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CN-121986120-A - Bispecific polypeptide molecules

CN121986120ACN 121986120 ACN121986120 ACN 121986120ACN-121986120-A

Abstract

The present invention relates to bispecific polypeptide molecules capable of simultaneously binding a disease-associated antigen and a cell surface antigen expressed on the surface of an immune effector cell (e.g. a T cell), thereby activating the immune effector cell.

Inventors

  • M. von Essen
  • HOYAL DAVID
  • P. Masson
  • E. Rahm
  • T. Kornfoss

Assignees

  • 埃纳拉生物有限公司

Dates

Publication Date
20260505
Application Date
20240705
Priority Date
20230705

Claims (20)

  1. 1. A bispecific polypeptide molecule capable of simultaneously binding a first antigen and a second antigen, wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: (A) The first polypeptide chain comprises (i) a first antigen binding domain, (ii) a first hinge domain subunit, (iii) a first Fc domain subunit, and (B) The second polypeptide chain comprises (i) a second antigen binding domain, (ii) a second hinge domain subunit, (iii) a second Fc domain subunit; Wherein the first and second hinge domain subunits are capable of forming a stable binding as a hinge domain and the first and second Fc domain subunits are capable of forming a stable binding as an Fc domain or Fc domain portion such that the two polypeptide chains are linked by covalent and/or non-covalent bonds between the hinge domain subunits and the Fc domain subunits, preferably wherein the first and second antigens are expressed on two different cells.
  2. 2. Bispecific polypeptide molecule according to claim 1, wherein the first antigen binding domain comprises a first binding region (VD 1) of an antibody variable domain and a second binding region (VD 2) of an antibody variable domain, and a first linker (LNK 1) connecting said domains, wherein the first binding region (VD 1) and the second binding region (VD 2) combine to form a first antigen binding site (VD 1) (VD 2).
  3. 3. The bispecific polypeptide molecule according to claim 2, wherein VD1 is at the N-terminus of VD2, or wherein VD2 is at the N-terminus of VD1, and wherein VD1 and VD2 are connected via a first linker LNK1, optionally wherein VD1 is at the N-terminus of VD2, and VD1 and VD2 are connected via LNK 1.
  4. 4. A bispecific polypeptide molecule according to claim 2 or claim 3, wherein the first binding region (VD 1) of the variable domain comprises an antibody variable light chain domain (VL) or an epitope-binding portion thereof, and the second binding region (VD 2) of the variable domain comprises an antibody variable heavy chain domain (VH) or an antigen-binding portion thereof, optionally wherein the variable light chain domain (VL) and/or variable heavy chain domain (VH) epitope or a respective binding portion thereof, may further comprise part or all of a corresponding antibody heavy chain constant region CL and/or CH 1.
  5. 5. The bispecific polypeptide molecule according to any one of claims 2 to 4, wherein the linker LNK1 (a) is a flexible linker of 3 to 20 amino acids and/or comprises small non-polar amino acids and/or small polar amino acids, optionally wherein LNK1 (a) comprises amino acids comprising glycine, glycine serine and/or threonine, or (b) comprises at least one sequence motif selected from GGGS, GGGGS, TVLRT, TVSSAS and TVLSSAS, or (c) comprises a sequence selected from ggggsggggggggggggs (SEQ ID No. 46) or GSADDAKKDAAKKDGKS (SEQ ID No. 47).
  6. 6. The bispecific polypeptide molecule of claim 5, wherein the linker LNK1 comprises all or part of an immunoglobulin (Ig) hinge sequence.
  7. 7. The bispecific polypeptide molecule according to any one of claims 2 to 6, wherein VD1 and VD2 comprise an engineered disulfide bond introducing a covalent bond between VD1 and VD2, wherein in the case of VL cysteine is introduced into framework region 4 (FR 4), in the case of VH cysteine is introduced into framework region 2 (FR 2), or in the case of VH cysteine is introduced into framework region 4 (FR 4), in the case of VL cysteine is introduced into framework region 2 (FR 2).
  8. 8. The bispecific polypeptide molecule according to claim 7, wherein the linker LNK1 comprises a first LNK1 cysteine residue (Cys) and a second LNK1 cysteine residue (Cys), wherein a first disulfide bond is formed between the introduced VH cysteine (Cys) and the first LNK1 Cys, and/or a second disulfide bond is formed between the introduced VL Cys and the second LNK1 Cys.
