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  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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  • C12N9/10—Transferases (2.)
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  • C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
  • C12Y204/99001—Beta-galactoside alpha-2,6-sialyltransferase (2.4.99.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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  • C07K2317/622—Single chain antibody (scFv)
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  • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

• Antibodies and antibody-like molecules that can multimerize, such as IgA and IgM antibodies, have emerged as promising drug candidates, e.g., in the fields of immuno- oncology and infectious diseases, allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951,134, 9,938,347, 10,351,631, 10,400,038, 10,570,191, 10,604,559, 10,618,978, 10,689,449, and 10,787,520, U.S. Patent Application Publication Nos.
• PK pharmacokinetics
• PD pharmacodynamics
• the IgG antibody class has a serum half-life of 20 days, whereas the half-lives for IgM and IgA antibodies are only about 5–8 days (Brekke, OH., and I. Sandlie, Nature Reviews Drug Discovery 2: 52-62 (2003)).
• One of the key determinants of PK of an antibody or other biotherapeutic is its level and type of glycosylation (Higel, F. et al. Eur. J. Pharm. Biopharm.139:123-131 (2019)).
• Sugar moieties and their derivatives covalently linked to specific residues on an antibody can determine how they are recognized by receptors such as asialo-glycoprotein (ASGP) receptor, which in turn determines how quickly they are cleared from systemic circulation.
• ASGP asialo-glycoprotein
• Each IgM heavy chain constant region has five sites of asparagine- (N-)linked glycosylation, and the J-chain has one N-linked glycosylation site.
• a pentameric, J- chain containing IgM contains up to 51 glycan moieties, which results in a complex glycosylation profile (Hennicke, J., et al., Anal. Biochem. 539:162-166 (2017)).
• each binding molecule including ten or twelve IgM-derived heavy chains, where the IgM- derived heavy chains include glycosylated IgM heavy chain constant regions each associated with a binding domain that specifically binds to a target, where each IgM heavy chain constant region includes at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, where an N-linked glycosylation motif includes the amino acid sequence N-X 1 -S/T, where N is asparagine, X 1 is any amino acid except proline, and S/T is serine or threonine, where 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 where the monoclonal population of binding molecules includes at least thirty-five (35) moles sia
• the monoclonal population of binding molecules includes 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 sialic acid per mole of binding molecule. In some embodiments, the monoclonal population of binding molecules includes at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 moles sialic acid per mole of binding molecule.
• the monoclonal population of binding molecules includes 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, about 60 to about 70 moles sialic acid per mole of binding molecule.
• the IgM heavy chain constant regions are human IgM heavy chain constant regions or variants thereof including five N-linked glycosylation motifs N- X 1 -S/T starting 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).
• motifs N1, N2, and N3 are occupied by complex glycans.
• the monoclonal population of binding molecules is produced by the method of cell line modification, in vitro glycoengineering, or any combination thereof.
• the cell line modification includes transfecting a cell line that produces the monoclonal population of binding molecules with a gene encoding a sialyltransferase, thereby producing a modified cell line that overexpresses the sialyltransferase.
• the sialyltransferase includes human beta- galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).
• the cell line modification further includes transfecting a cell line that produces the monoclonal population of binding molecules with a gene encoding a galactosyltransferase, thereby producing a modified cell line that overexpresses the galactosyltransferase.
• the galactosyltransferase includes human beta-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).
• the in vitro glycoengineering includes contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate.
• the sialyltransferase includes a soluble variant of human beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).
• ST6GAL1 human beta-galactoside alpha-2,6-sialyltransferase 1
• the soluble variant of ST6GAL1 includes amino acids x to 406 of SEQ ID NO: 3, where x is an integer from 27 to 120.
• the soluble variant of ST6GAL1 includes amino acids 120 to 406, 115 to 406, 110 to 406, 109 to 406, 105 to 406, 100 to 406, 95 to 406, 90 to 406, 89 to 406, 88 to 406, 87 to 406, 86 to 406, 85 to 406, 84 to 406, 83 to 406, 82 to 406, 81 to 406, 80 to 406, 75 to 406, 70 to 406, 65 to 406, 60 to 406, 55 to 406, 50 to 406, 45 to 406, 40 to 406, 35 to 406, 30 to 406, or 27 to 406 of SEQ ID NO: 3.
• the sialic acid substrate includes cytidine monophosphate-N-acetyl-neuraminic acid (CMP-NANA).
• CMP-NANA cytidine monophosphate-N-acetyl-neuraminic acid
• the mass ratio of binding molecule: sialic acid substrate is about 1:4 to about 40:1. In some embodiments, the mass ratio of binding molecule: sialyltransferase is about 80:1 to about 5000:1. In some embodiments, the mass ratio of binding molecule: sialyltransferase is about 500:1. In some embodiments, the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 500:62.5:1.
• the mass ratio of binding molecule: sialyltransferase is about 2000:1. In some embodiments, the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 2000:500:1. In some embodiments, the molar ratio of binding molecule: sialyltransferase is about 80:1. In some embodiments, the molar ratio of binding molecule: sialic acid substrate: sialyltransferase is about 80:500:1. [0014] In some embodiments, the contacting of the monoclonal population of binding molecules with the soluble sialyltransferase and the sialic acid substrate comprises at least 30 minutes of contact.
• the contacting comprises at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours of contact.
• the contacting of the monoclonal population of binding molecules with the soluble sialyltransferase and the sialic acid substrate occurs at about 2° C to about 40°C.
• the contacting occurs at 15°C to about 37°C, 15°C to about 30°C, or 15°C to about 25°C.
• the in vitro glycoengineering further includes contacting the monoclonal population of binding molecules with a galactosyltransferase and a galactose substrate.
• the galactosyltransferase includes a soluble variant of human beta-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).
• B4GALT4 human beta-1,4-galactosyltransferase 4
• the soluble variant of B4GALT4 includes amino acids x to 344 of SEQ ID NO: 4, where x is an integer from 39 to 120.
• the soluble variant of B4GALT4 includes amino acids 120 to 344, 115 to 344, 110 to 344, 105 to 344, 100 to 344, 95 to 344, 90 to 344, 85 to 344, 80 to 344, 75 to 344, 70 to 344, 65 to 344, 60 to 344, 55 to 344, 50 to 344, 45 to 344, 40 to 344, or 39 to 344 of SEQ ID NO: 4.
• the galactose substrate includes uridine-diphosphate- ⁇ -D-galactose (UDP- Gal).
• each binding molecule is multispecific, and two or more binding domains associated with the IgM heavy chain constant regions of each binding molecule specifically bind to different targets. In some embodiments, the binding domains associated with the IgM heavy chain constant regions of each binding molecule specifically bind to the same target. In some embodiments, the binding domains associated with the IgM heavy chain constant regions of each binding molecule are identical. [0017] In some embodiments, the binding domains are antibody-derived antigen-binding domains.
• each binding molecule is a pentameric or a hexameric IgM antibody including five or six bivalent IgM binding units, respectively, where each binding unit includes two IgM heavy chains each including a VH situated amino terminal to the variant IgM constant region, and two immunoglobulin light chains each including a light chain variable domain (VL) situated amino terminal to an immunoglobulin light chain constant region, and where the VH and VL combine to form an antigen-binding domain that specifically binds to the target.
• each antigen-binding domain of each binding molecule binds to the same target.
• each antigen- binding domain of each binding molecule is identical.
• the target is a target epitope, a target antigen, a target cell, a target organ, or a target virus.
• each binding molecule is pentameric and further includes a J-chain, or functional fragment thereof, or a functional variant thereof.
• the J-chain is a mature human J-chain including the amino acid sequence SEQ ID NO: 6 or a functional fragment thereof, or a functional variant thereof.
• the J-chain includes an N-linked glycosylation motif N-X1-S/T starting at amino acid positions corresponding to amino acid 49 of SEQ ID NO: 6 (motif N6).
• the J-chain is a functional variant J-chain including one or more single amino acid substitutions, deletions, or insertions relative to a reference J-chain identical to the variant J-chain except for the one or more single amino acid substitutions, deletions, or insertions, and where the monoclonal population of binding molecules exhibits an increased serum half-life upon administration to a subject animal relative to a reference IgM-derived binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the variant J-chain, and is administered using the same method to the same animal species.
• the variant J-chain or functional fragment thereof includes one, two, three, or four single amino acid substitutions, deletions, or insertions relative to the reference J-chain.
• the variant J-chain or functional fragment thereof 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. [0021] In some embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 6 is substituted with alanine (A). In some embodiments, the J-chain includes the amino acid sequence SEQ ID NO: 7.
• the J-chain or fragment or variant thereof is a modified J- chain further including a heterologous moiety, where the heterologous moiety is fused or conjugated to the J-chain or fragment or variant thereof.
• the heterologous moiety is a polypeptide fused to the J-chain or fragment or variant thereof.
• the heterologous polypeptide is fused to the J-chain or fragment or variant thereof via a peptide linker.
• the peptide linker includes at least 5 amino acids, but no more than 25 amino acids.
• the peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 43).
• the heterologous polypeptide is fused to the N-terminus of the J-chain or fragment or variant thereof or to the C-terminus of the J-chain or fragment or variant thereof.
• heterologous moieties that can be the same or different are fused to the N-terminus and C-terminus of the J-chain or fragment or variant thereof.
• the heterologous polypeptide includes a binding domain.
• the binding domain of the heterologous polypeptide is an antibody or antigen-binding fragment thereof.
• the antigen-binding fragment is a scFv fragment.
• the heterologous scFv fragment binds to CD3 ⁇ .
• the modified J-chain includes the amino acid sequence 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*), amino acids 20-420 of SEQ ID NO: 34 (VJH), amino acids 20-420 of SEQ ID NO: 35 (VJ*H), or SEQ ID NOs: 6 or 7 fused via a peptide linker to an anti-CD3 ⁇ scFv including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences 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, respectively.
• a pharmaceutical composition comprising the monoclonal population of binding molecules disclosed herein and a pharmaceutically acceptable excipient.
• a recombinant host cell that produces the monoclonal population of binding molecules disclosed herein.
• a method of producing the monoclonal population of binding molecules disclosed herein including culturing the host cell disclosed herein, and recovering the population of binding molecules.
• Also provided herein is a method for producing a monoclonal population of highly sialylated multimeric binding molecules, including providing a cell line that expresses the monoclonal population of binding molecules, culturing the cell line, and recovering the monoclonal population of binding molecules, where each binding molecule includes ten or twelve IgM-derived heavy chains, where the IgM-derived heavy chains include glycosylated IgM heavy chain constant regions each associated with a binding domain that specifically binds to a target, where each IgM heavy chain constant region includes at least three, at least four, or at least five asparagine(N)-linked glycosylation motifs, where an N- linked glycosylation motif includes the amino acid sequence N-X 1 -S/T, where N is asparagine, X 1 is any amino acid except proline, and S/T is serine or threonine, where on average at least one, at least two, or at least three of the N-linked glycosylation motifs on each I
• Also provided herein is a method for producing a monoclonal population of highly sialylated multimeric binding molecules, including providing a cell line that expresses the monoclonal population of binding molecules, culturing the cell line, and recovering the monoclonal population of binding molecules, where each binding molecule includes ten or twelve IgM-derived heavy chains, where the IgM-derived heavy chains include glycosylated IgM heavy chain constant regions each associated with a binding domain that specifically binds to a target, where each IgM heavy chain constant region includes at least three, at least four, or at least five asparagine(N)-linked glycosylation motifs, where an N- linked glycosylation motif includes the amino acid sequence N-X 1 -S/T, where N is asparagine, X 1 is any amino acid except proline, and S/T is serine or threonine, where on average at least one, at least two, or at least three of the N-linked glycosylation motifs on each I
• the cell line, culture conditions, recovery process, or a combination thereof is optimized to result in a monoclonal population of binding molecules including 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 sialic acid per mole of binding molecule.
• the cell line, recovery process, or a combination thereof is optimized to result in a monoclonal population of binding molecules including 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 sialic acid per mole of binding molecule. In some embodiments, the cell line, recovery process, or a combination thereof is optimized to result in a monoclonal population of binding molecules including at least 30, at least 35, at least 40, at least 45, at least 50, or at least 60 moles sialic acid per mole of binding molecule.
• the cell line, recovery process, or a combination thereof is optimized to result in a monoclonal population of binding molecules comprising 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, about 60 to about 70 moles sialic acid per mole of binding molecule.
• the IgM heavy chain constant regions are derived from human IgM heavy chain constant regions including five N-linked glycosylation motifs N- X1-S/T starting 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).
• one, two, or all three of motifs N1, N2, and N3 in the population of binding molecules are occupied by complex glycans on average.
• the provided cell line is modified to overexpress a sialyltransferase.
• the sialyltransferase includes human beta- galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1, SEQ ID NO: 3).
• the recovery process includes subjecting the monoclonal population of binding molecules to in vitro glycoengineering.
• the in vitro glycoengineering includes contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate.
• the sialyltransferase includes a soluble variant of human beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).
• ST6GAL1 human beta-galactoside alpha-2,6-sialyltransferase 1
• the soluble variant of ST6GAL1 includes amino acids x to 406 of SEQ ID NO: 3, where x is an integer from 27 to 120.
• the soluble variant of ST6GAL1 includes amino acids 120 to 406, 115 to 406, 110 to 406, 109 to 406, 105 to 406, 100 to 406, 95 to 406, 90 to 406, 89 to 406, 88 to 406, 87 to 406, 86 to 406, 85 to 406, 84 to 406, 83 to 406, 82 to 406, 81 to 406, 80 to 406, 75 to 406, 70 to 406, 65 to 406, 60 to 406, 55 to 406, 50 to 406, 45 to 406, 40 to 406, 35 to 406, 30 to 406, or 27 to 406 of SEQ ID NO: 3.
• the sialic acid substrate includes cytidine monophosphate (CMP)-N-acetyl- neuraminic acid (CMP-NANA).
• CMP cytidine monophosphate
• CMP-NANA cytidine monophosphate
• the mass ratio of binding molecule: sialic acid substrate is about 1:4 to about 40:1.
• the mass ratio of binding molecule: sialyltransferase is about 80:1 to about 10000:1.
• the mass ratio of binding molecule: sialyltransferase is about 500:1.
• the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 500:62.5:1.
• the mass ratio of binding molecule: sialyltransferase is about 2000:1. In some embodiments, the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 2000:500:1. In some embodiments, the molar ratio of binding molecule: sialyltransferase is about 80:1. In some embodiments, the molar ratio of binding molecule: sialic acid substrate: sialyltransferase is about 80:500:1. [0035] In some embodiments, the contacting of the monoclonal population of binding molecules with the soluble sialyltransferase and the sialic acid substrate comprises at least 30 minutes of contact.
• the contacting comprises at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours of contact.
• the contacting of the monoclonal population of binding molecules with the soluble sialyltransferase and the sialic acid substrate occurs at about 2°C to about 40°C.
• the contacting occurs at 15°C to about 37°C, 15°C to about 30°C, or 15°C to about 25°C.
• in vitro glycoengineering further includes contacting the monoclonal population of binding molecules with a galactosyltransferase and a galactose substrate.
• the galactosyltransferase includes a soluble variant of human beta-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).
• the galactose substrate includes uridine-diphosphate- ⁇ -D-galactose (UDP- Gal).
• the contacting with the galactosyltransferase and a galactose substrate occurs prior to or simultaneously with the contacting with the soluble sialyltransferase and a sialic acid substrate.
• FIG. 1A shows the structure of a “simple” glycan.
• FIG. 1B shows an exemplary structure of an oligomannose glycan.
• FIG.1C shows an exemplary structure of a complex glycan.
