mass ratio of the binding molecule to the sialyltransferase is about 80:1 to about 5000:1. [Invention 1032] Any method of the present invention 1029 to 1031, wherein contacting a monoclonal group of the binding molecule with the soluble sialyltransferase and the sialic acid substrate comprises contact at a temperature of about 2°C to about 40°C for at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours. [Brief explanation of the drawing]

[0039] [Figure 1] Figure 1A shows the structure of a "simple" glycan. Figure 1B shows an exemplary structure of an oligomannose glycan. Figure 1C shows an exemplary structure of a complex glycan. Figure 1D shows an exemplary structure of a hybrid glycan. Monosaccharides: dark circle = mannose; light circle = galactose; square = N-acetylglucosamine; diamond = N-acetylneuraminic acid (sialic acid or NANA); triangle = fucose. Derived from: Varki, A., and Schauer, R., Essentials of Glycobiology, 3rd Edition, Chapter 8, Consortium of Glycobiology (2009). [Figure 2] Figure 2A shows the structure of N-acetylneuraminic acid (sialic acid or NANA). Figure 2B shows the structure of cytidine monophosphate N-acetylneuraminic acid (CMP-NANA). [Figure 3A] This is a space-filling model of the human IgM heavy chain, showing the positions of the five N-linked glycosylation sites. [Figure 3B] This shows the alignment of the amino acid sequence of the constant region of the human IgM heavy chain (allele IGHM*04, SEQ ID NO: 2) with that of mouse (GenBank: CAC20701.1, SEQ ID NO: 46) and cynomolgus monkey (GenBank: EHH62210.1, amino acids 14-487 of SEQ ID NO: 47). Amino acids corresponding to the asparagine (N)-linked glycosylation motif are enclosed in squares. [Figure 4] This shows the amount of sialylation of anti-CD20×CD3 IGM-A resulting from treatment with various concentrations of shortened human α-2,6-sialyltransferase (ST6). [Figure 5] This shows the invitrocylylation of two different IgM antibodies, anti-DR5 IgM-B and anti-DR5 IgM-C. [Figure 6] This shows the pharmacokinetics of anti-CD20×CD3 IGM-A and anti-CD20×CD3 IGM-A-GEM antibodies in a mouse model. [Figure 7] This shows the SNA-I lectin labeling of subclones. Cells were labeled with SNA-1 lectin conjugated to fluorescein isothiocyanate (FITC). The geometric mean of the signal from 488 em / 530 ex measured by cytometer is shown for each subclone. [Figure 8] Figure 8A shows a reduced, denatured BioRad® CriterionTGX Stain-Free Precast gel visualized and imaged according to the manufacturer's instructions for use, for the purified protein from fermentation performed on a 2,6-sialyltransferase pool and two subclones (25 and 47). Figure 8B shows a Western blot of the same protein as in Figure 8A, using biotinylated SNA-I lectin. A streptavidin horseradish peroxidase fusion was used for blotting. [Figure 9] Figures 9A-9B show comparative fermentation data from a 3L bioreactor of anti-CD20×CD3 IGM-A producing cell lines. The two curves show the production run using a control parental cell line lacking the 2,6-sialyltransferase gene and subclone 25 possessing the 2,6-sialyltransferase gene. Figure 9A shows the viability of the cell line during the run (VCD). Figure 9B shows the viability of the cell line. Figure 9C shows the titer determined by size exclusion chromatography (SEC). Figure 9D shows the sialic acid ratio measured with purified IgM. [Figure 10] Figure 10A shows a plot of 96-well level screening for CHO cell clones transfected with 2,6-sialyltransferase. Figure 10B shows a plot of cytometry-based analysis of 2,6-sialic acid levels on the cell surface. [Figure 11]Figures 11A and 11B show the levels of 2,3-sialic acid and 2,6-sialic acid in untransfected cells, respectively. Figure 11C compares the levels of 2,3-sialic acid and 2,6-sialic acid in untransfected and transfected cells. [Figure 12] This shows T cell activation by various amounts of antibodies with sialic acid levels within a certain range. [Figure 13] Figures 13A-13B show the time course of sialylation of anti-CD20×CD3-IGM-A at various temperatures using different amounts of ST6 and CMP-NANA. [Figure 14] This shows the time course of sialylation of anti-CD20×CD3-IGM-A at room temperature using various amounts of ST6 and CMP-NANA. [Figure 15] This paper compares sialic acid levels and the resulting AUC0-∞ for various antibodies. [Figure 16] This shows the pharmacokinetics of anti-CD20×CD3 IGM-F (SA18) and anti-CD20×CD3 IGM-F-GEM (SA51) antibodies in a cynomolgus monkey model. [Figure 17] Figure 17A shows the relative number of B cells in cynomolgus monkeys at each time point after administration of anti-CD20×CD3-IGM-F (SA9 or 18) or anti-CD20×CD3-IGM-F-GEM (SA51). Figure 17B shows the day on which B cells in cynomolgus monkeys began to recover after administration of anti-CD20×CD3-IGM-F (SA9 or 18) or anti-CD20×CD3-IGM-F-GEM (SA51). [Modes for carrying out the invention]

[0040] Detailed explanation definition As used herein, the terms “a” or “an” refer to one or more entities; for example, “a binding molecule” is understood to represent one or more binding molecules. In such cases, the terms “a” (or “an”), “one or more,” and “at least one” may be used interchangeably herein.

[0041] Furthermore, when used herein, “and / or” should be understood as a specific disclosure of each of two particular features or components, with or without the other. Thus, when used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when used in phrases such as “A, B, and / or C,” the term “and / or” is intended to encompass 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).

[0042] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the relevant field of this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide general dictionaries of many of the terms used herein.

[0043] Units, prefixes, and symbols are shown in their Systeme International de Unites (SI) approved forms. Numerical ranges include the number defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino-carboxyl direction. The headings provided herein are not intended to limit the various embodiments or embodiments of the disclosure that can be obtained by referring to the entire specification. Thus, the terms defined immediately below are more fully defined by referring to the entire specification.

[0044] As used herein, the term “polypeptide” encompasses both the singular and plural forms “polypeptide” and is intended to refer to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or more of two or more amino acids and does not refer to a product of a specific length. Thus, peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or any other term used to refer to a chain or more of two or more amino acids is included in the definition of “polypeptide,” and the term “polypeptide” may be used in place of any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, and derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. Polypeptides may originate from biological sources or be produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. They can be produced by any method, including by chemical synthesis.

[0045] Polypeptides disclosed herein may have sizes of approximately 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. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure but can rather assume a number of different conformations are referred to as unfolded. As used herein, the term glycoprotein refers to a protein bound to at least one carbohydrate moiety, such as an amino acid, via an oxygen-containing or nitrogen-containing side chain of serine or asparagine. Asparagine(N)-linked glycans are described in more detail elsewhere in this disclosure.

[0046] An “isolated” polypeptide or its fragments, variants, or derivatives are intended to be a polypeptide not found in its natural environment. No specific level of purification is required. For example, an isolated polypeptide may be taken from its natural or natural environment. Recombinant polypeptides and proteins expressed in host cells, similar to naturally occurring or recombinant polypeptides separated, fractionated, or partially or substantially purified by any appropriate technique, are considered isolated when disclosed in this specification.

[0047] As used herein, the term “non-naturally occurring polypeptide” or any grammatical variation thereof is a conditional definition that expressly excludes, but only such, forms of polypeptides that are determined or may be determined or interpreted as “naturally occurring” by a judge, or by an administrative or judicial body.

[0048] Other polypeptides disclosed herein include fragments, derivatives, analogs, or variants of the polypeptides described herein, and any combination thereof. Where disclosed herein, the terms “fragment,” “variant,” “derivative,” and “analog” include any polypeptide that retains at least some of the properties of the corresponding native antibody or polypeptide, for example, specific binding to an antibody. Polypeptide fragments include, in addition to specific antibody fragments discussed elsewhere herein, proteolytic fragments and deletion fragments, for example. Polypeptide variants, for example, include the fragments described above, as well as polypeptides having modified amino acid sequences by amino acid substitutions, deletions, or insertions. In certain embodiments, variants may not exist in nature. Variants that do not exist in nature can be generated using mutagenesis techniques known in the art. Variant polypeptides may include conserved or non-conserved amino acid substitutions, deletions, or additions. Derivatives are polypeptides modified to exhibit additional features not found in the original polypeptide. Examples include fusion proteins. As used herein, the “derivative” of a polypeptide may also refer to the polypeptide of the present invention having one or more amino acids that have been chemically derivatized by the reaction of a functional side chain. Also included as “derivatives” are polypeptides comprising one or more derivatives of 20 standard amino acids. For example, 4-hydroxyproline may substitute for proline; 5-hydroxylysine may substitute for lysine; 3-methylhistidine may substitute for histidine; homoserine may substitute for serine; and ornithine may substitute for lysine.

[0049] A "conservative amino acid substitution" is one in which one amino acid is replaced by another amino acid having a similar side chain. The family of amino acid residues having similar side chains is defined in the art and includes amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substituting tyrosine with phenylalanine is a conservative substitution. In certain embodiments, conserved substitutions in the sequences of the polypeptides, binding molecules, and antibodies of this disclosure do not inhibit the binding of the polypeptides, binding molecules, or antigens to the antigens to which the antibodies bind. Methods for identifying conserved nucleotide and amino acid substitutions that do not preclude antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0050] The term "polynucleotide" is intended to encompass single and multiple nucleic acids and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The terms "nucleic acid" or "nucleic acid sequence" refer to any one or more nucleic acid segments present in a polynucleotide, such as fragments of DNA or RNA.

[0051] "Isolated" nucleic acids or polynucleotides refer to any form of nucleic acid or polynucleotide isolated from its natural environment. For example, gel-purified polynucleotides, or recombinant polynucleotides encoding polypeptides contained in vectors, would be considered "isolated." Also, polynucleotide segments manipulated to have restriction sites for cloning, such as PCR products, are also considered "isolated." Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or purified (partially or substantially) polynucleotides in non-natural solutions such as buffer or saline. Isolated RNA molecules include RNA transcripts of polynucleotides in vivo or in vitro, where the transcripts are not found in nature. Isolated polynucleotides or nucleic acids further include synthetically produced such molecules. Furthermore, polynucleotides or nucleic acids may be or contain regulatory elements such as promoters, ribosome-binding sites, or transcription terminators.

[0052] As used herein, the term “polynucleotide not found in nature” or any grammatical variation thereof is a conditional definition that expressly excludes, but only such, any form of nucleic acid or polynucleotide that may be determined or interpreted as “found in nature” by a judge, or by an administrative or judicial body.

[0053] As used herein, “coding region” refers to a portion of a nucleic acid consisting of codons that are translated into amino acids. “Stop codons” (TAG, TGA, or TAA) are not translated into amino acids and may be considered part of the coding region, but adjacent sequences such as promoters, ribosome-binding sites, transcription terminators, and introns are not part of the coding region. Two or more coding regions may reside on a single polynucleotide construct, e.g., a single vector, or on separate polynucleotide constructs, e.g., separate (different) vectors. Furthermore, any vector may contain a single coding region or two or more coding regions; for example, a single vector may separately code for an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. Additionally, vectors, polynucleotides, or nucleic acids may contain heterogeneous coding regions that are fused to or unfused to other coding regions. Heterogeneous coding regions include, but are not limited to, those that code for specific elements or motifs, such as secretory signaling peptides or heterogeneous functional domains.

[0054] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide containing a nucleic acid encoding a polypeptide may typically include promoters and / or other transcriptional or translational regulatory elements that operably bind to one or more coding regions. An operable binding is when a gene product, e.g., the coding region of a polypeptide, binds to one or more regulatory sequences in such a way that the expression of the gene product is influenced or controlled by those sequences. Two DNA fragments (such as a polypeptide coding region and a promoter bound to it) are said to be "operably linked" if the induction of promoter function results in the transcription of mRNA encoding a desired gene product, and the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to lead to gene product expression, or the ability of the DNA template to be transcribed. Therefore, a promoter is operably linked to a nucleic acid encoding a polypeptide if the promoter can result in the transcription of that nucleic acid. The promoter may be a cell-specific promoter that induces substantial transcription of DNA in a given cell. In addition to promoters, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably conjugated to polynucleotides to induce cell-specific transcription.

[0055] Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, cytomegalovirus (early promoter in combination with intron A), Simianvirus 40 (early promoter), and promoter and enhancer segments derived from retroviruses (such as Roussarcoma virus), and other transcriptional regulatory regions that function in vertebrate cells. Other transcriptional regulatory regions include regions derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, and other sequences that can regulate gene expression in eukaryotic cells. Further preferred transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters that can be induced by interferon or interleukin).

[0056] Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and picornavirus-derived elements (in particular, intra-sequence ribosome entry sites or IRESs, also known as CITE sequences).

[0057] In other embodiments, the polynucleotide may be in the form of RNA, such as messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0058] Polynucleotides and nucleic acid coding regions can be bound to additional coding regions encoding secretory peptides or signal peptides, which, as disclosed herein, direct the secretion of polynucleotides encoded by the polynucleotides. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein when the transport of growth protein chains across the rough endoplasmic reticulum is initiated. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells may have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full-length" polypeptide to produce a secretory or "mature" form of the polypeptide. In certain embodiments, native signal peptides, such as signal peptides of immunoglobulin heavy or light chains, are used, or functional derivatives of their sequences that retain the ability to direct polypeptide secretion operably bound thereto are used. Alternatively, heterologous mammalian signal peptides or functional derivatives 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 mouse β-glucuronidase.

[0059] As used herein, the term “binding molecule” in its broadest sense refers to a molecule that specifically binds to a receptor or target, such as an epitope or antigenic determinant. As further described herein, a binding molecule may include one or more “binding domains” as described herein, such as “antigen-binding domains” as described herein. Non-limiting examples of binding molecules are antibodies or antibody-like molecules described in detail herein that possess antigen-specific binding. In certain embodiments, a “binding molecule” includes antibodies or antibody-like molecules or antibody-derived molecules described in detail herein.

[0060] As used herein, the terms “binding domain” or “antigen-binding domain” (which may be used interchangeably) refer to a region of a binding molecule, such as an antibody or antibody-like molecule or antibody-derived molecule, that is necessary and sufficient to specifically bind to a target, such as an epitope, polypeptide, cell, or organ. For example, “Fv,” such as the heavy chain variable region and light chain variable region of an antibody, is considered a “binding domain” as either two distinct polypeptide subunits or a single chain. Other binding domains include, non-limitingly, the single-domain heavy chain variable region (VHH) of an antibody derived from a camel species, or the six immunoglobulin complementarity-determining regions (CDRs) expressed within a fibronectin scaffold. The “binding molecule” or “antibody” as described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more “antigen-binding domains.”

[0061] The terms “antibody” and “immunoglobulin” may be used interchangeably herein. An antibody (or any fragment, variant, or derivative thereof disclosed herein, e.g., an IgM-like antibody) comprises at least a variable region of the heavy chain (e.g., derived from a camel species), or at least variable domains of both the heavy and light chains. The basic immunoglobulin structures in vertebrate systems are relatively well understood. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise stated, the term “antibody” encompasses everything from small antigen-binding fragments of antibodies to full-sized antibodies, such as IgG antibodies containing two complete heavy chains and two complete light chains, IgA antibodies containing four complete heavy chains and four complete light chains and a J chain and / or secretory components, or IgM-derived binding molecules, such as IgM antibodies or IgM-like antibodies, containing 10 or 12 complete heavy chains and 10 or 12 complete light chains and optionally a J chain or a functional fragment or variant thereof.

[0062] The term “immunoglobulin” encompasses a broad class of polypeptides that can be biochemically distinguished. Those skilled in the art will understand that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4 or α1-α2) in between. It is the properties of this chain that determine the “isotype” of an antibody, such as IgG, IgM, IgA, IgD, or IgE, respectively. Subclasses (subtypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., are well-characterized and known to result in functional specialization. Modified versions of each of these immunoglobulins are readily recognizable to those skilled in the art in light of this disclosure and are therefore within the scope of this disclosure.

[0063] Light chains are classified as either κ or λ(κ,λ). Each heavy chain class can bind to either a kappa or lambda light chain. Generally, when immunoglobulins are expressed by, for example, hybridomas, B cells, or genetically modified host cells, the light and heavy chains are covalently bonded to each other, with the "tail" portions of the two heavy chains linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus of the branched ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of a particular antibody, such as an IgG antibody, includes two heavy chain subunits and two light chain subunits covalently bonded by disulfide bonds to form a "Y" structure, which is referred to herein as the "H2L2 structure" or "binding unit".

[0064] The term “binding unit” is used herein to refer to a portion of a binding molecule, such as an antibody, antibody-like molecule, or antigen-derived molecule, its antigen-binding fragment, or its multimerized fragment, which corresponds to a standard “H2L2” immunoglobulin structure, i.e., two heavy chains or fragments thereof, and two light chains or fragments thereof. In certain embodiments, for example, if the binding molecule is a bivalent IgG antibody or its antigen-binding fragment, the terms “binding molecule” and “binding unit” are equivalent. In other embodiments, for example, if the binding molecule is a multimer, such as 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, or any derivative thereof, the binding molecule comprises two or more “binding units.” Two in the case of an IgA dimer, and five or six in the case of an IgM pentamer or hexamer, respectively. A binding unit does not necessarily have to include the heavy and light chains of a full-length antibody, but is typically bivalent, i.e., it contains two “antigen-binding domains,” as defined above. As used herein, certain binding molecules provided in this disclosure are “dimers” and comprise two bivalent binding units comprising an IgA constant region or a polymerized fragment thereof. Certain binding molecules provided in this disclosure are “pentamers” or “hexamers” and comprise five or six bivalent binding units comprising an IgM constant region or a polymerized fragment or variant thereof. Binding molecules comprising two or more binding units, for example, two, five, or six binding units, such as antibodies or antibody-like molecules or antigen-derived binding molecules, are referred to herein as “multimers.”

[0065] As used herein, the term “J chain” refers to the J chain of an IgM or IgA antibody of any animal species, any functional fragment thereof, its derivatives, and / or variants thereof, including the mature human J chain whose amino acid sequence is presented as SEQ ID NO: 6. Various J chain variants and modified J chain derivatives are disclosed herein. As those skilled in the art will recognize, “functional fragment” or “functional variant” includes those fragments and variants that can bind to the constant region of the IgM heavy chain to form a pentameric IgM antibody.

[0066] The term “modified J chain” is used herein to refer to a derivative of a J chain polypeptide comprising a heterogeneous moiety, e.g., a heterogeneous polypeptide, e.g., an exogenous antigen-binding domain or a functional domain introduced into the J chain sequence. The introduction can be achieved by any means, including direct or indirect fusion of the heterogeneous polypeptide or other moiety, or by adhesion via a peptide or chemical linker. The term “modified human J chain” non-limitingly includes a natural human J chain of the amino acid sequence of SEQ ID NO: 6, or a functional fragment thereof, or a functional variant thereof, modified by the introduction of a heterogeneous moiety, e.g., a heterogeneous polypeptide, e.g., an exogenous binding domain. In certain embodiments, the heterogeneous moiety does not interfere with the efficient polymerization of IgM into pentamers and the binding of such polymers to targets. Exemplary modified J chains can be found, for example, in U.S. Patent Nos. 9,951,134, 10,400,038, and 10,618,978, and U.S. Patent Application Publication No. US-2019-0185570, each of which is incorporated herein by reference in its entirety.

[0067] As used herein, the term “IgM-derived binding molecule” collectively refers to natural IgM antibodies, IgM-like antibodies, and other IgM-derived binding molecules that contain a non-antibody-binding domain and / or a functional domain instead of an antibody-antigen-binding domain or its subunits, as well as any fragments thereof, e.g., polymerized fragments, variants, or derivatives thereof.

[0068] As used herein, the term “IgM-like antibody” generally refers to a variant antibody or antibody-derived conjugate molecule that still retains the ability to bind to, for example, a J chain to form a pentamer or hexamer. IgM-like antibodies or other IgM-derived conjugate molecules typically contain at least the Cμ4-tp domain of the IgM constant region, but may also contain heavy chain constant region domains from other antibody isotypes of the same or different species, e.g., IgG. IgM-like antibodies or other IgM-derived conjugate molecules may also be fragments lacking one or more constant region domains, as long as the IgM-like antibody is capable of forming hexamers and / or pentamers. Thus, IgM-like antibodies or other IgM-derived conjugate molecules may, for example, be hybrid IgM / IgG antibodies or “multimerized fragments” of IgM antibodies.

