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WO-2026093188-A1 - MULTIMERIZATION DOMAINS FOR THE GENERATION OF HOMOGENOUS MULTIMERS WITH IMMUNOGLOBULIN BACKBONE

WO2026093188A1WO 2026093188 A1WO2026093188 A1WO 2026093188A1WO-2026093188-A1

Abstract

The present invention relates to multimerization domains for the generation of homogenous multimers with immunoglobulin backbone, and polypeptides, conjugates and multimers comprising such multimerization domains. In particular, the present invention deals with multimers of IgM and IgG backbones having a tail domain which differs from the wild-type IgM tailpiece.

Inventors

  • LANGER, THOMAS
  • LEUSCHNER, Wulf Dirk
  • RAO, ERCOLE
  • SOMMERFELD, MARK
  • WEIL, Sandra

Assignees

  • SANOFI

Dates

Publication Date
20260507
Application Date
20251027
Priority Date
20241028

Claims (15)

  1. 1. A polypeptide comprising a first immunoglobulin domain (IGD1), a second immunoglobulin domain (IGD2) and a tail domain (TD), wherein the IGD1, IGD2 and TD have the following structure from N-terminus to C-terminus: IGD1 - IGD2 - TD and wherein (ii) IGD1 is selected from IgM CH3 and an immunoglobulin constant domain, in particular IgG CH2, IgD CH2, IgE CH3, IgAl CH2 or IgA2 CH2, comprising a substitution of an amino acid residue into a Cys residue at a position allowing the formation of a disulphide bond with a Cys of an IGD1 of another identical or different polypeptide according to claim 1, in particular a polypeptide comprised in a multimer according to claim 11 in an adjacent dimer; (ii) IGD2 is an immunoglobulin constant domain, in particular selected from IgM CH4, IgG CH3, IgAl CH3, IgA2 CH3, IgD CH3, and IgE CH4; and (iii) TD is selected from a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of homogenous multimers, in particular dodecamers, compared to a human wild-type IgM tail piece, and a non-mammalian IgA or IgM tail piece that is heterologous to the IGD1 and/or IGD2.
  2. 2. The polypeptide according to claim 1, wherein IGD1 is selected from IgM CH3 and IgG CH2 L328C, wherein the numbering of the amino acid position is according to EU numbering.
  3. 3. The polypeptide according to claim 1 or 2, wherein TD comprises or consists of the sequence KPXIX2X 3 X 4 VSX 5 X6X7X8X9X10X11 X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X 4 is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is A, S, G or N; wherein if X4 is N, then Xi is A, X2 is V or H, X3 is L, V or I, X5 is V, X7 is Y and/or X9 is E.
  4. 4. The polypeptide according to any one of claims 1 to 3, wherein the substitution in TD replaces an Asn residue, in particular an Asn residue at position 6 of SEQ ID NO: 1, at position 440 of the IgM constant domain (according to SEQ ID NO: 23), position 340 of the IgAl constant domain (according to SEQ ID NO: 24) or position 327 of the IgA2 constant domain (according to SEQ ID NO: 25), with another amino acid.
  5. 5. The polypeptide according to any one of claims 1 to 4, wherein TD has a sequence selected from (a) KPTLYQVSLVMSDTAGTCY (human IgM N440Q) (SEQ ID NO: 3), (b) KPTHVQVSVVMAEVDGTCY (human IgAl N340Q) (SEQ ID NO: 4), (c) KPTHIQVSVVMAEADGTCY (human IgA2 N327Q) (SEQ ID NO: 5), (d) KPAVLQVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, N440Q, L443V, M445Y, D447E) (SEQ ID NO: 6), (e) KPAVLNVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, L443V, M445Y, D447E) (SEQ ID NO: 7), (f) NQPNLVNLSLNVPQRCMAQ (trout IgM tail piece) (SEQ ID NO: 8), (g) PSFVNISLALMDTINSCQ (nurse shark IgM tail piece) (SEQ ID NO: 9), (h) NRJDLNMNINQDSKCSA (Nile tilapia IgM tail piece) (SEQ ID NO: 10), and (i) KPTNVNVSLVLSDTC (Xenopus laevis IgM tail piece) (SEQ ID NO: 11).
  6. 6. The polypeptide according to any one of claims 1 to 5, further comprising a dimerization domain (D), wherein the IGD1, IGD2, D and TD have the following structure from N- terminus to C-terminus: D - IGD1 - IGD2 - TD and D is selected from an immunoglobulin constant domain, an immunoglobulin hinge region and a peptide linker; in particular wherein D comprises a Cys residue at a position allowing the formation of a disulphide bond with a Cys of a D domain of another identical or different polypeptide according to claim 8: and/or is a third immunoglobulin domain (IGD3).
  7. 7. The polypeptide according to any one of claims 1 to 6, further comprising an effector domain (E), which comprises a diagnostic or therapeutic polypeptide or part thereof; in particular wherein E comprises one or more antigen binding sites; more particularly E comprises a single domain antibody or a single chain antibody.
  8. 8. A conjugate comprising a polypeptide according to any one of claims 1 to 7 covalently linked to a diagnostic or therapeutic agent (F).
  9. 9. A protein complex comprising or consisting of a first polypeptide according to any one of claims 1 to 7 and a second polypeptide comprising an immunoglobulin variable domain and an immunoglobulin constant domain.
  10. 10. A dimer comprising or consisting of two identical or different polypeptides according to any one of claims 1 to 7, two identical or different conjugates according to claim 8, a polypeptide according to any one of claims 1 to 7 and a conjugate according to claim 8, a polypeptide according to any one of claims 1 to 7 and a protein complex according to claim 9, or two identical or different protein complexes according to claim 9.
  11. 11. A multimer comprising or consisting of identical or different polypeptides according to the first aspect, identical or different conjugates according to the second aspect, identical or different protein complexes according to the third aspect, or identical or different dimers according to the fourth aspect, in particular a dodecamer comprising or consisting of twelve identical or different polypeptides according to any one of claims 1 to 7, twelve identical or different conjugates according to claim 8, twelve identical or different protein complexes according to claim 9, or six identical or different dimers according to claim 10.
  12. 12. One or more polynucleotides encoding the polypeptide according to any one of claims 1 to 7, the polypeptides of the protein complex according to claim 9, the polypeptides of the dimer according to claim 10, or the polypeptides of the multimer according to claim 11.
  13. 13. One or more vectors comprising the one or more polynucleotides according to claim 12.
  14. 14. A cell comprising the one or more polynucleotides according to claim 12 or the one more vectors according to claim 13.
  15. 15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound according to any of claims 1 to 7, the conjugate according to claim 8, the protein complex according to claim 9, the dimer according to claim 10, the multimer according to claim 11, the one or more polynucleotides according to claim 12, the one or more vectors according to claim 13, or the cell according to claim 14.