• FIG. 1D shows an exemplary structure of a hybrid glycan.
• FIG.3A is a space-filling model of a human IgM heavy chain, showing the positions of the five N-linked glycosylation sites.
• FIG. 3B shows an alignment of the human IgM heavy chain constant region amino acid sequence (allele IGHM*04, SEQ ID NO: 2) with those of mouse (GenBank: CAC20701.1, SEQ ID NO: 46) and cynomolgus monkey (amino acids 14 to 487 of GenBank: EHH62210.1, SEQ ID NO: 47). The amino acids corresponding to asparagine (N)-linked glycosylation motifs are boxed.
• FIG.4 shows the amount of sialylation of anti-CD20 x CD3 IGM-A resulting from treatment with varying concentrations of truncated human ⁇ -2,6-sialyltransferase (ST6).
• FIG. 5 shows in vitro sialylation of two different IgM antibodies, anti-DR5 IgM-B and anti-DR5 IgM-C.
• FIG. 6 shows the pharmacokinetics of anti-CD20 x CD3 IGM-A and anti-CD20 x CD3 IGM-A-GEM antibodies in a mouse model.
• FIG. 7 shows SNA-I lectin labeling of subclones. Cells were labelled with SNA-1 lectin conjugated to fluorescein isothiocyanate (FITC). The geomean of the signal from 488em/530ex measured by a cytometer is shown for each subclone.
• FITC fluorescein isothiocyanate
• FIG.8A shows a reduced, denatured BioRad® CriterionTGX Stain-Free Precast gel visualized and imaged according to manufacturer’s instructions, for purified proteins from fermentations carried out on the 2,6-sialyl transferase pool as well as 2 subclones (25 and 47).
• FIG. 8B shows western blot on the same proteins in FIG. 8A using a biotinylated SNA-I lectin. A streptavidin horseradish peroxidase fusion was used for the blot.
• FIGS. 9A-9B shows fermentation comparison data from a 3-L bioreactor for anti- CD20 x CD3 IGM-A producing cell lines.
• FIG.9A shows the viable cell density (VCD) over the course of the run.
• FIG. 9B shows the viability of the cell-lines.
• FIG. 9C shows the titer as determined by size exclusion chromatography (SEC).
• FIG.9D shows the ratio of the sialic acid measured on the purified IgM.
• FIG. 10A shows a plot of the screening at the 96-well level for CHO cell clones transfected with 2,6-sialyltransferase.
• FIG. 10A shows a plot of the screening at the 96-well level for CHO cell clones transfected with 2,6-sialyltransferase.
• FIG. 10B shows a plot of the cytometry based analysis of cell surface 2,6-sialic acid levels.
• FIGS. 11A and 11B show the 2,3-sialic acid and 2,6-sialic acid levels for untransfected cells, respectively.
• FIG.11C compares the 2,3-sialic acid and 2,6-sialic acid levels in untransfected and transfected cells.
• FIG.12 shows T cell activation with various amounts of antibodies with a range of sialic acid levels.
• FIGS. 13A-13B show a time course of sialylation of anti-CD20 x CD3 – IGM-A with various amounts of ST6 and CMP-NANA and at various temperatures.
• FIG. 13A-13B show a time course of sialylation of anti-CD20 x CD3 – IGM-A with various amounts of ST6 and CMP-NANA and at various temperatures.
• FIG. 13A-13B show a time course of sialylation of anti-
• FIG. 14 shows a time course of sialylation of anti-CD20 x CD3 – IGM-A with various amounts of ST6 and CMP-NANA and room temperature.
• FIG. 15 shows a comparison of sialic acid levels and the resulting AUC 0- ⁇ for various antibodies.
• FIG.16 shows the pharmacokinetics of anti-CD20 x CD3 IGM-F (SA 18) and anti- CD20 x CD3 IGM-F-GEM (SA 51) antibodies in a cynomolgus monkey model.
• FIG.17A shows the relative numbers of cynomolgus B cells at each time point after administration of anti-CD20 x CD3 – IGM-F (SA 9 or 18) or anti-CD20 x CD3 – IGM-F- GEM (SA 51).
• FIG. 17B shows the day that cynomolgus B cells began to recover after administration of anti-CD20 x CD3 – IGM-F (SA 9 or 18) or anti-CD20 x CD3 – IGM-F- GEM (SA 51).
• a or “an” entity refers to one or more of that entity; for example, "a binding molecule,” is understood to represent one or more binding molecules.
• the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
• “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
• the term and/or" as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
• polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
• polypeptide refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product.
• polypeptides dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of any of these terms.
• polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
• a polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
• a polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure.
• glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid, e.g., a serine or an asparagine.
• Asparagine (N)-linked glycans are described in more detail elsewhere in this disclosure.
• an isolated polypeptide can be removed from its native or natural environment.
• Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
• a non-naturally occurring polypeptide or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the polypeptide that are, or might be, determined or interpreted by a judge or an administrative or judicial body, to be “naturally-occurring.”
• Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof.
• fragment fragment
• fragment variant
• variant as disclosed herein include any polypeptides which retain at least some of the properties of the corresponding native antibody or polypeptide, for example, specifically binding to an antigen.
• Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein.
• Variants of, e.g., a polypeptide include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions.
• variants can be non-naturally occurring.
• Non-naturally occurring variants can be produced using art-known mutagenesis techniques.
• Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions, or additions.
• Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the original polypeptide. Examples include fusion proteins.
• a "derivative" of a polypeptide can also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those polypeptides that contain one or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5- hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine. [0063] A "conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain.
• Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
• basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glut
• substitution of a phenylalanine for a tyrosine is a conservative substitution.
• conservative substitutions in the sequences of the polypeptides, binding molecules, and antibodies of the present disclosure do not abrogate the binding of the polypeptide, binding molecule, or antibody containing the amino acid sequence, to the antigen to which the antibody binds.
• Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng.
• polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA).
• mRNA messenger RNA
• cDNA plasmid DNA
• pDNA plasmid DNA
• a polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
• PNA peptide nucleic acids
• nucleic acid or “nucleic acid sequence” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
• isolated nucleic acid or polynucleotide is intended any form of the nucleic acid or polynucleotide that is separated from its native environment.
• gel- purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be “isolated.”
• a polynucleotide segment e.g., a PCR product, which has been engineered to have restriction sites for cloning is considered to be “isolated.”
• Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a non-native solution such as a buffer or saline.
• Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not one that would be found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically.
• polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
• a non-naturally occurring polynucleotide or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the nucleic acid or polynucleotide that are, or might be, determined or interpreted by a judge, or an administrative or judicial body, to be “naturally-occurring.”
• a "coding region” is a portion of nucleic acid which consists of codons translated into amino acids.
• a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.
• Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
• any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region.
• a vector, polynucleotide, or nucleic acid can include heterologous coding regions, either fused or unfused to another coding region. Heterologous coding regions include without limitation, those encoding specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
• the polynucleotide or nucleic acid is DNA.
• a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
• An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
• Two DNA fragments are "operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
• a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
• the promoter can be a cell-specific promoter that directs substantial transcription of the DNA in predetermined cells.
• transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
• a variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
• transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit ß-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
• tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
• lymphokine-inducible promoters e.g., promoters inducible by interferons or interleukins.
• translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
• a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.
• mRNA messenger RNA
• ribosomal RNA RNA
• Polynucleotide and nucleic acid coding regions can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
• polypeptides secreted by vertebrate cells can 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 secreted or "mature” form of the polypeptide.
• the native signal peptide e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
• a heterologous mammalian signal peptide, or a functional derivative thereof can be used.
• the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ß-glucuronidase.
• TPA tissue plasminogen activator
• the term “binding molecule” refers in its broadest sense to a molecule that specifically binds to a receptor or target, e.g., an epitope or an antigenic determinant.
• a binding molecule can comprise one of more “binding domains,” e.g., “antigen-binding domains” described herein.
• a non-limiting example of a binding molecule is an antibody or antibody-like molecule as described in detail herein that retains antigen-specific binding.
• a “binding molecule” comprises an antibody or antibody-like or antibody-derived molecule as described in detail herein.
• binding domain or “antigen-binding domain” (can be used interchangeably) refer to a region of a binding molecule, e.g., an antibody or antibody-like, or antibody-derived molecule, that is necessary and sufficient to specifically bind to a target, e.g., an epitope, a polypeptide, a cell, or an organ.
• an “Fv,” e.g., a heavy chain variable region and a light chain variable region of an antibody, either as two separate polypeptide subunits or as a single chain, is considered to be a “binding domain.”
• Other antigen-binding domains include, without limitation, a single domain heavy chain variable region (VHH) of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold.
• a “binding molecule,” or “antibody” as described herein can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more “antigen-binding domains.”
• the terms “antibody” and “immunoglobulin” can be used interchangeably herein.
• An antibody (or a fragment, variant, or derivative thereof as disclosed herein, e.g., an IgM- like antibody) includes at least the variable domain of a heavy chain (e.g., from a camelid species) or at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood.
• antibody encompasses anything ranging from a small antigen-binding fragment of an antibody to a full sized antibody, e.g., an IgG antibody that includes two complete heavy chains and two complete light chains, an IgA antibody that includes four complete heavy chains and four complete light chains and includes a J-chain and/or a secretory component, or an IgM-derived binding molecule, e.g., an IgM antibody or IgM-like antibody, that includes ten or twelve complete heavy chains and ten or twelve complete light chains and optionally includes a J-chain or functional fragment or variant thereof.
• immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ 1- ⁇ 4 or ⁇ 1- ⁇ 2)). It is the nature of this chain that determines the "isotype" of the antibody as IgG, IgM, IgA IgD, or IgE, respectively.
• immunoglobulin subclasses e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , IgA 2 , etc. are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.
• Light chains are classified as either kappa or lambda ( ⁇ , ⁇ ). Each heavy chain class can be bound with either a kappa or lambda light chain.
• the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are expressed, e.g., by hybridomas, B cells or genetically engineered host cells.
• the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
• binding unit is used herein to refer to the portion of a binding molecule, e.g., an antibody, antibody-like molecule, or antibody-derived molecule, antigen-binding fragment thereof, or multimerizing fragment thereof, which corresponds to a standard “H2L2” immunoglobulin structure, i.e., two heavy chains or fragments thereof and two light chains or fragments thereof.
• the terms “binding molecule” and “binding unit” are equivalent.
• the binding molecule comprises two or more “binding units.” Two in the case of an IgA dimer, or five or six in the case of an IgM pentamer or hexamer, respectively.
• a binding unit need not include full-length antibody heavy and light chains, but will typically be bivalent, i.e., will include two “antigen-binding domains,” as defined above.
• certain binding molecules provided in this disclosure are “dimeric,” and include two bivalent binding units that include IgA constant regions or multimerizing fragments thereof.
• Certain binding molecules provided in this disclosure are “pentameric” or “hexameric,” and include five or six bivalent binding units that include IgM constant regions or multimerizing fragments or variants thereof.
• a binding molecule e.g., an antibody or antibody-like molecule or antibody-derived binding molecule, comprising two or more, e.g., two, five, or six binding units, is referred to herein as “multimeric.”
• J-chain refers to the J-chain of IgM or IgA antibodies of any animal species, any functional fragment thereof, derivative thereof, and/or variant thereof, including a mature human J-chain, the amino acid sequence of which is presented as SEQ ID NO: 6.
• Various J-chain variants and modified J-chain derivatives are disclosed herein.
• modified J-chain is used herein to refer to a derivative of a J-chain polypeptide comprising a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding domain or functional domain introduced into or attached to the J-chain sequence.
• the introduction can be achieved by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety or by attachment through a peptide or chemical linker.
• modified human J-chain encompasses, without limitation, a native sequence human J-chain of the amino acid sequence of SEQ ID NO: 6 or functional fragment thereof, or functional variant thereof, modified by the introduction of a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding domain.
• a heterologous moiety e.g., a heterologous polypeptide, e.g., an extraneous binding domain.
• the heterologous moiety does not interfere with efficient polymerization of IgM into a pentamer or IgA into a dimer, and binding of such polymers to a target.
• Exemplary modified J-chains can be found, e.g., in U.S. Patent Nos.9,951,134, 10,400,038, and 10,618,978, and in U.S.
• IgM-derived binding molecule refers collectively to native IgM antibodies, IgM-like antibodies, as well as other IgM-derived binding molecules comprising non-antibody binding and/or functional domains instead of an antibody antigen binding domain or subunit thereof, and any fragments, e.g., multimerizing fragments, variants, or derivatives thereof.
• IgM-like antibody refers generally to a variant antibody or antibody-derived binding molecule that still retains the ability to form hexamers or pentamers, e.g., in association with a J-chain.
• An IgM-like antibody or other IgM-derived binding molecule typically includes at least the C ⁇ 4-tp domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species.
• an IgM-like antibody or other IgM-derived binding molecule can likewise be an antibody fragment in which one or more constant regions are deleted, as long as the IgM-like antibody is capable of forming hexamers and/or pentamers.
• an IgM-like antibody or other IgM-derived binding molecule can be, e.g., a hybrid IgM/IgG antibody or can be a “multimerizing fragment” of an IgM antibody.
• valency refers to the number of binding domains, e.g., antigen-binding domains in given binding molecule, e.g., antibody, antibody-derived, or antibody-like molecule, or in a given binding unit.
• binding domains e.g., antigen-binding domains in given binding molecule, e.g., antibody, antibody-derived, or antibody-like molecule, or in a given binding unit.
• bivalent “tetravalent”, and “hexavalent” in reference to a given binding molecule, e.g., an IgM antibody, IgM-like antibody, other IgM-derived binding molecule, or multimerizing fragment thereof, denote the presence of two antigen- binding domains, four antigen-binding domains, and six antigen-binding domains, respectively.
• a typical IgM antibody, IgM-like antibody, or other IgM-derived binding molecule, where each binding unit is bivalent, can have 10 or 12 valencies.
• a bivalent or multivalent binding molecule, e.g., antibody or antibody-derived molecule can be monospecific, i.e., all of the antigen-binding domains are the same, or can be bispecific or multispecific, e.g., where two or more antigen-binding domains are different, e.g., bind to different epitopes on the same antigen, or bind to entirely different antigens.
• epitope includes any molecular determinant capable of specific binding to an antigen-binding domain of an antibody, antibody-like, or antibody-derived molecule.
• an epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have three-dimensional structural characteristics, and or specific charge characteristics.
• An epitope is a region of a target that is bound by an antigen-binding domain of an antibody.
• target is used in the broadest sense to include substances that can be bound by a binding molecule, e.g., antibody, antibody-like, or antibody-derived molecule.
• a target can be, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule, or a minimal epitope on such molecule.
• a “target” can, for example, be a cell, an organ, or an organism, e.g., an animal, plant, microbe, or virus, that comprises an epitope that can be bound by a binding molecule, e.g., antibody, antibody-like, or antibody-derived molecule.
• a binding molecule e.g., antibody, antibody-like, or antibody-derived molecule.
• variable domains of both the variable light (VL) and variable heavy (VH) chain portions determine antigen recognition and specificity.
• the constant region domains of the light chain (CL) and the heavy chain e.g., CH1, CH2, CH3, or CH4 confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
• the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino- terminus of the antibody.
• a “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or C ⁇ 1), an antibody heavy chain constant domain 2 (CM2 or C ⁇ 2), an antibody heavy chain constant domain 3 (CM3 or C ⁇ 3), and an antibody heavy chain constant domain 4 (CM4 or C ⁇ 4) that can include a tailpiece.