[0069] The terms “valence,” “divalent,” and “multivalent,” and their grammatical synonyms, refer to the number of antigen-binding domains in a given binding molecule, e.g., an antibody, antibody-derived molecule, or antibody-like molecule, or in a given binding unit. Therefore, the terms “divalent,” “tetravalent,” and “hexavalent” for a given binding molecule, e.g., an IgM antibody, an IgM-like antibody, another IgM-derived binding molecule, or a multimerized fragment thereof, indicate the presence of two antigen-binding domains, four antigen-binding domains, and six antigen-binding domains, respectively. A typical IgM antibody, or another IgM-derived binding molecule, where each binding unit is divalent, can have a valence of 10 or 12. Divalent or multivalent binding molecules, e.g., antibodies or antibody-derived molecules, may be monospecific, i.e., all antigen-binding domains may be the same; or they may be bispecific or multispecific, e.g., two or more antigen-binding domains may be different, e.g., binding to different epitopes on the same antigen or to completely different antigens.

[0070] The term "epitope" includes any molecular determinant that can specifically bind to the antigen-binding domain of an antibody, antibody-like molecule, or antibody-derived molecule. In certain embodiments, an epitope may include a chemically active surface grouping of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have three-dimensional structural properties and / or specific charge properties. An epitope is a region of the target to which the antigen-binding domain of an antibody is bound.

[0071] The term “target” is used in its broadest sense to include substances that can be bound by binding molecules, such as antibodies, antibody-like molecules, or antibody-derived molecules. A target may be, for example, a polypeptide, nucleic acid, carbohydrate, lipid, or other molecule, or a minimal epitope on such a molecule. Furthermore, a “target” may be a cell, organ, or organism, such as an animal, plant, microorganism, or virus, containing an epitope that can be bound by binding molecules, such as antibodies, antibody-like molecules, or antibody-derived molecules.

[0072] The light and heavy chains of antibodies, antibody-like molecules, or antibody-derived molecules are classified into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains in both the variable light chain (VL) and variable heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant region (CL) of the light chain and the constant region of the heavy chain (e.g., CH1, CH2, CH3, or CH4) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they become more distal to the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 (or, for example, CH4 in the case of IgM) domain and CL domain actually contain the carboxyl terminus of the heavy chain and light chain, respectively.

[0073] A "full-length IgM antibody heavy chain" is a polypeptide that contains, from the N-terminus to the C-terminus, 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) which may include a tailpiece.

[0074] As described above, variable regions (multiple) allow binding molecules, such as antibodies, antibody-like molecules, or antigen-derived molecules, to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH domains, or subsets of complementarity-determining regions (CDRs), of a binding molecule, such as an antibody, antibody-like molecule, or antibody-derived molecule, are combined to form an antigen-binding domain. More specifically, the antigen-binding domain can be defined by three CDRs on the VH and VL chains, respectively. Certain antibodies form larger structures. For example, IgM can form a pentamer or hexamer molecule containing five or six H2L2 binding units and optionally a J chain covalently bonded via a disulfide bond.

[0075] The six "complementarity-determining regions" or "CDRs" present in the antigen-binding domain of an antibody are short, discontinuous sequences of amino acids specifically positioned to form the antigen-binding domain, assuming the antibody's three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domain, called the "framework" region, exhibit less intermolecular variation. The framework region primarily employs a β-sheet structure, with the CDRs linking the β-sheet structure and, in some cases, forming loops that constitute part of the β-sheet structure. Thus, the framework region acts to form a scaffold, resulting in the positioning of the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its homologous epitope. Since the amino acids constituting the CDR and framework regions are defined in various different ways, those skilled in the art can easily identify any given heavy or light chain variable region (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), the full content of which is incorporated herein by reference).

[0076] Where there are two or more definitions of a term used and / or permitted in the art, the definitions used herein are intended to include all such meanings unless expressly stated otherwise. A specific example is the use of the term “complementarity-determining regions” (“CDR”) to describe discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described, for example, by Kabat et al., US 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 definitions by Kabat and Chothia include overlaps or subsets of amino acids when compared to each other. Nevertheless, the application of any definition (or other definition known to those skilled in the art) referring to the CDR of an antibody or its variant is intended to be within the scope of the terms defined and used herein unless otherwise indicated. Table 1 below shows, for comparison, the appropriate amino acids that encompass the CDRs as defined by each of the cited references above. The exact amino acid numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which amino acids contain a particular CDR by considering the amino acid sequence of the variable region of the antibody.

[0077] (Table 1) Definition of CDR * TIFF0007857221000001.tif33128 * The numbering of all CDR definitions in Table 1 follows the numbering convention established by Kabat et al. (see below).

[0078] Furthermore, the antibody variable region can be analyzed using, for example, the IMGT information system (imgt_dot_cines_dot_fr / ) (IMGT® / V-Quest) to identify the variable region segment, including the CDR (see, for example, Brochet et al., Nucl. Acids Res. 36:W503-508, 2008).

[0079] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can readily assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system presented in Kabat et al., US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). However, unless the use of the Kabat numbering system is expressly stated, sequential numbering is used for all amino acid sequences in this disclosure.

[0080] 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,” US Dept. of Health and Human Services (1991). IgM constant regions can be numbered sequentially (i.e., amino acid number 1 starts from the first amino acid of the constant region) or by using the Kabat numbering scheme. A comparison of the sequential numbering of two alleles in the human IgM constant region (presented herein as SEQ ID NO: 1 (allele IGHM*03) and SEQ ID NO: 2 (allele IGHM*04)) with the numbering by the Kabat system is described below. Underlined amino acid residues are not considered in the Kabat system (the double-underlined "X" below may be serine (S) (SEQ ID NO: 1) or glycine (G) (SEQ ID NO: 2)). Sequential (SEQ ID NO: 1 or SEQ ID NO: 2) / KABAT numbering key for IgM heavy chain TIFF0007857221000002.tif76132

[0081] Binding molecules, such as antibodies, antibody-like molecules, or antibody-derived molecules, antigen-binding fragments, variants, or derivatives thereof, and / or multimerization fragments thereof include, without limitation, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab’, and F(ab’)2, Fd, Fvs, single-chain Fv (scFv), single-chain antibodies, disulfide-bonded Fv (sdFv), fragments containing either the VL domain or the VH domain, and fragments generated by Fab expression libraries. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0082] "Specifically binds" generally means that a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, binds to an epitope through its antigen-binding domain, and the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule, such as an antibody, antibody-like molecule, or antibody-derived molecule, is said to "specifically bind" to an epitope when it binds to the epitope more readily through its antigen-binding domain than to a random unrelated epitope. The term "specificity" is used herein to limit the relative affinity of a particular binding molecule for a particular epitope. For example, binding molecule "A" can be considered to have higher specificity for a given epitope than binding molecule "B", or it can be said that binding molecule "A" binds to epitope "C" with higher specificity than to related epitope "D".

[0083] Binding molecules, such as the antibodies or fragments, variants, or derivatives thereof disclosed herein, are 5×10 -2 seconds -1 10 -2 seconds -1 5×10 -3 seconds -1 10 -3 seconds -1 5×10 -4 seconds -1 10 -4seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 , or 10 -7 seconds -1 It can be said that the target antigen binds at the following off-rate (k(off)).

[0084] The binding molecule, for example, the antibody or its fragment, variant, or derivative disclosed herein, is 10 times the target antigen. 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , 5×10 4 M -1 seconds -1 , 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5×10 6 M -1 seconds -1 , or 10 7 M -1 seconds -1 It can be said that the connections are made using the above on-rate (k(on)).

[0085] A binding molecule, such as an antibody or its fragment, variant, or derivative, is said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks the reference antibody or antigen-binding fragment from binding to that epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. A binding molecule can be said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0086] As used herein, the term "affinity" refers to a measure of the strength of binding between an individual epitope and one or more antigen-binding domains, such as the antigen-binding domains of an immunoglobulin molecule. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), pp. 27-28. As used herein, the term "binding strength" refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, for example, Harlow, pp. 29-34. Binding strength is related to both the affinity of individual antigen-binding domains within a population possessing a particular epitope, and the titer of the immunoglobulin-antigen binding. For example, the interaction between a bivalent monoclonal antibody and a highly repeating epitope structure, such as a polymer, is considered to have high binding strength. The interaction between a bivalent monoclonal antibody and receptors present at high densities on the cell surface is also considered to have high binding strength.

[0087] The binding molecules disclosed herein, such as antibodies, or their fragments, variants, or derivatives, can also be described or identified in terms of their cross-reactivity. As used herein, the term “cross-reactivity” refers to the ability of a binding molecule specific to one antigen, such as an antibody, or its fragments, variants, or derivatives, to react with a second antigen, and is a measure of the relevance 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 generally contain many of the same complementary structural features as the inducible epitope, and in some cases, can actually fit better than the original.

[0088] Binding molecules, such as antibodies or their fragments, variants, or derivatives, can also be described or specified in relation to their binding affinity to the antigen. For example, a binding molecule may have a binding affinity of 5 × 10⁻⁶. -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or 10 -15 Dissociation constants less than or equal to M, i.e., K D It can then bind to the antigen.

[0089] An "antigen-binding antibody fragment" containing a single-chain antibody or other binding domain may exist alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domain, J chain, or secretory component. The antigen-binding fragment may also include any combination of a variable region(s) and one or more of the hinge region, CH1, CH2, CH3, or CH4 domain, J chain, or secretory component. The binding molecule, e.g., antibody or its antigen-binding fragment, may be of any animal origin, including birds and mammals. The antibody may be, for example, a human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody. In another embodiment, the variable region may originate from cartilaginous fish (e.g., shark). As used herein, “human” antibody includes antibodies having the amino acid sequence of human immunoglobulins, and also includes antibodies isolated from a human immunoglobulin library or from animals transgenic to one or more human immunoglobulins, and may or may not express endogenous immunoglobulins, as described below and, for example, as described in U.S. Patent No. 5,939,598 by Kucherlapati et al. According to embodiments of the present disclosure, IgM antibodies, IgM-like antibodies, or other IgM-derived conjugated molecules, as provided herein, may include antigen-binding fragments of the antibody sufficient to enable the IgM antibody, IgM-like antibody, or other IgM-derived conjugated molecule to form a multimer, for example, a hexamer or pentamer, such as an scFv fragment. As used herein, such fragments include “multimerized fragments.”

[0090] As used herein, the term “heavy chain subunit” includes an amino acid sequence derived from an immunoglobulin heavy chain, and a binding molecule, e.g., an antibody, antibody-like molecule, or antibody-derived molecule containing a heavy chain subunit, may 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, antibody-derived molecule, or fragment thereof, e.g., a multimerized fragment, variant, or derivative, may, non-limitingly, include, in addition to the VH domain, a CH1 domain; a CH1 domain, hinge, and CH2 domain; a CH1 domain and CH3 domain; a CH1 domain, hinge, and CH3 domain; or a CH1 domain, hinge domain, CH2 domain, and CH3 domain. In certain embodiments, the conjugating molecule, e.g., an antibody, antibody-like molecule, or antigen-derived molecule, or a fragment thereof, e.g., a multimerized fragment, variant, or derivative, may include the CH3 domain and the CH4 domain in addition to the VH domain; or the CH3 domain, the CH4 domain, and the J chain. Furthermore, the conjugating molecule for use in this disclosure, e.g., an antibody, antibody-like molecule, or antibody-derived molecule, may lack certain constant region portions, e.g., all or part of the CH2 domain. Those skilled in the art will understand that such domains (e.g., heavy chain subunits) may be modified to have a different amino acid sequence from the original immunoglobulin molecule. According to embodiments of this disclosure, an IgM antibody, IgM-like antibody, or other IgM-derived conjugating molecule, as provided herein, includes a portion of the IgM heavy chain constant region sufficient to enable the IgM antibody, IgM-like antibody, or other IgM-derived conjugating molecule to form a multimer, e.g., a hexamer or a pentamer. As used herein, such fragment includes a “multimerized fragment”.

[0091] As used herein, the term “light chain subunit” includes an amino acid sequence derived from an immunoglobulin light chain. A light chain subunit includes at least a VL and may further include a CL (e.g., Cκ or Cλ) domain.

[0092] A binding molecule, such as an antibody, antibody-like molecule, antibody-derived molecule, its antigen-binding fragment, variant, or derivative, or its polymerized fragment, can be described or identified in terms of the target to which the binding molecule recognizes or specifically binds, such as an epitope(s) or part(s) of an antigen. A part of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an “epitope” or “antigenic determinant.” A target antigen may contain a single epitope or at least two epitopes, and may contain any number of epitopes depending on the size, conformation, and type of the antigen.

[0093] As used herein, the term “disulfide bond” includes, for example, a covalent bond formed between two sulfur atoms in a cysteine ​​residue of a polypeptide. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a second thiol group. The disulfide bond may be “intrachain,” i.e., bonded to a cysteine ​​residue in a single polypeptide or polypeptide subunit, or “interchain,” i.e., linking two separate polypeptide subunits, such as an antibody heavy chain and an antibody light chain, to the constant region and J chain of an antibody heavy chain, or an IgM or IgA antibody heavy chain.

[0094] As used herein, the term “chimeric antibody” refers to an antibody in which the immune response region or site is obtained or induced from a first species, and the constant region (which may be intact, partially, or modified) is obtained from a second species. In some embodiments, the target-binding region or site is of non-human origin (e.g., mouse or primate), and the constant region is human.

[0095] The terms "multispecific antibody" or "bispecific antibody" refer to an antibody, antibody-like molecule, or antibody-derived molecule that has antigen-binding domains for two or more different epitopes within a single antibody molecule. Other binding molecules added to a standard antibody structure may be constructed using two binding specificities. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines capable of secreting bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means (Strohlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). Bispecific antibodies may also be diabodies.

[0096] As used herein, the term “manipulated antibody” refers to an antibody in which the variable domain, constant region, and / or J chain are modified by at least partial substitution of one or more amino acids. In certain embodiments, an entire CDR from an antibody of known specificity can be transplanted into the framework region of a heterologous antibody. The replacement CDR may be derived from an antibody of the same class or subclass as the antibody from which the framework region originates, but the CDR can also be derived from an antibody of a different class, e.g., from an antibody of a different species. A manipulated antibody in which one or more “donor” CDRs from a non-human antibody with known specificity are transplanted into a human heavy or light chain framework region is referred to herein as a “humanized antibody.” In certain embodiments, not all CDRs are replaced with a complete CDR from the donor variable region, but the antigen-binding ability of the donor can still be transferred to the variable domain of the recipient. For example, considering the descriptions in U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, this falls well within the skill of a person skilled in the art, and functionally manipulated antibodies or humanized antibodies can be obtained by routine experiments or by trial and error.

[0097] As used herein, the term “manipulated” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or any combination thereof).

[0098] As used herein, the terms “conjugated,” “fused,” or “combined,” or other grammatical synonyms, may be used interchangeably. These terms refer to the joining 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 open reading frames (ORFs) of polynucleotides in a manner that preserves the translational reading frame of the original ORFs to form a continuous, longer ORF. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to polypeptides encoded by the original ORFs (these segments do not typically join in this manner in nature). Although the reading frame is thus constructed continuously across the fused segments, the segments may be separated physically or spatially, for example, by an in-frame linker sequence. For example, polynucleotides encoding immunoglobulin variable region CDRs can be fused in-frame, but can be separated by at least one polynucleotide encoding an immunoglobulin framework region or an additional CDR region, as long as the “fused” CDR is co-translated as part of a continuous polypeptide.

[0099] In the context of polypeptides, a "linear sequence" or "sequence" refers to the order of amino acids within a polypeptide from the amino terminus to the carboxyl terminus, where adjacent amino acids within the sequence are consecutive within the polypeptide's primary structure. A polypeptide portion that is "amino-terminal" or "N-terminal" relative to another part of the polypeptide is the first to appear in a continuous polypeptide chain. Similarly, a polypeptide portion that is "carboxyl-terminal" or "C-terminal" relative to another part of the polypeptide is the second to appear in a continuous polypeptide chain. For example, in a typical antibody, the variable region is "N-terminal" relative to the constant region, and the constant region is "C-terminal" relative to the variable region.

[0100] As used herein, the term “expression” refers to the process by which a gene produces a biochemical substance, such as a polypeptide. This process includes gene knockdown, as well as any expression of a functional presence of a gene within a cell, including but not limited to transient and stable expression. This includes, but is not limited to, the transcription of a gene into RNA, such as messenger RNA (mRNA), and the translation of such mRNA into polypeptides. If the final desired product is a biochemical substance, expression includes the creation of that biochemical substance and any precursors. Gene expression produces a “gene product.” As used herein, a gene product may be either a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from the transcript. Gene products as described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0101] The terms “N-linked oligosaccharide,” “N-linked sugar,” “N-linked glycan,” and other similar or grammatical variants refer to oligosaccharide chains linked to a peptide backbone via asparagine residues. All N-linked oligosaccharides have a common pentasaccharide core of Man3GlcNAc2, also called “simple oligosaccharides.” See Figure 1A. N-linked glycans can generally be classified into three types: (1) oligomannoses in which only mannose residues are linked to the core (Figure 1B); (2) complexes in which “antennas” initiated by N-acetylglucosaminyltransferase (GlcNAcT) are attached to the core (Figure 1C); and (3) hybrids in which only mannose residues are linked to the Manα1-6 arms of the core, with one or two antennas on the Manα1-3 arms (Figure 1D). For example, see Varki, A., and Schauer, R., Essentials of Glycobiology, 3D Edition, Chapter 8, Consortium of Glycobiology (2009).

[0102] The term "glycosyltransferase" refers to an enzyme capable of transferring a monosaccharide from a nucleotide sugar to an acceptor molecule such as an oligosaccharide. Examples of such glycosyltransferases include, but are not limited to, glucosyltransferases, mannosyltransferases, galactosyltransferases, and sialyltransferases. These enzymes are typically type II membrane proteins found in the Golgi organ of cells, with the active portion of the enzyme located in the Golgi lumen. In glycosyltransferase catalysis, the monosaccharide substrate units glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), galacturonic acid (GalUA), glucuronic acid (GlcUA), galacturonic acid (GalUA), and xylose are activated as uridine diphosphate (UDP)-α-D derivatives; arabinose is activated as a UDP-β-L derivative; mannose (Man) and fucose are activated as GDP-α-D and GDP-β-L derivatives, respectively; and sialic acid (=β-D-Neu5Ac;=Neu5Ac;=SA;=NANA) is activated as a CMP derivative of sialic acid. See, for example, U.S. Patent Application Publication No. US2017 / 0298405.

[0103] The term "sialic acid" refers to any member of the family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonunropyranos-1-onic acid, often abbreviated as Neu5Ac, NeuAc, or NANA). See Figure 2A, e.g., Varki, A., and Schauer, R., Essentials of Glycobiology, 3rd Edition, Chapter 14, Consortium of Glycobiology (2009).

[0104] Sialyltransferases (="ST") are glycosyltransferases that catalyze the transfer of sialic acid residues from a donor substrate to, for example, the terminal monosaccharide acceptor group of an N-linked glycan of a glycoprotein. Mammalian sialyltransferases, including the human ST species, use cytidine-5'-monophospho-N-acetylneuraminic acid (=CMP-β-D-Neu5Ac;=CMP-Neu5Ac;=CMP-NANA;=CMP-sialic acid;=CMP-SA, Figure 2B) as a common donor substrate. Other functional equivalents are known, including but not limited to azide-CMP-sialic acid used for glycan labeling by "click" chemistry. See, for example, Moh, et al., Anal. Biochem. 584:11385 (2019). The transfer and covalent bonding of sialic acid residues (or their functional equivalents) to receptor sites is also called "sialylating" or "sialylation."

[0105] Terminal sialic acid residues can be bound to galactose residues by various linkages, for example, (i) α2→3 (α2,3) linked to galactose or (ii) α2→6 (α2,6) linked to galactose. Sialyltransferase enzymes are generally named and classified according to their respective monosaccharide acceptor substrates and the position of the glycosidic bond they catalyze. Exemplary eukaryotic sialyltransferases include (i) ST3Gal (e.g., found in CHO cells) and (ii) ST6Gal (found in human cells). The abbreviation "ST3" specifically includes sialyltransferases that catalyze α2,3 sialylation. The abbreviation "ST6" specifically includes sialyltransferases that catalyze α2,6 sialylation.

[0106] The disaccharide moiety β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine (=Galβ1,4GlcNAc) is a frequent sialic acid acceptor of the antennae of N-linked glycans in glycoproteins. Furthermore, the terminal Galβ1,4GlcNAc moiety can be generated in certain target glycoproteins as a result of galactosyltransferase enzyme activity, e.g., human β-1,4-galactosyltransferase 4 (="hB4GALT4"). The enzyme β-galactoside-α2,6-sialyltransferase (="ST6Gal") can catalyze glycan or glycan branching, or α2,6-sialylation of the terminal Galβ1,4GlcNAc acceptor moiety of antennae.

