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JP-7857221-B2 - Highly sialized polymer-binding molecules

JP7857221B2JP 7857221 B2JP7857221 B2JP 7857221B2JP-7857221-B2

Inventors

  • キート ブルース
  • バリガ ラメシュ
  • アフメド サチ
  • カーリン ケヴィン
  • ヒントン ポール
  • スミス マーク
  • サイニ アブネシュ
  • トラン フエン
  • ピーターソン マーヴィン

Assignees

  • アイジーエム バイオサイエンシズ インコーポレイテッド

Dates

Publication Date
20260512
Application Date
20210105
Priority Date
20200106

Claims (20)

  1. A monoclonal population of multimeric IgM-binding molecules, each binding molecule comprising 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, the N-linked glycosylation motif comprising the amino acid sequence N- X1 -S/T, where N is asparagine and X The monoclonal population of the polymer IgM binding molecule wherein 1 is any amino acid other than proline, and S/T is serine or threonine, and at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region are occupied by a complex glycan, and the monoclonal population of the binding molecule contains at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 moles of sialic acid per mole of the binding molecule.
  2. A monoclonal population of a polymer IgM-binding molecule according to claim 1, comprising 40 to 70, 40 to 60, 40 to 55, 40 to 50, 50 to 70, or 60 to 70 moles of sialic acid per mole of the binding molecule.
  3. A monoclonal population of a multimeric IgM-binding molecule according to claim 1 or 2, wherein each IgM heavy chain constant region is a human IgM heavy chain constant region or a variant thereof, comprising five N-linked glycosylated motifs N-X1-S/T starting from amino acid positions corresponding to amino acid 46 (motif N1), amino acid 209 (motif N2), amino acid 272 (motif N3), amino acid 279 (motif N4), and amino acid 440 (motif N5) of SEQ ID NO: 1 (allele IGHM*03) or SEQ ID NO: 2 (allele IGHM*04).
  4. Each binding molecule is a pentameric or hexamer IgM antibody containing five or six divalent IgM binding units, and each binding unit comprises two IgM-derived heavy chains, each containing a heavy chain variable domain (VH) located at the amino-terminus of the variant IgM heavy chain constant region, and two immunoglobulin light chains, each containing a light chain variable domain (VL) located at the amino-terminus of the immunoglobulin light chain constant region, wherein the VH and VL bind to form an antibody-derived antigen-binding domain that specifically binds to the target, and furthermore, each of the five or six divalent IgM binding units specifically binds to the same target, as described in any one of claims 1 to 3.
  5. The monoclonal population of a multimeric IgM-binding molecule according to claim 4, wherein each binding molecule is a pentamer and further comprises a J chain or a functional fragment thereof or a functional variant, and optionally, the J chain is a mature human J chain, a functional fragment thereof, or a functional variant thereof, comprising the amino acid sequence shown in SEQ ID NO: 6.
  6. The monoclonal population of a polymeric IgM-binding molecule according to claim 5, wherein the variant J chain or its functional fragment contains an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J chain of SEQ ID NO: 6, and optionally, the amino acid corresponding to Y102 of SEQ ID NO: 6 is substituted with alanine (A).
  7. The monoclonal population of the polymeric IgM-binding molecule according to claim 6, wherein the J chain contains the amino acid sequence shown in SEQ ID NO: 7.
  8. The monoclonal population of a polymer IgM- binding molecule according to any one of claims 5 to 7, wherein the J chain or its functional fragment or functional variant is a modified J chain further comprising a heterogeneous portion, the heterogeneous portion being a polypeptide fused to the J chain or its functional fragment or functional variant, and optionally via a peptide linker comprising at least 5 but no more than 25 amino acids.