• VH antibody heavy chain variable domain
• CM1 or C ⁇ 1 an antibody heavy chain constant domain 1
• CM2 or C ⁇ 2 an antibody heavy chain constant domain 2
• CM3 or C ⁇ 3 an antibody heavy chain constant domain 4
• variable region(s) allow a binding molecule, e.g., antibody, antibody-like, or antibody-derived molecule, to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of a binding molecule, e.g., an antibody, antibody-like, or antibody-derived molecule, combine to form the antigen-binding domain. More specifically, an antigen-binding domain can be defined by three CDRs on each of the VH and VL chains. Certain antibodies form larger structures.
• IgM can form a pentameric or hexameric molecule that includes five or six H2L2 binding units and optionally a J-chain covalently connected via disulfide bonds.
• the six “complementarity determining regions” or “CDRs” present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment.
• the remainder of the amino acids in the antigen-binding domain referred to as "framework" regions, show less inter- molecular variability.
• the framework regions largely adopt a E-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the E-sheet structure.
• framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
• the antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
• CDR complementarity determining region
• Antibody variable domains can also be analyzed, e.g., using the IMGT information system (imgt_dot_cines_dot_fr/) (IMGT®/V-Quest) to identify variable region segments, including CDRs. (See, e.g., Brochet et al., Nucl. Acids Res.36:W503-508, 2008).
• IMGT®/V-Quest IMGT information system
• Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
• One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself.
• Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive numbering is used for all amino acid sequences in this disclosure. [0093] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al.
• IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region, or by using the Kabat numbering scheme.
• SEQ ID NO: 1 allele IGHM*03
• SEQ ID NO: 2 allele IGHM*04
• Binding molecules e.g., antibodies, antibody-like, or antibody-derived molecules, antigen-binding fragments, variants, or derivatives thereof, and/or multimerizing fragments thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain
• a binding molecule e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen- binding domain, and that the binding entails some complementarity between the antigen- binding domain and the epitope.
• a binding molecule e.g., antibody, antibody-like, or antibody-derived molecule, is said to "specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
• binding molecule "A” can be deemed to have a higher specificity for a given epitope than binding molecule "B,” or binding molecule "A” can be said to bind to epitope "C” with a higher specificity than it has for related epitope “D.”
• a binding molecule e.g., an antibody or fragment, variant, or derivative thereof disclosed herein can be said to bind a target antigen with an off rate (k(off)) of less than or equal to 5 X 10 -2 sec -1 , 10 -2 sec -1 , 5 X 10 -3 sec -1 , 10 -3 sec -1 , 5 X 10 -4 sec -1 , 10 -4 sec -1 , 5 X 10 -5 sec -1 , or 10 -5 sec -1 5 X 10 -6 sec -1 , 10 -6 sec -1 , 5
• a binding molecule e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a target antigen with an on rate (k(on)) of greater than or equal to 10 3 M -1 sec -1 , 5 X 10 3 M -1 sec -1 , 10 4 M -1 sec -1 , 5 X 10 4 M -1 sec -1 , 10 5 M -1 sec -1 , 5 X 10 5 M -1 sec -1 , 10 6 M -1 sec -1 , or 5 X 10 6 M -1 sec -1 or 10 7 M -1 sec -1 .
• k(on) on rate
• a binding molecule e.g., an antibody or fragment, variant, or derivative thereof is said to competitively inhibit 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, to some degree, binding of the reference antibody or antigen-binding fragment to the epitope.
• Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays.
• a binding molecule can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
• the term "affinity” refers to a measure of the strength of the binding of an individual epitope with one or more antigen-binding domains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28.
• the term “avidity” refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, e.g., Harlow at pages 29-34.
• Avidity is related to both the affinity of individual antigen-binding domains in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. An interaction between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity. [0100] Binding molecules, e.g., antibodies or fragments, variants, or derivatives thereof as disclosed herein can also be described or specified in terms of their cross-reactivity.
• cross-reactivity refers to the ability of a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances.
• a binding molecule is cross reactive if it binds to an epitope other than the one that induced its formation.
• the cross-reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.
• a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof can also be described or specified in terms of their binding affinity to an antigen.
• a binding molecule can bind to an antigen with a dissociation constant or KD no greater than 5 x 10 -2 M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10- 15 M, or 10 -15 M.
• Antigen-binding antibody fragments including single-chain antibodies or other antigen-binding domains can exist alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domains, J-chain, or secretory component. Also included are antigen-binding fragments that can include any combination of variable region(s) with one or more of a hinge region, CH1, CH2, CH3, or CH4 domains, a J-chain, or a secretory component. Binding molecules, e.g., antibodies, or antigen-binding fragments thereof can be from any animal origin including birds and mammals.
• the antibodies can be, e.g., human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
• the variable region can be condricthoid in origin (e.g., from sharks).
• "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and can in some instances express endogenous immunoglobulins and some not, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
• an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule is able to form a multimer, e.g., a hexamer or a pentamer.
• such a fragment comprises a “multimerizing fragment.”
• the term “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain, a binding molecule, e.g., an antibody, antibody-like, or antibody-derived molecule comprising a heavy chain subunit can include at least one of: 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.
• a binding molecule e.g., an antibody, antibody-like, or antibody-derived molecule, or fragment, e.g., multimerizing fragment, variant, or derivative thereof can include without limitation, in addition to a VH domain: a CH1 domain; a CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain.
• a binding molecule e.g., an antibody, antibody-like, or antibody-derived molecule, or fragment, e.g., multimerizing fragment, variant, or derivative thereof can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, and a J-chain.
• a binding molecule e.g., an antibody, antibody-like, or antibody-derived molecule, for use in the disclosure can lack certain constant region portions, e.g., all or part of a CH2 domain.
• an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein comprises sufficient portions of an IgM heavy chain constant region to allow the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule to form a multimer, e.g., a hexamer or a pentamer.
• such a fragment comprises a “multimerizing fragment.”
• the term “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain.
• the light chain subunit includes at least a VL, and can further include a CL (e.g., C ⁇ or C ⁇ ) domain.
• Binding molecules e.g., antibodies, antibody-like molecules, antibody-derived molecules, antigen-binding fragments, variants, or derivatives thereof, or multimerizing fragments thereof can be described or specified in terms of the epitope(s) or portion(s) of a target, e.g., a target antigen that they recognize or specifically bind.
• a target antigen can comprise a single epitope or at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
• disulfide bond includes the covalent bond formed between two sulfur atoms, e.g., in cysteine residues of a polypeptide.
• the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
• Disulfide bonds can be “intra-chain,” i.e., linking to cysteine residues in a single polypeptide or polypeptide subunit, or can be “inter-chain,” i.e., linking two separate polypeptide subunits, e.g., an antibody heavy chain and an antibody light chain, to antibody heavy chains, or an IgM or IgA antibody heavy chain constant region and a J- chain.
• the term “chimeric antibody” refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial, or modified) is obtained from a second species.
• the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.
• multispecific antibody or “bispecific antibody” refer to an antibody, antibody-like, or antibody-derived molecule that has antigen-binding domains for two or more different epitopes within a single antibody molecule.
• Other binding molecules in addition to the canonical antibody structure can be constructed with 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 that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Ströhlein and Heiss, Future Oncol.
• a bispecific antibody can also be a diabody.
• engineered antibody refers to an antibody in which a variable domain, constant region, and/or J-chain is altered by at least partial replacement of one or more amino acids.
• entire CDRs from an antibody of known specificity can be grafted into the framework regions of a heterologous antibody.
• CDRs can also be derived from an antibody of different class, e.g., from an antibody from a different species.
• an engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity are grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody.”
• a humanized antibody In certain embodiments not all of the CDRs are replaced with the complete CDRs from the donor variable region and yet the antigen- binding capacity of the donor can still be transferred to the recipient variable domains.
• the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or some combination of these techniques).
• the terms “linked,” “fused” or “fusion” or other grammatical equivalents can be used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means.
• an "in-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs.
• ORFs polynucleotide open reading frames
• a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence.
• polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.
• a "linear sequence" or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
• a portion of a polypeptide that is “amino-terminal” or “N-terminal” to another portion of a polypeptide is that portion that comes earlier in the sequential polypeptide chain.
• a portion of a polypeptide that is “carboxy-terminal” or “C- terminal” to another portion of a polypeptide is that portion that comes later in the sequential polypeptide chain.
• the variable domain is “N-terminal” to the constant region
• the constant region is “C-terminal” to the variable domain.
• the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript.
• a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript.
• Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
• N-linked oligosaccharide “N-linked sugar,” “N-linked glycan” or other similar or grammatical variants, denote oligosaccharide chains that are linked to a peptide backbone via an asparagine residue.
• N-linked oligosaccharides have a common pentasaccharide core of Man3GlcNAc2, also called a “simple oligosaccharide,” see FIG. 1A.
• N-linked glycans can be generally classified into three types: (1) oligomannose, in which only mannose residues are attached to the core (FIG. 1B); (2) complex, in which “antennae” initiated by N-acetylglucosaminyltransferases (GlcNAcTs) are attached to the core (FIG. 1C); and (3) hybrid, in which only mannose residues are attached to the Man ⁇ 1–6 arm of the core and one or two antennae are on the Man ⁇ 1–3 arm (FIG.
• glycosyltransferase denotes an enzyme capable of transferring a monosaccharide moiety from a nucleotide sugar to an acceptor molecule such as an oligosaccharide.
• examples of such glycosyltransferases include, but not limited to glucosyltransferases, mannosyltransferases, galactosyltransferases, and sialyltransferases.
• sialic acid denotes any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-l-onic acid (often abbreviated as Neu5Ac, NeuAc, or NANA).
• Neu5Ac NeuAc
• NANA NANA
• Other functional equivalents are known, including, but not limited to azido-CMP-sialic acid to be used for glycan labelling by “click” chemistry. See, e.g., Moh, et al., Anal. Biochem. 584:11385 (2019).
• a terminal sialic acid residue can be coupled to a galactose residue by various linkages, e.g., (i) ⁇ 2 ⁇ 3 ( ⁇ 2,3) linked to a galactose or (ii) ⁇ 2 ⁇ 6 ( ⁇ 2,6) linked to a galactose.
• a sialyltransferase enzyme is generally named and classified according to its respective monosaccharide acceptor substrate and according to the position of the glycosidic bond it catalyzes.
• Exemplary eukaryotic sialyltransferases include (i) ST3Gal (found, e.g., in CHO cells) and (ii) ST6Gal found in human cells.
• ST3 specifically encompasses the sialyltransferases catalyzing an ⁇ 2,3 sialylation.
• ST6 specifically encompasses the sialyltransferases catalyzing an ⁇ 2,6 sialylation.
• ST6Gal enzymes catalyzes transfer of a Neu5Ac residue to the C6 hydroxyl group of a free galactosyl residue being part of terminal Gal ⁇ 1,4GlcNAc in a glycan or an antenna of a glycan, thereby forming in the glycan a terminal sialic acid residue ⁇ 2 ⁇ 6 linked to the galactosyl residue of the Gal ⁇ 1,4GlcNAc moiety.
• the wild-type polypeptide of human ⁇ -galactoside- ⁇ -2,6-sialyltransferase I (hST6Gal-I, UniProtKB/Swiss-Prot: P15907.1), is presented as SEQ ID NO: 3.
• Mammalian sialyltransferases share with other mammalian Golgi-resident glycosyltransferases a type II architecture with a cytoplasmic N-terminal tail, a transmembrane region, a stem region of variable length, and a C-terminal catalytic domain in the lumen of the Golgi apparatus.
• the cytoplasmic region of hST6GAL-1 includes amino acids 1-9 of SEQ ID NO: 3, the transmembrane region includes amino acids 10-26 of SEQ ID NO: 3, and the luminal region includes amino acids 27-406 of SEQ ID NO: 3.
• a soluble variant of hST6Gal-I would lack at least the transmembrane region and could further lack the N-terminal cytoplasmic region, and some portion of the luminal region, provided that the enzyme retains catalytic activity.
• a soluble variant of ST6GAL1 can include amino acids x to 406 of SEQ ID NO: 3, where x is an integer from 27 to 120.
• a soluble variant of ST6GAL1 can include amino acids 120 to 406, 115 to 406, 110 to 406, 109 to 406, 105 to 406, 100 to 406, 95 to 406, 90 to 406, 89 to 406, 88 to 406, 87 to 406, 86 to 406, 85 to 406, 84 to 406, 83 to 406, 82 to 406, 81 to 406, 80 to 406, 75 to 406, 70 to 406, 65 to 406, 60 to 406, 55 to 406, 50 to 406, 45 to 406, 40 to 406, 35 to 406, 30 to 406, or 27 to 406 of SEQ ID NO: 3.
• the cytoplasmic region of hB4GALT4 includes amino acids 1-12 of SEQ ID NO: 4, the transmembrane region includes amino acids 13-38 of SEQ ID NO: 4, and the luminal region includes amino acids 39-344 of SEQ ID NO: 4.
• a soluble variant of hB4GALT4 would lack at least the transmembrane region and could further lack the N- terminal cytoplasmic region, and some portion of the luminal region, provided that the enzyme retains catalytic activity.
• a soluble variant of hB4GALT4 can include amino acids x to 344 of SEQ ID NO: 4, wherein x is an integer from 39 to 120.
• a soluble variant of hB4GALT4 can include amino acids 120 to 344, 115 to 344, 110 to 344, 105 to 344, 100 to 344, 95 to 344, 90 to 344, 85 to 344, 80 to 344, 75 to 344, 70 to 344, 65 to 344, 60 to 344, 55 to 344, 50 to 344, 45 to 344, 40 to 344, or 39 to 344 of SEQ ID NO: 4.
• Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt or slow the progression of an existing diagnosed pathologic condition or disorder.
• “prevent,” “prevention,” “avoid,” “deterrence” and the like refer to prophylactic or preventative measures that prevent the development of an undiagnosed targeted pathologic condition or disorder.
• “those in need of treatment” can include those already with the disorder and/or those prone to have the disorder.
• the terms “serum half-life” or “plasma half-life” refer to the time it takes (e.g., in minutes, hours, or days) following administration for the serum or plasma concentration of a drug, e.g., a binding molecule such as an antibody, antibody-like, or antibody-derived molecule or fragment, e.g., multimerizing fragment thereof as described herein, to be reduced by 50%.
• the alpha half-life, ⁇ half- life, or t 1/2 ⁇ which is the rate of decline in plasma concentrations due to the process of drug redistribution from the central compartment, e.g., the blood in the case of intravenous delivery, to a peripheral compartment (e.g., a tissue or organ), and the beta half-life, ⁇ half- life, or t 1/2 ⁇ which is the rate of decline due to the processes of excretion or metabolism.
• AUC area under the plasma drug concentration-time curve
• ⁇ area under the plasma drug concentration-time curve
• MRT mean residence time
• subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject. In certain embodiments the subject is a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
• Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.
• a subject that would benefit from therapy refers to a subset of subjects, from amongst all prospective subjects, which would benefit from administration of a given therapeutic agent, e.g., a binding molecule such as an antibody, comprising one or more antigen-binding domains.
• a binding molecule such as an antibody, comprising one or more antigen-binding domains.
• binding molecules e.g., antibodies, can be used, e.g., for a diagnostic procedure and/or for treatment or prevention of a disease.
• IgM antibodies, IgM-like antibodies, other IgM-derived binding molecules, and populations of such molecules [0129]
• IgM is the first immunoglobulin produced by B cells in response to stimulation by antigen. Naturally-occurring IgM is naturally present at around 1.5 mg/ml in serum with a half-life of about 5 days. IgM is a pentameric or hexameric molecule and thus includes five or six binding units. An IgM binding unit typically includes two light and two heavy chains.