[0107] The activity of the ST6Gal enzyme catalyzes the transfer of a free galactosyl residue, which is part of the terminal Galβ1,4GlcNAc of the Neu5Ac residue in the glycan or the antenna of the glycan, to a C6 hydroxyl group, thereby forming a terminal sialic acid residue that is α2→6 linked to the galactosyl residue of the Galβ1,4GlcNAc moiety in the glycan.

[0108] The wild-type polypeptide of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I, UniProtKB / Swiss-Prot:P15907.1) is shown as SEQ ID NO: 3. Mammalian sialyltransferases share a type II structure with glycosyltransferases present in the Golgi apparatus of other mammals, comprising a cytoplasmic N-terminal tail, a transmembrane domain, a variable-length stem domain, and a C-terminal catalytic domain in the lumen of the Golgi organ. The cytoplasmic domain of hST6GAL-1 contains amino acids 1-9 of SEQ ID NO: 3, the transmembrane domain contains amino acids 10-26 of SEQ ID NO: 3, and the lumen domain contains amino acids 27-406 of SEQ ID NO: 3. Soluble variants of hST6Gal-I may lack at least the transmembrane domain and, provided the enzyme retains catalytic activity, may also lack parts of the N-terminal cytoplasmic domain and lumen domain. In certain embodiments, the soluble variant of ST6GAL1 may comprise amino acids x through 406 of SEQ ID NO: 3, where x is an integer from 27 to 120. For example, soluble variants of ST6GAL1 may include amino acids 120-406, 115-406, 110-406, 109-406, 105-406, 100-406, 95-406, 90-406, 89-406, 88-406, 87-406, 86-406, 85-406, 84-406, 83-406, 82-406, 81-406, 80-406, 75-406, 70-406, 65-406, 60-406, 55-406, 50-406, 45-406, 40-406, 35-406, 30-406, or 27-406 of SEQ ID NO: 3. U.S. Patent Application No. 2017 / 0298405 reports that amino acids 90-109 of SEQ ID NO: 3 confer additional sialidase activity to the enzyme in the presence of free CMP.

[0109] The wild-type polypeptide of human β-1,4-galactosyltransferase 4 (hB4GALT4, UniProtKB / Swiss-Prot:O60513.1) is shown as SEQ ID NO: 4. This enzyme similarly possesses a type II structure comprising a cytoplasmic N-terminal region, a transmembrane region, a variable-length stem region, and a C-terminal catalytic region in the lumen of the Golgi organ. The cytoplasmic region of hB4GALT4 contains amino acids 1-12 of SEQ ID NO: 4, the transmembrane region contains amino acids 13-38 of SEQ ID NO: 4, and the lumen region contains amino acids 39-344 of SEQ ID NO: 4. Soluble variants of hB4GALT4 may lack at least the transmembrane region and, provided that the enzyme retains catalytic activity, a portion of the N-terminal cytoplasmic region and the lumen region. In certain embodiments, soluble variants of hB4GALT4 may contain amino acids x-344 of SEQ ID NO: 4, where x is an integer from 39 to 120. For example, soluble variants of hB4GALT4 may include amino acids 120-344, 115-344, 110-344, 105-344, 100-344, 95-344, 90-344, 85-344, 80-344, 75-344, 70-344, 65-344, 60-344, 55-344, 50-344, 45-344, 40-344, or 39-344 of SEQ ID NO: 4.

[0110] Terms such as “to treat,” “to cure,” “to alleviate,” or “to alleviate” refer to therapeutic means that cure, slow down, reduce, or halt or slow the progression of symptoms of an existing diagnosed pathological condition or disorder. Terms such as “to prevent,” “prevent,” “avoid,” or “block” refer to preventive or precautionary measures that prevent the onset of an undiagnosed targeted pathological condition or disorder. Thus, “those who require treatment” include those who already have a disorder and / or are prone to developing a disorder.

[0111] As used herein, the terms “serum half-life” or “plasma half-life” refer to the time (e.g., minutes, hours, or days) it takes for the concentration of a drug, such as an antibody, antibody-like molecule, or antibody-derived molecule or fragment, such as the multimerized fragment described herein, in serum or plasma to decrease by 50% after administration. Two types of half-lives may be described below: the rate of decrease in plasma concentration due to the redistribution process of the drug from the central compartment, e.g., blood, to the peripheral compartment (e.g., tissue or organ) in the case of intravenous delivery; the alpha half-life, α half-life, or t half-life. 1 / 2 α, and the rate of decrease due to excretion or metabolic processes, beta half-life, β half-life, or t 1 / 2 β.

[0112] As used herein, the term “Area under the plasma drug concentration-time curve” or “AUC” reflects the actual physical exposure to a drug after administration of a given dose and is expressed in mg*h / L units. This area under the curve is measured, for example, from 0 hours (t0) to infinity (∞) and depends on the rate of drug elimination from the body and the dose administered.

[0113] As used herein, the terms “Mean Residence Time” or “MRT” refer to the average length of time a drug remains in the body.

[0114] "Subject," "individual," "animal," "patient," or "mammal" means any subject. In certain embodiments, the subject is a mammalian subject for which diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domesticated animals, livestock, and animals from zoos, sports, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0115] As used herein, the term “subjects that would benefit from a therapeutic agent” refers to a subset of subjects from among all anticipated subjects that would benefit from the administration of a given therapeutic agent, such as a binding molecule including one or more antigen-binding domains, such as an antibody. Such binding molecules, such as antibodies, can be used, for example, for diagnostic procedures and / or for the treatment or prevention of disease.

[0116] IgM antibodies, IgM-like antibodies, IgM-derived binding molecules, and groups of these molecules. IgM is the first immunoglobulin produced by B cells in response to antigenic stimulation. Naturally occurring IgM is found in serum at approximately 1.5 mg / ml and has a half-life of 5 days. IgM is a pentameric or hexamer molecule and therefore contains 5 or 6 binding units. An IgM binding unit typically contains two light chains and two heavy chains. The IgG heavy chain constant region contains three heavy chain constant domains (CH1, CH2, and CH3), but the heavy (μ) constant region of IgM further contains a fourth constant domain (CH4) and a C-terminal "tailpiece" (tp). The human IgM constant region typically contains the amino acid sequence of SEQ ID NO: 1 (e.g., GenBank accession numbers pir||S37768, CAA47708.1, and CAA47714.1, identical to allele IGHM*03) or SEQ ID NO: 2 (e.g., GenBank accession number sp|P01871.4, identical to allele IGHM*04). The human Cμ1 region is located in the range of approximately amino acids 5 to 102 of SEQ ID NO: 1 or 2; the human Cμ2 region is located in the range of approximately amino acids 114 to 205 of SEQ ID NO: 1 or 2; the human Cμ3 region is located in the range of approximately amino acids 224 to 319 of SEQ ID NO: 1 or 2; the Cμ4 region is located in the range of approximately amino acids 329 to 430 of SEQ ID NO: 1 or 2; and the tailpiece is located in the range of approximately amino acids 431 to 453 of SEQ ID NO: 1 or 2.

[0117] Other forms and alleles of the human IgM constant region with slight sequence variations exist, including, but are not limited to, GenBank accession numbers CAB37838.1 and pir||MHHU. Amino acid substitutions, insertions, and / or deletions at the positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 as described elsewhere in this disclosure and claimed may also be incorporated into alternative human IgM sequences and IgM constant region amino acid sequences of other species.

[0118] Human IgM constant regions, as well as certain non-human primate IgM constant regions, typically contain five naturally occurring asparagine (N)-linked glycosylation motifs or sites. See Figures 3A and 3B. As used herein, “N-linked glycosylation motif” contains or consists of the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, for example, Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.), Oxford University Press, USA. N-linked glycosylation motifs arise in the human IgM heavy chain constant region of SEQ ID NO: 1 or SEQ ID NO: 2, starting at positions 46 ("N1"), 209 ("N2"), 272 ("N3"), 279 ("N4"), and 440 ("N5"). These five motifs are conserved in the non-human primate IgM heavy chain constant region, and four of the five are conserved in the mouse IgM heavy chain constant region. See, for example, Figure 3B.

[0119] Studies on recombinant and serum-derived human IgM have shown that the N1, N2, and N3 motifs of the human IgM heavy chain are primarily decorated with complex N-glycans, though not always, and the N4 and N5 motifs are primarily decorated with oligomannose-type N-glycans, though not always. See, for example, Moh, ESX, et al., J. Am. Soc. Mass Spectrom. 27:1143-1155 (2016) and Hennicke, J., et al., Anal. Biochem. 539:162-166 (2017).

[0120] Each IgM heavy chain constant region may be associated with a binding domain, such as an antigen-binding domain, such as scFv or VHH, or a subunit of an antigen-binding domain, such as a VH region. In other embodiments, the binding domain may be a non-antibody-binding domain, such as a receptor ectodomain, its ligand or receptor-binding fragment, a cytokine or its receptor-binding fragment, a growth factor or its receptor-binding fragment, a neurotransmitter or its receptor-binding fragment, a peptide or protein hormone or its receptor-binding fragment, an immune checkpoint modulator ligand or its receptor-binding fragment, or a receptor-binding fragment of an extracellular matrix protein. See, for example, PCT application publication number WO2020 / 086745, which is incorporated herein by reference in its entirety.

[0121] The five IgM-binding units can form complexes with additional small polypeptide chains (J chains), or their functional fragments, variants, or derivatives, to form pentameric IgM antibodies or IgM-like antibodies. The precursor form of the human J chain is shown as SEQ ID NO: 5. The signal peptide extends from amino acid 1 to approximately amino acid 22 in SEQ ID NO: 5, while the mature human J chain extends from approximately amino acid 23 to approximately amino acid 159 in SEQ ID NO: 5. The mature human J chain contains the amino acid sequence of SEQ ID NO: 6.

[0122] Exemplary variants and modified J chains are provided elsewhere in this specification. In the absence of a J chain, an IgM antibody or IgM-like antibody is typically assembled into a hexamer containing up to 12 antigen-binding domains. With a J chain, an IgM antibody or IgM-like antibody is typically assembled into a pentamer containing up to 10 antigen-binding domains, or more if the J chain is a modified J chain containing one or more heterologous polypeptides with additional antigen-binding domains. The assembly of five or six IgM-binding units into a pentameric or hexamer IgM antibody or IgM-like antibody is considered to involve Cμ4 and a tailpiece domain. See, for example, Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Therefore, the pentameric or hexamer IgM antibodies provided in this disclosure typically contain at least Cμ4 and / or a tailpiece domain (collectively referred to herein as Cμ4-tp). Therefore, the "multimerized fragment" of the IgM heavy chain constant region includes at least the Cμ4-tp domain. The IgM heavy chain constant region may additionally 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 or other IgM heavy chain domains. In certain embodiments, the IgM-derived binding molecules provided herein, e.g., IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules may include the complete IgM heavy (μ) chain constant domain, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, or its variants, derivatives, or analogs, e.g., those provided herein.

[0123] In certain embodiments, the Disclosure provides a monoclonal population of multimers, e.g., pentamers or hexamers, of binding molecules, each binding molecule comprising 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region, each bound to a binding domain that specifically binds to a target. These embodiments are described in detail elsewhere in the Disclosure. In certain embodiments, the Disclosure provides a monoclonal population of IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules, comprising 5 or 6 divalent binding units, each binding unit comprising two IgM or IgM-like heavy chain constant regions, or merized fragments or variants thereof, each bound to an antigen-binding domain or a subunit thereof. In certain embodiments, the two IgM heavy chain constant regions contained in each binding unit are human heavy chain constant regions.

[0124] If the monoclonal population of IgM antibodies, IgM-like antibodies, other IgM-derived binding molecules, or multimer-binding molecules provided herein is a pentamer, then the molecules included in the monoclonal population of IgM antibodies, IgM-like antibodies, other IgM-derived binding molecules, or multimer-binding molecules typically further include a J chain, or a functional fragment or variant thereof. In certain embodiments, the J chain is a modified J chain or a variant thereof further including one or more heterogeneous moieties bound to the J chain, as described elsewhere in this specification. In certain embodiments, the J chain may be mutated to affect, for example, enhance the serum half-life of the monoclonal population of IgM antibodies, IgM-like antibodies, other IgM-derived binding molecules, or multivalent binding molecules provided herein, as discussed elsewhere in this specification. In certain embodiments, the J chain may be mutated to affect glycosylation, as discussed elsewhere in this disclosure.

[0125] The IgM heavy chain constant region may include one or more of the following: a Cμ1 domain or a fragment or variant thereof, a Cμ2 domain or a fragment or variant thereof, a Cμ3 domain or a fragment or variant thereof, and / or a Cμ4 domain or a fragment or variant thereof, provided that the constant region can perform a desired function in IgM, an IgM-like antibody, or another IgM-derived binding molecule, for example, by associating with a second IgM constant region to form a binding unit having one, two, or more antigen-binding domains, and / or by associating with another binding unit (the J chain in the case of a pentamer) to form a hexamer or pentamer. In certain embodiments, each of the two IgM heavy chain constant regions or fragments or variants within an individual binding unit includes a Cμ4 domain or a fragment or variant thereof, a tailpiece (tp) or a fragment or variant thereof, or any combination of the Cμ4 domain, TP, or fragments or variants thereof. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants within each binding unit further comprise a Cμ3 domain or a fragment or variant thereof, a Cμ2 domain or a fragment or variant thereof, a Cμ1 domain or a fragment or variant thereof, or any combination thereof.

[0126] Modified J chain In certain embodiments, pentameric IgM-derived binding molecules, such as IgM or IgM-like antibodies, provided herein may be modified, for example, by introducing heterologous moieties, or two or more heterologous moieties, such as polypeptides, without impeding their ability to bind to binding targets of monoclonal populations of IgM antibodies, IgM-like antibodies, other IgM-derived binding molecules, or multimer-binding molecules. See, for example, U.S. Patents 9,951,134, 10,400,038, and 10,618,978, and U.S. Patent Application Publication No. US-2019-0185570. Each of these is incorporated herein by reference in whole. Accordingly, monoclonal populations of multimer-binding molecules containing IgM antibodies, IgM-like antibodies, or multimer-binding molecules containing multimer IgM or IgM-like antibodies as described elsewhere herein may include a modified J chain or functional fragment or variant containing a heterologous moiety, such as a heterologous polypeptide, which is introduced into the J chain or its fragment or variant, for example, by fusion or chemical conjugate. In certain embodiments, the heterologous moiety may be a peptide or polypeptide sequence fused in frame with the J chain or chemically conjugated with the J chain or its fragment or variant. In certain embodiments, the heterologous polypeptide is fused to the J chain or its functional fragment via a linker, such as a peptide linker typically consisting of at least 5 amino acids but 25 amino acids or less. In certain embodiments, the peptide linker consists of GGGGS (SEQ ID NO: 41), GGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGS (SEQ ID NO: 43), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 44), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 45). In certain embodiments, the heterogeneous portion may be a chemical portion conjugated to the J chain.The heterogeneous portion bound to the J chain may include, but is not limited to, a binding portion, such as an antibody or its antigen-binding fragment, such as a single-chain Fv(scFv) molecule, a cytokine, such as IL-2 or IL-15 (see, for example, PCT application publication number WO2020 / 086745, which is incorporated herein by reference in its entirety), an IgM antibody, an IgM-like antibody, another IgM-derived binding molecule, or a stabilizing peptide capable of increasing the human half-life of a monoclonal population of a multimer-binding molecule, such as serum albumin (HSA) or an HSA-binding molecule, or a heterogeneous chemical portion such as a polymer or cytotoxin.

[0127] In some embodiments, the modified J chain may include an antigen-binding domain, which may be, non-limited, a polypeptide capable of specifically binding to a target antigen. In certain embodiments, the antigen-binding domain bound to the modified J chain may be an antibody or its antigen-binding fragment as described elsewhere herein. In certain embodiments, the antigen-binding domain may be, for example, an scFv antigen-binding domain or a single-strand binding domain derived from a camelid antibody or a condricthoid antibody. The antigen-binding domain may 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 bound IgM or IgA antibody. Non-limited insertion sites may be at or near the C-terminus, at or near the N-terminus, or at an internal position accessible based on the three-dimensional structure of the J chain. In certain embodiments, the antigen-binding domain may be introduced into the mature human J chain of SEQ ID NO: 6 between cysteine ​​residues 92 and 101 of SEQ ID NO: 6. In further embodiments, the antigen-binding domain may be introduced into the human J chain of SEQ ID NO: 6 at or near a glycosylation site. In further embodiments, the antigen-binding domain may be introduced into the human J chain of SEQ ID NO: 15 within approximately 10 amino acid residues from the C-terminus or within approximately 10 amino acid residues from the N-terminus.

[0128] In certain embodiments, variant J chains or functional fragments of IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules provided herein include an amino acid substitution at the amino acid position corresponding to amino acid Y102 of a mature wild-type human J chain (SEQ ID NO: 6). “Amino acid corresponding to amino acid Y102 of a mature wild-type human J chain” means an amino acid in the sequence of any species' J chain that is homologous to Y102 in the human J chain. See, for example, U.S. Patent Application Publication No. US 2020-0239572, which is incorporated herein by reference in its entirety. The position corresponding to Y102 in SEQ ID NO: 6 is conserved in at least 43 other species' J chain amino acid sequences. See, for example, Figure 4 of U.S. Patent No. 9,951,134, which is incorporated herein by reference. A specific mutation at the position corresponding to Y102 in SEQ ID NO: 6 can inhibit the binding of certain immunoglobulin receptors, such as human or mouse Fcαμ receptors, mouse Fcμ receptors, and / or human or mouse high molecular weight Ig receptors (pIg receptors), to IgM pentamers containing mutant J chains. Monoclonal populations of IgM antibodies, IgM-like antibodies, other IgM-derived binding molecules, or multimatured binding molecules containing a mutation at the amino acid corresponding to Y102 in SEQ ID NO: 6 have improved serum half-lives compared to monoclonal populations of corresponding antibodies, antibody-like molecules, binding molecules, or binding molecules administered to the same species in the same manner, which are identical except for the substitution when administered to animals. In certain embodiments, the amino acid corresponding to Y102 in SEQ ID NO: 6 may be substituted with any amino acid. In certain embodiments, the amino acid corresponding to Y102 in SEQ ID NO: 6 may be substituted with alanine (A), serine (S), or arginine (R). In certain embodiments, the amino acid corresponding to Y102 in SEQ ID NO: 6 may be substituted with alanine. In certain embodiments, the J chain or its functional fragment or variant is a variant human J chain, referred to herein as "J*", and comprises the amino acid sequence of SEQ ID NO: 7.

[0129] A highly sialized population of IgM-derived binding molecules This disclosure provides a monoclonal population of multimer-binding molecules, each binding molecule comprising 10 or 12 IgM-derived heavy chains, each containing a binding domain that binds to a target and a glycosylated IgM heavy chain constant region or a multimerized fragment thereof, each bound to a target. In certain embodiments, each IgM heavy chain constant region comprises at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, the N-linked glycosylation motifs comprising the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine. In certain embodiments, at least one, at least two, at least three, at least four, or at least five of the N-linked glycosylation motifs on each IgM heavy chain constant region are occupied by complex glycans as defined elsewhere herein. The constant region of human or non-human primate IgM heavy chain typically contains five N-linked glycosylation motifs N1-N5, although, as mentioned above, N4 and N5 are typically occupied primarily by oligomannose-type oligosaccharides rather than complex oligosaccharides, but not always. Therefore, in certain embodiments, at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region (e.g., N1, N2, and N3) are occupied by complex glycans.

[0130] In certain embodiments, the monoclonal population of binding molecules provided by this disclosure contains a level of sialylation higher than that observed or measured for IgM antibodies in normal circulation; that is, the monoclonal population of binding molecules provided contains levels of sialylation that do not exist in nature. When measured by the inventors (see, for example, Example 4), the average level of sialylation for human IgM antibodies isolated from normal circulation is about 30–32 moles of sialic acid per mole of IgM. Accordingly, this disclosure provides monoclonal populations of such multimer-binding molecules containing at least 33 moles, at least 34 moles, or at least 35 moles of sialic acid per mole of binding molecule. Sialic acid residues are typically terminal monosaccharides of complex glycans, and a single oligosaccharide glycan may contain, for example, 1, 2, 3, or 4 sialic acid monosaccharides, depending on the number of oligosaccharide antennas. In certain embodiments, the monoclonal population of the binding molecule provided may include higher levels of sialylation, for example, the monoclonal population of the binding molecule may contain at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 124, at least 130, at least 140, or 146 moles of sialic acid per mole of binding molecule. In some embodiments, the monoclonal population of the binding molecule may contain 33 to 146 moles of sialic acid per mole of binding molecule, for example, 33 to 130, 33 to 120, 33 to 110, 33 to 100, 33 to 90, 33 to 80, 33 to 70, 33 to 60, 33 to 50, 35 to 130, 35 to 120, 35 to 110, 35 to 100, Contains 35-90, 35-80, 35-70, 35-60, 35-50, 45-130, 45-120, 45-110, 45-100, 45-90, 45-80, 45-70, 45-60, 45-50, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, or 50-60 moles of sialic acid.In some embodiments, the monoclonal population of binding molecules is approximately 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 40-45, 40-50, 40-55, 40-65, 40-65, and 40 The conjugate molecules contain approximately 70, 45-50, 45-55, 45-60, 45-65, 45-70, 50-55, 50-60, 50-65, 50-70, 55-60, 55-65, 55-70, 60-65, 60-70, or approximately 65-70 moles of sialic acid. In some embodiments, the monoclonal population of the conjugate molecule contains approximately 40-55 moles of sialic acid per mole of conjugate molecule. As shown in the examples herein, monoclonal populations of conjugate molecules having sialic acid levels exceeding 35 moles per mole of conjugate molecule improved the pharmacokinetic properties of the conjugate molecule compared to the same conjugate molecule having lower levels of sialic acid. In certain cases, it may be desirable to prepare and use monoclonal populations of the binding molecule, where the sialic acid level is not the maximum possible level, such as about 40 to 55 moles of sialic acid per mole of binding molecule. Such molecules may have other desirable properties, such as different solubility, ease of preparation, and / or immunogenicity.