  9. 9. The bispecific polypeptide molecule according to claim 8, wherein the linker LNK1 comprises the sequence CPPC (SEQ ID No. 52), and/or 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 polypeptide molecule according to any one of claims 1 to 9, wherein the first antigen binding domain comprises, or consists of, a single chain Fv (scFv).
  11. 11. The bispecific polypeptide molecule of any one of the preceding claims, wherein the first antigen binding domain is capable of specifically binding to a cell surface antigen expressed on the surface of an immune effector cell (optionally a human immune effector cell), optionally wherein the immune effector cell expresses an activating receptor, and wherein the first antigen binding domain binds to the activating receptor, resulting in activation of the immune effector cell.
  12. 12. The bispecific polypeptide molecule of 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 polypeptide molecule according to claim 11 or 12, wherein the activated receptor is selected from the group consisting of CD3, such as CD3y, CD35 and CD3E chain , 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, CD1 17, 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 polypeptide molecule according to any one of claims 1 to 13, wherein the first binding region (VD 1) of the variable domain and the second binding region (VD 2) of the variable domain are derived from a humanized anti-CD 3 antibody or a variant thereof.
  15. 15. The bispecific polypeptide molecule according to claim 14, wherein the humanized anti-CD 3 antibody variant is a humanized anti-CD 3 antibody variant UCHT1, optionally UCHT1 v.9, wherein the variable domain (VD 1) comprises an antibody variable light chain domain (VL) of SEQ ID No.1 or an epitope-binding portion thereof, and wherein the second binding region (VD 2) of the variable domain comprises an antibody variable heavy chain domain (VH) of SEQ ID No.2 or an epitope-binding portion thereof.
  16. 16. The bispecific polypeptide molecule according to any one of claims 1to 15, wherein the first antigen binding domain or the first antigen binding site (VD 1) (VD 2) binds or specifically binds to a first antigen with an affinity (KD) of about 100 μm or less.
  17. 17. The bispecific polypeptide molecule according to any one of the preceding claims, wherein the second antigen binding domain comprises a first binding region (VR 1) of a TCR variable domain and a second binding region (VR 2) of a TCR variable domain, and a second linker (LNK 2) connecting the domains, wherein the first binding region (VR 1) and the second binding region (VR 2) combine to form a second antigen binding site (VR 1) (VR 2).
  18. 18. The bispecific polypeptide molecule according to claim 17, wherein the first binding region (VR 1) of the variable domain comprises a TCR alpha chain variable domain (vα) or an antigen or MHC-related peptide epitope binding portion thereof, the second binding region (VR 2) of the variable domain comprises a TCR beta chain variable domain (vβ) or an antigen or MHC-related peptide epitope binding portion thereof, optionally wherein the first binding region (VR 1) of the variable domain may further comprise part or all of a TCR alpha chain constant domain cα, preferably cα is linked or fused to the C-terminus of a vα domain, and/or the second binding region (VR 2) of the variable domain may further comprise part or all of a TCR beta chain constant domain cβ, preferably cβ is linked or fused to the C-terminus of a vβ domain.
  19. 19. The bispecific polypeptide molecule according to claim 18, wherein VR1 is at the N-terminus of VR2 or VR2 is at the N-terminus of VR1, and wherein VR1 and VR2 are linked by LNK2, optionally wherein VR2 is at the N-terminus of VR1 and VR2 and VR1 are linked by LNK 2.
  20. 20. The bispecific polypeptide molecule according to any one of claims 17 to 19, wherein the linker LNK2 (a) is a flexible linker of 3 to 20 amino acids and/or comprises small non-polar amino acids and/or small polar amino acids, optionally wherein LNK2 (a) comprises amino acids comprising glycine, glycine serine and/or threonine, or (b) comprises at least one sequence motif selected from GGGS, GGGGS, TVLRT, TVSSAS and TVLSSAS, or (c) comprises a sequence selected from ggggsgggggggggggggs (SEQ ID No. 46) or GSADDAKKDAAKKDGKS (SEQ ID No. 47).