• IgG heavy chain constant region contains three heavy chain constant domains (CH1, CH2 and CH3)
• the heavy ( ⁇ ) constant region of IgM additionally contains a fourth constant domain (CH4) and includes a C-terminal “tailpiece.”
• the human IgM constant region typically comprises the amino acid sequence SEQ ID NO: 1 (identical to, e.g., GenBank Accession Nos. pir
• the human C ⁇ 1 region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO: 1 or SEQ ID NO: 2; the human C ⁇ 2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO: 1 or SEQ ID NO: 2, the human C ⁇ 3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO: 1 or SEQ ID NO: 2, the C ⁇ 4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO: 1 or SEQ ID NO: 2, and the tailpiece ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO: 1 or SEQ ID NO: 2.
• Human IgM constant regions, and also certain non-human primate IgM constant regions, as provided herein typically include five (5) naturally-occurring asparagine (N)- linked glycosylation motifs or sites. See FIGs. 3A and 3B.
• an N-linked glycosylation motif comprises or consists of the amino acid sequence N-X1-S/T, wherein 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, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA.
• N-linked glycosylation motifs occur in the human IgM heavy chain constant regions 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 non- human primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. See, e.g., FIG.3B.
• N1, N2, and N3 motifs on the human IgM heavy chain are decorated predominantly, but not invariably with complex-type N-glycans, where the N4 and N5 motifs are decorated predominantly, but not invariably with oligomannose type N-glycans. See, e.g., Moh, E.S.X., et al., J. Am. Soc. Mass Spectrom. 27:1143-1155 (2016) and Hennicke, J., et al., Anal. Biochem.539:162-166 (2017).
• Each IgM heavy chain constant region can be associated with a binding domain, e.g., an antigen-binding domain, e.g., a scFv or VHH, or a subunit of an antigen-binding domain, e.g., a VH region.
• a binding domain e.g., an antigen-binding domain, e.g., a scFv or VHH
• a subunit of an antigen-binding domain e.g., a VH region.
• the binding domain can be a non- antibody binding domain, e.g., a receptor ectodomain, a ligand or receptor-binding fragment thereof, a cytokine or receptor-binding fragment thereof, a growth factor or receptor binding fragment thereof, a neurotransmitter or receptor binding fragment thereof, a peptide or protein hormone or receptor binding fragment thereof, an immune checkpoint modulator ligand or receptor-binding fragment thereof, or a receptor-binding fragment of an extracellular matrix protein.
• a non- antibody binding domain e.g., a receptor ectodomain, a ligand or receptor-binding fragment thereof, a cytokine or receptor-binding fragment thereof, a growth factor or receptor binding fragment thereof, a neurotransmitter or receptor binding fragment thereof, a peptide or protein hormone or receptor binding fragment thereof, an immune checkpoint modulator ligand or receptor-binding fragment thereof, or a receptor-binding fragment of an extracellular matrix protein.
• IgM binding units can form a complex with an additional small polypeptide chain (the J-chain), or a functional fragment, variant, or derivative thereof, to form a pentameric IgM antibody or IgM-like antibody.
• the precursor form of the human J-chain is presented as SEQ ID NO: 5.
• the signal peptide extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 5, and the mature human J-chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO: 5.
• the mature human J-chain includes the amino acid sequence SEQ ID NO: 6.
• an IgM antibody or IgM-like antibody typically assembles into a hexamer, comprising up to twelve antigen-binding domains.
• an IgM antibody or IgM-like antibody typically assembles into a pentamer, comprising up to ten antigen- binding domains, or more, if the J-chain is a modified J-chain comprising one or more heterologous polypeptides comprising additional antigen-binding domain(s).
• the assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody or IgM-like antibody is thought to involve the C ⁇ 4 and tailpiece domains.
• a pentameric or hexameric IgM antibody provided in this disclosure typically includes at least the C ⁇ 4 and/or tailpiece domains (also referred to herein collectively as C ⁇ 4-tp).
• a “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the C ⁇ 4-tp domains.
• An IgM heavy chain constant region can additionally include a C ⁇ 3 domain or a fragment thereof, a C ⁇ 2 domain or a fragment thereof, a C ⁇ 1 domain or a fragment thereof, and/or other IgM heavy chain domains.
• an IgM-derived binding molecule e.g., an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein can include a complete IgM heavy ( ⁇ ) chain constant domain, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, or a variant, derivative, or analog thereof, e.g., as provided herein.
• a complete IgM heavy ( ⁇ ) chain constant domain e.g., SEQ ID NO: 1 or SEQ ID NO: 2
• a variant, derivative, or analog thereof e.g., as provided herein.
• the disclosure provides a monoclonal population of multimeric, e.g., pentameric or hexameric binding molecules, where each binding molecule includes ten or twelve IgM-derived heavy chains, and where the IgM-derived heavy chains comprise glycosylated IgM heavy chain constant regions each associated with a binding domain that specifically binds to a target.
• the disclosure provides a monoclonal population of IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules that include five or six bivalent binding units, where each binding unit includes two IgM or IgM-like heavy chain constant regions or multimerizing fragments or variants thereof, each associated with an antigen-binding domain or subunit thereof.
• the two IgM heavy chain constant regions included in each binding unit are human heavy chain constant regions.
• the IgM antibody, IgM-like antibody, other IgM-derived binding molecule, or monoclonal population of multimeric binding molecules provided in this disclosure is pentameric
• the IgM antibody, IgM-like antibody, other IgM-derived binding molecule, or the molecules contained in a monoclonal population of multimeric binding molecules typically further include a J-chain, or functional fragment or variant thereof.
• the J-chain is a modified J-chain or variant thereof that further comprises one or more heterologous moieties attached to the J-chain, as described elsewhere herein.
• the J-chain can be mutated to affect, e.g., enhance, the serum half- life of the IgM antibody, IgM-like antibody, other IgM-derived binding molecule, or monoclonal population of multimeric binding molecules provided herein, as discussed elsewhere in this disclosure.
• the J-chain can be mutated to affect glycosylation, as discussed elsewhere in this disclosure.
• An IgM heavy chain constant region can include one or more of a C ⁇ 1 domain or fragment or variant thereof, a C ⁇ 2 domain or fragment or variant thereof, a C ⁇ 3 domain or fragment or variant thereof, and/or a C ⁇ 4 domain or fragment or variant thereof, provided that the constant region can serve a desired function in the IgM antibody, IgM- like antibody, or other IgM-derived binding molecule, e.g., associate with second IgM constant region to form a binding unit with one, two, or more antigen-binding domain(s), and/or associate with other binding units (and in the case of a pentamer, a J-chain) to form a hexamer or a pentamer.
• the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each comprise a C ⁇ 4 domain or fragment or variant thereof, a tailpiece (tp) or fragment or variant thereof, or a combination of a C ⁇ 4 domain and a TP or fragment or variant thereof.
• the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each further comprise a C ⁇ 3 domain or fragment or variant thereof, a C ⁇ 2 domain or fragment or variant thereof, a C ⁇ 1 domain or fragment or variant thereof, or any combination thereof.
• the J-chain of a pentameric IgM-derived binding molecule e.g., an IgM antibody or IgM-like antibody as provided herein can be modified, e.g., by introduction of a heterologous moiety, or two or more heterologous moieties, e.g., polypeptides, without interfering with the ability of IgM antibody, IgM-like antibody, other IgM-derived binding molecule, or monoclonal population of multimeric binding molecules to assemble and bind to its binding target(s). See U.S. Patent Nos. 9,951,134, 10,400,038, and 10,618,978, and U.S. Patent Application Publication No.
• an IgM antibody, IgM-like antibody, other IgM-derived binding molecule, or monoclonal population of multimeric binding molecules provided by this disclosure, including multispecific IgM or IgM-like antibodies as described elsewhere herein, can comprise a modified J-chain or functional fragment or variant thereof comprising a heterologous moiety, e.g., a heterologous polypeptide, introduced, e.g., fused or chemically conjugated, into the J-chain or fragment or variant thereof.
• a heterologous moiety e.g., a heterologous polypeptide
• the heterologous moiety can be a peptide or polypeptide sequence fused in frame to the J-chain or chemically conjugated to the J-chain or fragment or variant thereof.
• the heterologous polypeptide is fused to the J-chain or functional fragment thereof via a peptide linker, e.g., a peptide linker, typically consisting of least 5 amino acids, but no more than 25 amino acids.
• the peptide linker consists of
• the heterologous moiety can be a chemical moiety conjugated to the J-chain.
• Heterologous moieties to be attached to a J-chain can include, without limitation, a binding moiety, e.g., an antibody or antigen-binding fragment thereof, e.g., a single chain Fv (scFv) molecule, a cytokine, e.g., IL-2 or IL-15 (see, e.g., PCT Application Publication No.
• a binding moiety e.g., an antibody or antigen-binding fragment thereof, e.g., a single chain Fv (scFv) molecule
• a cytokine e.g., IL-2 or IL-15
• a stabilizing peptide that can increase the half-life of the IgM antibody, IgM-like antibody, other IgM- derived binding molecule, or monoclonal population of multimeric binding molecules, e.g., human serum albumin (HSA) or an HSA binding molecule, or a heterologous chemical moiety such as a polymer or a cytotoxin.
• HSA human serum albumin
• HSA binding molecule e.g., a heterologous chemical moiety such as a polymer or a cytotoxin.
• a modified J-chain can comprise an antigen-binding domain that can include without limitation a polypeptide capable of specifically binding to a target antigen.
• an antigen-binding domain associated with a modified J- chain can be an antibody or an antigen-binding fragment thereof, as described elsewhere herein.
• the antigen-binding domain can be a scFv antigen-binding domain or a single-chain antigen-binding domain derived, e.g., from a camelid or condricthoid antibody.
• the antigen-binding domain can be introduced into the J-chain at any location that allows the binding of the antigen-binding domain to its binding target without interfering with J-chain function or the function of an associated IgM or IgA antibody.
• Insertion locations include but are not limited to at or near the C-terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain, is accessible.
• the antigen-binding domain can be introduced into the mature human J-chain of SEQ ID NO: 6 between cysteine residues 92 and 101 of SEQ ID NO: 6.
• the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 6 at or near a glycosylation site.
• the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 6 within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from the N-terminus.
• the J-chain of the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J-chain (SEQ ID NO: 6).
• an amino acid corresponding to amino acid Y102 of the mature wild-type human J-chain is meant the amino acid in the sequence of the J-chain of any species which is homologous to Y102 in the human J-chain.
• an IgM antibody, IgM-like antibody, other IgM- derived binding molecule, or monoclonal population of multimeric binding molecules comprising a mutation at the amino acid corresponding to Y102 of SEQ ID NO: 6 has an improved serum half-life when administered to an animal than a corresponding antibody, antibody-like molecule, binding molecule, or monoclonal population of binding molecules that is identical except for the substitution, and which is administered to the same species in the same manner.
• the amino acid corresponding to Y102 of SEQ ID NO: 6 can be substituted with any amino acid.
• the amino acid corresponding to Y102 of SEQ ID NO: 6 can be substituted with alanine (A), serine (S) or arginine (R).
• the amino acid corresponding to Y102 of SEQ ID NO: 6 can be substituted with alanine.
• the J-chain or functional fragment or variant thereof is a variant human J-chain referred to herein as “J*,” and comprises the amino acid sequence SEQ ID NO: 7.
• J* human J-chain referred to herein as “J*,” and comprises the amino acid sequence SEQ ID NO: 7.
• each IgM heavy chain constant region includes at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, where an N-linked glycosylation motif comprises the amino acid sequence N-X1-S/T, where N is asparagine, X1 is any amino acid except proline, and S/T is serine or threonine.
• N is asparagine
• X1 is any amino acid except proline
• S/T is serine or threonine.
• 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 a complex glycan as defined elsewhere herein.
• a human or non-human primate IgM heavy chain constant region typically includes five N-linked glycosylation motifs N1 to N5, as noted earlier N4 and N5 are typically, but not invariably, occupied by oligomannose-type oligosaccharides as opposed to complex oligosaccharides.
• N4 and N5 are typically, but not invariably, occupied by oligomannose-type oligosaccharides as opposed to complex oligosaccharides.
• at least three of the N-linked glycosylation motifs (e.g., N1, N2, and N3) on each IgM heavy chain constant region are occupied by a complex glycan.
• the monoclonal population of binding molecules provided by this disclosure comprises a level of sialylation that is greater than that observed or measured for IgM antibodies in normal circulation, i.e., the provided monoclonal population of binding molecules includes a non-naturally-occurring level of sialylation.
• the average level of sialylation for human IgM antibodies isolated from normal circulation is about 30-32 moles sialic acid per mole of IgM.
• this disclosure provides a monoclonal population of multimeric binding molecules as noted above, that includes at least thirty-three (33), at least thirty-four (34), or at least of at least thirty-five (35) moles sialic acid per mole of the binding molecules.
• Sialic acid residues are typically the terminal monosaccharides on complex glycans, and a single oligosaccharide glycan can include, e.g., one, two, three, or four sialic acid monosaccharides depending on the number of antennae on the oligosaccharide.
• the provided monoclonal population of binding molecules can include higher levels of sialylation, e.g., the monoclonal population of binding molecules can include 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 sialic acid per mole of binding molecule.
• the monoclonal population of binding molecules includes 33-146 moles sialic acid per mole of binding molecule, such as 33-130, 33-120, 33-110, 33-100, 33-90, 33-80, 33-70, 33-60, 33-50, 35-130, 35-120, 35-110, 35-100, 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 sialic acid per mole of binding molecule.
• sialic acid per mole of binding molecule such as 33-130, 33-120, 33-110, 33-100, 33-90, 33-80, 33-70, 33-60, 33-50, 35-130, 35-120, 35-110, 35-100, 35-90, 35-80, 35-70, 35
• the monoclonal population of binding molecules comprises about 35 to about 40, about 35 to about 45, about 35 to about 50, about 35 to about 55, about 35 to about 60, about 35 to about 65, about 35 to about 70, about 40 to about 45, about 40 to about 50, about 40 to about 55, about 40 to about 60, about 40 to about 65, about 40 to about 70, about 45 to about 50, about 45 to about 55, about 45 to about 60, about 45 to about 65, about 45 to about 70, about 50 to about 55, about 50 to about 60, about 50 to about 65, about 50 to about 70, about 55 to about 60, about 55 to about 65, about 55 to about 70, about 60 to about 65, about 60 to about 70, or about 65 to about 70 moles sialic acid per mole of binding molecule.
• the monoclonal population of binding molecules comprises about 40 to about 55 moles sialic acid per mole of binding molecule.
• monoclonal population of binding molecules with sialic acid levels above 35 moles of sialic acid per mole of binding molecule have improved pharmacokinetic properties of the binding molecules compared to the same binding molecules with lower levels of sialic acid.
• Such molecules may have other desirable properties, such as a different solubility, ease of manufacturability, and/or immunogenicity.
• each IgM-derived heavy chain in the provided population of binding molecules includes a glycosylated IgM or IgM-derived heavy chain constant region or multimerizing fragment or derivative thereof, which can be a full-length IgM heavy chain constant region, a multimerizing fragment of an IgM heavy chain constant region, or a hybrid constant region that includes at least the minimal portion of an IgM heavy chain constant region required for multimerization, associated with a binding domain, e.g., an antibody antigen-binding domain, that specifically binds to a target of interest.
• a binding domain e.g., an antibody antigen-binding domain
• the IgM heavy chain constant regions are derived from human IgM heavy chain constant regions that include up to five N-linked glycosylation motifs N-X1-S/T starting 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).