[0131] As provided by this disclosure, each IgM-derived heavy chain in the provided population of binding molecules comprises a glycosylated IgM or IgM-derived heavy chain constant region or a multimerized fragment or derivative thereof, which may be a full-length IgM heavy chain constant region, a multimerized fragment of the IgM heavy chain constant region, or a hybrid constant region comprising at least the minimum portion of the IgM heavy chain constant region required for multimerization, bound to a binding domain (e.g., an antibody-antigen binding domain) that specifically binds to a target of interest. In certain embodiments, the IgM heavy chain constant region is derived from a human IgM heavy chain constant region comprising up to five N-linked glycosylated motifs N-X1-S / T starting from amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04). The binding domain that binds to the target may be, for example, an antigen-binding domain or a subunit of an antigen-binding domain, such as the variable region (VH) of the heavy chain of an antibody. This disclosure relates to a binding molecule that binds to any target of interest.

[0132] The monoclonal populations of binding molecules provided herein can be generated by in vitro glycotechnology of the monoclonal populations of binding molecules during downstream processing, including but not limited to modification of cell lines expressing the population of binding molecules, or by any combination thereof, or by many different methods.

[0133] In certain embodiments, a highly sialized monoclonal population of the provided multimer-binding molecule is generated via cell line modification. Cell line modification for increasing the sialization of a monoclonal population of a binding molecule, as provided by this disclosure, includes, but is not limited to, transfecting a cell line that generates a monoclonal population of a binding molecule with one or more genes encoding glycosyltransferases, e.g., galactosyltransferase (see, for example, Figures 1C and 1D, for providing acceptor residues for sialic acid residues via α-2,6 and / or α-2,3 linkages), and / or further sialyltransferases, to generate a cell line that overexpresses these enzymes (glycosyltransferase "knock-in"), thereby improving and / or enhancing the cell line's ability to facilitate the transfer of sialic acid monosaccharides from CMP-NANA substrates or derivatives thereof to compatible acceptor oligosaccharides. Other cell line modifications include deletion or "knock-out" of sialidase enzymes normally produced by the cell line. Methods for “knocking in” various glucosyltransferases are described in the examples and are otherwise well known to those skilled in the art. Similarly, methods for “knocking out” genes encoding sialidases in cell lines, for example, are readily available to those skilled in the art.

[0134] An exemplary sialyltransferase is human β-galactoside α-2,6-sialyltransferase 1 (SEQ ID NO: 3), also known as ST6GAL1. Other sialyltransferases that can be "knocked in" include human β-galactoside α-2,6-sialyltransferase-II (ST6GALII) and any of the four β-galactoside α2-3-sialyltransferases (ST3GAL-I-IV). An exemplary galactosyltransferase is human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).

[0135] In certain embodiments, a highly sialized monoclonal population of a provided polymer-binding molecule is generated via glycoengineering, for example, by adding sialic acid residues to the monoclonal population of the binding molecule during downstream processing, thereby generating a monoclonal population of, for example, a glycoengineered IgM antibody, an IgM-like antibody, or an IgM-derived binding molecule (GEM). In certain embodiments, in vitro glycoengineering involves contacting the monoclonal population of the binding molecule with a soluble sialyltransferase (or a soluble sialyltransferase attached to a solid support) and a sialic acid substrate (e.g., a substrate comprising cytidine monophosphate (CMP)-N-acetylneuraminic acid (CMP-NANA)) under conditions in which the sialic acid moves from CMP-NANA to a galactose residue on a complex glycan on the population of the binding molecule. Contact may occur in one or more steps of protein purification, after which the soluble sialyltransferase can be removed by subsequent purification steps or by separating the cluster of binding molecules from the solid support to which the enzyme is bound.

[0136] In certain embodiments, the sialyltransferase variant used for GEM production may be a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3). For example, the sialyltransferase may be a variant of ST6GAL1 that excludes the transmembrane region of SEQ ID NO: 3 (e.g., excluding amino acids 10-26 of SEQ ID NO: 3), or both the cytoplasmic and transmembrane regions of SEQ ID NO: 3 (e.g., excluding amino acids 1-9 of SEQ ID NO: 3 and amino acids 10-26 of SEQ ID NO: 3), but maintains the catalytic activity of the protein. In certain embodiments, the soluble variant of ST6GAL1 comprises amino acids x through 406 of SEQ ID NO: 3, where x is an integer from 27 to 120. For example, soluble variants of ST6GAL1 may include amino acids 120-406, 115-406, 110-406, 109-406, 105-406, 100-406, 95-406, 90-406, 89-406, 88-406, 87-406, 86-406, 85-406, 84-406, 83-406, 82-406, 81-406, 80-406, 75-406, 70-406, 65-406, 60-406, 55-406, 50-406, 45-406, 40-406, 35-406, 30-406, or 27-406 of SEQ ID NO: 3. In certain embodiments, the sialic acid substrate includes cytidyl monophosphate (CMP)-N-acetylneuraminic acid (=CMP-β-D-Neu5Ac;=CMP-Neu5Ac;=CMP-NANA;=CMP-sialic acid;=CMP-SA, Figure 2B). Functional derivatives include, but are not limited to, azide-CMP-sialic acid used for glycan labeling by "click" chemistry.

[0137] The inventors observed that efficient and high-level sialylation of IgM antibodies can be performed with low concentrations of the soluble variant of ST6GAL1, compared to the higher amounts required for glycoengineering of IgG antibodies, despite the presence of numerous glycans (51 in the case of pentamers and 60 in the case of hexamers). For example, efficient sialylation of IgM antibodies was performed with a mass ratio of approximately 5000:1 or 2000:1 between IgM antibody and soluble sialyltransferase, and with a mass ratio of approximately 5000:2500:1 or 2000:500:1 between IgM antibody, sialic acid substrate and soluble sialyltransferase (providing an excess amount of sialic acid substrate). This is calculated as a molar ratio of approximately 200:1 or 80:1 between IgM antibody and soluble sialyltransferase, or approximately 200:2500:1 or 80:500:1 between IgM antibody, sialic acid substrate, and soluble sialyltransferase. In certain embodiments, the molar ratio of IgM antibody to sialyltransferase is at least approximately 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 105:1, 110:1, 115:1, 120:1, 125:1, 130:1, 135:1, 140:1, 145:1, 150:1, 175:1, or 200:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase can be about 80:1 to about 5000:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase can be about 80:1 to about 100:1, about 80:1 to about 250:1, about 80:1 to about 500:1, about 80:1 to about 750:1, about 80:1 to about 1000:1, about 80:1 to about 1250:1, about 80:1 to about 1500:1, about 80:1 to about 1750:1, about 80:1 to about 2000:1, about 80:1 to about 2500:1, about 80:1 to about 3000:1, and about 8 0:1~approx. 3500:1, approx. 80:1~approx. 4000:1, approx. 80:1~approx. 4500:1, approx. 250:1~approx. 500:1, approx. 250:1~approx. 750:1, approx. 250:1~approx. 1000:1, approx. 250:1~approx. 1250:1, approx. 250:1~approx. 1500:1, approx. 250:1~approx. 1750:1, approx. 250:1~approx. 2000:1, approx. 250:1~approx. 2500:1, approx. 250:1~approx. 3000:1, approx. 250:1~approx. 3500:1,Approximately 250:1 to approximately 4000:1, approximately 250:1 to approximately 4500:1, approximately 250:1 to approximately 5000:1, approximately 500:1 to approximately 750:1, approximately 500:1 to approximately 1000:1, approximately 500:1 to approximately 1250:1, approximately 500:1 to approximately 1500:1, approximately 500:1 to approximately 1750:1, approximately 500:1 to approximately 2000:1, approximately 500:1 to approximately 2500:1, approximately 500:1 to approximately 3000:1, approximately 500:1 to approximately 3500:1, approximately 500:1 to approximately 4000:1, approximately 500:1 to approximately 4500:1, approximately 500:1~approx. 5000:1, approx. 1000:1~approx. 1250:1, approx. 1000:1~approx. 1500:1, approx. 1000:1~approx. 1750:1, approx. 1000:1~approx. 2000:1, approx. 1000:1~approx. 2500:1, approx. 1000:1~approx. 3000:1, approx. 1000:1~approx. 3500:1, approx. 1000:1~approx. 4000:1, approx. 1000:1~approx. 4500:1, approx. 1000:1~approx. 5000:1, approx. 1500:1~approx. 1750:1, approx. 1500:1~approx. 2000:1, approx. 150 0:1~approx. 2500:1, approx. 1500:1~approx. 3000:1, approx. 1500:1~approx. 3500:1, approx. 1500:1~approx. 4000:1, approx. 1500:1~approx. 4500:1, approx. 1500:1~approx. 5000:1, approx. 2000:1~approx. 2500:1, approx. 2000:1~approx. 3000:1, approx. 2000:1~approx. 3500:1, approx. 2000:1~approx. 4000:1, approx. 2000:1~approx. 4500:1, approx. 2000:1~approx. 5000:1, approx. 2500:1~approx. 3000:1, approx. 2500: The ratios can be 1 to approximately 3500:1, approximately 2500:1 to approximately 4000:1, approximately 2500:1 to approximately 4500:1, approximately 2500:1 to approximately 5000:1, approximately 3000:1 to approximately 3500:1, approximately 3000:1 to approximately 4000:1, approximately 3000:1 to approximately 4500:1, approximately 3000:1 to approximately 5000:1, approximately 3500:1 to approximately 4000:1, approximately 3500:1 to approximately 4500:1, approximately 3500:1 to approximately 5000:1, approximately 4000:1 to approximately 4500:1, or approximately 4000:1 to approximately 5000:1. This is in contrast to the much larger amounts of enzyme required for the invitrocylylation of IgG antibodies, and a molar ratio of 3:1 IgG antibody to sialyltransferase is recommended. for example,Please refer to Malik, S., and Thomann, M., (2016) In Vitro Glycoengineering - Suitability for BioPharma manufacturing, Application Note, available at custombiotech.roche.com.

[0138] The inventors also observed that, despite the large number of glycans (51 in the case of pentamers and 60 in the case of hexamers), efficient and high-level sialylation of IgM antibodies can be performed with low concentrations of sialic acid substrates compared to the higher amounts required for glycosylation of IgG antibodies. In some embodiments, the mass ratio of sialic acid substrate to sialyltransferase is approximately 1:4 to approximately 3000:1, for example, approximately 1:4 to approximately 1:1, approximately 1:4 to approximately 5:1, approximately 1:4 to approximately 50:1, approximately 1:4 to approximately 100:1, approximately 1:4 to approximately 500:1, approximately 1:4 to approximately 1000:1, approximately 1:4 to approximately 1500:1, approximately 1:4 to approximately 2000:1, approximately 1:4 to approximately 2500:1, approximately 1:1 to approximately 5:1, approximately 1:1 to approximately 10:1, approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 100:1, and approximately 1:1 to approximately 500:1. , approximately 1:1 to approximately 1000:1, approximately 1:1 to approximately 1500:1, approximately 1:1 to approximately 2000:1, approximately 1:1 to approximately 2500:1, approximately 1:1 to approximately 3000:1, approximately 2:1 to approximately 5:1, approximately 2:1 to approximately 10:1, approximately 2:1 to approximately 50:1, approximately 2:1 to approximately 100:1, approximately 2:1 to approximately 500:1, approximately 2:1 to approximately 1000:1, approximately 2:1 to approximately 1500:1, approximately 2:1 to approximately 2000:1, approximately 2:1 to approximately 2500:1, approximately 2:1 to approximately 3000:1, approximately 5:1 to approximately 10:1, approximately 5:1 to approximately 50:1, approximately 5:1 to approximately 10 0:1, approximately 5:1~approximately 500:1, approximately 5:1~approximately 1000:1, approximately 5:1~approximately 1500:1, approximately 5:1~approximately 2000:1, approximately 5:1~approximately 2500:1, approximately 5:1~approximately 3000:1, approximately 10:1~approximately 50:1, approximately 10:1~approximately 100:1, approximately 10:1~approximately 500:1, approximately 10:1~approximately 1000:1, approximately 10:1~approximately 1500:1, approximately 10:1~approximately 2000:1, approximately 10:1~approximately 2500:1, approximately 10:1~approximately 3000:1, approximately 50:1~approximately 100:1, approximately 50:1~approximately 500:1, approximately 5 0:1~approx. 1000:1, approx. 50:1~approx. 1500:1, approx. 50:1~approx. 2000:1, approx. 50:1~approx. 2500:1, approx. 50:1~approx. 3000:1, approx. 100:1~approx. 500:1, approx. 100:1~approx. 1000:1, approx. 100:1~approx. 1500:1, approx. 100:1~approx. 2000:1, approx. 100:1~approx. 2500:1, approx. 100:1~approx. 3000:1, approx. 500:1~approx. 1000:1, approx. 500:1~approx. 1500:1, approx. 500:1~approx. 2000:1, approx. 500:1~approx. 2500:1,It could be approximately 500:1 to 3000:1, approximately 1000:1 to 1500:1, approximately 1000:1 to 2000:1, approximately 1000:1 to 2500:1, approximately 1000:1 to 3000:1, approximately 1500:1 to 2000:1, approximately 1500:1 to 2500:1, approximately 1500:1 to 3000:1, approximately 2000:1 to 2500:1, approximately 2000:1 to 3000:1, or approximately 2500:1 to 3000:1. In some embodiments, the mass ratio of the binding molecule to sialyltransferase may be about 80:1, about 100:1, about 250:1, about 500:1, about 750:1, about 1000:1, about 1250:1, about 1500:1, about 1750:1, about 2000:1, about 2500:1, about 3000:1, about 3500:1, about 4000:1, about 4500:1, or about 5000:1; and / or the mass ratio of the sialic acid substrate to sialyltransferase may be about 5:1, about 10:1, about 50:1, about 100:1, about 500:1, about 1000:1, about 1500:1, about 2000:1, about 2500:1, or about 3000:1.

[0139] In some embodiments, the antibody:sialic acid substrate mass ratio is approximately 1:1 to approximately 40:1, for example, approximately 1:1 to approximately 2:1, approximately 1:1 to approximately 4:1, approximately 1:1 to approximately 6:1, approximately 1:1 to approximately 8:1, approximately 1:1 to approximately 10:1, approximately 1:1 to approximately 15:1, approximately 1:1 to approximately 20:1, approximately 2:1 to approximately 4:1, approximately 2:1 to approximately 6:1, approximately 2:1 to approximately 8:1, approximately 2:1 to approximately 10:1, approximately 2:1 to approximately 15:1, approximately 2:1 to approximately 20:1, approximately 2:1 to approximately 40:1, approximately 4:1 to approximately 6:1, approximately 4:1 to approximately 8:1, approximately 4:1 to approximately 10:1, approximately 4:1 to approximately 15:1, approximately 4:1 to approximately 20:1, approximately 4:1 to approximately 40:1, approximately 6:1 to approximately 8:1, approximately 6:1 to approximately 10:1, approximately 6:1 to approximately 15:1, approximately 6:1 to approximately 20:1, approximately 6:1 to approximately 40:1, approximately 8:1 to approximately 10:1, approximately 8:1 to approximately 15:1, approximately 8:1 to approximately 20:1, approximately 8:1 to approximately 40:1, approximately 10:1 to approximately 15:1, approximately 10:1 to approximately 20:1, approximately 10:1 to approximately 40:1, approximately 15:1 to approximately 20:1, approximately 15:1 to approximately 40:1, or possibly approximately 20:1 to approximately 40:1.

[0140] The inventors have further observed that efficient and high-level sialylation of IgM antibodies can be carried out over a wider temperature range and longer duration than that used for glycoengineering of IgG antibodies. In some embodiments, in vitro glycoengineering involves contacting a monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate for at least 30 minutes, for example, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 48 hours. In several embodiments, contact can last for approximately 30 minutes to 48 hours, for example, approximately 30 minutes to 4 hours, approximately 30 minutes to 5 hours, approximately 30 minutes to 6 hours, approximately 30 minutes to 7 hours, approximately 30 minutes to 10 hours, approximately 30 minutes to 12 hours, approximately 30 minutes to 18 hours, approximately 30 minutes to 24 hours, approximately 30 minutes to 36 hours, approximately 2 hours to 48 hours, approximately 3 hours to 6 hours, approximately 3 hours to 10 hours, approximately 3 hours to 12 hours, approximately 3 hours to 18 hours, approximately 3 hours to 24 hours, approximately 3 hours to 36 hours, approximately 3 hours to 48 hours, approximately 4 hours to 10 hours, approximately 4 hours to 12 hours, approximately 4 hours to 18 hours, approximately 4 hours to 24 hours, approximately 4 hours to 36 hours, approximately 4 hours to 48 hours, approximately 5 hours to Approximately 10 hours, approximately 5 to 12 hours, approximately 5 to 18 hours, approximately 5 to 24 hours, approximately 5 to 36 hours, approximately 5 to 48 hours, approximately 7 to 10 hours, approximately 7 to 12 hours, approximately 7 to 18 hours, approximately 7 to 24 hours, approximately 7 to 36 hours, approximately 7 to 48 hours, approximately 10 to 18 hours, approximately 10 to 24 hours The intervals between these periods are approximately 10 to 36 hours, 10 to 48 hours, 12 to 18 hours, 12 to 24 hours, 12 to 36 hours, 12 to 48 hours, 18 to 24 hours, 18 to 36 hours, 18 to 48 hours, 24 to 36 hours, 24 to 48 hours, or 36 to 48 hours.

[0141] In some embodiments, in vitro glycotechnology involves transferring a monoclonal collection of binding molecules with a soluble sialyltransferase and a sialic acid substrate at temperatures ranging from approximately 2°C to approximately 40°C, for example, approximately 2°C to approximately 37°C, 2°C to approximately 30°C, 2°C to approximately 25°C, 2°C to approximately 22°C, 2°C to approximately 20°C, 2°C to approximately 10°C, approximately 4°C to approximately 40°C, approximately 4°C to approximately 37°C, 4°C to approximately 30°C, 4°C to approximately 25°C, 4°C to approximately 22°C, 4°C to approximately 20°C, 4°C to approximately 10°C, approximately 10°C to approximately 40°C, and approximately 1 The method includes contact at temperatures of 0°C to approximately 37°C, 10°C to approximately 30°C, 10°C to approximately 25°C, 10°C to approximately 22°C, 10°C to approximately 20°C, approximately 20°C to approximately 40°C, approximately 20°C to approximately 37°C, 20°C to approximately 30°C, 20°C to approximately 25°C, 20°C to approximately 22°C, approximately 22°C to approximately 40°C, approximately 22°C to approximately 37°C, 22°C to approximately 30°C, 22°C to approximately 25°C, approximately 25°C to approximately 40°C, approximately 25°C to approximately 37°C, 25°C to approximately 30°C, approximately 30°C to approximately 40°C, or approximately 30°C to approximately 37°C. In some embodiments, in vitro glycotechnology includes contacting a monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate at temperatures of approximately 15°C to approximately 25°C.

