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CN-121986114-A - Anti-RTK antibodies and uses thereof

CN121986114ACN 121986114 ACN121986114 ACN 121986114ACN-121986114-A

Abstract

The present invention relates to RTK agonists, particularly FGFR2b agonists, FGFR1b agonists and DDR1 agonists, and methods of using the same.

Inventors

  • Ko tokuyuki
  • NOGUCHI TAKAAKI
  • YANG QIUYING

Assignees

  • 中外制药株式会社

Dates

Publication Date
20260505
Application Date
20240927
Priority Date
20230929

Claims (20)

  1. 1. An agonistic antigen binding molecule that binds to a Receptor Tyrosine Kinase (RTK) protein, wherein the molecule is an agonist of the RTK protein, wherein the molecule comprises a first antigen binding domain that binds to a first epitope of a first RTK protein and a second antigen binding domain that binds to a second epitope of the first RTK protein or a second RTK protein, wherein the first epitope resides in the first domain of the RTK protein and the second epitope resides in the second domain of the RTK protein.
  2. 2. The agonistic antigen binding molecule of claim 1, wherein the RTK protein is a human fibroblast growth factor receptor 2 (FGFR 2) protein, wherein the molecule comprises a first antigen binding domain that binds a first epitope of an extracellular domain (ECD) of a first FGFR2 protein and a second antigen binding domain that binds a second epitope of the extracellular domain (ECD) of the first FGFR2 protein or a second FGFR2 protein, wherein the first epitope and the second epitope are different from each other.
  3. 3. The molecule of claim 2, wherein the first antigen binding domain binds a first FGFR2 protein and the second antigen binding domain binds a second FGFR2 protein.
  4. 4. A molecule according to claim 2 or 3 which is a dual site antigen binding molecule.
  5. 5. The molecule of any one of claims 2-4, wherein the human FGFR2 protein is a human FGFR2b protein.
  6. 6. The molecule of any one of claims 2-5, wherein each of the extracellular domains is independently selected from an immunoglobulin (Ig) -like domain of an FGFR2 protein.
  7. 7. The molecule of any one of claims 2-6, wherein the first antigen binding domain binds to a first domain of the FGFR2 protein and the second antigen binding domain binds to a second domain of the FGFR2 protein, wherein the first domain and the second domain are the same.
  8. 8. The molecule of any one of claims 2-6, wherein the first antigen binding domain binds to a first domain of the FGFR2 protein and the second antigen binding domain binds to a second domain of the FGFR2 protein, wherein the second domain is different from the first domain.
  9. 9. The molecule of any one of claims 2-8, wherein the first antigen binding domain and the second antigen binding domain bind to an Ig-like domain of the FGFR2 protein independently of each other, the Ig-like domain selected from the group consisting of IgI, igII, igIII, igIIIb and IgIIIc of FGFR 2.
  10. 10. The molecule of any one of claims 6 to 9, wherein the first antigen binding domain and the second antigen binding domain, independently of each other, bind to an Ig-like domain of FGFR2 in a manner selected from the group consisting of: (i) Wherein the first antigen binding domain and the second antigen binding domain bind to IgIIIb and IgIIIb domains of FGFR2 independently of each other; (ii) Wherein the first antigen binding domain and the second antigen binding domain bind to IgIIIb and IgIII domains of FGFR2 independently of each other; (iii) Wherein the first antigen binding domain and the second antigen binding domain bind to IgIIIb and IgII domains of FGFR2 independently of each other, and (Iv) Wherein the first antigen binding domain and the second antigen binding domain bind to IgIIIb and IgI domains of FGFR2 independently of each other.
  11. 11. The molecule of any one of claims 6 to 10, wherein (i) the first antigen binding domain binds to IgIIIb domains of FGFR2, or wherein (ii) the first antigen binding domain specifically binds to IgIIIb of FGFR2 and exhibits less than 10% or less than 5% or 0% cross-reactivity with IgIIIc of FGFR2 and less than 10% or less than 5% or 0% cross-reactivity with IgI and IgII of FGFR 2.
  12. 12. The molecule of any one of claims 4 to 11, wherein binding of the first antigen binding domain and the second antigen binding domain to FGFR2b protein results in the following agonist activity: (i) Wherein the molecule activates signaling in a target cell via binding of FGFR2b expressed on the cell surface in the target cell to a FGFR2b protein, thereby exhibiting agonist activity, and wherein the activated signal in the target cell is measured by increasing expression of an intracellular protein downstream of FGFR2b by at least 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10% or more compared to expression of the intracellular protein prior to binding of the molecule to the FGFR2b protein expressed on the cell surface in the target cell, in particular wherein the intracellular protein downstream of FGFR2b is selected from the group consisting of pAKT, ppcg 1, pFRS, pPAK4, pRAS, pMAPK1/pERK2 and pMAPK/pERK 1, or (Ii) Wherein the molecule induces proliferation of the target cell via binding of FGFR2b expressed on the cell surface in the