• the binding domain that binds to a target can be, e.g., an antigen-binding domain or a subunit of an antigen-binding domain, e.g., the heavy chain variable region (VH) of an antibody.
• This disclosure relates to binding molecules that bind to any target of interest.
• the monoclonal population of binding molecules provided by this disclosure can be produced in a number of different ways, including, but not limited to, modifications to the cell line expressing the population of binding molecules, by in vitro glycoengineering of the monoclonal population of binding molecules during downstream processing, or any combination of these, or other methods.
• the provided highly sialylated monoclonal population of multimeric binding molecules is produced via cell line modification.
• Cell line modifications to increase sialylation of a monoclonal population of binding molecules as provided by this disclosure include, without limitation, transfecting the cell line that produces the monoclonal population of binding molecules with one or more genes encoding glycosyltransferases, e.g., galactosyltransferases (to provide an acceptor residue for a sialic acid residue via an alpha-2,6 and/or alpha-2,3 linkage, see, e.g., FIGs.
• glycosyltransferases e.g., galactosyltransferases (to provide an acceptor residue for a sialic acid residue via an alpha-2,6 and/or alpha-2,3 linkage, see, e.g., FIGs.
• sialyltransferases to produce cell lines that overexpress these enzymes (glycosyltransferase “knock-ins”), thereby improving and/or increasing the capacity of the ability of the cell line to facilitate the transfer of sialic acid monosaccharides from a CMP- NANA substrate or a derivative thereof to a compatible acceptor oligosaccharide.
• Other cell line modifications include the 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 by persons of ordinary skill in the art.
• sialyltransferase is human beta-galactoside alpha-2,6- sialyltransferase 1, also referred to as ST6GAL1 (SEQ ID NO: 3).
• Other sialyltransferases that can be “knocked in” include human beta-galactoside alpha-2,6-sialyltransferase-II (ST6GALII), and any of the four beta-galactoside ⁇ 2–3-sialyltransferases (ST3GAL-I- IV).
• an exemplary galactosyltransferase is human beta-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).
• the provided highly sialylated monoclonal population of multimeric binding molecules is produced via glycoengineering, to produce, e.g., a monoclonal population of glycoengineered IgM antibodies, IgM-like antibodies, or IgM- derived binding molecules (GEMs), e.g., via the addition of sialic acid residues to the monoclonal population of binding molecules during downstream processing.
• the in vitro glycoengineering comprises contacting the monoclonal population of binding molecules 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-acetyl-neuraminic acid (CMP-NANA)), under conditions where sialic acid is transferred from CMP-NANA to a galactose residue on complex glycans on the population of binding molecules.
• a sialic acid substrate e.g., a substrate comprising cytidine monophosphate (CMP)-N-acetyl-neuraminic acid (CMP-NANA)
• the contacting can occur during one or more steps of protein purification, after which the soluble sialyltransferase can be removed by a subsequent purification step, or by separating the population of binding molecules from the solid support to which the enzyme is attached.
• the sialyltransferase variant to be used in production of GEMs can be a soluble variant of human beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).
• the sialyltransferase can be a variant of ST6GAL1 that excludes the transmembrane region of SEQ ID NO: 3 (e.g., excluding amino acids 10 to 26 of SEQ ID NO: 3), or both the cytoplasmic and transmembrane regions of SEQ ID NO: 3 (e.g., excluding amino acids 1 to 9 of SEQ ID NO: 3 and amino acids 10 to 26 of SEQ ID NO: 3), but maintains the catalytic activity of the protein.
• the soluble variant of ST6GAL1 comprises amino acids x to 406 of SEQ ID NO: 3, wherein x is an integer from 27 to 120.
• the soluble variant of ST6GAL1 can include amino acids 120 to 406, 115 to 406, 110 to 406, 109 to 406, 105 to 406, 100 to 406, 95 to 406, 90 to 406, 89 to 406, 88 to 406, 87 to 406, 86 to 406, 85 to 406, 84 to 406, 83 to 406, 82 to 406, 81 to 406, 80 to 406, 75 to 406, 70 to 406, 65 to 406, 60 to 406, 55 to 406, 50 to 406, 45 to 406, 40 to 406, 35 to 406, 30 to 406, or 27 to 406 of SEQ ID NO: 3.
• CMP cytidine monophosphate
• Functional derivatives include but are not limited to azido-CMP-sialic acid to be used for glycan labelling by “click” chemistry.
• IgM antibodies can be carried out with low concentrations of the soluble variant of ST6GAL1, relative to the higher amounts required for glycoengineering of IgG antibodies.
• efficient sialylation of IgM antibodies has been carried out with a mass ratio of IgM antibody to soluble sialyltransferase of about 5000:1 or 2000:1, and an IgM antibody to sialic acid substrate to soluble sialyltransferase ratio of about 5000:2500:1 or 2000:500:1 (an excess amount of the sialic acid substrate is provided).
• the molar ratio of IgM antibody to sialyltransferase is at least about 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.
• the mass ratio of binding molecule: sialyltransferase can be about 80:1 to about 5000:1. In some embodiments, the mass ratio of binding molecule: 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, about 80:1 to about 3500:1, about 80:1 to about 4000:1, about 80:1 to about 4500:1, about 250:1 to about 500:1, about 250:1 to about 750:1, about 250:1 to about 1000:1, about 250:1 to about 1250:1, about 250:1 to about 1500:1, about 250:1 to about 1750:1, about 250:1 to about 5000:1.
• the inventors have also observed that despite the large numbers of glycans (51 for pentamers, 60 for hexamers), efficient and high-level sialylation of IgM antibodies can be carried out with low concentrations of the sialic acid substrate, relative to the higher amounts required for glycoengineering of IgG antibodies.
• the mass ratio of sialic acid substrate: sialyltransferase can be about 1:4 to about 3000:1, such as about 1:4 to about 1:1, about 1:4 to about 5:1, about 1:4 to about 50:1, about 1:4 to about 100:1, about 1:4 to about 500:1, about 1:4 to about 1000:1, about 1:4 to about 1500:1, about 1:4 to about 2000:1, about 1:4 to about 2500:1, about 1:1 to about 5:1, about 1:1 to about 10:1, about 1:1 to about 50:1, about 1:1 to about 100:1, about 1:1 to about 500:1, about 1:1 to about 1000:1, about 1:1 to about 1500:1, about 1:1 to about 2000:1, about 1:1 to about 2500:1, about 1:1 to about 3000:1, about 2:1 to about 5:1, about 2:1 to about 10:1, about 2:1 to about 50:1, about 2:1 to about 100:1, about 2:1 to about 500:1, about 2:1 to about 1000:1, about 2:1 to about 1500:1, about 2:1 to about 2000:1, about 2:1 to about 2500:1, about 1:1 to about
• the mass ratio of binding molecule: sialyltransferase can 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 sialic acid substrate: sialyltransferase can 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.
• the mass ratio of antibody: sialic acid substrate can be about 1:1 to about 40:1, such as about 1:1 to about 2:1, about 1:1 to about 4:1, about 1:1 to about 6:1, about 1:1 to about 8:1, about 1:1 to about 10:1, about 1:1 to about 15:1, about 1:1 to about 20:1, about 2:1 to about 4:1, about 2:1 to about 6:1, about 2:1 to about 8:1, about 2:1 to about 10:1, about 2:1 to about 15:1, about 2:1 to about 20:1, about 2:1 to about 40:1, about 4:1 to about 6:1, about 4:1 to about 8:1, about 4:1 to about 10:1, about 4:1 to about 15:1, about 4:1 to about 20:1, about 4:1 to about 40:1, about 6:1 to about 8:1, about 6:1 to about 10:1, about 6:1 to about 15:1, about 6:1 to about 20:1, about 6:1 to about 40:1, about 8:1 to about 10:1, about 8:1 to about 15:1, about 8:1 to about 20:1, about 6:1 to about 40:1, about 8:1 to about 10:1, about 8:1 to
• the in vitro glycoengineering comprises contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate for at least 30 minutes, such as 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.
• the contacting occurs for about 30 minutes to about 48 hours, such as about 30 minutes to about 4 hours, about 30 minutes to about 5 hours, about 30 minutes to about 6 hours, about 30 minutes to about 7 hours, about 30 minutes to about 10 hours, about 30 minutes to about 12 hours, about 30 minutes to about 18 hours, about 30 minutes to about 24 hours, about 30 minutes to about 36 hours, about 2 hours to about 48 hours, about 3 hours to about 6 hours, about 3 hours to about 10 hours, about 3 hours to about 12 hours, about 3 hours to about 18 hours, about 3 hours to about 24 hours, about 3 hours to about 36 hours, about 3 hours to about 48 hours, about 4 hours to about 10 hours, about 4 hours to about 12 hours, about 4 hours to about 18 hours, about 4 hours to about 24 hours, about 4 hours to about 36 hours, about 4 hours to about 48 hours, about 5 hours to about 10 hours, about 5 hours to about 12 hours, about 5 hours to about 18 hours, about 5 hours to about 24 hours, about 5 hours to about 36 hours, about 5 hours to about 48 hours, about 7 hours
• the in vitro glycoengineering comprises contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate at a temperature at about 15 °C to about 25 °C.
• in vitro sialylation can be enhanced by ensuring a sufficient number of galactose acceptor residues are present on the complex glycans of the provided monoclonal population of IgM, IgM-like, or IgM-derived binding molecules.
• ST6GAL1 transfers a sialic acid monosaccharide from CMP-NANA to a galactose acceptor residue on the molecule’s glycans via an ⁇ -2,6 linkage.
• production of GEMs can further include contacting the monoclonal population of binding molecules with a galactosyltransferase, e.g., a soluble variant of beta-1,4- galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4), and a galactose substrate, e.g., uridine-diphosphate- ⁇ -D-galactose (UDP-Gal), either prior to or simultaneously with the contacting with the sialyltransferase and sialic acid substrate.
• a galactosyltransferase e.g., a soluble variant of beta-1,4- galactosyltransferase 4 (B4GALT4) (S
• the sialyltransferase can be a variant of B4GALT4 that excludes the transmembrane region of SEQ ID NO: 4 (e.g., excluding amino acids 13 to 38 of SEQ ID NO: 4), or both the cytoplasmic and transmembrane regions of SEQ ID NO: 4 (e.g., excluding amino acids 1 to 12 of SEQ ID NO: 4 and amino acids 13 to 38 of SEQ ID NO: 4), but maintains the catalytic activity of the protein.
• the soluble variant of B4GALT4 comprises amino acids x to 344 of SEQ ID NO: 4, wherein x is an integer from 39 to 120.
• the soluble variant of B4GALT4 comprises amino acids 120 to 344, 115 to 344, 110 to 344, 105 to 344, 100 to 344, 95 to 344, 90 to 344, 85 to 344, 80 to 344, 75 to 344, 70 to 344, 65 to 344, 60 to 344, 55 to 344, 50 to 344, 45 to 344, 40 to 344, or 39 to 344 of SEQ ID NO: 4.
• the galactose substrate comprises UDP- Gal.
• IgM heavy chain constant regions in the provided monoclonal population of binding molecules are each associated with a binding domain or subunit thereof, e.g., an antibody antigen-binding domain, e.g., a scFv, a VHH or the VH subunit of an antibody antigen- binding domain, where the binding domain specifically binds to a target of interest.
• the target is a target epitope, a target antigen, a target cell, a target organ, or a target virus.
• Targets can include, without limitation, tumor antigens, other oncologic targets, immuno-oncologic 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 diseases and neuroinflammatory diseases, and any combination thereof.
• 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 diseases and neuroinflammatory diseases
• Exemplary targets and binding domains that bind to such targets are provided elsewhere herein, and can be found in, e.g., U.S. Patent Application Publication Nos. US 2019-0330360, US 2019-0338040, US 2019-0338041, US 2019-0330374, US 2019- 0185570, US 2019-0338031, or US 2020-0239572, in PCT Publication Nos.
• the provided population of multimeric binding molecules is multispecific, e.g., bispecific, trispecific, or tetraspecific, where two or more binding domains associated with the IgM heavy chain constant regions of each binding molecule specifically bind to different targets.
• the binding domains of the provided population of multimeric binding molecules all specifically bind to the same target.
• the binding domains of the provided population of multimeric binding molecules are identical. In such cases the population of multimeric binding molecules can still be bispecific, if, for example, a binding domain with a different specificity is part of a modified J-chain as described elsewhere herein.
• the binding domains are antibody-derived antigen-binding domains, e.g., a scFv associated with the IgM heavy chain constant regions or a VH subunit of an antibody binding domain associated with the IgM heavy chain constant regions.
• each binding molecule is a pentameric or a hexameric IgM antibody comprising five or six bivalent IgM binding units, respectively, wherein each binding unit comprises two IgM heavy chains each comprising a VH situated amino terminal to the variant IgM constant region, and two immunoglobulin light chains each comprising a light chain variable domain (VL) situated amino terminal to an immunoglobulin light chain constant region, and wherein the VH and VL combine to form an antigen-binding domain that specifically binds to the target.
• each antigen-binding domain of each binding molecule binds to the same target.
• each antigen-binding domain of each binding molecule is identical.
• the target is a tumor-specific antigen, i.e., a target antigen that is largely expressed only on tumor or cancer cells, or that may be expressed only at undetectable levels in normal healthy cells of an adult.
• the target is a tumor-associated antigen, i.e., a target antigen that is expressed on both healthy and cancerous cells but is expressed at a much higher density on cancerous cells than on normal healthy cells.
• Exemplary tumor-specific or tumor-associated antigens include, without limitation, B-cell maturation antigen (BCMA), CD19, CD20, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2, also called ErbB2), HER3 (ErbB3), receptor tyrosine-protein kinase ErbB4, cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed cell death protein 1 (PD-1), Programmed death-ligand 1 (PD- L1), vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR1), VEGFR2, CD52, CD30, prostate-specific membrane antigen (PSMA), CD38, ganglioside GD2, self- ligand receptor of the signaling lymphocytic activation molecule family member 7 (SLAMF7), platelet-derived growth factor receptor A (PDGFRA), CD22, FLT3 (CD135), CD123, MUC-16, carcinoembryonic antigen-related cell adhe
• tumor associated or tumor-specific antigens include, without limitation: 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, TNFalpha, IL-23, IL-17, CD80, CD86, CD3, CEA, Muc16, PSCA, CD44, c-Kit, DDR1, DDR2, RSPO1, RSPO2, RSPO3, RSPO4, BMP family polypeptides, BMPR1a,
• the monoclonal population of multimeric binding molecules comprises a population of pentameric or hexameric IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules that each include five or six bivalent IgM binding units, respectively.
• each binding unit includes two IgM heavy chains as described herein, each having a VH situated amino terminal to the variant IgM constant region, and two immunoglobulin light chains each having a light chain variable domain (VL) situated amino terminal to an immunoglobulin light chain constant region, e.g., a kappa or lambda constant region.
• VH and VL combine to form an antigen-binding domain that specifically binds to the target of interest.
• each antibody or binding molecule can further include a J-chain, or functional fragment thereof, or a functional variant thereof, as described elsewhere herein.
• the J-chain can be a mature human J-chain that includes the amino acid sequence SEQ ID NO: 6 or a functional fragment thereof, or a functional variant thereof.
• a functional fragment or a “functional variant” in this context includes those fragments and variants that can associate with IgM binding units, e.g., IgM heavy chain constant regions, to form a pentameric IgM antibody, IgM-like antibody, or IgM-derived binding molecule.
• IgM binding units e.g., IgM heavy chain constant regions
• the J-chain of a pentameric IgM-derived binding molecule e.g., an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein is a functional variant J-chain that includes one or more single amino acid substitutions, deletions, or insertions relative to a reference J-chain identical to the variant J-chain except for the one or more single amino acid substitutions, deletions, or insertions.