[0142] In certain embodiments, invitrocylylation can be enhanced by ensuring that a sufficient number of galactose acceptor residues are present on the complex glycan of the provided monoclonal population of IgM, IgM-like, or IgM-derived binding molecules. ST6GAL1 transfers the sialic acid monosaccharide from CMP-NANA to galactose acceptor residues on the glycan of the molecule via α-2,6 linkages. To ensure a sufficient number of acceptor galactose residues on the glycan present in the monoclonal population of binding molecules, GEM generation may further involve contacting the monoclonal population of binding molecules with a galactosyltransferase, e.g., a soluble variant of β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4), and a galactose substrate, e.g., uridine-diphosphate-α-D-galactose (UDP-Gal), either before or simultaneously with contact with the sialyltransferase and sialic acid substrate. For example, sialyltransferase may be a variant of B4GALT4 that excludes the transmembrane region of SEQ ID NO: 4 (e.g., excluding amino acids 13-38 of SEQ ID NO: 4), or both the cytoplasmic and transmembrane regions of SEQ ID NO: 4 (e.g., excluding amino acids 1-12 of SEQ ID NO: 4 and amino acids 13-38 of SEQ ID NO: 4), while maintaining the catalytic activity of the protein. In certain embodiments, a soluble variant of B4GALT4 comprises amino acids x through 344 of SEQ ID NO: 4, where x is an integer from 39 to 120. For example, soluble variants of B4GALT4 include amino acids 120-344, 115-344, 110-344, 105-344, 100-344, 95-344, 90-344, 85-344, 80-344, 75-344, 70-344, 65-344, 60-344, 55-344, 50-344, 45-344, 40-344, or 39-344 of SEQ ID NO: 4. In certain embodiments, the galactose substrate includes UDP-Gal.

[0143] Each IgM heavy chain constant region in the monoclonal population of the provided binding molecules is bound to a binding domain or its subunits, such as an antibody-antigen binding domain, e.g., scFv, VHH, or the VH subunit of an antibody-antigen binding domain, and the binding domain specifically binds to the target of interest. In certain embodiments, the target is a target epitope, a target antigen, a target cell, a target organ, or a target virus. Targets include, but are not limited to, tumor antigens, other oncological targets, immunoneoplastic targets such as immune checkpoint inhibitors, infectious disease targets such as viral antigens expressed on the surface of infected cells, target antigens involved in blood-brain barrier transport, target antigens involved in neurodegenerative and neuroinflammatory diseases, and any combination thereof. Exemplary targets and binding domains that bind to such targets are provided elsewhere in this Specified, for example, U.S. Patent Application Publication Nos. US2019-0330360, US2019-0338040, US2019-0338041, US2019-0330374, US2019-0185570, US2019-0338031, or US2020-0239572, PCT Publication Nos. WO2018 / 017888, WO2018 / 017889, WO2018 / 017761, WO These can be found in 2018 / 017763, or WO2018 / 187702, and WO2019 / 165340, or U.S. Patents 9,951,134, 9,938,347, 8,377,435, 9,458,241, 9,409,976, 10,400,038, 10,351,631, 10,570,191, 10,604,559, 10,618,978, 10,689,449, or 10,787,520. Each of these applications and patents is incorporated herein by reference in its entirety for all purposes.

[0144] In certain embodiments, the population of multimer-binding molecules provided is multispecific, e.g., bispecific, triplicate, or tetraspecific, where two or more binding domains bound to the IgM heavy chain constant region of each binding molecule specifically bind to different targets. In certain embodiments, all binding domains of the provided population of multimer-binding molecules specifically bind to the same target. In certain embodiments, the binding domains of the provided population of multimer-binding molecules are identical. In such cases, for example, if the binding domains having different specificities are part of a modified J chain as described elsewhere herein, the population of multimer-binding molecules may still be bispecific. In certain embodiments, the binding domain is an antigen-binding domain derived from an antibody, e.g., an scFv bound to the IgM heavy chain constant region or a VH subunit of an antibody-binding domain bound to the IgM heavy chain constant region.

[0145] In certain embodiments, each binding molecule is a pentameric or hexamer IgM antibody containing five or six divalent IgM binding units, each binding unit containing two IgM heavy chains, each containing a VH located at the amino-terminus of the variant IgM constant region, and two immunoglobulin light chains, each containing a light chain variable domain (VL) located at the amino-terminus of the immunoglobulin light chain constant region, wherein the VH and VL bind to form an antigen-binding domain that specifically binds to the target. In certain embodiments, each antigen-binding domain of each binding molecule binds to the same target. In certain embodiments, each antigen-binding domain of each binding molecule is identical.

[0146] In certain embodiments, the target is a tumor-specific antigen, i.e., a target antigen that is largely expressed only on tumor or cancer cells, or expressed only at undetectable levels on normal, healthy adult cells. In certain embodiments, the target is a tumor-associated antigen, i.e., a target antigen that is expressed on both healthy and cancerous cells, but at much higher densities on cancerous cells than on normal, healthy cells. Exemplary tumor-specific and tumor-associated antigens include, but are not limited to, B-cell maturation antigen (BCMA), CD19, CD20, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2, also known as ErbB2), HER3 (ErbB3), receptor tyrosine protein kinase ErbB4, cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR1), VEGFR2, CD52, CD30, and prostate-specific membrane Examples include the antigen (PSMA), CD38, ganglioside GD2, autoligand receptor for signaling lymphocyte activator family member 7 (SLAMF7), platelet-derived growth factor receptor A (PDGFRA), CD22, FLT3 (CD135), CD123, MUC-16, carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM-1), mesothelin, tumor-associated calcium signaling transducer 2 (Trop-2), glypican-3 (GPC-3), human blood group H type 1 trisaccharide (Globo-H), sialyl Tn antigen (STn antigen), and CD33. Those skilled in the art will understand that although these target antigens appear in the literature under many different names, these well-known therapeutic targets can be easily identified using online databases, such as EXPASY.org.

[0147] Other tumor-related or tumor-specific antigens include, but are not limited to, DLL4, Notch1, Notch2, Notch3, Notch4, JAG1, JAG2, c-Met, IGF-1R, Patched, Hedgehog family polypeptides, WNT family polypeptides, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, IL-6, TNFα, IL-23, IL-17, CD80, CD86, CD3, CEA, Muc16, PSCA, CD44, c-Kit, DDR1, DDR2, RSPO1, RSPO 2. Examples include RSPO3, RSPO4, BMP family polypeptides, BMPR1a, BMPR1b, or TNF superfamily receptor proteins, such as TNFR1(DR1), TNFR2, TNFR1 / 2, CD40(p50), Fas(CD95, Apo1, DR2), CD30, 4-1BB(CD137, ILA), TRAILR1(DR4, Apo2), DR5(TRAILR2), TRAILR3(DcR1), TRAILR4(DcR2), OPG(OCIF), TWEAKR(FN14), LIGHTR(HVEM), DcR3, DR3, EDAR, and XEDAR.

[0148] In certain embodiments, the monoclonal population of multimer-binding molecules comprises a population of pentameric or hexamer IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules, each containing five or six divalent IgM-binding units. According to certain embodiments, each binding unit comprises two IgM heavy chains described herein, each having a VH located at the amino-terminal end of the variant IgM constant region, and two immunoglobulin light chains, each having a light chain variable domain (VL) located at the amino-terminal end of the immunoglobulin light chain constant region, e.g., the kappa or lambda constant region. The provided VH and VL bind to form an antigen-binding domain that specifically binds to the target of interest. In certain embodiments, the five or six IgM-binding units are identical.

[0149] In these embodiments, where the collection of multimeric IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules is a pentamer, each antibody or binding molecule may further comprise a J chain, a functional fragment thereof, or a functional variant thereof, as described elsewhere in this specification. For example, the J chain may be a mature human J chain comprising the amino acid sequence of SEQ ID NO: 6, a functional fragment thereof, or a functional variant thereof. Those skilled in the art will recognize that “functional fragment” or “functional variant” in this context includes those fragments and variants that can associate with an IgM binding unit, e.g., the constant region of the IgM heavy chain, to form a pentameric IgM antibody, an IgM-like antibody, or an IgM-derived binding molecule.

[0150] In certain embodiments, the J chain of a pentameric IgM-derived conjugated molecule, such as an IgM antibody, IgM-like antibody, or other IgM-derived conjugated molecule provided herein, is a functional variant J chain containing one or more single amino acid substitutions, deletions, or insertions compared 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. For example, a specific amino acid substitution, deletion, or insertion can result in an IgM-derived conjugated molecule that, when administered to a target animal, exhibits an increased serum half-life compared to a reference IgM-derived conjugated molecule that is identical to the reference J chain except for one or more single amino acid substitutions, deletions, or insertions, and is administered to the same animal species using the same method. In certain embodiments, the variant J chain may contain one, two, three, or four single amino acid substitutions, deletions, or insertions compared to the reference J chain.

[0151] As described in detail elsewhere in this specification, in certain embodiments, a variant J chain or functional fragment of a pentameric IgM-derived binding molecule provided herein, e.g., an IgM antibody, an IgM-like antibody, or another IgM-derived binding molecule, includes an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J chain (SEQ ID NO: 6). Y102 can be substituted with any amino acid, such as alanine. In certain embodiments, the variant human J chain may include the amino acid sequence of SEQ ID NO: 7. A J chain having the amino acid sequence of SEQ ID NO: 7 may be referred to as "J*" in some cases.

[0152] A pentameric IgM-derived binding molecule having either a variant or wild-type amino acid sequence, for example, the J chain or fragment of an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule provided herein, may be a “modified J chain” further comprising a heterologous moiety, the heterologous moiety fused or conjugated to the J chain or its fragment or variant. Exemplary but non-limiting heterologous moieties are provided, for example, in U.S. Patent Nos. 9,951,134 and 10,618,978, and U.S. Patent Application Publication No. 2019 / 0185570, which are incorporated herein by reference. In certain embodiments, the heterologous moiety is a polypeptide fused to or within the J chain or its fragment or variant. The heterologous polypeptide may optionally be fused to or within the J chain or its fragment or variant via a peptide linker. Any suitable linker may be used, for example, the peptide linker may include at least 5 amino acids, at least 10 amino acids, at least 20 amino acids, at least 30 amino acids or more, etc. In certain embodiments, the peptide linker contains 25 or fewer amino acids. In certain embodiments, the peptide linker may consist of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids. In certain embodiments, the peptide linker contains glycine and serine, for example, (GGGGS)n (SEQ ID NO: 48), where N may be 1, 2, 3, 4, 5, or more. In certain embodiments, the peptide linker consists of GGGGS (SEQ ID NO: 41), GGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGS (SEQ ID NO: 43), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 44), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 45). In certain embodiments, the heterologous polypeptide may be fused to the N-terminus of the J chain or its fragment or variant, to the C-terminus of the J chain or its fragment or variant, or to both the N-terminus and C-terminus of the J chain or its fragment or variant. In certain embodiments, the heterologous polypeptide may be fused internally within the J chain.In certain embodiments, the heterologous polypeptide may be a binding domain, such as an antigen-binding domain. For example, the heterologous polypeptide may be an antibody, an antibody subunit, or an antigen-binding fragment of an antibody, such as an scFv fragment. In certain embodiments, the binding domain, such as an scFv fragment, may bind to effector cells, such as T cells or NK cells. In certain embodiments, the binding domain, such as an scFv fragment, may specifically bind to CD3 on cytotoxic T cells, such as CD3ε. In certain special embodiments, the modified J chain of the pentameric IgM-derived binding molecule provided herein includes a J chain containing an anti-CD3εscFv antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 36 (V15J) or SEQ ID NO: 37 (V15J*), or the six complementarity-determining regions of the mouse antibody SP34 (VH=SEQ ID NO: 14VL=SEQ ID NO: 18), specifically the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 15, 16, 17, 19, 20, and 21, respectively, for example, a modified J chain SJ* comprising the amino acid sequence of SEQ ID NO: 39. Other humanized SP35 antibodies include the VH and VL or scFv sequences of A-55 (sequences 22, 23, and 24 in WO2018208864, respectively), A-56 (sequences 25, 26, and 27 in WO2018208864, respectively), or A-57 (sequences 28, 29, and 30 in WO2018208864, respectively), which are incorporated into the modified J chains A-55-J* (sequence number 31), A-56-J* (sequence number 32), and A-57-J* (sequence number 33). In certain embodiments, the modified J chains provided herein may further include additional heterologous moieties attached to the opposite end of the J chain, for example, an anti-CD3ε scFv binding domain. For example, the modified J chain may further include human serum albumin protein. Examples include, but are not limited to, VJH (sequence number 34) and VJ*H (sequence number 35).

[0153] IgM-derived binding molecules with extended serum half-lives The highly sialized IgM antibodies, IgM-like antibodies, or monoclonal populations of IgM-derived binding molecules provided herein can be further manipulated to enhance their serum half-lives. Exemplary IgM heavy chain constant region mutations that can extend the serum half-life of IgM-derived binding molecules are disclosed in U.S. Patent Application Publication No. 2020-0239572, the contents of which are incorporated herein by reference in their entirety. For example, variant IgM heavy chain constant region mutations of highly sialized IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules provided herein may include amino acid substitutions at amino acid positions corresponding to amino acids S401, E402, E403, R344, and / or E345 of the wild-type human IgM constant region (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). "Amino acids corresponding to amino acids S401, E402, E403, R344, and / or E345 in the wild-type human IgM constant region" means amino acids in the sequence of the IgM constant region of any species that are homologous to S401, E402, E403, R344, and / or E345 in the human IgM constant region. In certain embodiments, the amino acids corresponding to S401, E402, E403, R344, and / or E345 in SEQ ID NO: 1 or SEQ ID NO: 2 may be substituted with any amino acid, such as alanine.

[0154] The wild-type J chain typically contains one N-linked glycosylation site. In certain embodiments, variant J chains or functional fragments of pentameric IgM-derived binding molecules provided herein contain a mutation in the asparagine (N)-linked glycosylation motif N-X1-S / T, starting from the amino acid position corresponding to amino acid 49 (motif N6) of a mature human J chain (SEQ ID NO: 42) or J* (SEQ ID NO: 43), where N is asparagine, X1 is any amino acid except proline, and S / T is serine or threonine, and this mutation prevents glycosylation in that motif. As demonstrated in U.S. Patent Application Publication No. 2020-0239572, a mutation that prevents glycosylation at this site can result in a population of IgM-derived conjugate molecules provided herein, such as IgM antibodies, IgM-like antibodies, or other IgM-derived conjugate molecules, which, when administered to a target animal, are identical except for the mutation that prevents glycosylation in the variant J chain and exhibit an increased serum half-life compared to a reference IgM-derived conjugate molecule administered to the same animal species using the same method.

[0155] For example, in certain embodiments, a variant J chain or functional fragment of a binding molecule containing a J chain provided herein may include an amino acid substitution at the corresponding amino acid position of amino acid N49 or S51 in SEQ ID NO: 6 or SEQ ID NO: 7, provided that the amino acid corresponding to S51 is not substituted with threonine (T), or the variant J chain includes an amino acid substitution at the corresponding amino acid positions of both amino acid N49 and S51 in SEQ ID NO: 6 or SEQ ID NO: 7. In certain embodiments, the corresponding position of amino acid N49 in SEQ ID NO: 6 or SEQ ID NO: 7 is substituted with any amino acid, e.g., alanine (A), glycine (G), threonine (T), serine (S), or aspartic acid (D). In certain embodiments, the position corresponding to N49 in SEQ ID NO: 6 or SEQ ID NO: 7 may be substituted with alanine (A). In certain embodiments, the position corresponding to N49 in SEQ ID NO: 6 or SEQ ID NO: 7 may be substituted with aspartic acid (D).

[0156] Variant human IgM constant region with reduced CDC activity The monoclonal populations of IgM-derived binding molecules provided herein, such as IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules, can be manipulated to exhibit reduced CDC activity against cells in the presence of complement, compared to a reference population of IgM antibodies or IgM-like antibodies that are identical to the corresponding reference human IgM constant region, except for mutations conferring reduced complement-dependent cytotoxicity (CDC) activity. These CDC mutations can be combined with any mutations to confer an increase in serum half-life, as provided herein. "Corresponding reference human IgM constant region" means a human IgM constant region or a portion thereof, such as the Cμ3 domain, that is identical to the variant IgM constant region, except for modifications or multiple modifications in the constant region that affect CDC activity. In certain embodiments, the variant human IgM constant region includes one or more amino acid substitutions, for example, in the Cμ3 domain, compared to the wild-type human IgM constant region, as described, for example, in PCT publication number WO / 2018 / 187702, which is incorporated herein in its entirety by reference. Assays for measuring CDC are well known to those skilled in the art, and an exemplary assay is described, for example, in PCT publication number WO / 2018 / 187702.

[0157] In certain embodiments, the variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, the variant IgM constant region provided herein includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, the variant IgM constant region provided herein includes a combination of substitutions corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and at P313 of SEQ ID NO: 1 or SEQ ID NO: 2. These proline residues can be independently substituted with any amino acid, e.g., alanine, serine, or glycine. In certain embodiments, the variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 22 or SEQ ID NO: 23. Lysine residues can be independently substituted with any amino acid, e.g., alanine, serine, glycine, or aspartic acid. In certain embodiments, the variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution with aspartate corresponding to the wild-type human IgM constant region at position K315 in SEQ ID NO: 22 or SEQ ID NO: 23.

[0158] host cell In certain embodiments, the Disclosure provides host cells capable of generating a highly sialylated monoclonal population of binding molecules as provided herein. In certain embodiments, such host cells overexpress ST6GAL1 and / or B4GALT4. The Disclosure also provides a method for generating a monoclonal population of binding molecules as provided herein, the method comprising culturing the provided host cells and recovering the population of binding molecules.

[0159] Method for generating a highly sialylated population of IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules. The Disclosure further provides a method for generating a monoclonal population of highly sialylated multimer-binding molecules as described in detail herein, the method comprising providing a cell line expressing the monoclonal population of binding molecules, culturing the cell line, and recovering the monoclonal population of binding molecules. In certain embodiments, each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region or a multimerized fragment thereof, each bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, each N-linked glycosylation motif comprising the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine. As provided herein, on average, at least one, at least two, or at least three N-linked glycosylation motifs on each IgM heavy chain constant region in the population are occupied by complex glycans, and cell lines, culture conditions, harvesting processes, or combinations thereof are optimized to enrich complex glycans containing at least one, two, at least three, or four sialic acid-terminal monosaccharides per glycan.

[0160] In certain embodiments, cell lines, culture conditions, recovery processes, or combinations thereof can be optimized according to a method provided to yield a monoclonal population of binding molecules containing at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 124, at least 130, at least 140, or at least 146 moles of sialic acid per mole of binding molecules. In certain embodiments, cell lines, recovery processes, or combinations thereof are optimized according to a method provided to yield a monoclonal population of binding molecules containing at least 30, at least 35, at least 40, at least 45, at least 50, or at least 60 moles of sialic acid per mole of binding molecules. In some embodiments, the monoclonal population of binding molecules is approximately 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 40-45, 40-50, 40-55, 40-65, 40-65, and 40 The molecules contain approximately 70, 45-50, 45-55, 45-60, 45-65, 45-70, 50-55, 50-60, 50-65, 50-70, 55-60, 55-65, 55-70, 60-65, 60-70, or approximately 65-70 moles of sialic acid. In some embodiments, the monoclonal population of binding molecules contains approximately 40-55 moles of sialic acid per mole of binding molecule. According to the provided method, the IgM heavy chain constant region may be derived from the human IgM heavy chain constant region, comprising five N-linked glycosylated motifs N-X1-S / T that start at amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04).In certain embodiments, on average, one, two, or all three of the motifs N1, N2, and N3 in the group of binding molecules are occupied by a complex glycan that can be sialylated by the provided method.

[0161] In certain embodiments, cell lines cultured according to the provided method are modified to overexpress sialyltransferase. In certain embodiments, the overexpressed sialyltransferase is 2,6-sialyltransferase. In certain embodiments, the overexpressed sialyltransferase is human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1). In certain embodiments, the overexpressed sialyltransferase is 2,3-sialyltransferase. Cell lines cultured according to the provided method may also be modified to overexpress galactosyltransferase. In certain embodiments, the overexpressed galactosyltransferase is human β-1,4-galactosyltransferase 4 (B4GALT4). Cell lines cultured according to the provided method can also be modified to overexpress UDP-GlcNAc2 2-epimerase / ManNAc kinase enzyme (GNE), including R263 or R266 mutations such as Q, W, or L mutations; α-mannosidase II; N-acetylglucosaminyltransferase-II (GNT-II); N-acetylglucosaminyltransferase-IV (GNT-IV); N-acetylglucosaminyltransferase-V (GNT-V); CMP-sialic acid synthase (CMP-SAS), CMP-sialic acid transporter (CMP-SAT), or any combination thereof. In certain embodiments, cell lines cultured according to the provided method can also be modified to block the expression of specific sialidases. In certain embodiments, cell lines cultured according to the provided method can also be modified to block the expression of neuraminidases.