  9. The monoclonal population of a polymeric IgM-binding molecule according to claim 8, wherein the heterogeneous polypeptide comprises an scFv fragment, and optionally, the scFv fragment is bound to CD3ε.
  10. A pharmaceutical composition comprising a monoclonal population of a polymer IgM-binding molecule as described in any one of claims 1 to 9, and a pharmaceutically acceptable excipient.
  11. Recombinant host cells that generate a monoclonal population of a multimeric IgM-binding molecule as described in any one of claims 1 to 9.
  12. The recombinant host cell according to claim 11, wherein the cell is transfected with one or more genes encoding glycosyltransferases, and optionally, the one or more genes encoding galactosyltransferases that provide acceptor residues for sialic acid residues via α-2,6 and/or α-2,3 linkages.
  13. The recombinant host cell according to claim 11 or 12, wherein the cell is transfected with one or more genes encoding sialyltransferase, thereby improving and/or enhancing the cell's ability to promote the transfer of sialic acid monosaccharides from a CMP-NANA substrate or its derivative to a compatible acceptor oligosaccharide.
  14. The recombinant host cell according to claim 13, wherein the sialyltransferase comprises any of the following: human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1, SEQ ID NO: 3), human β-galactoside α-2,6-sialyltransferase-II (ST6GALII), or four β-galactoside α2-3-sialyltransferases (ST3GAL-I-IV).
  15. The recombinant host cell according to any one of claims 12 to 14, wherein the galactosyltransferase comprises human β-1,4-galactosyltransferase 4 (B4GALT4).
  16. A method for generating a monoclonal population of a highly sialylated multimeric IgM-binding molecule according to any one of claims 1 to 9, comprising: providing a cell line expressing the monoclonal population of the IgM-binding molecule; culturing the cell line; and recovering the monoclonal population of the IgM-binding molecule, wherein each binding molecule comprises 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least 3, at least 4, or at least 5 asparagine (N)-linked glycosylation motifs, each N-linked glycosylation motif comprising the amino acid sequence N- X1 -S/T, where N is asparagine and X The method wherein 1 is any amino acid other than proline, and S/T is serine or threonine, and on average at least one, at least two, or at least three of the N-linked glycosylation motifs on each IgM heavy chain constant region in the population are occupied by complex glycans, and the cell line, harvesting process, or a combination thereof is optimized to concentrate complex glycans containing at least one, two, three, or four sialic acid-terminal monosaccharides per glycan, thereby yielding a monoclonal population of highly sialylated IgM-binding molecules containing at least 35, at least 40, at least 45, at least 50, at least 60, or at least 65 moles of sialic acid per mole of binding molecule, or 40–70, 40–60, 40–55, 40–50, 50–70, or 60–70 moles of sialic acid per mole of binding molecule.
  17. The method according to claim 16, wherein the cell line is modified to overexpress sialyltransferase.
  18. The method according to claim 16 or 17, wherein the recovery process comprises subjecting the monoclonal population of IgM-binding molecules to in vitro glycosylation, and the in vitro glycosylation comprises contacting the monoclonal population of IgM-binding molecules with a soluble sialyltransferase and a sialic acid substrate.
  19. The method according to claim 17 or 18, wherein the sialyltransferase comprises a soluble variant of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (SEQ ID NO: 3), and/or the sialic acid substrate comprises cytidine monophosphate (CMP)-N-acetylneuraminic acid (CMP-NANA).
  20. The method according to claim 18, wherein the mass ratio of the binding molecule to the sialic acid substrate is 1:4 to 40:1, and/or the mass ratio of the binding molecule to the sialyltransferase is 80:1 to 5000:1.