• certain amino acid substitutions, deletions, or insertions can result in the IgM-derived binding molecule exhibiting an increased serum half-life upon administration to a subject animal relative to a reference IgM-derived binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the variant J-chain, and is administered using the same method to the same animal species.
• the variant J-chain can include one, two, three, or four single amino acid substitutions, deletions, or insertions relative to the reference J-chain.
• the variant J-chain or functional fragment thereof of a pentameric IgM-derived binding molecule comprises 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, for example alanine.
• the variant human J-chain can include the amino acid sequence SEQ ID NO: 7.
• J-chain having the amino acid sequence of SEQ ID NO: 7 can in some instances be referred to as “J*.”
• the J-chain or fragment of a pentameric IgM-derived binding molecule e.g., an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein, having either a variant or wild type amino acid sequence, can be a “modified J-chain” that further include a heterologous moiety, wherein the heterologous moiety is fused or conjugated to the J-chain or fragment or variant thereof.
• Exemplary, but non-limiting heterologous moieties are provided, e.g., in U.S. Patent Nos.
• the heterologous moiety is a polypeptide fused to or within the J-chain or fragment or variant thereof.
• the heterologous polypeptide can in some instances be fused to or within the J-chain or fragment or variant thereof via a peptide linker.
• Any suitable linker can be used, for example the peptide linker can include at least 5 amino acids, at least ten amino acids, and least 20 amino acids, at least 30 amino acids or more, and so on. In certain embodiments the peptide linker includes no more than 25 amino acids.
• the peptide linker can consist of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids.
• the peptide linker comprises glycines and serines, e.g., (GGGGS)n (SEQ ID NO: 48), where N can be 1, 2, 3, 4, 5, or more.
• the peptide linker consists of GGGGS (SEQ ID NO: 41), GGGGSGGGGS (SEQ ID NO: 42),
• the heterologous polypeptide can be fused to the N-terminus of the J-chain or fragment or variant thereof, the C-terminus of the J-chain or fragment or variant thereof, or to both the N-terminus and C-terminus of the J-chain or fragment or variant thereof.
• the heterologous polypeptide can be fused internally within the J- chain.
• the heterologous polypeptide can be a binding domain, e.g., an antigen binding domain.
• the heterologous polypeptide can be an antibody, a subunit of an antibody, or an antigen-binding fragment of an antibody, e.g., a scFv fragment.
• the binding domain, e.g., scFv fragment can bind to an effector cell, e.g., a T cell or an NK cell.
• the binding domain, e.g., scFv fragment can specifically bind to CD3 on cytotoxic T cells, e.g., to CD3 ⁇ .
• humanized SP35 antibodies include the VH and VL or scFv sequences of A-55 (SEQ ID NOs 22, 23, and 24, respectively, WO2018208864), A-56 (SEQ ID NOs 25, 26, and 27, respectively, WO2018208864), or A-57 (SEQ ID NOs 28, 29, and 30, respectively, WO2018208864), incorporated into the modified J-chains A-55-J* (SEQ ID NO: 31), A- 56-J* (SEQ ID NO: 32), and A-57-J* (SEQ ID NO: 33).
• a modified J-chain as provided herein can further include an additional heterologous moiety attached, e.g., on the opposite end of the J-chain from the anti-CD3 ⁇ scFv binding domain.
• the modified J-chain can further include the human serum albumin protein. Examples include, but are not limited to, VJH (SEQ ID NO: 34) and VJ*H (SEQ ID NO: 35).
• VJH SEQ ID NO: 34
• VJ*H SEQ ID NO: 35
• IgM-derived Binding molecules with enhanced serum half-life [0166] A monoclonal population of highly sialylated IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules as provided herein can be further engineered to have enhanced serum half-life.
• variant IgM heavy chain constant region mutations that can enhance serum half-life of an IgM-derived binding molecule are disclosed in US Patent Application Publication No. US 2020-0239572, which is incorporated by reference herein in its entirety.
• variant IgM heavy chain constant regions of the population of highly sialylated IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules as provided herein can include an amino acid substitution at an amino acid position corresponding to amino acid S401, E402, E403, R344, and/or E345 of a wild-type human IgM constant region (e.g., SEQ ID NO: 1 or SEQ ID NO: 2).
• an amino acid corresponding to amino acid S401, E402, E403, R344, and/or E345 of a wild-type human IgM constant region is meant the amino acid in the sequence of the IgM constant region of any species which is homologous to S401, E402, E403, R344, and/or E345 in the human IgM constant region.
• the amino acid corresponding to S401, E402, E403, R344, and/or E345 of SEQ ID NO: 1 or SEQ ID NO: 2 can be substituted with any amino acid, e.g., alanine.
• Wild-type J-chains typically include one N-linked glycosylation site.
• a variant J-chain or functional fragment thereof of a pentameric IgM- derived binding molecule as provided herein includes a mutation within the asparagine(N)- linked glycosylation motif N-X1-S/T, e.g., starting at the amino acid position corresponding to amino acid 49 (motif N6) of the mature human J-chain (SEQ ID NO: 6) or J* (SEQ ID NO: 7), wherein N is asparagine, X1 is any amino acid except proline, and S/T is serine or threonine, and wherein the mutation prevents glycosylation at that motif.
• N asparagine
• X1 is any amino acid except proline
• S/T is serine or threonine
• mutations preventing glycosylation at this site can result in the population of IgM-derived binding molecules, e.g., an IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules as provided herein, exhibiting an increased serum half-life upon administration to a subject animal relative to a reference IgM-derived binding molecule that is identical except for the mutation or mutations preventing glycosylation in the variant J-chain, and is administered in the same way to the same animal species.
• IgM-derived binding molecules e.g., an IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules as provided herein
• the variant J-chain or functional fragment thereof of a pentameric IgM-derived binding molecule as provided herein can include an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 SEQ ID NO: 6 or SEQ ID NO: 7, provided that the amino acid corresponding to S51 is not substituted with threonine (T), or wherein the variant J-chain comprises amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 6 or SEQ ID NO: 7.
• the position corresponding to N49 of 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).
• alanine A
• G glycine
• T threonine
• S serine
• D aspartic acid
• the position corresponding to N49 of SEQ ID NO: 6 or SEQ ID NO: 7 can be substituted with alanine (A).
• the position corresponding to N49 of SEQ ID NO: 6 or SEQ ID NO: 7 can be substituted with aspartic acid (D).
• a monoclonal population of IgM-derived binding molecules e.g., IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules as provided herein, can be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity to cells in the presence of complement, relative to a reference population of IgM antibodies or IgM-like antibodies with corresponding reference human IgM constant regions identical, except for the mutations conferring reduced CDC activity.
• CDC complement-dependent cytotoxicity
• corresponding reference human IgM constant region is meant a human IgM constant region or portion thereof, e.g., a C ⁇ 3 domain, that is identical to the variant IgM constant region except for the modification or modifications in the constant region affecting CDC activity.
• the variant human IgM constant region includes one or more amino acid substitutions, e.g., in the C ⁇ 3 domain, relative to a wild-type human IgM constant region as described, e.g., in PCT Publication No. WO/2018/187702, which is incorporated herein by reference in its entirety.
• Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described e.g., in PCT Publication No.
• a 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.
• the variant IgM constant region as provided herein contains 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.
• the variant IgM constant region as provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at positions P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and P313 of SEQ ID NO: 1 or SEQ ID NO: 2.
• proline residues can be independently substituted with any amino acid, e.g., with alanine, serine, or glycine.
• a 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.
• the lysine residue can be independently substituted with any amino acid, e.g., with alanine, serine, glycine, or aspartic acid.
• a 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 with aspartic acid.
• this disclosure provides a host cell that is capable of producing the highly sialylated monoclonal population of binding molecules as provided herein. In certain aspects such a host cell overexpresses ST6GAL1 and/or B4GALT4. The disclosure also provides a method of producing the monoclonal population of binding molecules as provided herein, where the method comprises culturing the provided host cell, and recovering the population of binding molecules.
• This disclosure further provides a method for producing a monoclonal population of highly sialylated multimeric binding molecules as described in detail in this disclosure, where the method includes: providing a cell line that expresses the monoclonal population of binding molecules, culturing the cell line, and recovering the monoclonal population of binding molecules.
• each binding molecule comprises ten or twelve IgM-derived heavy chains, wherein the IgM-derived heavy chains comprise glycosylated IgM heavy chain constant regions, or multimerizing fragments thereof, each associated with a binding domain that specifically binds to a target, wherein 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, wherein and wherein an N- linked glycosylation motif comprises the amino acid sequence N-X 1 -S/T, where N is asparagine, X 1 is any amino acid except proline, and S/T is serine or threonine.
• 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 is/are occupied by complex glycans, and wherein the cell line, culture conditions, recovery process, or a combination thereof is optimized to enrich for complex glycans comprising at least one, two, at least three, or four sialic acid terminal monosaccharides per glycan.
• the cell line, culture conditions, recovery process, or a combination thereof can be optimized according to the provided method to result in a monoclonal population of binding molecules comprising 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 sialic acid per mole of binding molecule.
• the cell line, recovery process, or a combination thereof can be optimized according to the provided method to result in a monoclonal population of binding molecules comprising at least 35, at least 40, at least 45, at least 50, or at least 60 moles sialic acid per mole of binding molecule.
• the monoclonal population of binding molecules comprises about 35 to about 40, about 35 to about 45, about 35 to about 50, about 35 to about 55, about 35 to about 60, about 35 to about 65, about 35 to about 70, about 40 to about 45, about 40 to about 50, about 40 to about 55, about 40 to about 60, about 40 to about 65, about 40 to about 70, about 45 to about 50, about 45 to about 55, about 45 to about 60, about 45 to about 65, about 45 to about 70, about 50 to about 55, about 50 to about 60, about 50 to about 65, about 50 to about 70, about 55 to about 60, about 55 to about 65, about 55 to about 70, about 60 to about 65, about 60 to about 70, or about 65 to about 70 moles sialic acid per mole of binding molecule.
• the monoclonal population of binding molecules comprises about 40 to about 55 moles sialic acid per mole of binding molecule.
• the IgM heavy chain constant regions can be derived from human IgM heavy chain constant regions comprising five N-linked glycosylation motifs N-X1- S/T starting 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).
• the cell line cultured according to provided method is modified to overexpress a sialyltransferase.
• the overexpressed sialyltransferase is a 2,6-sialyl transferase.
• the overexpressed sialyltransferase is human beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1).
• the overexpressed sialyltransferase is a 2,3-sialyl transferase.
• the cell line cultured according to provided method can also be modified to overexpress a galactosyltransferase.
• the overexpressed galactosyltransferase is human beta-1,4-galactosyltransferase 4 (B4GALT4).
• the cell line cultured according to provided method can also be modified to overexpress an UDP-GlcNAc 2- Epimerase/ManNAc Kinase enzyme (GNE), such as GNE comprising an R263 or R266 mutation, such as a Q, W, or L mutation; an ⁇ -mannosidase II; an N- Acetylglucosaminyltransferase-II (GNT-II); an N-Acetylglucosaminyltransferase-IV (GNT-IV); an N-Acetylglucosaminyltransferase-V (GNT-V); a CMP-sialic acid synthase (CMP-SAS), a CMP-sialic acid transporter (CMP-SAT), or any combination thereof.
• GNE UDP-GlcNAc 2- Epimerase/ManNAc Kinase enzyme
• the cell line cultured according to provided method can also be modified to block expression of certain sialidases. In certain embodiments the cell line cultured according to provided method can also be modified to block expression of a neuraminidase.
• the recovery process includes subjecting the monoclonal population of multimeric binding molecules to glycoengineering during downstream processing, to produce, e.g., a monoclonal population of glycoengineered IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules, or “GEMs.”
• the GEMs are highly sialylated, e.g., possessing at least 35 moles sialic acid per mole of binding molecule.
• the production of GEMs includes contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate.
• the soluble sialyltransferase can be a soluble variant of human beta-galactoside alpha-2,6- sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).
• ST6GAL1 human beta-galactoside alpha-2,6- sialyltransferase 1
• the soluble variant of ST6GAL1 includes amino acids x to 406 of SEQ ID NO: 3, wherein x is an integer from 27 to 120.
• the soluble variant of ST6GAL1 can include amino acids 120 to 406, 115 to 406, 110 to 406, 109 to 406, 105 to 406, 100 to 406, 95 to 406, 90 to 406, 89 to 406, 88 to 406, 87 to 406, 86 to 406, 85 to 406, 84 to 406, 83 to 406, 82 to 406, 81 to 406, 80 to 406, 75 to 406, 70 to 406, 65 to 406, 60 to 406, 55 to 406, 50 to 406, 45 to 406, 40 to 406, 35 to 406, 30 to 406, or 27 to 406 of SEQ ID NO: 3.
• the sialic acid substrate can include cytidine monophosphate (CMP)-N- acetyl-neuraminic acid (CMP-NANA), or a derivative thereof.
• CMP cytidine monophosphate
• CMP-NANA cytidine monophosphate
• the inventors have discovered that the production of highly sialylated IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules requires much less enzyme than a comparable method for sialylating IgG.
• the mass ratio of binding molecule: sialyltransferase can be about 80:1 to about 5000:1.
• the mass ratio of binding molecule: 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, about 80:1 to about 3500:1, about 80:1 to about 4000:1, about 80:1 to about 4500:1, about 80:1 to about 5000:1, about 250:1 to about 500:1, about 250:1 to about 750:1, about 250:1 to about 1000:1, about 250:1 to about 1250:1, about 250:1 to about 1500:1, about 250:1 to about 1750:1, about 250:1 to about 2000:1, about 250:1 to about 2500:1, about 250:1 to about 3000:1, about
• the molar ratio of binding molecule: sialyltransferase can 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.
• the mass ratio of binding molecule: sialic acid substrate: sialyltransferase can be about 2000:500:1.
• the molar ratio of binding molecule: sialyltransferase can 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 to sialyltransferase can be about 80:1.
• production of GEMs can further include contacting the monoclonal population of binding molecules with a galactosyltransferase, e.g., a soluble variant of human beta-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4) and a galactose substrate, e.g., uridine-diphosphate- ⁇ -D- galactose (UDP-Gal).
• B4GALT4 human beta-1,4-galactosyltransferase 4
• UDP-Gal uridine-diphosphate- ⁇ -D- galactose
• the mass ratio of sialic acid substrate: sialyltransferase can be about 5:1 to about 3000:1, such as 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, about 10:1 to about 500:1, about 10:1 to about 1000:1, about 10:1 to about 1500:1, about 10:1 to about 2000:1, about 10:1 to about 2500:1, about 10:1 to about 3000:1, about 50:1 to about 100:1, about 50:1 to about 500:1, about 50:1 to about 1000:1, about 50:1 to about 1500:1, about 50:1 to about 2000:1, about 50:1 to about 2500:1, about 50:1 to about 3000:1, about 50:1 to about 100:1,
• the mass ratio of antibody: sialic acid substrate can be about 1:1 to about 40:1, such as about 1:1 to about 2:1, about 1:1 to about 4:1, about 1:1 to about 6:1, about 1:1 to about 8:1, about 1:1 to about 10:1, about 1:1 to about 15:1, about 1:1 to about 20:1, about 2:1 to about 4:1, about 2:1 to about 6:1, about 2:1 to about 8:1, about 2:1 to about 10:1, about 2:1 to about 15:1, about 2:1 to about 20:1, about 2:1 to about 40:1, about 4:1 to about 6:1, about 4:1 to about 8:1, about 4:1 to about 10:1, about 4:1 to about 15:1, about 4:1 to about 20:1, about 4:1 to about 40:1, about 6:1 to about 8:1, about 6:1 to about 10:1, about 6:1 to about 15:1, about 6:1 to about 20:1, about 6:1 to about 40:1, about 8:1 to about 10:1, about 8:1 to about 15:1, about 8:1 to about 20:1, about 6:1 to about 40:1, about 8:1 to about 10:1, about 8:1 to
• the method comprises contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate for at least 30 minutes, such as 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.