[0162] In certain embodiments of the provided method, the recovery process includes subjecting a monoclonal population of the polymer-binding molecule to glycoengineering during downstream processing to produce, for example, a glycoengineered IgM antibody, an IgM-like antibody, or an IgM-derived binding molecule, i.e., a “GEM”. In certain embodiments, the GEM is highly sialized, for example, possessing at least 35 moles of sialic acid per mole of binding molecule. The production of the GEM is described in detail elsewhere herein, and any and all embodiments of the production of the GEM may be included in the provided method. In certain embodiments, the production of the GEM includes contacting a monoclonal population of the binding molecule with a soluble sialyltransferase and a sialic acid substrate. In certain embodiments, the sialyltransferase may include a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3). In a particular embodiment, the soluble variant of ST6GAL1 comprises amino acids x through 406 of SEQ ID NO: 3, where x is an integer from 27 to 120. For example, soluble variants of ST6GAL1 may include amino acids 120-406, 115-406, 110-406, 109-406, 105-406, 100-406, 95-406, 90-406, 89-406, 88-406, 87-406, 86-406, 85-406, 84-406, 83-406, 82-406, 81-406, 80-406, 75-406, 70-406, 65-406, 60-406, 55-406, 50-406, 45-406, 40-406, 35-406, 30-406, or 27-406 of SEQ ID NO: 3. In certain embodiments, the sialic acid substrate may include cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA) or a derivative thereof.

[0163] As described elsewhere in this specification, the inventors have found that the production of highly sialized IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules requires far fewer enzymes than equivalent methods for sializing IgG. For example, the binding molecule:sialyltransferase mass ratio can range from about 80:1 to about 5000:1. In some embodiments, the mass ratio of the binding molecule to the sialyltransferase is approximately 80:1 to 100:1, approximately 80:1 to 250:1, approximately 80:1 to 500:1, approximately 80:1 to 750:1, approximately 80:1 to 1000:1, approximately 80:1 to 1250:1, approximately 80:1 to 1500:1, approximately 80:1 to 1750:1, approximately 80:1 to 2000:1, approximately 80:1 to 2500:1, approximately 80:1 to 3000:1, approximately 80:1 to 3500:1, and approximately 80:1 to 40 00:1, approximately 80:1 to approximately 4500:1, approximately 80:1 to approximately 5000:1, approximately 250:1 to approximately 500:1, approximately 250:1 to approximately 750:1, approximately 250:1 to approximately 1000:1, approximately 250:1 to approximately 1250:1, approximately 250:1 to approximately 1500:1, approximately 250:1 to approximately 1750:1, approximately 250:1 to approximately 2000:1, approximately 250:1 to approximately 2500:1, approximately 250:1 to approximately 3000:1, approximately 250:1 to approximately 3500:1, approximately 250:1 to approximately 4000:1, approximately 250:1 to approximately 4500:1 , approximately 250:1 to approximately 5000:1, approximately 500:1 to approximately 750:1, approximately 500:1 to approximately 1000:1, approximately 500:1 to approximately 1250:1, approximately 500:1 to approximately 1500:1, approximately 500:1 to approximately 1750:1, approximately 500:1 to approximately 2000:1, approximately 500:1 to approximately 2500:1, approximately 500:1 to approximately 3000:1, approximately 500:1 to approximately 3500:1, approximately 500:1 to approximately 4000:1, approximately 500:1 to approximately 4500:1, approximately 500:1 to approximately 5000:1, approximately 1000:1 to approximately 1250: 1. Approximately 1000:1 to 1500:1, approximately 1000:1 to 1750:1, approximately 1000:1 to 2000:1, approximately 1000:1 to 2500:1, approximately 1000:1 to 3000:1, approximately 1000:1 to 3500:1, approximately 1000:1 to 4000:1, approximately 1000:1 to 4500:1, approximately 1000:1 to 5000:1, approximately 1500:1 to 1750:1, approximately 1500:1 to 2000:1, approximately 1500:1 to 2500:1, approximately 1500:1 to 3000:1,Approximately 1500:1 to approximately 3500:1, approximately 1500:1 to approximately 4000:1, approximately 1500:1 to approximately 4500:1, approximately 1500:1 to approximately 5000:1, approximately 2000:1 to approximately 2500:1, approximately 2000:1 to approximately 3000:1, approximately 2000:1 to approximately 3500:1, approximately 2000:1 to approximately 4000:1, approximately 2000:1 to approximately 4500:1, approximately 2000:1 to approximately 5000:1, approximately 2500:1 to approximately 3000:1, approximately 2500:1 to approximately 3500:1, approximately 250 0:1 to approximately 4000:1, approximately 2500:1 to approximately 4500:1, approximately 2500:1 to approximately 5000:1, approximately 3000:1 to approximately 3500:1, approximately 3000:1 to approximately 4000:1, approximately 3000:1 to approximately 4500:1, approximately 3000:1 to approximately 5000:1, approximately 3500:1 to approximately 4000:1, approximately 3500:1 to approximately 4500:1, approximately 3500:1 to approximately 5000:1, approximately 4000:1 to approximately 4500:1, or approximately 4000:1 to approximately 5000:1. In certain embodiments, the molar ratio of the binding molecule to the sialyltransferase may be approximately 200:1, 175:1, 150:1, 155:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or 50:1. For example, the mass ratio of the binding molecule to the sialic acid substrate to the sialyltransferase may be approximately 2000:500:1. In certain embodiments, the molar ratio of binding molecule:sialyltransferase may be about 200:1, 175:1, 150:1, 155:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or 50:1. In certain embodiments, the molar ratio of binding molecule:sialyltransferase may be about 80:1. As described elsewhere in this specification, the generation of GEMs involves a monoclonal population of binding molecules, galactosyltransferase, e.g., a soluble variant of human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4), and a galactose substrate.For example, this may further include contact with uridine diphosphate-α-D-galactose (UDP-Gal). Contact with galactosyltransferase and the galactose substrate may occur before or simultaneously with contact with soluble sialyltransferase and the sialic acid substrate.

[0164] In some embodiments, the mass ratio of sialic acid substrate to sialyltransferase is about 5:1 to about 3000:1, for example, about 5:1 to about 10:1, about 5:1 to about 50:1, about 5:1 to about 100:1, about 5:1 to about 500:1, about 5:1 to about 1000:1, about 5:1 to about 1500:1, about 5:1 to about 2000:1, about 5:1 to about 2500:1, about 10:1 to about 50:1, about 10:1 to about 100:1, approximately 10:1 to approximately 500:1, approximately 10:1 to approximately 1000:1, approximately 10:1 to approximately 1500:1, approximately 10:1 to approximately 2000:1, approximately 10:1 to approximately 2500:1, approximately 10:1 to approximately 3000:1, approximately 50:1 to approximately 100:1, approximately 50:1 to approximately 500:1, approximately 50:1 to approximately 1000:1, approximately 50:1 to approximately 1500:1, approximately 50:1 to approximately 2000:1, approximately 50:1 to approximately 2500:1, approximately 50:1~approx. 3000:1, approx. 100:1~approx. 500:1, approx. 100:1~approx. 1000:1, approx. 100:1~approx. 1500:1, approx. 100:1~approx. 2000:1, approx. 100:1~approx. 2500:1, approx. 100:1~approx. 3000:1, approx. 500:1~approx. 1000:1, approx. 500:1~approx. 1500:1, approx. 500:1~approx. 2000:1, approx. 500:1~approx. 2500:1, approx. 500:1~approx. 30 00:1, approximately 1000:1 to approximately 1500:1, approximately 1000:1 to approximately 2000:1, approximately 1000:1 to approximately 2500:1, approximately 1000:1 to approximately 3000:1, approximately 1500:1 to approximately 2000:1, approximately 1500:1 to approximately 2500:1, approximately 1500:1 to approximately 3000:1, approximately 2000:1 to approximately 2500:1, approximately 2000:1 to approximately 3000:1, or possibly approximately 2500:1 to approximately 3000:1.

[0165] In some embodiments, the antibody:sialic acid substrate mass ratio is approximately 1:1 to approximately 40:1, for example, approximately 1:1 to approximately 2:1, approximately 1:1 to approximately 4:1, approximately 1:1 to approximately 6:1, approximately 1:1 to approximately 8:1, approximately 1:1 to approximately 10:1, approximately 1:1 to approximately 15:1, approximately 1:1 to approximately 20:1, approximately 2:1 to approximately 4:1, approximately 2:1 to approximately 6:1, approximately 2:1 to approximately 8:1, approximately 2:1 to approximately 10:1, approximately 2:1 to approximately 15:1, approximately 2:1 to approximately 20:1, approximately 2:1 to approximately 40:1, approximately 4:1 to approximately 6:1, approximately 4:1 to approximately 8:1, approximately 4:1 to approximately 10:1, approximately 4:1 to approximately 15:1, approximately 4:1 to approximately 20:1, approximately 4:1 to approximately 40:1, approximately 6:1 to approximately 8:1, approximately 6:1 to approximately 10:1, approximately 6:1 to approximately 15:1, approximately 6:1 to approximately 20:1, approximately 6:1 to approximately 40:1, approximately 8:1 to approximately 10:1, approximately 8:1 to approximately 15:1, approximately 8:1 to approximately 20:1, approximately 8:1 to approximately 40:1, approximately 10:1 to approximately 15:1, approximately 10:1 to approximately 20:1, approximately 10:1 to approximately 40:1, approximately 15:1 to approximately 20:1, approximately 15:1 to approximately 40:1, or possibly approximately 20:1 to approximately 40:1.

[0166] In some embodiments, the method involves contacting a monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate for at least 30 minutes, for example, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 48 hours. In several embodiments, contact can last for approximately 30 minutes to 48 hours, for example, approximately 30 minutes to 4 hours, approximately 30 minutes to 5 hours, approximately 30 minutes to 6 hours, approximately 30 minutes to 7 hours, approximately 30 minutes to 10 hours, approximately 30 minutes to 12 hours, approximately 30 minutes to 18 hours, approximately 30 minutes to 24 hours, approximately 30 minutes to 36 hours, approximately 2 hours to 48 hours, approximately 3 hours to 6 hours, approximately 3 hours to 10 hours, approximately 3 hours to 12 hours, approximately 3 hours to 18 hours, approximately 3 hours to 24 hours, approximately 3 hours to 36 hours, approximately 3 hours to 48 hours, approximately 4 hours to 10 hours, approximately 4 hours to 12 hours, approximately 4 hours to 18 hours, approximately 4 hours to 24 hours, approximately 4 hours to 36 hours, approximately 4 hours to 48 hours, approximately 5 hours to Approximately 10 hours, approximately 5 to 12 hours, approximately 5 to 18 hours, approximately 5 to 24 hours, approximately 5 to 36 hours, approximately 5 to 48 hours, approximately 7 to 10 hours, approximately 7 to 12 hours, approximately 7 to 18 hours, approximately 7 to 24 hours, approximately 7 to 36 hours, approximately 7 to 48 hours, approximately 10 to 18 hours, approximately 10 to 24 hours The intervals between these periods are approximately 10 to 36 hours, 10 to 48 hours, 12 to 18 hours, 12 to 24 hours, 12 to 36 hours, 12 to 48 hours, 18 to 24 hours, 18 to 36 hours, 18 to 48 hours, 24 to 36 hours, 24 to 48 hours, or 36 to 48 hours.

[0167] In some embodiments, the method involves transferring a monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate at temperatures ranging from approximately 2°C to approximately 40°C, for example, approximately 2°C to approximately 37°C, 2°C to approximately 30°C, 2°C to approximately 25°C, 2°C to approximately 22°C, 2°C to approximately 20°C, 2°C to approximately 10°C, approximately 4°C to approximately 40°C, approximately 4°C to approximately 37°C, 4°C to approximately 30°C, 4°C to approximately 25°C, 4°C to approximately 22°C, 4°C to approximately 20°C, 4°C to approximately 10°C, approximately 10°C to approximately 40°C, and approximately 10°C to This includes contact at temperatures of approximately 37°C, 10°C to approximately 30°C, 10°C to approximately 25°C, 10°C to approximately 22°C, 10°C to approximately 20°C, approximately 20°C to approximately 40°C, approximately 20°C to approximately 37°C, 20°C to approximately 30°C, 20°C to approximately 25°C, 20°C to approximately 22°C, approximately 22°C to approximately 40°C, approximately 22°C to approximately 37°C, 22°C to approximately 30°C, approximately 22°C to approximately 25°C, approximately 25°C to approximately 40°C, approximately 25°C to approximately 37°C, 25°C to approximately 30°C, approximately 30°C to approximately 40°C, or approximately 30°C to approximately 37°C.

[0168] This disclosure employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, unless otherwise indicated, and such techniques are within the scope of the skill of those skilled in the art. Such techniques are adequately described in the literature. For example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DN Glover and BD Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 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 Edition (Wiley-Blackwell); Woodward, J., Immobilized Cells And Enzymes (IRL Press) (1985);Perbal (1988) A Practical Guide To Molecular Cloning;2d Edition (Wiley-Interscience);Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory);SCSee 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).

[0169] The general principles of antibody engineering are described, for example, in Strohl, WR, and LM Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). The general principles of protein engineering are described, for example, in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press). The general principles of immunology are described, for example, in Abbas and Lichtman (2017), Cellular and Molecular Immunology 9th Edition (Elsevier). Furthermore, standard immunological methods known in this field can be found, for example, 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).

[0170] All references cited above, and all references cited herein, are incorporated herein in their entirety by reference.

[0171] Exemplary Embodiments The embodiments provided include the following:

[0172] Embodiment 1. A monoclonal population of polymer-binding molecules, wherein each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, each N-linked glycosylation motif comprising the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine, and at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region are occupied by a complex glycan, and the monoclonal population of binding molecules comprises at least 35 moles of sialic acid per mole of binding molecule.

[0173] Embodiment 2. A monoclonal population of the binding molecule described in Embodiment 1, comprising at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 moles of sialic acid per mole of binding molecule.

[0174] Embodiment 3. A monoclonal population of the binding molecules described in Embodiment 1, containing approximately 40 to 70 moles, 40 to 60 moles, 40 to 55 moles, 40 to 50 moles, 50 to 70 moles, and 60 to 70 moles of sialic acid per mole of binding molecules.

[0175] Embodiment 4. A monoclonal population of binding molecules according to any one of Embodiments 1 to 3, wherein the IgM heavy chain constant region comprises five N-linked glycosylated motifs N-X1-S / T starting from amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04).

[0176] Embodiment 5. A monoclonal collection of the binding molecule described in Embodiment 4, wherein motifs N1, N2, and N3 are occupied by a complex glycan.

[0177] Embodiment 6. A monoclonal population of the binding molecules described in any one of Embodiments 1 to 5, generated by cell line modification, in vitro glycotechnology, or any combination thereof.

[0178] Embodiment 7. The monoclonal population of the binding molecule according to Embodiment 6, wherein the cell line modification includes transfecting a cell line that generates a monoclonal population of the binding molecule with a gene encoding sialyltransferase, thereby generating a modified cell line that overexpresses sialyltransferase.

[0179] Embodiment 8. A monoclonal population of the binding molecule described in Embodiment 7, wherein the sialyltransferase includes human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).

[0180] Embodiment 9. The monoclonal population of the binding molecule according to Embodiment 7 or Embodiment 8, further comprising transfecting a cell line that generates a monoclonal population of the binding molecule with a gene encoding galactosyltransferase, thereby generating a modified cell line that overexpresses galactosyltransferase.

[0181] Embodiment 10. A monoclonal population of the binding molecule described in Embodiment 9, wherein the galactosyltransferase includes human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).

[0182] Embodiment 11. A monoclonal group of binding molecules according to any one of Embodiments 6 to 10, wherein in vitro glycosylation comprises contacting the monoclonal group of binding molecules with a soluble sialyltransferase and a sialic acid substrate.

[0183] Embodiment 12. A monoclonal population of the binding molecules according to Embodiment 11, wherein the sialyltransferase comprises a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).

[0184] Embodiment 13. A monoclonal population of the binding molecules according to Embodiment 12, wherein the soluble variant of ST6GAL1 comprises amino acids x to 406 of SEQ ID NO: 3, where x is an integer from 27 to 120.

[0185] Embodiment 14. The monoclonal population of the binding molecules according to Embodiment 13, wherein the soluble variant of ST6GAL1 comprises amino acids 120-406, 115-406, 110-406, 109-406, 105-406, 100-406, 95-406, 90-406, 89-406, 88-406, 87-406, 86-406, 85-406, 84-406, 83-406, 82-406, 81-406, 80-406, 75-406, 70-406, 65-406, 60-406, 55-406, 50-406, 45-406, 40-406, 35-406, 30-406, or 27-406 of SEQ ID NO: 3.

[0186] Embodiment 15. A monoclonal population of the binding molecules according to any one of Embodiments 11 to 14, wherein the sialic acid substrate comprises cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA).

[0187] Embodiment 16. A monoclonal population of the binding molecules according to any one of Embodiments 11 to 15, wherein the mass ratio of the binding molecule to the sialic acid substrate is about 1:4 to about 40:1.

[0188] Embodiment 17. A monoclonal population of the binding molecules according to any one of Embodiments 11 to 16, wherein the mass ratio of the binding molecule to the sialyltransferase is about 80:1 to about 5000:1.

[0189] Embodiment 18. A monoclonal collection of the binding molecules described in any one of Embodiments 11 to 17, wherein the mass ratio of binding molecules to sialyltransferase is approximately 2000:1.

[0190] Embodiment 19. A monoclonal collection of the binding molecule described in Embodiment 18, wherein the mass ratio of binding molecule:sialic acid substrate:sialyltransferase is approximately 2000:500:1.

[0191] Embodiment 20. A monoclonal collection of the binding molecules according to any one of Embodiments 11 to 17, wherein the molar ratio of binding molecules to sialyltransferase is approximately 80:1.

[0192] Embodiment 21. A monoclonal population of the binding molecule described in Embodiment 20, wherein the molar ratio of binding molecule:sialic acid substrate:sialyltransferase is approximately 80:500:1.

[0193] Embodiment 22. A monoclonal collection of the binding molecules according to any one of Embodiments 11 to 17, wherein the mass ratio of binding molecules to sialyltransferase is approximately 500:1.

[0194] Embodiment 23. A monoclonal collection of the binding molecule described in Embodiment 22, wherein the mass ratio of binding molecule:sialic acid substrate:sialyltransferase is approximately 500:62.5:1.

[0195] Embodiment 24. A monoclonal group of binding molecules according to any one of Embodiments 11 to 23, wherein contact of the monoclonal group of binding molecules with the soluble sialyltransferase and the sialic acid substrate includes contact for at least 30 minutes.

[0196] Embodiment 25. A monoclonal population of binding molecules according to Embodiment 24, wherein the contact includes contact for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours.

[0197] Embodiment 26. A monoclonal group of binding molecules according to any one of Embodiments 11 to 25, wherein the contact of the monoclonal group of binding molecules with the soluble sialyltransferase and the sialic acid substrate occurs at a temperature of approximately 2°C to approximately 40°C.

[0198] Embodiment 27. A monoclonal group of binding molecules according to Embodiment 26, wherein the contact occurs at 15°C to about 37°C, 15°C to about 30°C, or 15°C to about 25°C.

[0199] Embodiment 28. A monoclonal group of binding molecules according to any one of Embodiments 11 to 27, wherein in vitro glycosylation comprises contacting the monoclonal group of binding molecules with a soluble galactosyltransferase and a galactose substrate.

[0200] Embodiment 29. A monoclonal population of the binding molecule according to Embodiment 28, wherein the galactosyltransferase comprises a soluble variant of human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).

[0201] Embodiment 30. A monoclonal population of the binding molecule according to Embodiment 29, wherein the soluble variant of B4GALT4 comprises amino acids x to 344 of SEQ ID NO: 4, where x is an integer from 39 to 120.

[0202] Embodiment 31. A monoclonal population of the binding molecule described in Embodiment 30, wherein the soluble variant of B4GALT4 includes amino acids 120-344, 115-344, 110-344, 105-344, 100-344, 95-344, 90-344, 85-344, 80-344, 75-344, 70-344, 65-344, 60-344, 55-344, 50-344, 45-344, 40-344, or 39-344 of SEQ ID NO: 4.

[0203] Monoclonal population of binding molecules according to any one of embodiments 28 to 31, wherein the galactose substrate comprises uridine diphosphate-α-D-galactose (UDP-Gal).

[0204] Monoclonal population of binding molecules according to any one of embodiments 28 to 32, wherein contacting the galactosyltransferase and the galactose substrate occurs before or simultaneously with contacting the soluble sialyltransferase and the sialic acid substrate.

[0205] Monoclonal population of binding molecules according to any one of embodiments 1 to 33, wherein each binding molecule is multispecific and two or more binding domains that bind to the IgM heavy chain constant region of each binding molecule specifically bind to different targets.

[0206] Monoclonal population of binding molecules according to any one of embodiments 1 to 33, wherein the binding domains that bind to the IgM heavy chain constant region of each binding molecule specifically bind to the same target.

[0207] Monoclonal population of binding molecules according to embodiment 35, wherein the binding domains that bind to the IgM heavy chain constant region of each binding molecule are identical.

