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EP-4739395-A2 - BISPECIFIC POLYPEPTIDE MOLECULE

EP4739395A2EP 4739395 A2EP4739395 A2EP 4739395A2EP-4739395-A2

Abstract

The present invention relates to bispecific polypeptide molecules capable of simultaneously binding to a disease associated antigen and a cell surface antigen expressed on the surface of an immune effector cell such as a T-cell resulting in the activation of the immune effector cell.

Inventors

  • VON ESSEN, Magdalena
  • HOWIE, Duncan
  • MASON, PETER
  • LAM, Emily
  • CORNFORTH, Terri

Assignees

  • Enara Bio Limited

Dates

Publication Date
20260513
Application Date
20240705

Claims (20)

  1. Claims 1. A bispecific polypep^de molecule capable of simultaneously binding afirst and second an^gen, wherein the bispecific polypep^de molecule comprises afirst polypep^de chain and a second polypep^de chain, wherein (A) thefirst polypep^de chain comprises: (i) afirst an^gen-binding domain, (ii) afirst hinge domain subunit, (iii) afirst Fc domain subunit; and (B) the second polypep^de chain comprises: (i) a second an^gen-binding domain, ii) a second hinge domain subunit, (iii) a second Fc domain subunit; wherein thefirst and second hinge domain subunits are capable of forming a stable associa^on as a hinge domain and thefirst and second Fc domain subunits are capable of forming a stable associa^on as an Fc domain or Fc domain por^on, such that the two polypep^de chains are connected by covalent and/or non-covalent bonds between the hinge domain subunits and Fc-domain subunits, preferably wherein thefirst and second an^gens are expressed on two dis^nct cells.
  2. 2. The bispecific polypep^de molecule according to claim 1 wherein thefirst an^gen-binding domain comprises afirst binding region of a variable domain (VD1) of an an^body and a second binding region of a variable domain (VD2) of an an^body and afirst linker (LNK1) connec^ng said domains wherein thefirst binding region (VD1) and the second binding region (VD2) associate to form afirst an^gen binding site (VD1)(VD2).
  3. 3. The bispecific polypep^de molecule according to claim 2, wherein VD1 is N-terminal to VD2 or wherein VD2 is N terminal to VD1, and wherein VD1 and VD2 are connected by the first linker LNK1, op^onally wherein VD1 is N-terminal to VD2 and VD1 and VD2 are connected by LNK1.
  4. 4. The bispecific polypep^de molecule according to either claim 2 or claim 3, wherein the first binding region of a variable domain (VD1) comprises an an^body variable light domain (VL) or epitope binding por^on thereof and the second binding region of a variable domain (VD2) comprises an an^body variable heavy domain (VH) or an^gen binding por^on thereof, op^onally wherein said variable light domain (VL) and/or variable heavy domain (VH) epitope or respec^ve binding por^on thereof, may further comprise part or all of a respec^ve an^body heavy chain constant domain, CL and/or CH1.
  5. 5. The bispecific polypep^de molecule according to any one of claims 2 to 4, wherein the linker LNK1 (a) is aflexible linker of between 3 and 20 amino acids and/or comprising of small, non-polar and/or small polar amino acids, op^onally wherein LNK1 (a) comprises amino acids including glycine, or glycine serine and/or threonine or (b) comprises at least one sequence mo^f selected from GGGS, GGGGS, TVLRT, TVSSAS, and TVLSSAS or (c) comprises a sequence selected from GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO.46) or GSADDAKKDAAKKDGKS, (SEQ ID NO.47).
  6. 6. The bispecific polypep^de molecule according to claim 5 wherein the linker LNK1 comprises all or part of an immunoglobulin (Ig) hinge sequence.
  7. 7. The bispecific polypep^de molecule according to any of claims 2 to 6 wherein VD1 and VD2 comprise an engineered disulphide bridge introducing a covalent bond between VD1 and VD2, wherein cysteines are introduced into framework region 4 (FR4) in case of VL and framework region 2 (FR2) in case of VH or are introduced into framework region 4 (FR4) in case of VH and framework region 2 (FR2) in case of VL.
  8. 8. The bispecific polypep^de molecule according to claim 7, wherein the linker LNK1 comprises afirst LNK1 cysteine residue (Cys) and a second LNK1 cysteine residue (Cys), wherein afirst disulphide bond is formed between the introduced VH cysteine (Cys) and a first LNK1 Cys and/or a second disulphide bond is formed between the introduced VL Cys and a second LNK1 Cys.
