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EP-4735476-A1 - CHARGE PAIR MUTATIONS TO ENABLE CORRECT HEAVY-LIGHT CHAIN PAIRING

EP4735476A1EP 4735476 A1EP4735476 A1EP 4735476A1EP-4735476-A1

Abstract

This application relates to the facilitation of selective binding between modified VH and VL domains by introducing amino acids of complementary charges in the two domains at novel locations within these domains. These alterations are particularly useful in designing and generating multispecific antibodies, such as bispecific antibodies, in which the charge pair mutations in VH and VL domains can facilitate desired pairing between particular heavy and light chain polypeptides. These charge pair mutations can be used on their own, or in combination with additional strategies to promote correct pairing of polypeptide chains.

Inventors

  • RILEY, TIMOTHY PATRICK

Assignees

  • Amgen Inc.

Dates

Publication Date
20260506
Application Date
20240626

Claims (20)

  1. 1. An isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are bound together, wherein the VH and VL comprise at least one of the following sets of charged amino acids: a. the VH comprises a charged amino acid at position number 39, and the VL comprises a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH; b. the VH comprises a charged amino acid at position number 105, and the VL comprises a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH; or c. the VH comprises a charged amino acid at position number 91, and the VL comprises a charged ammo acid at position number 38 that is complementary in charge to the amino acid at position 91 of the VH; and wherein the position numbers of the charged amino acids in the VH and VL domains refer to their positions according to the Kabat numbering scheme.
  2. 2. The isolated protein of claim 1, wherein the VH and VL comprise the set of charged amino acids in (a).
  3. 3. The isolated protein of claim 2, wherein the VH comprises a positively charged amino acid at position 39 and the VL comprises a negatively charged amino acid at position 85.
  4. 4. The isolated protein of claim 3, wherein the VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) a lysine at VH39 and an aspartic acid at VL85, (ii) a lysine at VH39 and a glutamic acid at VL85. (iii) an arginine at VH39 and an aspartic acid at VL85, or (iv) an arginine at VH39 and a glutamic acid at VL85.
  5. 5. The isolated protein of claim 2, wherein the VH comprises a negatively charged amino acid at position 39 and the VL comprises a positively charged amino acid at position 85.
  6. 6. The isolated protein of claim 5, wherein the VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) an aspartic acid at VH39 and a lysine at VL85, (ii) a glutamic acid at VH39 and a lysine at VL85, (iii) an aspartic acid at VH39 and an arginine at VL85, or (iv) a glutamic acid at VH39 and an arginine at VL85.
  7. 7. The isolated protein of any of claims 1-6, wherein the VH and VL comprise the set of charged amino acids in (b).
  8. 8. The isolated protein of claim 7, wherein the VH comprises a positively charged amino acid at position 105 and the VL comprises a negatively charged amino acid at position 42.
  9. 9. The isolated protein of claim 8, wherein the VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) a lysine at VH105 and an aspartic acid at VL42, (ii) a lysine at VH105 and a glutamic acid at VL42, (hi) an arginine at VH105 and an aspartic acid at VL42, or (iv) an arginine at VH105 and a glutamic acid at VL42.
  10. 10. The isolated protein of claim 7, wherein the VH comprises a negatively charged amino acid at position 105 and the VL comprises a positively charged amino acid at position 42.
  11. 11. The isolated protein of claim 10, wherein the VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) an aspartic acid at VH105 and a lysine at VL42, (ii) a glutamic acid at VH105 and a lysine at VL42. (iii) an aspartic acid at VH105 and an arginine at VL42, or (iv) a glutamic acid at VH105 and an arginine at VL42.
  12. 12. The isolated protein of any of claims 1-11, wherein the VH and VL comprise the set of charged amino acids in (c).
  13. 13. The isolated protein of claim 12, wherein the VH comprises a positively charged amino acid at position 91 and the VL comprises a negatively charged amino acid at position 38.
  14. 14. The isolated protein of claim 13, wherein the VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) a lysine at VH91 and an aspartic acid at VL38, (ii) a lysine at VH91 and a glutamic acid at VL38, (iii) an arginine at VH91 and an aspartic acid at VL38, or (iv) an arginine at VH91 and a glutamic acid at VL38.
  15. 15. The isolated protein of claim 12, wherein the VH comprises a negatively charged amino acid at position 91 and the VL comprises a positively charged amino acid at position 38.
  16. 1 . The isolated protein of claim 15, wherein the VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) an aspartic acid at VH91 and a lysine at VL38, (ii) a glutamic acid at VH91 and a lysine at VL38, (iii) an aspartic acid at VH91 and an arginine at VL38, or (iv) a glutamic acid at VH91 and an arginine at VL38.
  17. 17. The isolated protein of any of claims 1-16, wherein the isolated protein comprises an antibody heavy chain comprising the VH and an antibody light chain comprising the VL.
  18. 18. The isolated protein of claim 17, wherein the isolated protein is an antibody.
  19. 19. The isolated protein of claim 18, wherein the antibody is an IgG antibody.
  20. 20. The isolated protein of claim 19, wherein the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody.