[0208] Monoclonal population of binding molecules according to any one of embodiments 34 to 36, wherein the binding domain is an antigen-binding domain derived from an antibody.

[0209] Embodiment 38. A monoclonal population of binding molecules according to Embodiment 37, wherein each binding molecule is a pentameric or hexamer IgM antibody containing five or six divalent IgM binding units, and each binding unit contains two IgM heavy chains each containing a VH located at the amino-terminal end of the variant IgM constant region, and two immunoglobulin light chains each containing a light chain variable domain (VL) located at the amino-terminal end of the immunoglobulin light chain constant region, and the VH and VL bind to form an antigen-binding domain that specifically binds to the target.

[0210] Embodiment 39. A monoclonal collection of binding molecules as described in Embodiment 38, wherein each antigen-binding domain of each binding molecule binds to the same target.

[0211] Embodiment 40. A monoclonal collection of binding molecules as described in Embodiment 39, wherein each antigen-binding domain of each binding molecule is identical.

[0212] Embodiment 41. A monoclonal population of binding molecules according to any one of Embodiments 1 to 40, wherein the target is a target epitope, a target antigen, a target cell, a target organ, or a target virus.

[0213] Embodiment 42. A monoclonal collection of binding molecules according to any one of Embodiments 1 to 41, wherein each binding molecule is a pentamer and further comprises a J chain or a functional fragment thereof or a functional variant thereof.

[0214] Embodiment 43. A monoclonal population of the binding molecule according to Embodiment 42, wherein the J chain is a mature human J chain containing the amino acid sequence of SEQ ID NO: 6, or a functional fragment thereof, or a functional variant thereof.

[0215] Embodiment 44. A monoclonal population of the binding molecule according to Embodiment 43, comprising the N-linked glycosylation motif N-X1-S / T (motif N6) in which the J chain starts at a position corresponding to amino acid 49 of SEQ ID NO: 6.

[0216] Embodiment 45. A functional variant J chain comprising one or more single amino acid substitutions, deletions, or insertions compared to a reference J chain identical to the variant J chain except for the substitution, deletion, or insertion of one or more single amino acids, wherein the monoclonal population of the binding molecule is identical to the variant J chain except for the substitution, deletion, or insertion of one or more single amino acids, and exhibits an increased serum half-life upon administration to a target animal compared to a binding molecule derived from reference IgM administered to the same animal species using the same method.

[0217] Embodiment 46. A monoclonal population of the binding molecule according to Embodiment 45, wherein the variant J chain or its functional fragment comprises one, two, three, or four single amino acid substitutions, deletions, or insertions compared to the reference J chain.

[0218] Embodiment 47. A monoclonal population of the binding molecule according to Embodiment 45 or Embodiment 46, wherein the variant J chain or its functional fragment contains an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J chain of SEQ ID NO: 6.

[0219] Embodiment 48. A monoclonal population of the binding molecule according to Embodiment 47, wherein the amino acid corresponding to Y102 in SEQ ID NO: 6 is substituted with alanine (A).

[0220] Embodiment 49. A monoclonal population of the binding molecule described in Embodiment 48, wherein the J chain contains the amino acid sequence of SEQ ID NO: 7.

[0221] Embodiment 50. A monoclonal group of binding molecules according to any one of Embodiments 42 to 49, wherein the J chain or a fragment or variant thereof is a modified J chain further comprising a heterogeneous portion, and the heterogeneous portion is fused to or conjugated to the J chain or a fragment or variant thereof.

[0222] Embodiment 51. A monoclonal group of the binding molecule according to Embodiment 50, wherein the heterogeneous portion is a polypeptide fused to the J chain or a fragment or variant thereof.

[0223] Embodiment 52. A monoclonal collection of the binding molecule according to Embodiment 51, wherein the heterogeneous polypeptide is fused to the J chain or a fragment or variant thereof via a peptide linker.

[0224] Embodiment 53. A monoclonal group of binding molecules according to Embodiment 52, wherein the peptide linker comprises at least 5 but 25 or fewer amino acids.

[0225] Embodiment 54. A monoclonal group of the binding molecule described in Embodiment 52 or Embodiment 53, wherein the peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 43).

[0226] Embodiment 55. A monoclonal collection of binding molecules according to any one of Embodiments 51 to 54, wherein the heterogeneous polypeptide is fused to the N-terminus of the J chain or a fragment or variant thereof, or to the C-terminus of the J chain or a fragment or variant thereof.

[0227] Embodiment 56. A monoclonal collection of the binding molecule according to any one of Embodiments 51 to 55, wherein heterogeneous parts, which may be the same or different, are fused to the N-terminus and C-terminus of the J chain or a fragment or variant thereof.

[0228] Embodiment 57. A monoclonal collection of binding molecules according to any one of Embodiments 51 to 56, wherein the heterogeneous polypeptide includes a binding domain.

[0229] Embodiment 58. A monoclonal population of binding molecules according to Embodiment 57, wherein the antigen-binding domain of the heterologous polypeptide is an antibody or an antigen-binding fragment thereof.

[0230] Embodiment 59. A monoclonal population of the binding molecule according to Embodiment 58, wherein the antigen-binding fragment is an scFv fragment.

[0231] Embodiment 60. A monoclonal population of the binding molecule described in Embodiment 59, wherein the heterogeneous scFv fragment is bound to CD3ε.

[0232] Embodiment 61. A monoclonal population of the binding molecule according to Embodiment 60, wherein the modified J chain includes amino acids 20-420 (VJH) of SEQ ID NO: 36 (V15J), SEQ ID NO: 37 (V15J*), SEQ ID NO: 38 (SJ*), SEQ ID NO: 31 (A-55-J*), SEQ ID NO: 32 (A-56-J*), SEQ ID NO: 33 (A-57-J*), SEQ ID NO: 34, amino acids 20-420 (VJ*H) of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO: 6 or 7, which is fused via a peptide linker to the scFv of anti-CD3ε containing the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, including SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0233] Embodiment 62. A pharmaceutical composition comprising a monoclonal group of binding molecules described in any one of Embodiments 1 to 61 and a pharmaceutically acceptable excipient.

[0234] Embodiment 63. Recombinant host cells that generate a monoclonal population of the binding molecule described in any one of Embodiments 1 to 61.

[0235] Embodiment 64. A method for generating a monoclonal population of binding molecules according to any one of Embodiments 1 to 61, comprising culturing the host cells according to Embodiment 62 and recovering the population of binding molecules.

[0236] Embodiment 65. A method for generating a monoclonal population of a highly sialylated multimer-binding molecule, comprising: providing a cell line expressing the monoclonal population of the binding molecule; culturing the cell line; and recovering the monoclonal population of the binding molecule, wherein each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, and each IgM heavy chain constant region comprising at least 3, at least 4, or at least 5 asparagine (N)-linked glycosylated motifs. The method wherein the N-linked glycosylation motif comprises the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine, and on average, at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region in the population are occupied by complex glycans, and the cell line, harvesting process, or combination thereof is optimized to concentrate complex glycans containing at least one, two, three, or four sialic acid-terminal monosaccharides per glycan.

[0237] Embodiment 66. The method according to Embodiment 65, wherein the cell line, recovery process, or combination thereof is optimized to yield a monoclonal population of binding molecules containing at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, or at least 65 moles of sialic acid per mole of binding molecule.

[0238] Embodiment 67. The method according to Embodiment 66, wherein the cell line, recovery process, or combination thereof is optimized to yield a monoclonal population of binding molecules containing about 40 to about 70, about 40 to about 60, about 40 to about 55, about 40 to about 50, about 50 to about 70, and about 60 to about 70 moles of sialic acid per mole of binding molecule.

[0239] Embodiment 68. The method according to any one of Embodiments 65 to 67, wherein the IgM heavy chain constant region is derived from a human IgM heavy chain constant region comprising five N-linked glycosylated motifs N-X1-S / T that start from amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04).

[0240] Embodiment 69. The method according to Embodiment 68, wherein, on average, one, two, or all three of the motifs N1, N2, and N3 in the group of binding molecules are occupied by a complex glycan.

[0241] Embodiment 70. The method according to any one of Embodiments 65 to 69, wherein the provided cell line is modified to overexpress sialyltransferase.

[0242] Embodiment 71. The method according to Embodiment 70, wherein the sialyltransferase comprises human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).

[0243] Embodiment 72. The method according to any one of Embodiments 65 to 71, wherein the recovery process comprises subjecting the monoclonal population of the binding molecules to in vitro glycosylation.

[0244] Embodiment 73. The method according to Embodiment 72, wherein the in vitro glycosylation comprises contacting a monoclonal population of the binding molecule with a soluble sialyltransferase and a sialic acid substrate.

[0245] Embodiment 74. The method according to Embodiment 73, wherein the sialyltransferase comprises a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).

[0246] Embodiment 75. The method according to Embodiment 74, wherein the soluble variant of ST6GAL1 comprises amino acids x to 406 of SEQ ID NO: 3, where x is an integer from 27 to 120.

[0247] Embodiment 76. The method according to Embodiment 75, wherein the soluble variant of ST6GAL1 includes amino acids 120-406, 115-406, 110-406, 109-406, 105-406, 100-406, 95-406, 90-406, 89-406, 88-406, 87-406, 86-406, 85-406, 84-406, 83-406, 82-406, 81-406, 80-406, 75-406, 70-406, 65-406, 60-406, 55-406, 50-406, 45-406, 40-406, 35-406, 30-406, or 27-406 of SEQ ID NO: 3.

[0248] Embodiment 77. The method according to any one of Embodiments 73 to 75, wherein the sialic acid substrate comprises cytidine monophosphate (CMP)-N-acetylneuraminic acid (CMP-NANA).

[0249] Embodiment 78. The method according to any one of Embodiments 73 to 77, wherein the mass ratio of the binding molecule to the sialic acid substrate is about 1:4 to about 40:1.

[0250] Embodiment 79. The method according to any one of Embodiments 73 to 78, wherein the mass ratio of the binding molecule to the sialyltransferase is about 80:1 to about 10000:1.

[0251] Embodiment 80. The method according to any one of Embodiments 73 to 79, wherein the mass ratio of the binding molecule to the sialyltransferase is approximately 2000:1.

[0252] Embodiment 81. The method according to Embodiment 80, wherein the mass ratio of binding molecule:sialic acid substrate:sialyltransferase is approximately 2000:500:1.

[0253] Embodiment 82. The method according to any one of Embodiments 73 to 79, wherein the molar ratio of the binding molecule to the sialyltransferase is approximately 80:1.

[0254] Embodiment 83. The method according to Embodiment 82, wherein the molar ratio of binding molecule:sialic acid substrate:sialyltransferase is approximately 80:500:1.

[0255] Embodiment 84. The method according to any one of Embodiments 73 to 79, wherein the mass ratio of the binding molecule to the sialyltransferase is approximately 500:1.

[0256] Embodiment 85. The method according to Embodiment 84, wherein the mass ratio of binding molecule:sialic acid substrate:sialyltransferase is approximately 500:62.5:1.

[0257] Embodiment 86. The method according to any one of Embodiments 73 to 85, wherein the contact of the monoclonal group of the binding molecule with the soluble sialyltransferase and the sialic acid substrate includes contact for at least 30 minutes.

[0258] Embodiment 87. The method according to Embodiment 86, wherein the contact includes contact for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours.

[0259] Embodiment 88. The method according to any one of Embodiments 73 to 87, wherein the contact of the monoclonal group of the binding molecule with the soluble sialyltransferase and the sialic acid substrate occurs at a temperature of approximately 2°C to approximately 40°C.

[0260] Embodiment 89. The method according to Embodiment 88, wherein the contact occurs at a temperature of 15°C to approximately 37°C, 15°C to approximately 30°C, or 15°C to approximately 25°C.

[0261] Embodiment 90. The method according to any one of Embodiments 73 to 77, wherein in vitro glycosylation comprises contacting a monoclonal population of the binding molecule with a soluble galactosyltransferase and a galactose substrate.

[0262] Embodiment 91. The method according to Embodiment 90, wherein the galactosyltransferase comprises a soluble variant of human β-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).

[0263] Embodiment 92. The method according to Embodiment 90 or Embodiment 91, wherein the galactose substrate comprises uridine diphosphate-α-D-galactose (UDP-Gal).

[0264] Embodiment 93. The method according to any one of Embodiments 90 to 92, wherein the contact with the galactosyltransferase and galactose substrate occurs before or simultaneously with the contact with the soluble sialyltransferase and sialic acid substrate.

[0265] The following examples are provided for illustrative purposes only and are not limiting. [Examples]

[0266] Example 1 - Materials and Method Population of IgM antibodies Unless otherwise noted below, these experiments were performed on a monoclonal population of the IgM bispecific antibody CD20×CD3 IGM-A, comprising an IgM heavy chain containing the wild-type human IgM constant region (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) and the anti-CD20 VH region of SEQ ID NO: 8, a light chain containing the anti-CD20 VL region of SEQ ID NO: 9, and a modified J chain binding to CD3 containing amino acids 20-420 of SEQ ID NO: 34. CD20×CD3 IGM-A is described in detail in U.S. Patent Application Publication No. US-2018-0265596-A1, which is incorporated in its entirety herein by reference. Glycoengineered IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules are referred to as “GEMs” throughout the examples, regardless of the glycoengineering method.

[0267] Glycoengineering of IgM antibody populations Along with various controls, as shown in the following examples, varying amounts of abbreviated human α-2,6 sialyltransferase ("Abbreviated Human ST6" available from Roche Diagnostics, Inc. (material no. 07012250103 or material no. 08098174103)) or from Agilent (part no. GKT-S26) were added to 20 μl reaction solutions containing a partially purified monoclonal population of IgM antibody, e.g., anti-CD20×CD3 IGM-A, and varying amounts of sodium cytidine-5'-monophospho-N-acetylneuraminate (CMP-NANA) dissolved in 50 mM Tris acetate (pH 7.5). Unless otherwise indicated, the reaction was carried out at 37°C for 8 hours. The reaction was stopped by freezing at -20°C. The IgM population treated with ST6 was further purified, e.g., by anion exchange chromatography and / or mixed-mode chromatography, before further analysis.

[0268] Determination of total sialic acid The Agilent AdvanceBio Total Sialic Acid Quantitation Kit measures sialic acid (N-acetylneuraminic acid (NANA)) released from glycoproteins. This assay utilizes an enzyme-conjugated reaction in which free sialic acid is oxidized to produce an intermediate, which stoichiometrically reacts with a probe to produce a product detectable by absorbance (OD=530nm) or fluorescence (excitation / emission (Ex / Em)=530 / 590nm). The kit measures sialic acid in a linear range from 40 pmol to 1,000 pmol, with a detection sensitivity of 0.15 mg / ml of IgM antibody. The kit was used according to the manufacturer's recommendations. Briefly, samples were digested with sialidase A for 2 hours. Bovine fetuin control protein was used as a positive control, with an expected range of 9.6–13.9 mol / mol. Sialic acid standards were prepared using the following pmol concentrations for fluorescence measurements: 1,000, 500, 250, and 0 pmol. Next, conversion and colorimetric mixtures were prepared according to Table 2 below.

[0269] (Table 2) Sialic acid quantitative assay TIFF0007857221000003.tif31139

[0270] When sialic acid is released by sialidase A digestion, N-acetylneuramine aldolase catalyzes the reaction to form pyruvate. The reaction then proceeds through an additional step using pyruvate oxidase as a catalyst to form hydrogen peroxide, which forms a 1:1 complex with a dye to form a fluorescent reporter dye. This dye can be read by fluorescence detection (Ex / Em = 530 / 590 nm). This then correlates to yield the sialic acid level mol / mol from the sialic acid standard curve.

[0271] Example 2: Effect of α-2,6-sialyltransferase concentration on sialylation of IgM antibody populations As described in Example 1, various amounts of cleaved human ST6 were added to 20 μl of a reaction solution containing 60 μg of anti-CD20×CD3 IGM-A (3 mg / ml) and 30 μg of cytidine-5'-monophospho-N-acetylneuraminate sodium salt (CMP-NANA, 1.5 mg / ml). The resulting sialylation was quantified as described in Example 1, and the amount of sialylation compared to the concentration of cleaved human ST6 is shown in Figure 4.

[0272] These results show that a low ST6 concentration of 1.5 μg / ml (a molar ratio of IgM to cleaved human ST6 of 80:1) can be used to produce an SA level of 40 mol / mol, and increasing the cleaved human ST6 concentration to 30 μg / ml or higher (a molar ratio of IgM to cleaved human ST6 of approximately 4:1 or higher) can produce SA levels exceeding 60 mol / mol.

[0273] Example 3: Siarylation of other IgM antibodies To determine whether the invitrocyarylation procedure developed above could be applied to other IgM antibodies, two other CHO cell lines expressing recombinant IgM antibodies, pentameric anti-DR5 IGM-B (VH: SEQ ID NO: 10, VL: SEQ ID NO: 11, see U.S. Patent No. 7,521,048) and hexameric anti-DR5 IGM-C (VH: SEQ ID NO: 12, VL: SEQ ID NO: 13, see U.S. Patent No. 7,790,165), were sialylated and analyzed as described in Example 1. Each 20 μl reaction contained 0.28 μg of cleaved human ST6 (final concentration 14 μg / mL) and 60 μg of anti-DR5 IGM-B or anti-DR5 IGM-C (approximately 8:1 molar ratio of IgM to cleaved human ST6), with 30 μg of CMP-NANA used. A control reaction without cleaved human ST6 was also performed. The resulting sialylation was determined as described in Example 1, and the amount of sialylation under each condition is shown in Figure 5 and Table 3.

[0274] (Table 3) Siarylation of anti-DR5 antibodies TIFF0007857221000004.tif25128

[0275] Example 4: Siarylation level of human serum IgM The sialylation level of human serum IgM (obtained from Sigma, catalog number I8260-25mg) was determined using a sialic acid (NANA) assay kit (Agilent AdvanceBio Total Sialic Acid Determination Kit), as described in Example 1.

[0276] When sialic acid is released by sialidase A digestion, N-acetylneuramine aldolase catalyzes the reaction to form pyruvate. The reaction then proceeds through an additional step using pyruvate oxidase as a catalyst to form hydrogen peroxide, which forms a 1:1 complex with a dye to form a fluorescent reporter dye. This dye can be read by fluorescence detection (Ex / Em = 530 / 590 nm). This then correlates to the sialic acid level mol / mol from the sialic acid standard curve. The resulting amount of sialylation is shown in Table 4.

[0277] (Table 4) Sialic acid content of human serum IgM TIFF0007857221000005.tif11128

[0278] Example 5: Effect of increasing sialylation of IGM antibody The glycosylated IgM CD20×CD3 IGM-A ("anti-CD20×CD3 IGM-A-GEM") material used in the experiments of this example contained approximately 37 moles of sialic acid per mole of IgM and was prepared as described in Example 1. Unglycosylated IGM CD20×CD3 IGM-A contained approximately 14 moles of sialic acid per mole of IgM.

[0279] Complement-dependent cytotoxicity CD20-expressing Ramos cells (ATCC catalog number CRL-1596) were cultured in RPMI (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Gibco catalog number 16140-071). Ramos cells (50,000) were seeded in 96-well plates at a volume of 10 ul / well. The cells were treated with serial dilutions of two different lots of in vitro sialylated anti-CD20×CD3 IGM or anti-CD20×CD3 IGM-A ("anti-CD20×CD3 IGM-A-GEM"), as described in Example 1. All antibody dilutions were performed in RPMI medium supplemented with 10% heat-inactivated serum. Human serum complement (Quidel catalog number A113) was added to the antibody-treated cells at a final concentration of 5% at a volume of 10 ul / well. The reaction mixture was incubated at 37°C for 4 hours. An amount of CELLTITER GLO® (Promega catalog number G7572) reagent equal to the amount of cell culture medium present in each well was added. The plate was shaken for 2 minutes, incubated at room temperature for 10 minutes, and luminescence was measured using an Envision multimode reader (Perkin Elmer) with an integration time of 0.1 seconds per well. Data were analyzed using GraphPad Prism and a 4-parameter fit with upper and lower values ​​fixed to 100% and 0% feasibility, respectively. Half of the maximum response (EC) was used. 50 The concentrations of antibodies that produce the desired effect were calculated for each condition and are shown in Table 5. Invitrocylylation did not have a noticeable effect on complement-dependent cytotoxicity.