  9. 9. The bispecific polypep^de molecule according to claim 8, wherein the linker LNK1 comprises the sequence CPPC (SEQ ID No.52) and/or the LNK1 comprises an amino acid sequence selected from any of: GGGSGGSGGCPPCGGSGG (SEQ ID NO.17), GGGSDDSGGCPPCGGKGG (SEQ ID NO.18), and GGAAGGSGGCPPCGGSGG(SEQ ID NO.19).
  10. 10. The bispecific polypep^de molecule according to any of claims 1 to 9, wherein thefirst an^gen-binding domain comprises or consists of a single chain Fv (scFv).
  11. 11. The bispecific polypep^de molecule according to any preceding claim, wherein thefirst an^gen-binding domain is capable of specifically binding to a cell surface an^gen expressed on the surface of an immune effector cell, op^onally human immune effector cell, op^onally wherein said immune effector cell expresses an ac^va^ng receptor and wherein thefirst an^gen-binding domain binds to the activating receptor resulting in immune effector cell activation.
  12. 12. The bispecific polypep^de molecule according to claim 11 wherein the immune effector cell is a T- cell, a CD4+ T-cell, a CD8+ T-cell, a natural killer cell, a macrophage, a granulocyte, or a dendritic cell, optionally a CD8+ T-cell.
  13. 13. The bispecific polypep^de molecule according to claim 11 or 12, wherein activating receptor is selected from the group consis^ng of: CD3, such as the CD3y, CD35, and CD3E chains, CD4, CD7, CD8, CD10, CD11 b, CD11 c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41 , CD41 b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61 , CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FCERI, TCRa/β and TCRy/δ, HLA-DR.
  14. 14. The bispecific polypep^de molecule according to any of claims 1 to 13, wherein thefirst binding region of a variable domain (VD1) and the second binding region of a variable domain (VD2) are derived from the humanised an^-CD3 an^body or variant thereof.
  15. 15. The bispecific polypep^de molecule according to claim 14, wherein the humanised an^- CD3 an^body variant is a humanised an^-CD3 an^body variant, UCHT1, op^onally UCHT1 v.9, wherein the variable domain (VD1) comprises an an^body variable light domain (VL) of SEQ ID No.1. or epitope binding por^on thereof and wherein the second binding region of a variable domain (VD2) comprises an an^body variable heavy domain (VH) of SEQ ID No.2, or epitope binding por^on thereof.
  16. 16. The bispecific polypep^de molecule according to any of claims 1 to 15, wherein said first antigen binding domain or said first antigen binding site (VD1 )(VD2) binds or specifically binds the first antigen with an affinity (KD) of about 100 μΜ or less.
  17. 17. The bispecific polypep^de molecule according to any preceding claim, wherein the second an^gen-binding domain comprises afirst binding region of a variable domain (VR1) of a TCR and a second binding region of a variable domain (VR2) of a TCR and a second linker (LNK2) connec^ng said domains wherein thefirst binding region (VR1) and the second binding region (VR2) associate to form a second an^gen binding site (VR1 )(VR2).
  18. 18. The bispecific polypep^de molecule according to claim 17 wherein thefirst binding region of a variable domain (VR1) comprises a TCR α chain variable domain (Vα) or an^gen or MHC-associated pep^de epitope binding por^on thereof and the second binding region of a variable domain (VR2) comprises a TCR β chain variable domain (Vβ) or an^gen or MHC- associated pep^de epitope binding por^on thereof, op^onally wherein thefirst binding region of a variable domain (VR1) may further comprise part or all of a TCR α chain constant domain, Cα, preferably linked to or fused to the C-terminus of the Vα domain and/or the second binding region of a variable domain (VR2) may further comprise part or all of TCR β chain constant domain, Cβ, preferably linked to or fused to the C-terminus of the Vβ domain.
  19. 19. The bispecific polypep^de molecule according to claim 18, wherein VR1 is N-terminal to VR2 or VR2 is N terminal to VR1; and wherein VR1 and VR2 are connected by LNK2, op^onally wherein VR2 is N-terminal to VR1 and VR2 and VR1 are connected by LNK2.
  20. 20. The bispecific polypep^de molecule according to any one of claims 17 to 19, wherein the linker LNK2 (a) is aflexible linker of between 3 and 20 amino acids and/or comprising of small, non-polar and/or small polar amino acids, op^onally wherein LNK2 (a) comprises amino acids including glycine, or glycine serine and/or threonine or (b) comprises at least one sequence mo^f selected from GGGS, GGGGS, TVLRT, TVSSAS, and TVLSSAS or (c) comprises a sequence selected from GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO.46) or GSADDAKKDAAKKDGKS, (SEQ ID NO.47).