• a soluble sialyltransferase and a sialic acid substrate for at least 30 minutes, such as 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.
• the contacting occurs for about 30 minutes to about 48 hours, such as about 30 minutes to about 4 hours, about 30 minutes to about 5 hours, about 30 minutes to about 6 hours, about 30 minutes to about 7 hours, about 30 minutes to about 10 hours, about 30 minutes to about 12 hours, about 30 minutes to about 18 hours, about 30 minutes to about 24 hours, about 30 minutes to about 36 hours, about 2 hours to about 48 hours, about 3 hours to about 6 hours, about 3 hours to about 10 hours, about 3 hours to about 12 hours, about 3 hours to about 18 hours, about 3 hours to about 24 hours, about 3 hours to about 36 hours, about 3 hours to about 48 hours, about 4 hours to about 10 hours, about 4 hours to about 12 hours, about 4 hours to about 18 hours, about 4 hours to about 24 hours, about 4 hours to about 36 hours, about 4 hours to about 48 hours, about 5 hours to about 10 hours, about 5 hours to about 12 hours, about 5 hours to about 18 hours, about 5 hours to about 24 hours, about 5 hours to about 36 hours, about 5 hours to about 48 hours, about 7 hours
• This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, 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); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 1-4; Gait, ed.
• a monoclonal population of multimeric binding molecules each binding molecule comprising ten or twelve IgM-derived heavy chains, wherein the IgM- derived heavy chains comprise glycosylated IgM heavy chain constant regions each associated with a binding domain that specifically binds to a target, wherein 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, wherein an N-linked glycosylation motif comprises the amino acid sequence N-X1-S/T, wherein N is asparagine, X1 is any amino acid except proline, and S/T is serine or threonine, wherein 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 wherein the monoclonal population of binding molecules comprises at least thirty-five (35) moles sialic acid per mole of the binding
• Embodiment 2 The monoclonal population of binding molecules of embodiment 1, comprising at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 moles sialic acid per mole of binding molecule.
• Embodiment 3 The monoclonal population of binding molecules of embodiment 1, comprising 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, about 60 to about 70 moles sialic acid per mole of binding molecule.
• the IgM heavy chain constant regions are human IgM heavy chain constant regions or variants thereof comprising five N-linked glycosylation motifs N-X1-S/T starting 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
• Embodiment 6 The monoclonal population of binding molecules of any one of embodiments 1 to 5, produced by the method of cell line modification, in vitro glycoengineering, or any combination thereof.
• Embodiment 7. The monoclonal population of binding molecules of embodiment 6, wherein the cell line modification comprises transfecting a cell line that produces the monoclonal population of binding molecules with a gene encoding a sialyltransferase, thereby producing a modified cell line that overexpresses the sialyltransferase.
• Embodiment 9 The monoclonal population of binding molecules of embodiment 7 or embodiment 8, wherein the cell line modification further comprises transfecting a cell line that produces the monoclonal population of binding molecules with a gene encoding a galactosyltransferase, thereby producing a modified cell line that overexpresses the galactosyltransferase.
• Embodiment 10 Embodiment 10.
• B4GALT4 human beta-1,4- galactosyltransferase 4
• Embodiment 11 The monoclonal population of binding molecules of any one of embodiments 6 to 10, wherein in vitro glycoengineering comprises contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate.
• ST6GAL1 human beta- galactoside alpha-2,6-sialyltransferase 1
• Embodiment 13 The monoclonal population of binding molecules of embodiment 12, wherein the soluble variant of ST6GAL1 comprises amino acids x to 406 of SEQ ID NO: 3, wherein x is an integer from 27 to 120.
• CMP-NANA cytidine monophosphate-N-acetyl-neuraminic acid
• Embodiment 16 The monoclonal population of binding molecules of any one of embodiments 11 to 15, wherein the mass ratio of binding molecule: sialic acid substrate is about 1:4 to about 40:1.
• Embodiment 17 The monoclonal population of binding molecules of any one of embodiments 11 to 16, wherein the mass ratio of binding molecule: sialyltransferase is about 80:1 to about 5000:1.
• Embodiment 19 The monoclonal population of binding molecules of embodiment 18, wherein the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 2000:500:1.
• Embodiment 20 The monoclonal population of binding molecules of any one of embodiments 11 to 17, wherein the molar ratio of binding molecule: sialyltransferase is about 80:1.
• Embodiment 21 Embodiment 21.
• Embodiment 22 The monoclonal population of binding molecules of any one of embodiments 11 to 17, wherein the mass ratio of binding molecule: sialyltransferase is about 500:1.
• Embodiment 23 The monoclonal population of binding molecules of embodiment 22, wherein the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 500:62.5:1.
• Embodiment 24 The monoclonal population of binding molecules of embodiment 20, wherein the molar ratio of binding molecule: sialic acid substrate: sialyltransferase is about 80:500:1.
• Embodiment 22 The monoclonal population of binding molecules of any one of embodiments 11 to 17, wherein the mass ratio of binding molecule: sialyltransferase is about 500:1.
• Embodiment 23 The monoclonal population of binding molecules of embodiment 22, wherein the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 500:62.5:1.
• Embodiment 25 The monoclonal population of binding molecules of embodiment 24, wherein the contacting comprises at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours of contact.
• Embodiment 26 Embodiment 26.
• Embodiment 27 The monoclonal population of binding molecules of embodiment 26, wherein the contacting occurs at 15° C to about 37° C, 15° C to about 30° C, or 15° C to about 25° C.
• Embodiment 28 Embodiment 28.
• Embodiment 29 The monoclonal population of binding molecules of embodiment 28, wherein the galactosyltransferase comprises a soluble variant of human beta-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).
• B4GALT4 human beta-1,4-galactosyltransferase 4
• Embodiment 31 The monoclonal population of binding molecules of embodiment 29, wherein the soluble variant of B4GALT4 comprises amino acids x to 344 of SEQ ID NO: 4, wherein x is an integer from 39 to 120.
• Embodiment 31 The monoclonal population of binding molecules of embodiment 30, wherein the soluble variant of B4GALT4 comprises amino acids 120 to 344, 115 to 344, 110 to 344, 105 to 344, 100 to 344, 95 to 344, 90 to 344, 85 to 344, 80 to 344, 75 to 344, 70 to 344, 65 to 344, 60 to 344, 55 to 344, 50 to 344, 45 to 344, 40 to 344, or 39 to 344 of SEQ ID NO: 4.
• Embodiment 32 The monoclonal population of binding molecules of any one of embodiments 28 to 31, wherein the galactose substrate comprises uridine-diphosphate- ⁇ - D-galactose (UDP-Gal).
• Embodiment 33 The monoclonal population of binding molecules of any one of embodiments 28 to 32, wherein the contacting with the galactosyltransferase and the galactose substrate occurs prior to or simultaneously with the contacting with the soluble sialyltransferase and sialic acid substrate.
• Embodiment 34 Embodiment 34.
• Embodiment 35 The monoclonal population of binding molecules of any one of embodiments 1 to 33, wherein each binding molecule is multispecific, and wherein two or more binding domains associated with the IgM heavy chain constant regions of each binding molecule specifically bind to different targets.
• Embodiment 35 The monoclonal population of binding molecules of any one of embodiments 1 to 33, wherein the binding domains associated with the IgM heavy chain constant regions of each binding molecule specifically bind to the same target.
• Embodiment 36 The monoclonal population of binding molecules of embodiment 35, wherein the binding domains associated with the IgM heavy chain constant regions of each binding molecule are identical.
• Embodiment 37 Embodiment 37.
• Embodiment 38 The monoclonal population of binding molecules of embodiment 37, wherein each binding molecule is a pentameric or a hexameric IgM antibody comprising five or six bivalent IgM binding units, respectively, wherein each binding unit comprises two IgM heavy chains each comprising a VH situated amino terminal to the variant IgM constant region, and two immunoglobulin light chains each comprising a light chain variable domain (VL) situated amino terminal to an immunoglobulin light chain constant region, and wherein the VH and VL combine to form an antigen-binding domain that specifically binds to the target.
• VL light chain variable domain
• Embodiment 39 The monoclonal population of binding molecules of embodiment 38, wherein each antigen-binding domain of each binding molecule binds to the same target.
• Embodiment 40 The monoclonal population of binding molecules of embodiment 39, wherein each antigen-binding domain of each binding molecule is identical.
• Embodiment 41 The monoclonal population of binding molecules of 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.
• Embodiment 42 Embodiment 42.
• each binding molecule is pentameric and further comprises a J-chain, or functional fragment thereof, or a functional variant thereof.
• Embodiment 43 The monoclonal population of binding molecules of embodiment 42, wherein the J-chain is a mature human J-chain comprising the amino acid sequence SEQ ID NO: 6 or a functional fragment thereof, or a functional variant thereof.
• Embodiment 44 The monoclonal population of binding molecules of embodiment 43, wherein the J-chain comprises an N-linked glycosylation motif N-X1-S/T starting at amino acid positions corresponding to amino acid 49 of SEQ ID NO: 6 (motif N6).
• Embodiment 45 The monoclonal population of binding molecules of any one of embodiments 42 to 44, wherein the J-chain is a functional variant J-chain comprising one or more single amino acid substitutions, deletions, or insertions relative to a reference J- chain identical to the variant J-chain except for the one or more single amino acid substitutions, deletions, or insertions, and wherein the monoclonal population of binding molecules exhibits an increased serum half-life upon administration to a subject animal relative to a reference IgM-derived binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the variant J-chain, and is administered using the same method to the same animal species.
• Embodiment 46 Embodiment 46.
• Embodiment 47 The monoclonal population of binding molecules of embodiment 45 or embodiment 46, wherein the variant J-chain or functional fragment thereof comprises 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.
• Embodiment 48 The monoclonal population of binding molecules of embodiment 47, wherein the amino acid corresponding to Y102 of SEQ ID NO: 6 is substituted with alanine (A).
• Embodiment 49 Embodiment 49.
• Embodiment 50 The monoclonal population of binding molecules of any one of embodiments 42 to 49, wherein the J-chain or fragment or variant thereof is a modified J- chain further comprising a heterologous moiety, wherein the heterologous moiety is fused or conjugated to the J-chain or fragment or variant thereof.
• Embodiment 51 The monoclonal population of binding molecules of embodiment 50, wherein the heterologous moiety is a polypeptide fused to the J-chain or fragment or variant thereof.
• Embodiment 52 Embodiment 52.
• Embodiment 53 The monoclonal population of binding molecules of embodiment 52, wherein the peptide linker comprises at least 5 amino acids, but no more than 25 amino acids.
• Embodiment 54 The monoclonal population of binding molecules of embodiment 52 or embodiment 53, wherein the peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 43).
• Embodiment 55 The monoclonal population of binding molecules of embodiment 52 or embodiment 53, wherein the peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 43).
• Embodiment 56 The monoclonal population of binding molecules of any one of embodiments 51 to 54, wherein the heterologous polypeptide is fused to the N-terminus of the J-chain or fragment or variant thereof or to the C-terminus of the J-chain or fragment or variant thereof.
• Embodiment 56 The monoclonal population of binding molecules of any one of embodiments 51 to 55, wherein heterologous moieties that can be the same or different are fused to the N-terminus and C-terminus of the J-chain or fragment or variant thereof.
• Embodiment 57 The monoclonal population of binding molecules of any one of embodiments 51 to 56, wherein the heterologous polypeptide comprises a binding domain.
• Embodiment 58 Embodiment 58.
• the monoclonal population of binding molecules of embodiment 60 wherein the modified J-chain comprises the amino acid sequence 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*), amino acids 20-420 of SEQ ID NO: 34 (VJH), amino acids 20-420 of SEQ ID NO: 35 (VJ*H), or SEQ ID NOs: 6 or 7 fused via a peptide linker to an anti-CD3 ⁇ scFv comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences comprising 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, respectively.
• Embodiment 62 A pharmaceutical composition comprising the monoclonal population of binding molecules of any one of embodiments 1 to 61 and a pharmaceutically acceptable excipient.
• Embodiment 63 A recombinant host cell that produces the monoclonal population of binding molecules of any one of embodiments 1 to 61.
• Embodiment 64 A method of producing the monoclonal population of binding molecules of any one of embodiments 1 to 61, comprising culturing the host cell of embodiment 62, and recovering the population of binding molecules.
• Embodiment 65 A method of producing the monoclonal population of binding molecules of any one of embodiments 1 to 61, comprising culturing the host cell of embodiment 62, and recovering the population of binding molecules.
• a method for producing a monoclonal population of highly sialylated multimeric binding molecules comprising providing a cell line that expresses the monoclonal population of binding molecules, culturing the cell line, and recovering the monoclonal population of binding molecules, wherein each binding molecule comprises ten or twelve IgM-derived heavy chains, wherein the IgM-derived heavy chains comprise glycosylated IgM heavy chain constant regions each associated with a binding domain that specifically binds to a target, wherein each IgM heavy chain constant region comprises at least three, at least four, or at least five asparagine(N)-linked glycosylation motifs, wherein an N-linked glycosylation motif comprises the amino acid sequence N- X1-S/T, wherein N is asparagine, X1 is any amino acid except proline, and S/T is serine or threonine, wherein on average at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy
• Embodiment 66 The method of embodiment 65, wherein the cell line, recovery process, or a combination thereof is optimized to result in a monoclonal population of binding molecules comprising at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, or at least 65 moles sialic acid per mole of binding molecule.
• Embodiment 67 The method of embodiment 66, wherein the cell line, recovery process, or a combination thereof is optimized to result in a monoclonal population of binding molecules comprising 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, about 60 to about 70 moles sialic acid per mole of binding molecule.
• Embodiment 68 The method of any one of embodiments 65 to 67, wherein the IgM heavy chain constant regions are derived from human IgM heavy chain constant regions comprising five N-linked glycosylation motifs N-X1-S/T starting 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).
• Embodiment 69 Embodiment 69.
• Embodiment 70 The method of any one of embodiments 65 to 69, wherein the provided cell line is modified to overexpress a sialyltransferase.
• Embodiment 71 The method of embodiment 70, wherein the sialyltransferase comprises human beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1, SEQ ID NO: 3).
• Embodiment 72 comprises human beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1, SEQ ID NO: 3).
• Embodiment 73 The method of embodiment 72, wherein the in vitro glycoengineering comprises contacting the monoclonal population of binding molecules with a soluble sialyltransferase and a sialic acid substrate.
• Embodiment 74 The method of embodiment 73, wherein the sialyltransferase comprises a soluble variant of human beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3).
• ST6GAL1 human beta-galactoside alpha-2,6-sialyltransferase 1
• Embodiment 76 The method of embodiment 75, wherein the soluble variant of ST6GAL1 comprises amino acids 120 to 406, 115 to 406, 110 to 406, 109 to 406, 105 to 406, 100 to 406, 95 to 406, 90 to 406, 89 to 406, 88 to 406, 87 to 406, 86 to 406, 85 to 406, 84 to 406, 83 to 406, 82 to 406, 81 to 406, 80 to 406, 75 to 406, 70 to 406, 65 to 406, 60 to 406, 55 to 406, 50 to 406, 45 to 406, 40 to 406, 35 to 406, 30 to 406, or 27 to 406 of SEQ ID NO: 3.