Description

CHARGE PAIR MUTATIONS TO ENABLE CORRECT HEAVY-LIGHT CHAIN PAIRING REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0001] The present application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The computer readable format copy of the Sequence Listing, which was created on June 5, 2024. is named 10572-W001-SEC_ST26 and is 8.69 kilobytes in size. TECHNICAL FIELD [0002] The disclosure relates to the generation of multispecific antibodies. For example, the disclosure relates to the use of charge pair mutations in the variable regions of heavy and light chain polypeptide sequences to facilitate correct pairing between particular desirable heavy and light chain polypeptides. BACKGROUND [0003] Multispecific antibodies such as bispecific antibodies are an exciting generation of biotherapeutics, enabling simultaneous or sequential targeting of two or more unique epitopes located on the same, or distinct, targets. This dual-recognition capability enables diverse applications such as recruiting immune cells to kill tumor cells, crosslinking distinct cell surface receptors, or improving tissue specificity. (Labrijn AF et al., Nat. Rev. Drug Discov. 18:585-608 (2019); Lu RM et al., J. Biomed. Sci. 27: 1 (2020); Fan G et al., J. Hematol. Oncol. 8: 130 (2015)) For example, Amgen's Bispecific T-cell Engager (BiTE®) creates an artificial immune synapse between cytotoxic T cells and target tumor cells, by simultaneously binding a CD3 epitope on the surface of T cells and a tumor- associated antigen. (Wolf E et al. , Drug Discov. Today 10: 1237-44 (2005); Kantarjian H et al., N. Engl. J. Med. 376:836-47 (2017)) As of 2019, over 100 bispecific formats have been reported, with over 85 in development and three receiving US Food and Drug Administration approval. (Labrijn AF et al.. Nat. Rev. Drug Discov. 18:585-608 (2019); Brinkmann U and Kontermann RE, MAbs 9: 182-212 (2017); Wang Q et al.. Antibodies (Basel) 8(3):43 (2019)) [0004] There are two major design strategies to generate bispecific molecules. The first approach is to encode two or more unique fragment variable (Fv) sequences on the same polypeptide chain(s), as with formats such as BiTEs®, IgG-scFv, or DVD-Ig. (Wang Q et al. , Antibodies (Basel) 8(3):43 (2019); Spiess C et al.. Mol Immunol 67:95-106 (2015)) These single-chain formats bypass the challenges associated with assembling multiple polypeptide chains into a single molecule, but they also tend to show poor yields and suboptimal stability. Alternative strategies utilize biophysics and engineering to ‘steer’ individual chains to the correct orientation, while simultaneously disfavoring mispaired scenarios. Examples include technologies like Knob-into-Holes (KiH), Charge Pair Mutations (CPMs) and Strand-Exchange Engineered Domains (SEEDbody) and have been utilized in molecules requiring hetero-Fc pairing. (Davis JH et al., Protein Eng. Des. Sei. 23: 195-202 (2010); Dillon M et al. , MAbs 9:213-30 (2017); Gunasekaran K et al., J. Biol. Chem. 285: 19637-46 (2010); Ridgway JB et al. , Protein Eng. 9:617-21 (1996)). This second approach enables the production of molecules that more closely mimic the native structures of IgG molecules, allowing for increased stability and more diverse format design. (Wang Q et al., Antibodies (Basel) 8(3):43 (2019)) However, these engineering strategies are often imperfect, and chain-mispairing continues to pose a challenge. (Ha JH et al., Front. Immunol. 7:394 (2016)) [0005] Mispairing between heavy and light chains is a significant concern in the generation of multispecific antibodies. For example, if the incorrect light chain (LC) pairs with the undesired heavy chain (HC), the resulting mispaired molecule translates to an impurity that must be removed. Multiple protein engineering strategies have been proposed to address HC-LC pairing (Krah, S et al., Nat. Biotechnol ., 39(B): 167-173 (2017)), but due to the variability of the Fv interface, a solution designed for one molecule may not necessarily be applicable to the next. Accordingly, there is a need in the art for improved methods for facilitating the pairing between particular desirable heavy and light chain polypeptides. And there is a need in particular for pairings within the Fv interface. SUMMARY [0006] This application relates to the facilitation of selective binding between modified VH and VL domains by introducing amino acids of complementary charges in the two domains at novel locations within these domains. For example, alterations may be made at VH position 39 and VL position 85, at VH position 105 and VL position 105, or VH position 91 and VL position 38. These alterations are particularly useful in designing and generating multispecific antibodies, such as bispecific antibodies, in which the charge pair mutations in VH and VL domains can facilitate desired pairing between particular heavy and light chain polypeptide