Description

BISPECIFIC POLYPEPTIDE MOLECULE The present inven^on relates to bispecific polypep^de molecules capable of simultaneously binding to a disease associated an^gen and a cell surface an^gen expressed on the surface of an immune effector cell such as a T-cell resul^ng in the ac^va^on of the immune effector cell. The inven^on further relates to polynucleo^des encoding the bispecific polypep^de molecules, and vectors and host cells containing these nucleic acids. The inven^on further relates to methods for producing the bispecific polypep^de molecules, and methods of their use in the treatment of disease. Background of the invention There has been a focus in the field of cancer therapy over the past several decades on the development of bispecific molecules which are dual activity molecules combing a first binding domain specific for an epitope on tumour cells and a second binding domain specific for an epitope on immune effector cells. Various arrangements of binding domains have been proposed with the intention being to redirect the activity of immune effector cells to the site of tumour and to offer promising immune-stimulatory agents to treat cancer. Different formats have been proposed for bispecific molecules, including formats with or without IgG Fc regions, combining symmetric or asymmetric designs based on IgG-derived or TCR-derived component parts. Epitope binding regions of such bispecific molecules generally incorporate either antibody or TCR derived binding domains. The discovery and production of single-chain connected variable domains of antibodies (scFvs) led to the development of bispecific antibody derived molecules like the BiTE®. BiTE molecules, such as Blinatumomab, a BiTE targeting CD19, (Baeuerle, P.A.; Reinhardt, C. “bispecific T-cell engaging antibodies for cancer therapy”, Cancer Res.2009, 69, 4941-4944) have been promoted for cancer therapy. These molecules co-engage the CD3E subunit on T cells and a surface antigen on the tumour cell and thereby trigger T cell-mediated killing of the tumour. Concurrent engagement of the target cell antigen and CD3 leads to activation of polyclonal cytotoxic T-cells, resulting in target cell lysis. Being a small bispecific molecule, the BiTE, has a very short serum half-life, its size also makes it difficult to produce and purify due to aggregation. The chains of BiTEs an^body derived binding domains are connected by internal linker molecules, providingflexibility to the construct and favourable an^gen- binding kine^cs for the specific an^gens targeted. Dual-affinity re-targe^ng molecules (DARTs) have a similar basic structure but include a disulphide linker for addi^onal inter- chain stability. Bispecific (BiKEs) and trispecific killer cell engagers (TriKEs) consist of either two (BiKE) or three (TriKE) variable an^gen binding regions and ac^vate natural killer cells either by binding to CD16 and op^onally containing an IL15 cross-linker [Allen, C., Life 2021, 11(6), 465]. DART molecules proved highly effective at redirected T-cell killing of B-cell lymphoma. DART molecules proved to be more potent in directing B-cell lysis than a single- chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences. Use of IgG Fc in bispecific constructs has been employed to overcome issues of short serum half life. Originally flexible linker peptides were fused to the C termini of the heavy chains of IgG to permit the attachment of single-chain variable domains with different binding specificities to form a tetravalent bispecifics with increased productivity levels and more simplified purification (Coloma, M.J. and Morrison, S.L. (1997), “design and production of novel tetravalent bispecific antibodies”, Nat. Biotechnol.15, 159-163). For example, the addition of Fc to the Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule to form DART-Fc- achieved a significantly extended half-life whilst maintaining high potency for its cancer target. IgG-based bispecific formats have been generally improved by incorporation of engineered Fc mutations to facilitate the hetero-dimerization of two differing CH3-domains thereby connecting two different polypeptide chains which may have different binding functions optimally combined into the one therapeutic molecule; (Ridgway JB, et al. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng.1996 Jul;9(7):617-21). This Fc format was further improved by the additional introduction of cysteine-residues to form a stabilising disulphide-bond between the heterodimeric CH3- domains. Inclusion of the Fc into a bispecific format provided the advantage of accessing an interaction of the Fc-part of the bispecific with the human Fc- receptor FcRn. This prolonged the half-life of the IgG format through interac^on with neonatal FcRn. Further Fc mutations were developed to ensure that the recombinant molecule is capable of binding the target molecule without triggering significant complement depe