Description

Cross-reference of related applications This application claims the rights to U.S. Provisional Patent Application Serial Number 62/957,745, filed on 6 January 2020, which is incorporated herein by reference in its entirety. Sequence Listing This application includes a sequence listing, which is filed electronically in ASCII format and is incorporated in its entirety herein by reference. An ASCII copy prepared on 5 January 2021 is named 028WO1-Sequence-Listing and is 92,335 bytes in size. Background Antibodies and antibody-like molecules that can be multimerized, such as IgA and IgM antibodies, are emerging as promising drug candidates in fields such as immuno-oncology and infectious diseases, enabling improved specificity, enhanced binding affinity, and the ability to bind to multiple binding targets. For example, see U.S. Patent No. 9,951,134 (Patent Document 1), No. 9,938,347 (Patent Document 2), No. 10,351,631 (Patent Document 3), No. 10,400,038 (Patent Document 4), No. 10,570,191 (Patent Document 5), No. 10,604,559 (Patent Document 6), No. 10,618,978 (Patent Document 7), No. 10,689,449 (Patent Document 8), and No. 10,787,520 (Patent Document 9), U.S. Patent Application Publication No. 2019-0330. See Patent Document No. 374 (Patent Document 10), Patent Document No. 2019-0330360 (Patent Document 11), Patent Document No. 2019-0338040 (Patent Document 12), Patent Document No. 2019-0338041 (Patent Document 13), Patent Document No. 2019-0185570 (Patent Document 14), and Patent Document No. 2019-0002566 (Patent Document 15), Patent Document No. 2020-0239572 (Patent Document 16), as well as PCT Publications WO2018/187702 (Patent Document 17) and WO2019/165340 (Patent Document 18). The contents of these are incorporated herein by reference in their entirety. The pharmacokinetics (PK) and pharmacodynamics (PD) of multivalent antibodies are complex and depend on both the translated and posttranslational structures of monoclonal antibodies, as well as the physiological systems they target. Furthermore, different antibody classes are typically processed within a target via different cells and physiological systems. For example, the serum half-life of the IgG antibody class is 20 days, while the half-lives of IgM and IgA antibodies are only about 5–8 days (Brekke, OH., and I. Sandlie, Nature Reviews Drug Discovery 2:52–62 (2003) (Non-Patent Literature 1)). One of the key determinants of the pharmacokinetic (PK) of antibodies or other biotherapeutic agents is their level and type of glycosylation (Higel, F. et al. Eur. J. Pharma. Biopharm. 139:123-131 (2019) (Non-Patent Literature 2)). The sugar moieties and their derivatives covalently bound to specific residues of an antibody determine how they are recognized by receptors such as asialoglycoprotein (ASGP) receptors, and in turn, how quickly they are cleared from the systemic circulation. Each IgM heavy chain constant region has five asparagine-(N-) linked glycosylation sites, and the J chain has one N- linked glycosylation site. Therefore, the J chain of a pentamer containing IgM can contain up to 51 glycan moieties, resulting in a complex glycosylation profile (Hennicke, J., et al., Anal. Biochem. 539:162-166 (2017) (Non-Patent Literature 3)). This glycan complexity can make the production of uniformly glycosylated materials difficult. Despite advancements in the design of multimeric antibodies, the need to manipulate the physical, pharmacokinetic, and pharmacodynamic properties of these molecules remains. Strength rating 9,951,134U.S. Patent No. 9,938,347Strength rating 10,351,631U.S. Patent No. 10,400,038Strength rating 10,570,191Strength rating 10,604,559Strength No. 10,618,978Strength rating 10,689,449U.S. Patent No. 10,787,520U.S. Patent Application Publication No. 2019-0330374U.S. Patent Application Publication No. 2019-0330360U.S. Patent Application Publication No. 2019-0338040U.S. Patent Application Publication No. 2019-0338041U.S. Patent Application Publication No. 2019-0185570U.S. Patent Application Publication No. 2019-0002566U.S. Patent Application Publication No. 2020-0239572WO2018/187702WO2019/165340 Brekke, OH. , and I. Sandlie, Nature Reviews Drug Discovery 2:52-62 (2003)Higel, F. et al. Eur. J. Pharm. Biopharm. 139:123-131 (2019)Hennicke, J. , et al. , Anal. Biochem. 539:162-166 (2017) Summary Provided herein is a monoclonal population of polymer-binding molecules, each binding molecule comprising 10 or 12 IgM-derived heavy chains, each IgM-derived heavy chain comprising a glycosylated IgM heavy chain constant region bound to a binding domain that specifically binds to a target, each IgM heavy chain constant region comprising at least one, at least two, at least three, at least four, or at least five asparagine (N)-linked glycosylation motifs, each N-linked glycosylation motif comprising the amino acid sequence N- X1 -S/T, where N is asparagine, X1 is any amino acid other than proline, and S/T is serine or threonine, and at least one, at le