• Embodiment 77 The method of any one of embodiments 73 to 75, wherein the sialic acid substrate comprises cytidine monophosphate (CMP)-N-acetyl-neuraminic acid (CMP-NANA).
• Embodiment 78 The method of any one of embodiments 73 to 77, wherein the mass ratio of binding molecule: sialic acid substrate is about 1:4 to about 40:1.
• Embodiment 79 The method of any one of embodiments 73 to 78, wherein the mass ratio of binding molecule: sialyltransferase is about 80:1 to about 10000:1.
• Embodiment 80 Embodiment 80.
• Embodiment 81 The method of embodiment 80, wherein the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 2000:500:1.
• Embodiment 82 The method of any one of embodiments 73 to 79, wherein the molar ratio of binding molecule: sialyltransferase is about 80:1.
• Embodiment 83 The method of embodiment 82, wherein the molar ratio of binding molecule: sialic acid substrate: sialyltransferase is about 80:500:1.
• Embodiment 84 The method of any one of embodiments 73 to 79, wherein the mass ratio of binding molecule: sialyltransferase is about 500:1.
• Embodiment 85 The method of embodiment 84, wherein the mass ratio of binding molecule: sialic acid substrate: sialyltransferase is about 500:62.5:1.
• Embodiment 86 The method of any one of embodiments 73 to 85, wherein the contacting of the monoclonal population of binding molecules with the soluble sialyltransferase and the sialic acid substrate comprises at least 30 minutes of contact.
• Embodiment 87 Embodiment 87.
• Embodiment 86 wherein the contacting comprises at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 24 hours, 36 hours, or 48 hours of contact.
• Embodiment 88 The method of any one of embodiments 73 to 87, wherein the contacting of the monoclonal population of binding molecules with the soluble sialyltransferase and the sialic acid substrate occurs at about 2° C to about 40° C.
• Embodiment 89 The method of embodiment 88, wherein the contacting occurs at 15° C to about 37° C, 15° C to about 30° C, or 15° C to about 25° C.
• Embodiment 90 Embodiment 90.
• Embodiment 91 The method of embodiment 90, wherein the galactosyltransferase comprises a soluble variant of human beta-1,4-galactosyltransferase 4 (B4GALT4) (SEQ ID NO: 4).
• Embodiment 92 The method of embodiment 90 or embodiment 91, wherein the galactose substrate comprises uridine-diphosphate- ⁇ -D-galactose (UDP-Gal).
• Embodiment 93 The method of any one of embodiments 90 to 92, wherein the contacting with the galactosyltransferase and a galactose substrate occurs prior to or simultaneously with the contacting with the soluble sialyltransferase and a sialic acid substrate.
• the following examples are offered by way of illustration and not by way of limitation.
• Example 1 Materials and Methods Population of IgM antibodies [0279] Except as noted below, these experiments were carried out on a monoclonal population of the IgM bispecific antibody CD20 x CD3 IGM-A, which includes IgM heavy chains comprising wild-type human IgM constant regions (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) and an anti-CD20 VH region of SEQ ID NO: 8, light chains comprising the antiCD20 VL region of SEQ ID NO: 9, and a modified J-chain that binds to CD3 comprising amino acids 20-420 of SEQ ID NO: 34.
• CD20 x CD3 IGM-A is described in detail in U.S. Patent Application Publication No.
• Glycoengineered IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules are referred to as “GEMs” throughout the Examples regardless of the method of glycoengineering. Glycoengineering of IgM antibody populations [0280] Along with various controls, varying amounts of a truncated version of human ⁇ -2,6 sialyltransferase as indicated in the Examples below (“truncated human ST6,” available from Roche Diagnostics, Inc.
• the ST6 treated IgM populations were further purified, e.g., by anion exchange chromatography and/or mixed mode chromatography, prior to further analyses.
• Total Sialic Acid Quantitation [0281] The Sialic Acid (NANA) Assay Kit (Agilent AdvanceBio Total Sialic Acid Quantitation Kit) measures free or released sialic acid (N-acetylneuraminic acid (NANA)) from glycoproteins.
• the kit measures sialic acid in the linear range of 40 pmol-1,000 pmol with a detection sensitivity of 0.15 mg/ml concentration for IgM antibodies. The kit was used according to manufacturers’ recommendations. Briefly, samples were digested with sialidase A for 2 hours. A 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 with the following pmol for fluorescent measurement: 1,000, 500, 250 and 0 pmol. Conversion and Developer mix was then prepared according to the Table 2 below. Table 2: Sialic Acid Quantitation Assay [0282] Once sialic acid is released by the sialidase A digestion, N-acetylneuraminic aldolase catalyzes the reaction to form pyruvic acid. The reaction then goes through an additional step with pyruvate oxidase as the catalyst to form hydrogen peroxide which forms a 1:1 complex with the dye to form a fluorescence reporter dye.
• Example 2 Effect of ⁇ -2,6 sialyltransferase concentration on sialylation of IgM antibody populations [0283] Varying amounts of truncated human ST6 were added to a 20 ⁇ l reaction solution comprising 60 ⁇ g of anti-CD20 x CD3 IGM-A (3 mg/ml) and 30 ⁇ g of cytidine-5'- monophospho-N-acetylneuraminic acid sodium salt (CMP-NANA, 1.5 mg/ml) as described in Example 1.
• CMP-NANA cytidine-5'- monophospho-N-acetylneuraminic acid sodium salt
• Example 3 Sialylation of other IgM antibodies [0285] To determine if the in vitro sialylation procedures 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 Example 1.
• the reaction then goes through an additional step with pyruvate oxidase as the catalyst forming hydrogen peroxide which forms a 1:1 complex with the dye to form a fluorescence reporter dye.
• T cell activation (TCA) by anti-CD20 x CD3 IGM-A-GEM or anti-CD20 x CD3 IGM-A was determined using a luminescence-based readout in the presence of antigen-positive Jurkat based reporter cells.
• the mixture was incubated for 16h at 37 oC with 5% CO 2 .
• the cell mixtures were then mixed with 20 ⁇ L lysis buffer containing luciferin (Promega, CELLTITER-GLO®) to measure luciferase reporter activity. Light output was measured by EnVision plate reader.
• EC 50 was determined by 4 parameter curve fit using Prism software. [0291] The EC50 was calculated for each condition and is shown in Table 6. In vitro sialylation had no appreciable effect on T cell activation.
• Table 6 T cell Activation B-cell killing in vitro
• a CD19+CD20+ B cell line was labeled with a cell tracing dye (Oregon Green 488, ThermoFisher, Cat# C34555), and then co-cultured with primary human CD8+ T cells (Precision for Medicine, Cat# 84300; Negatively selected) with serial dilutions of anti-CD20 x CD3 IGM-A or anti-CD20 x CD3 IGM-A-GEM for 48 hours at 37 oC, 5% CO 2 .
• Cells were harvested and stained with 7-AAD (BD Biosciences, Cat# 559925) and analyzed by flow cytometry to assess viable B cells.
• 7-AAD BD Biosciences, Cat# 559925
• EC 50 was calculated for each condition and is shown in Table 7. In vitro sialylation had no appreciable effect on the ability of the antibody to kill B cells.
• Table 7 T-Cell dependent B Cell Killing Pharmacokinetics [0293] Pharmacokinetic parameters were measured for various IgM antibodies in an in vivo mouse model as follows. Balb/c mice were injected with 5 mg/kg of either anti-CD20 x CD3 IGM-A or anti-CD20 x CD3 IGM-A-GEM antibody via intravenous infusion. Blood samples were collected at 10 or 12 time points total for each antibody, with 2 mice per time point.
• Each mouse was bled once through the facial vein (100 ⁇ L) and then another time by terminal cardiac puncture (max obtainable, ⁇ 500 ⁇ L).
• a standard ELISA assay was used to measure the serum concentration of each antibody in the blood at each time point. Quality metrics were verified on all ELISAs, and PK parameters, including T 1/2-alpha , T 1/2-beta , and the area under the concentration curve from time zero to infinity (AUC 0- ⁇ , measured in ⁇ g/ml*hr) were derived using standard curve fitting techniques (Win Non Lin, Phoenix Software).
• the PK results, including area under the curve (AUC) are presented in FIG.6.
• Example 6 Cell line engineering to increase sialylation
• P15907.1), and a hygromycin marker selection was generated by standard methods.
• the vector was electroporated into a stable CHO subclone expressing anti-CD20 x CD3 IGM-A. After selection and recovery, the resulting pool was subcloned by limiting dilution into 384-well plates.
• Phenotypic screening was done by labelling 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 directly after being washed in FACS buffer. The fluorescence levels were detected, and the results are shown in FIG. 7. Only 4 of 60 subclones generated a signal above that of HEK293 cells, which were used as a positive control. Two subclones 25 and 47 were picked for further studies. [0295] To detect the presence of ST6 in the genome of the subclones 25 and 47, QPCR analysis was done using the primers in Table 8. Table 8: Primers used in a QPCR assay.
• FIG.8B The resulting image is shown in FIG.8B.
• the selected subclones had detectable levels of 2,6-sialic acid; whereas the stable pool of CHO cells from which the subclones originated did not.
• Subclone 25 was expanded for a 3-liter bioreactor production run so that a comparison study could be made against the parental anti-CD20 x CD3 IGM-A produced in cells which did not have the 2,6-sialyltransferase gene.
• FIGS. 9A-D shows how the cultures performed in terms of viable cell density (FIG. 9A), cell-viability (FIG. 9B), production of anti-CD20 x CD3 IGM-A (FIG.
• Example 7 2,6-Sialic Acid knock-in parental cell line [0300] A vector for stable insertion of alpha-2,6-sialyltransferase (NCBI Reference Sequence: NP_775324.1) and a hygromycin marker selection was generated by a commercial vendor. The vector was electroporated into a CHO suspension cell-line. The resulting stable pool was cloned, and 384 clones were expanded and screened by cytometry. Cell surface-based labeling was done with a 2,6-sialic acid specific lectin (SNA-1) which was chemically conjugated to a Cy5 dye, the results of this assay are shown in FIG. 10A.
• SNA-1 2,6-sialic acid specific lectin
• Table 9 shows the result of 7-day fed batch fermentations comparing the parental cell-line to the two 2,6-sialyltransferase clones.
• Sialic acid content was determined from the protein purified after the harvest.
• IgMs were transfected into 2B4 and the parental cell-line and two of these IgMs were also transfected into 2C2.
• Table 9 Harvest titer data and sialic acid levels on purified proteins from the harvested fermentation.
• 2,6-sialic acid and 2,3-sialic acid levels were measured on the cell surface by cytometry. Measurement was made using fluorescently conjugated lectins which are specific to either form of sialic acid.
• FIGS. 11A and 11B show the 2,3-sialic acid and 2,6-sialic acid levels for untransfected cells, respectively.
• FIG. 11C compares the 2,3-sialic acid and 2,6-sialic acid levels in untransfected and IgM #4 transfected parental and 2B4 cells.
• FIG.11A indicates that the CHO parental cell had higher 2,3-sialic acid levels then HEK293 cells or the clones transfected with 2,6-sialyltransferase.
• the data shown in FIG. 11B indicates the 2,6-sialic acid levels on the clones was elevated well above the level of the CHO parental.
• FIG. 11C indicates that the IgM transfected cell-lines retained high levels of 2,6-sialic acid.
• Example 8 in vitro sialylation under various conditions [0304] Varying amounts of truncated human ST6 were added to a reaction solution comprising an IgM antibody and cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt (CMP-NANA, 1.5 mg/ml) in ratios as shown in Table 10. The durations and temperatures for each reaction, and the resulting sialylation (quantitated as described in Example 1) are also shown in Table 10. Room temperature (RT) is 15 to 25 °C. Table 10: in vitro sialylation conditions and results
• the SA levels over 48 hours or over the first 15 hours are plotted in FIGS.13A and 13B, respectively.
• the SA levels of the antibodies from Conditions 32-36 were monitored at set timepoints throughout the reaction.
• the SA levels over 48 hours are plotted in FIG. 14.
• Antibody:CMP-NANA:ST-6 mass ratios of 100:50:1 achieved sialyation of >60 mol/mol SA by 18 h at RT and did not drop significantly for up to 48 hours at RT.
• Antibody:CMP- NANA:ST-6 mass ratios of 250:125:1 resulted in a slower rise in SA levels and reached >60 mol/mol SA after 36 h. For all ratios, no significant desialylation was observed at RT up to 36 hrs. At antibody:CMP-NANA:ST-6 mass ratios of 5000:2500:1, 40 mol/mol SA was achieved, and the antibody population was not sialylated to the maximum extent possible.
• Example 9 Pharmacokinetics of IgM antibodies with high sialic acid levels [0309] Pharmacokinetic parameters were measured for various IgM antibodies in an in vivo mouse model as follows.
• Balb/c mice were injected with 5 mg/kg of anti-CD20 x CD3 IGM-A, anti-CD20 x CD3 IGM-A-GEM antibodies of various sialic acid levels, anti- CD20 x CD3 IGM-F, anti-CD20 x CD3 IGM-F-GEM antibodies of various sialic acid levels, or human serum IgM via intravenous infusion.
• Blood samples were collected at 10 or 12 time points total for each antibody, with at least 2 mice per time point. Each mouse was bled once through the facial vein (100 ⁇ L) and then another time by terminal cardiac puncture (max obtainable, ⁇ 500 ⁇ L). A standard ELISA assay was used to measure the serum concentration of each antibody in the blood at each time point.
• PK parameters including T1/2-alpha, T1/2-beta, and the area under the concentration curve from time zero to infinity (AUC 0- ⁇ , measured in ⁇ g/ml*hr) were derived using standard curve fitting techniques (Win Non Lin, Phoenix Software). The sialic acid levels and the resulting AUC 0- ⁇ for each antibody are shown in FIG.15.
• Example 10 IgM antibodies with high sialic acid levels activity in cynomolgus monkeys [0310] Pharmacokinetic parameters and cellular markers were measured for IgM antibodies in an in vivo cynomolgus monkey model as follows.
• Cynomolgus primates were injected with 10 mg/kg of anti-CD20 x CD3 IGM-F (SA 18 mol/mol) (2 animals), anti-CD20 x CD3 IGM-F (SA 9 mol/mol) (2 animals), or anti-CD20 x CD3 IGM-F-GEM (SA 51 mol/mol) (4 animals) antibodies. Blood samples were collected at 12 time points total for each antibody. A standard ELISA assay was used to measure the serum concentration of each antibody in the blood at each time point.
• PK parameters including T 1/2-alpha , T 1/2-beta , and the area under the concentration curve from time zero to infinity (AUC 0- ⁇ , measured in ⁇ g/ml*hr) were derived using standard curve fitting techniques (Win Non Lin, Phoenix Software). Flow cytometry was used to measure cellular markers.
• the PK results for anti-CD20 x CD3 IGM-F (SA 18 mol/mol) and 2 of the 4 anti-CD20 x CD3 IGM-F-GEM (SA 51 mol/mol) treated animals are presented in FIG. 16.
• the AUC 0- ⁇ for the high sialic acid antibody was twofold higher than for the low sialic acid antibody.
• Example 11 in vitro sialylation with multiple enzymes
• Sialylation only was completed by mixing 120 ⁇ g anti-CD20 x CD3 IGM-A antibody with 1 mol/mol SA or 21 mol/mol SA, 60 ⁇ g of CMP NANA, and 20 ⁇ g ST6 (a mass ratio of IgM:CMP NANA:ST6 of 6:3:1). The sample was then incubated for 24 hours at 37 °C.

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