target cell to FGFR2b protein, thereby exhibiting agonist activity, wherein induction of proliferation of the target cell is measured by an increase in ATP expression in the target cell in the presence of the molecule of at least 1.5-fold, or 2-fold, or 2.5-fold or more, as compared to ATP expression in the target cell in the absence of the molecule, wherein the ATP expression is proportional to the concentration of live target cells, In particular wherein the target cell is a lung epithelial cell, optionally wherein the lung epithelial cell is selected from alveolar type II (AT 2) cells, or (Iii) Wherein receptor dimerization is detected in the presence of the molecule in an in vitro cell assay, wherein receptor dimerization is indicative of agonist activity, or (Iv) Wherein phosphorylation of the FGFR2b protein is detected in an in vitro cell assay in the presence of the molecule, wherein FGFR2b protein phosphorylation is indicative of agonist activity.
  13. 13. The agonistic antigen binding molecule of claim 1, wherein the RTK protein is a human fibroblast growth factor receptor 1 (FGFR 1) protein, wherein the molecule comprises a first antigen binding domain that binds a first epitope of the extracellular domain (ECD) of a first FGFR1 protein and a second antigen binding domain that binds a second epitope of the extracellular domain (ECD) of the first FGFR1 protein or a second FGFR1 protein, wherein the first epitope and the second epitope are different from each other.
  14. 14. The agonistic antigen binding molecule of claim 1, wherein the RTK protein is a human discotic domain receptor 1 (DDR 1) protein, wherein the molecule comprises a first antigen binding domain that binds a first epitope of an extracellular domain (ECD) of a first DDR1 protein and a second antigen binding domain that binds a second epitope of an extracellular domain (ECD) of the first DDR1 protein or a second DDR1 protein, wherein the first epitope and the second epitope are different from each other.
  15. 15. The molecule of any one of claims 1 to 14, wherein the first antigen binding domain and the second antigen binding domain are each independently selected from Fab, fab ', F (ab)' 2, scFab, scFv, fv, VHH, or single domain antibodies, or wherein the first antigen binding domain and the second antigen binding domain are each Fab, fab ', F (ab)' 2, scFab, scFv, fv, VHH, or single domain antibodies, the first antigen binding domain and the second antigen binding domain being linked by at least one disulfide bond.
  16. 16. The molecule of any one of claims 1 to 15, wherein the first antigen binding domain and the second antigen binding domain are characterized by having at least one disulfide bond formed between amino acid residues that are not in the hinge region.
  17. 17. The molecule of claim 16, wherein the at least one disulfide bond is an engineered disulfide bond not present in wild-type IgG, optionally wherein the at least one disulfide bond is formed between a CH1 region, CL region, VH or VHH region, or VL region of the first antigen binding domain and a CH1 region, CL region, VH or VHH region, or VL region of the second antigen binding domain, particularly wherein the at least one disulfide bond is formed between a group selected from the group consisting of: [i] amino acid residues in the CH1 region of the first antigen binding domain and amino acid residues in the CH1 region of the second antigen binding domain; [ ii ] amino acid residues in the CL region of the first antigen binding domain and amino acid residues in the CL region of the second antigen binding domain; (iii) amino acid residues in the CH1 region of the first antigen binding domain and amino acid residues in the CL region of the second antigen binding domain; [ iv ] amino acid residues in the VH or VHH region of the first antigen binding domain and amino acid residues in the VH or VHH region of the second antigen binding domain; [ v ] amino acid residues in the VL region of the first antigen-binding domain and amino acid residues in the VL region of the second antigen-binding domain, and [ Vi ] amino acid residues in the VH or VHH region of the first antigen binding domain and amino acid residues in the VL region of the second antigen binding domain.
  18. 18. The molecule according to any one of claims 1 to 17, wherein the molecule comprises an antibody constant region, in particular (i) wherein the antibody constant region is a human antibody constant region, or (ii) wherein the antibody constant region is selected from a human IgG1, igG2, igG3 or IgG4 constant region.
  19. 19. The molecule of any one of claims 1 to 18, wherein the molecule is characterized by two antigen binding domains, or wherein the molecule is a bivalent molecule, or wherein the first and second antigen binding domains are each a monovalent Fab or a monovalent VHH, or are each a monovalent scFab.
  20. 20. The molecule of any one of claims 1 to 19, wherein the molecule comprises more than two antigen binding domains, or wherein the molecule is a trivalent, tetravalent, or multivalent molecule, optionally wherein the molecule comprises a third antigen binding domain that binds the same epitope on the same domain as the first antigen binding domain or the second antigen binding domain.