Description

Sanofi PAT23105 MULTIMERIZATION DOMAINS FOR THE GENERATION OF HOMOGENOUS MULTIMERS WITH IMMUNOGLOBULIN BACKBONE The present invention relates to multimerization domains for the generation of homogenous multimers with immunoglobulin backbone, and polypeptides, conjugates and multimers comprising such multimerization domains. BACKGROUND OF THE INVENTION Naturally occurring IgM molecules consist of five IgM monomers and a Joining-chain (J-chain), forming a pentamer: IgM(5)J. The formation of the IgM(5)J-complex is dependent on C-terminal sequences of the IgM heavy chains called the tailpiece (TP). The TPs and the J- chain form an intermolecular beta-sheet network. Incorporation of the J-chain into this network is the bottleneck of recombinant expression of IgM(5)J. The function of the J-chain is binding to the polymeric immunoglobulin receptor (plgR). Upon binding to the plgR on the basolateral side of epithelial cells, the IgM(5)J complex is transported to the apical face of the epithelial cell and secreted, resulting a short half-life of IgM(5)J molecules. Recombinant IgM can also be expressed without J-chain, resulting in a mixture of IgM(5) pentamers and IgM(6) hexamers, which is highly undesired for biopharmaceutical products which often require high homogeneity. There remains a need in the art for an effective way to produce homogenous multimeric IgM molecules. A multimeric IgM backbone may also be useful as a platform for the development of novel biotherapeutics. Producing homogenous multimeric immunoglobulin-based molecules without the J-chain would significantly simplify protein production and lead to an enhanced plasma half-life due to lack of binding to plgR. Surprisingly, the inventors identified variant mammalian TPs and non-mammalian TPs that, when fused to IgM heavy chain sequences, lead to an increase in the formation of one type of multimers, usually hexamers, and a decrease in the formation of other multimers, thus promoting homogenous hexamerization. Besides production of homogenous multimeric proteins, the expression yields using these TP sequences were considerably high, which is a prerequisite for usage in the development of biotherapeutics. Thus, the polypeptides of the present invention provide inter alia for one or more of the following advantages: (i) homogenous formation of multimers; (ii) increased formation of hexamers; (iii) decreased formation of pentamers; (iv) simplified recombinant production; (v) high expression yields; (vi) increased half-life, (vii) no epithelial transcytosis, (viii) no secretion. SUMMARY OF THE INVENTION In a first aspect, the invention relates to a polypeptide comprising a first immunoglobulin domain (IGD1), a second immunoglobulin domain (IGD2) and a tail domain (TD), wherein the IGD1, IGD2 and TD have the following structure from N-terminus to C- terminus: IGD1 - IGD2 - TD and wherein IGD1 is selected from IgM CH3 and an immunoglobulin constant domain comprising a substitution of an amino acid residue into a Cys residue at a position allowing the formation of a disulphide bond with a Cys of an IGD1 of another identical or different polypeptide; IGD2 is an immunoglobulin constant domain; and TD is selected from a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of homogenous multimers of the polypeptide compared to a human wild-type IgM tail piece, and a non-mammalian IgA or IgM tail piece that is heterologous to the IGD1 and/or IGD2. In a second aspect, the invention relates to a conjugate comprising a polypeptide according to the first aspect covalently linked to a diagnostic or therapeutic agent (F). In a third aspect, the invention relates to a protein complex comprising or consisting of a first polypeptide according to the first aspect and a second polypeptide comprising an immunoglobulin variable domain and an immunoglobulin constant domain. In a fourth aspect, the invention relates to a dimer comprising or consisting of two identical or different polypeptides according to the first aspect, two identical or different conjugates according to the second aspect, a polypeptide according to the first aspect and a conjugate according to the second aspect, a polypeptide according to the first aspect and a protein complex according to the third aspect, a conjugate according to the second aspect and a protein complex according to the third aspect, or two identical or different protein complexes according to the third aspect. In a fifth aspect, the invention relates to a multimer comprising or consisting of identical or different polypeptides according to the first aspect, identical or different conjugates according to the second aspect, identical or different protein complexes according to the third aspect, or identical or different dimers according to the fourth aspect, in particular a dodecamer comprising or consisting of twelve identical or different polypeptides according to the first aspect, twelve identi