[0280] (Table 5) CDC activity TIFF0007857221000006.tif15128

[0281] T cell activation T cell activation (TCA) by anti-CD20×CD3 IGM-A-GEM or anti-CD20×CD3 IGM-A was determined using luminescence-based readout in the presence of antigen-positive Jurkat-based reporter cells. Manipulated Jurkat T cells (Promega J1601 part number J131A) and Ramos cells were cultured in RPMI (Invitrogen) supplemented with 10% thermoinactivated fetal bovine serum (Gibco catalog number 16140-071). Ramos cells (7500 cells / well, 10 μl volume) were added to a white 384-well assay plate. Next, serial dilutions of anti-CD20×CD3 IGM-A-GEM or anti-CD20×CD3 IGM-A were added to the Ramos cells in 10 μl volumes. Manipulated Jurkat cells (25000 cells / well, 20 μl volume) were added to the mixture to a final volume of 40 μL. The mixture was incubated with 5% CO2 at 37°C for 16 hours. The cell mixture was then mixed with 20 μL of lysis buffer containing luciferin (Promega, CELLTITER-GLO®) to measure luciferase reporter activity. Optical output was measured using an EnVision plate reader. EC 50 This was determined by a four-parameter curve fit using Prism software.

[0282] EC 50 The results for each condition were calculated and are shown in Table 6. Invitrocyalilation did not have a detectable effect on T cell activation.

[0283] (Table 6) T cell activation TIFF0007857221000007.tif15128

[0284] In vitro B cell killing The Ramos, CD19+CD20+B cell line was labeled with a cell-tracking dye (Oregon Green 488, ThermoFisher, catalog number C34555) and then co-cultured with serially diluted anti-CD20×CD3IGM-A or anti-CD20×CD3 IGM-A-GEM and primary human CD8+ T cells (Precision for Medicine, catalog number 84300; negative selection) at 37°C and 5% CO2 for 48 hours. Cells were harvested, stained with 7-AAD (BD Biosciences, catalog number 559925), and analyzed by flow cytometry to evaluate viable B cells. EC 50 EC was calculated for each condition and shown in Table 7. In vitro sialylation did not have a perceptible impact on the ability of the antibody to kill B cells.

[0285] (Table 7) T cell-dependent B cell killing TIFF0007857221000008.tif15128

[0286] Pharmacokinetics Pharmacokinetic parameters were measured for various IgM antibodies in an in vivo mouse model as follows. Balb / c mice were injected intravenously with either 5 mg / kg of anti-CD20×CD3IGM-A or anti-CD20×CD3IGM-A-GEM antibody. Blood samples were collected at a total of 10 or 12 time points for each antibody, and 2 mice were used at each time point. Each mouse was bled once (100 μL) from the facial vein and then again (maximum obtainable, approximately 500 μL) by terminal cardiac puncture. Serum concentrations of each antibody in the blood at each time point were measured using a standard ELISA assay. Quality metrics were for all ELISAs, and T 1 / 2-α T 1 / 2-β and the area under the concentration curve from time zero to infinity (AUC 0-∞The PK parameters, including those measured in μg / ml*hr units, were derived using a standard curve fitting method (Win Non Lin, Phoenix Software). The PK results, including the area under the curve (AUC), are shown in Figure 6.

[0287] Example 6: Cell line manipulation to increase sialylation Vectors containing the GACACC Kozak sequence, the sequence encoding α-2,6-sialyltransferase (ST6) SEQ ID NO: 3 (NCBI reference sequence: sp|P15907.1), and a hygromycin marker selection were generated by standard methods. The vectors were electroporated into stable CHO subclones expressing anti-CD20×CD3 IGM-A. After selection and harvesting, the resulting pool was subcloned by limiting dilution into 384-well plates. Phenotypic screening was performed by labeling the subcloned cells with SNA-1 conjugated to fluorescein isothiocyanate (FITC). SNA-1 is a lectin specific to 2,6-sialic acid. The cells themselves were labeled immediately after washing with FACS buffer. Fluorescence levels were detected, and the results are shown in Figure 7. Only 4 of the 60 subclones produced a signal exceeding the signal of the HEK293 cells used as a positive control. Two subclones, 25 and 47, were harvested for further study.

[0288] To detect the presence of ST6 in the genomes of subclones 25 and 47, QPCR analysis was performed using the primers shown in Table 8.

[0289] (Table 8) Primers used in QPCR assay TIFF0007857221000009.tif11147

[0290] These primers are represented by the coding sequence (CDS) of the ST6 gene. Both subclones are 38 cycles long. 目 The CHO cell control showed a positive reaction up to that point, but not the other cells.

[0291] Western blotting was performed to detect 2,6-linked sialic acid in the anti-CD20×CD3 IGM-A antibody expressed and purified from small-scale fermentation. The reduced denatured gel (BioRad® CriterionTGX stain-free precast gel) was visualized and imaged according to the manufacturer's instructions for use. The resulting images are shown in Figure 8A. The gel was then treated with biotinylated SNA-I lectin and streptavidin horseradish peroxidase fusion protein and imaged. The resulting images are shown in Figure 8B.

[0292] The selected subclones possessed detectable levels of 2,6-sialic acid. However, the stable pool of CHO cells from which the subclones originated did not.

[0293] Subclone 25 was expanded and grown for 3-liter bioreactor production so that it could be used for comparative studies against the parental anti-CD20×CD3 IGM-A produced in cells lacking the 2,6-sialyltransferase gene. Figures 9A–D show how the culture was performed in terms of viable cell density (Figure 9A), cell viability (Figure 9B), anti-CD20×CD3 IGM-A production (Figure 9C), and moles of sialic acid per mole of anti-CD20×CD3 IGM-A (Figure 9D). By day 8, subclone 25 produced 25% less anti-CD20×CD3 IGM-A than the parental cell line (Figure 9C), but the sialic acid content more than doubled and remained elevated (Figure 9D). Data at day 12 showed that subclone 25 produced 500 μg / ml (Figure 9C), and the sialic acid content of IgM was 37 mol / mol (Figure 9D).

[0294] Example 7: 2,6-sialic acid knock-in parental cell line Vectors for the stable insertion of α-2,6-sialyltransferase (NCBI reference sequence: NP_775324.1) and hygromycin marker selection were generated by a commercial vendor. The vectors were electroporated into CHO suspension cell lines. The resulting stable pool was cloned, and 384 clones were expanded and screened by cytometry. Cell surface-based labeling was performed using 2,6-sialic acid-specific lectin (SNA-1) chemically conjugated to Cy5 dye. The results of this assay are shown in Figure 10A. Based on the screening, 48 clones that grew well and had high lectin labeling levels were expanded into 24 deep-well plates and shaken at 300 RPM in an incubator at 37°C, 5% CO2, and 80% humidity. After the 48 clones expanded in deep-well plates, the top 22 were transferred to shaking flasks and the same 2,6-sialic acid screening was performed.

[0295] The 22 selected clones were screened again when the cell density was between 1 million and 4 million cells / ml. The lectin labeling levels in this case, along with the corresponding viability of the cultures at the time they were assayed, are shown in Figure 10B.

[0296] Based on shaking flask analysis, six clones were selected for evaluation by first transfecting them with 2–4 control IgMs. The parental CHO cell line was also transfected with the same 4 control IgMs and used as a basis for comparison. After transfection, four of the six selected clones either failed to recover post-transfection or pre-transfection, or had proliferation problems. The two clones that survived after transfection (2B4 and 2C2) showed high titers in all but one case and, in all cases, showed higher sialic acid content than the IgMs in the parental CHO cell line. Table 9 shows the results of a 7-day batch fermentation comparing the parental cell line with the two 2,6-sialyltransferase clones. Sialic acid content was determined from purified protein after harvest. Four different IgMs were transfected into 2B4 and the parental cell line, and two of these IgMs were also transfected into 2C2.

[0297] (Table 9) Recovery titer data and sialic acid levels of proteins purified from recovered fermented products TIFF0007857221000010.tif44152

[0298] To further characterize the clones, 2,6-sialic acid and 2,3-sialic acid levels were measured on the cell surface by cytometry. Measurements were performed using fluorescently labeled lectins specific to either form of sialic acid. At the time of labeling, all viability was above 95%. HEK293 cells, CHO parental cell lines, selected clones, and IgM-transfected clones were all analyzed using the same method. Figures 11A and 11B show the 2,3-sialic acid and 2,6-sialic acid levels of untransfected cells, respectively. Figure 11C compares the 2,3-sialic acid and 2,6-sialic acid levels of untransfected parental cells and 2B4 cells with those of IgM-4 transfected parental cells and 2B4 cells. The data shown in Figure 11A indicate that CHO parental cells had higher 2,3-sialic acid levels than HEK293 cells or clones transfected with 2,6-sialyltransferase. The data shown in Figure 11B indicates that 2,6-sialic acid levels in the clones were significantly elevated compared to those of the CHO parent. Figure 11C shows that IgM-transfected cell lines retain high levels of 2,6-sialic acid.

[0299] Example 8: Invitrocylylation under various conditions Various amounts of cleaved human ST6 were added to reaction solutions containing IgM antibody and cytidine-5'-monophospho-N-acetylneuraminate sodium salt (CMP-NANA, 1.5 mg / ml) in the ratios shown in Table 10. The duration and temperature of each reaction, as well as the resulting sialylation (quantified as described in Example 1), are also shown in Table 10. Room temperature (RT) is 15-25°C.

[0300] (Table 10) Invitrocyalilation conditions and results TIFF0007857221000011.tif180148

[0301] When antibodies under conditions 1 and 2 were compared using size exclusion chromatography (SEC), dynamic light scattering (DLS), hybrid gel, reduced gel, and the CDC and TCA assays described in Example 3, in vitro sialylation did not alter the SEC profile, dynamic radius, or antibody mobility, and the antibodies exhibited similar TCA and CDC activity (data not shown).

[0302] Antibodies from conditions 19-23 were also compared by the TCA assay described in Example 3, and the data are shown in Figure 12. All assayed antibodies showed similar TCA activity.

[0303] The SA levels of the antibodies under conditions 29–31 were monitored at set points during the reaction. SA levels over 48 hours or over the first 15 hours are plotted in Figures 13A and 13B, respectively. At a mass ratio of 100:50:1 antibody:CMP-NANA:ST-6, maximum sialylation was reached at 1 hour, with little difference observed between 37°C and 15°C.

[0304] The SA levels of the antibodies under conditions 32–36 were monitored at set points during the reaction. SA levels over 48 hours are plotted in Figure 14. At a mass ratio of 100:50:1 antibody:CMP-NANA:ST-6, sialization exceeding 60 mol / mol SA was achieved by 18 hours RT, and did not decrease significantly up to 48 hours RT. At a mass ratio of 250:125:1 antibody:CMP-NANA:ST-6, the increase in SA levels was slower, exceeding 60 mol / mol SA after 36 hours. No significant desialization was observed at RT up to 36 hours for all ratios. At a mass ratio of 5000:2500:1 antibody:CMP-NANA:ST-6, 40 mol / mol SA was achieved, and the antibody population was not sialized to the maximum possible extent.

[0305] Example 9: Pharmacokinetics of IgM antibodies with high sialic acid levels Pharmacokinetic parameters were measured for various IgM antibodies in an in vivo mouse model as follows: Balb / c mice were intravenously injected with anti-CD20×CD3 IGM-A, anti-CD20×CD3 IGM-A-GEM antibodies, anti-CD20×CD3 IGM-F, anti-CD20×CD3 IGM-F-GEM antibodies, or 5 mg / kg of human serum IgM at various sialic acid levels. Blood samples were collected at a total of 10 or 12 time points for each antibody, with at least two mice used at each time point. Each mouse was bled once from the facial vein (100 μL) and then again by terminal cardiac puncture (maximum obtainable, approximately 500 μL). Serum concentrations of each antibody in the blood at each time point were measured using a standard ELISA assay. Quality metrics were used for all ELISAs, and T 1 / 2-α , T 1 / 2-β , and the area under the concentration curve from zero time to infinity (AUC) 0-∞ PK parameters, including those measured in μg / ml*hr units, were derived using a standard curve fitting method (Win Non Lin, Phoenix Software). The sialic acid levels and resulting AUC obtained for each antibody are also shown. 0-∞ This is shown in Figure 15.

[0306] Example 10: IgM antibody exhibiting high sialic acid level activity in cynomolgus monkeys Pharmacokinetic parameters and cellular markers were measured for IgM antibodies in an in vivo cynomolgus monkey model as follows: Cynomolgus monkey primates were injected with 10 mg / kg of either anti-CD20×CD3 IGM-F (SA 18 mol / mol) (2 animals), anti-CD20×CD3 IGM-F (SA 9 mol / mol) (2 animals), or anti-CD20×CD3 IGM-F-GEM (SA 51 mol / mol) (4 animals). Blood samples were collected at a total of 12 time points for each antibody. Serum concentrations of each antibody in the blood at each time point were measured using a standard ELISA assay. Quality metrics were used for all ELISAs, and T1 / 2-α , T 1 / 2-β , and the area under the concentration curve from time zero to infinity (AUC 0-∞ , measured in μg / ml*hr units), were derived using standard curve fitting techniques (Win Non Lin, Phoenix Software). Cell markers were measured using flow cytometry. Animals treated with anti-CD20×CD3 IGM-F (SA 18 mol / mol) and four anti-CD20×CD3 IGM-F-GEM (SA 51 mol / mol) are shown in Figure 16. The AUC of the high sialic acid antibody 0-∞ was twice that of the low sialic acid antibody. The relative number of B cells at each time point is shown in Figure 17A, and the day when B cells began to recover is shown in Figure 17B.

[0307] Example 11: In Vitro Sialylation by Multiple Enzymes The combination of galactosylation and sialylation was compared to sialylation alone.

[0308] Sialylation was only completed by mixing 120 μg of anti-CD20×CD3 IGM-A antibody with 1 mol / mol SA or 21 mol / mol SA, 60 μg of CMP NANA, and 20 μg of ST6 (mass ratio of IgM:CMP NANA:ST6 of 6:3:1). The sample was then incubated at 37°C for 24 hours.

[0309] The combination of galactosylation and sialylation was completed by mixing 1 μg of β-1,4-galactosyltransferase, 60 μg of UDP-galactose, and 60 μg of anti-CD20×CD3 IGM-A antibody with 1 mol / mol SA or 21 mol / mol SA (mass ratio of IgM:UDP-galactose:β-1,4-galactosyltransferase of 60:60:1). The mixture was incubated at 37°C for 7 hours. Then, 30 μg of CMP NANA and 1 μg of ST6 were added to the mixture and incubated at 37°C for 20 hours.

[0310] The resulting sialic acid levels are shown in Table 11.

[0311] (Table 11) Sialic acid levels after sialylation with or without galactosylation TIFF0007857221000012.tif42142

[0312] The scope and width of this disclosure should not be limited by any of the exemplary embodiments or examples described above, but should be defined solely in accordance with the following claims and their equivalents.

[0313] (Table 12) Sequences in this disclosure TIFF0007857221000013.tif167158TIFF0007857221000014.tif220158TIFF0007857221000015.tif221158TIFF0007857221000016.tif215158

Claims

1. A monoclonal population of multimeric IgM-binding molecules, each binding molecule comprising 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, wherein the N-linked glycosylation motifs are of the amino acid sequence N-X １ - Includes S / T, where N is asparagine and X １ The monoclonal population of the polymer IgM binding molecule is any amino acid other than proline, and S / T is serine or threonine, and at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region are occupied by a complex glycan, and the monoclonal population of the binding molecule contains at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 moles of sialic acid per mole of the binding molecule.

2. A monoclonal population of a polymer IgM-binding molecule according to Claim 1, comprising 40 to 70, 40 to 60, 40 to 55, 40 to 50, 50 to 70, or 60 to 70 moles of sialic acid per mole of the binding molecule.

3. Each IgM heavy chain constant region starts at the amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04), forming five N-linked glycosylated motifs N-X. １ A monoclonal population of a multimer IgM-binding molecule according to claim 1 or 2, which is a human IgM heavy chain constant region or a variant thereof, containing -S / T.

4. Each binding molecule is a pentameric or hexamer IgM antibody containing five or six divalent IgM binding units, and each binding unit contains two IgM-derived heavy chains, each containing a heavy chain variable domain (VH) located at the amino terminus of the variant IgM heavy chain constant region, and two immunoglobulin light chains, each containing a light chain variable domain (VL) located at the amino terminus of the immunoglobulin light chain constant region, wherein the VH and VL bind to form an antibody-derived antigen-binding domain that specifically binds to the target, and further, each of the five or six divalent IgM binding units specifically binds to the same target, wherein the monoclonal population of multimeric IgM binding molecules according to any one of claims 1 to 3.

5. The monoclonal population of a multimeric IgM-binding molecule according to claim 4, wherein each binding molecule is a pentamer and further comprises a J chain or a functional fragment thereof or a functional variant, and optionally the J chain is a mature human J chain comprising the amino acid sequence shown in SEQ ID NO: 6, or a functional fragment thereof, or a functional variant thereof.

6. The monoclonal population of a polymeric IgM-binding molecule according to claim 5, wherein the variant J chain or its functional fragment includes an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J chain of SEQ ID NO: 6, and optionally the amino acid corresponding to Y102 of SEQ ID NO: 6 is substituted with alanine (A).

7. The monoclonal population of the polymer IgM-binding molecule according to claim 6, wherein the J chain comprises the amino acid sequence shown in SEQ ID NO:

7.

8. The monoclonal population of a polymer IgM-binding molecule according to any one of claims 5 to 7, wherein the J chain or its functional fragment or functional variant is a modified J chain further comprising a heterogeneous portion, the heterogeneous portion being a polypeptide fused to the J chain or its functional fragment or functional variant, and optionally via a peptide linker comprising at least 5 but no more than 25 amino acids.

9. The monoclonal population of a polymer IgM-binding molecule according to claim 8, wherein the heterogeneous polypeptide comprises an scFv fragment, and optionally the scFv fragment is bound to CD3ε.

10. A pharmaceutical composition comprising a monoclonal group of a polymer IgM-binding molecule as described in any one of claims 1 to 9, and a pharmaceutically acceptable excipient.

11. Recombinant host cells that generate a monoclonal population of a multimeric IgM-binding molecule according to any one of claims 1 to 9.

12. The recombinant host cell according to claim 11, wherein the cell is transfected with one or more genes encoding glycosyltransferase, and optionally, the one or more genes encoding galactosyltransferase that provides acceptor residues for sialic acid residues via α-2,6 and / or α-2,3 linkages.

13. Recombinant host cells according to claim 11 or 12, wherein the cells are transfected with one or more genes encoding sialyltransferase, thereby improving and / or enhancing the cell's ability to promote the transfer of sialic acid monosaccharides from CMP-NANA substrates or derivatives thereof to compatible acceptor oligosaccharides.

14. The recombinant host cell according to claim 13, wherein the sialyltransferase comprises any of the following: human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1, SEQ ID NO: 3), human β-galactoside α-2,6-sialyltransferase-II (ST6GALII), or four β-galactoside α2-3-sialyltransferases (ST3GAL-I-IV).

15. The recombinant host cell according to any one of claims 12 to 14, wherein the galactosyltransferase comprises human β-1,4-galactosyltransferase 4 (B4GALT4).

16. A method for generating a monoclonal population of a highly sialylated polymer IgM-binding molecule according to any one of claims 1 to 9, comprising: providing a cell line expressing the monoclonal population of IgM-binding molecules; culturing the cell line; and recovering the monoclonal population of IgM-binding molecules, wherein each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least 3, at least 4, or at least 5 asparagine (N)-linked glycosylation motifs, and the N-linked glycosylation motifs having an amino acid sequence N-X １ - Includes S / T, where N is asparagine and X １ The method wherein is any amino acid other than proline, and S / T is serine or threonine, and on average at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region in the population are occupied by complex glycans, and the cell line, harvesting process, or a combination thereof is optimized to concentrate complex glycans containing at least one, two, three, or four sialic acid-terminal monosaccharides per glycan, thereby yielding a monoclonal population of highly sialylated IgM-binding molecules containing at least 35, at least 40, at least 45, at least 50, at least 60, or at least 65 moles of sialic acid per mole of binding molecule, or 40–70, 40–60, 40–55, 40–50, 50–70, or 60–70 moles of sialic acid per mole of binding molecule.

17. The method according to claim 16, wherein the cell line is modified to overexpress sialyltransferase.

18. The method according to claim 16 or 17, wherein the recovery process comprises subjecting the monoclonal population of IgM-binding molecules to in vitro glycosylation, and the in vitro glycosylation comprises contacting the monoclonal population of IgM-binding molecules with a soluble sialyltransferase and a sialic acid substrate.

19. The method according to claim 17 or 18, wherein the sialyltransferase comprises a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3), and / or the sialic acid substrate comprises cytidine monophosphate (CMP)-N-acetylneuraminic acid (CMP-NANA).

20. The method according to claim 18, wherein the mass ratio of the binding molecule to the sialic acid substrate is 1:4 to 40:1, and / or the mass ratio of the binding molecule to the sialyltransferase is 80:1 to 5000:

1.

21. The method according to any one of claims 18 to 20, wherein the monoclonal population of the IgM-binding molecule is contacted with the soluble sialyltransferase and the sialic acid substrate for at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours at a temperature in the range of 2°C to 40°C.

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