Description

Bispecific polypeptide molecules The present invention relates to bispecific polypeptide molecules capable of simultaneously binding a disease-associated antigen and a cell surface antigen expressed on the surface of an immune effector cell (e.g. a T cell), thereby activating the immune effector cell. The invention also relates to polynucleotides encoding the bispecific polypeptide molecules, as well as vectors and host cells containing these nucleic acids. The invention also relates to a method for producing the bispecific polypeptide molecule and to a method for using the bispecific polypeptide molecule in the treatment of diseases. Background The focus in the field of cancer therapy has been on the development of bispecific molecules, which are dual active molecules, combining a first binding domain specific for an epitope on a tumor cell and a second binding domain specific for an epitope on an immune effector cell. Various arrangements of binding domains have been proposed with the aim of redirecting the activity of immune effector cells to the tumor site and providing promising immunostimulants for the treatment of cancer. For bispecific molecules, different forms have been proposed, including forms with or without IgG Fc regions, combinations based on symmetrical or asymmetrical designs of IgG-derived or TCR-derived components. The epitope binding region of such bispecific molecules typically incorporates an antibody or TCR-derived binding domain. The discovery and production of single chain linked variable domains (scFv) of antibodies has led to the development of bispecific antibody-derived molecules, such as BiTE. BiTE molecules, such as Blinatumomab, a CD19 targeting BiTE,(Baeuerle, P.A.; Reinhardt, C. "bispecific T-cell engaging antibodies for cancer therapy", Cancer Res. 2009, 69, 4941-4944), have been promoted for cancer treatment. These molecules co-engage the CD3E subunit on T cells and the surface antigen on tumor cells, triggering T cell mediated tumor killing. Simultaneous engagement of the target cell antigen with CD3 results in activation of polyclonal cytotoxic T cells, thereby resulting in lysis of the target cells. BiTE is a small bispecific molecule with a very short serum half-life and is difficult to produce and purify due to its size and easy aggregation. The chains of BiTE antibody-derived binding domains are linked by internal linker molecules, providing flexibility to the construct and favorable antigen binding kinetics for the specific antigen targeted. Dual affinity redirect molecules (DARTs) have a similar basic structure, but contain disulfide linkers for additional interchain stability. Bispecific (BiKE) and trispecific killer cell engagers (TriKE) consist of two (BiKE) or three (TriKE) variable antigen binding regions that activate natural killer cells by binding to CD16 and optionally comprising an IL15 cross-linker [ Allen, c., life 2021, 11 (6), 465]. DART molecules have been demonstrated to be highly effective in redirecting T-cell killing B-cell lymphomas. DART molecules have been shown to be more efficient in directed B cell lysis than single chain bispecific antibodies with the same CD19 and CD3 antibody Fv sequences. The use of IgG Fc in bispecific constructs can overcome the problem of short serum half-life. Initially, a flexible linker peptide was fused to the C-terminus of an IgG heavy chain to allow ligation of single-chain variable domains with different binding specificities, thereby forming tetravalent bispecific polypeptide molecules with increased production levels and a more simplified purification process (Coloma, M.J. and Morrison, S.L.(1997), "design and production of novel tetravalent bispecific antibodies", Nat. Biotechnol. 15, 159-163)., e.g., fc added to anti-P-cadherin/anti-CD 3 bispecific DART molecules, forming DART-Fc, with a significant half-life extension while maintaining high efficiency for cancer targets. Bispecific polypeptide molecular formats based on IgG are generally improved by incorporating engineered Fc mutations to promote heterodimerization of two different CH3 domains, thereby linking two polypeptide chains that may have different binding functions, and optimally combining them into 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). by additional introduction of cysteine residues to form stable disulfide bonds between heterodimeric CH3 domains, further improving the Fc format. Incorporating Fc into the form of a bispecific polypeptide molecule provides the advantage that the interaction of the Fc portion of the bispecific polypeptide molecule with the human Fc receptor FcRn can be achieved. By interacting with neonatal FcRn, the half-life of IgG forms is prolonged. Fc mutations have further been developed in order to ensure that recombinant molecules can bind to target molecules without causing significant