Description

Anti-RTK antibodies and uses thereof Technical Field The present invention relates to RTK agonists, particularly FGFR2b agonists, FGFR1b agonists and DDR1 agonists, and methods of using the same. Background Receptor Tyrosine Kinases (RTKs) play a vital role in various cellular processes. RTKs are known to be activated by ligand-induced dimerization. Dimerization results in kinase activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor, as well as activation of downstream signaling of various pathways. One of the members of the RTK includes a family of Fibroblast Growth Factor Receptors (FGFRs) expressed by various mammalian cells. The FGFR family consists of FGFR1, FGFR2, FGFR3 and FGFR4, and includes the high affinity receptor (NPL 1) of many different FGF ligands. This receptor is a transmembrane tyrosine kinase, and FGFR signaling drives the downstream pathway (NPL 2) necessary for cell proliferation, differentiation, survival and migration. Binding of FGF ligand to receptor induces dimerization of FGF: FGFR complex and activation of FGF downstream signaling (NPL 3). Members of the FGF family are potent stimulators of tissue repair and have been shown to contribute to many tissue regeneration processes, such as maintaining cell stem properties, inducing cell dedifferentiation, stimulating cell proliferation, inhibiting cell senescence, inhibiting cell death, modulating inflammation, stimulating angiogenesis, and enhancing protease expression, among others. FGF ligands perform these diverse functions by binding to and activating the FGFR family of RTKs. FGFR2 has two splice subtypes, FGFR2b, which is normally located in epithelial cells, and FGFR2c (NPL 1), which is normally located in mesenchymal cells. FGFR2b is a high affinity receptor for FGF ligands such as FGF7 and FGF10, while FGFR2c is a high affinity receptor (NPL 3) for FGF ligands such as FGF 2. Although various therapeutic agents targeting FGFR2 have been developed, such as anticancer drugs targeting FGFR2 overexpressing cells, these drugs are inhibitors of FGFR2, and there remains a significant unmet medical need for the development of FGFR2 agonists. FGFR1 also has two splice subtypes, FGFR1b is expressed in brain, tongue, glandular prepuce, vas deferens and skin, and FGFR1c is expressed in various tissues (NPL 8). FGFR1b is a receptor for FGF ligands such as FGF1 and FGF2, and FGFR1b is also a weak affinity receptor (NPL 9) for FGF7 and FGF 10. FGFR1c is shown to bind to various types of FGF ligands (including FGF1, FGF2, FGF4, FGF8, FGF9, FGF19, FGF21, and FGF 23) (NPL 10, NPL 11). In particular, FGF19, FGF21 and FGF23 act on β -Klotho complexed with FGFR1 c. FGFR1 is also evaluated as a therapeutic target for both anti-cancer and chronic diseases (such as obesity and diabetes) inhibitors and agonists. In particular, there are several reports of agonism of FGFR1, including homodimerization-based agonists for both FGFR1b and FGFR1c, and heterodimerization-based agonists (NPL 12, NPL 13, NPL 14) for FGFR1c and β -Klotho, however, there are no reports on strong FGFR1 b-specific agonists that can exhibit better agonistic activity than the natural ligands. Another member of the RTK includes the discotic domain receptor (discoidin domain receptor, DDR) family. The DDR family includes DDR1 and DDR2.DDR1 is expressed predominantly in epithelial cells, smooth muscle cells, and in some cases in fibroblasts, while DDR2 is expressed in cells of mesenchymal origin (NPL 15). When collagen binding occurs at the extracellular portion of the receptor, both DDR1 and DDR2 are activated and then initiate downstream signaling pathways, including receptor phosphorylation (NPL 16). Collagen is an important ECM component that interacts with various cellular receptors in tissues, and thus DDR is involved in a variety of important functions including regulation of cell proliferation, survival, differentiation, cell aggregation, adhesion, migration and invasion (NPL 16). While therapeutic applications of DDR have focused mainly on anti-fibrosis and anti-cancer therapies because of the overexpression of DDR by various cancer cells, some reports indicate that DDR activation regenerates cartilage tissue and heals wounds (NPL 17, NPL 18), suggesting that DDR agonism also has the potential to regenerate various tissues. However, as with FGFR1 and FGFR2, therapeutic agonist antibodies specific for DDR have not been successfully identified. [ Quotation list ] [ Patent literature ] [PTL 1] WO2012021841A2 [PTL 2] WO2021247718A1 [PTL 3] WO2022143728A1 [PTL 4] WO2022087243A1 [PTL 5] WO2021157679A1 [ Non-patent literature ] [NPL 1] Danilo Ranieri, Benedetta Rosato, Monica Nanni, Alessandra Magenta, Francesca Belleudi, Maria Rosaria Torrisi, Expression of the FGFR2 mesenchymal splicing variant in epithelial cells drives epithelial-mesenchymal transition. Oncotarget. 2016 2 Months and 2 days a year, 7 (5): 5440-60. [NPL 2] Susan