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CN-122011204-A - Bifunctional fusion proteins and uses thereof

CN122011204ACN 122011204 ACN122011204 ACN 122011204ACN-122011204-A

Abstract

The present invention relates to the field of biological medicine, to bifunctional fusion proteins and their uses, more specifically to polypeptide constructs comprising an antigen binding domain capable of specifically binding RANKL, and a PTH peptide, and their related uses for disease treatment.

Inventors

  • XU XIANG
  • WANG SHANSHAN
  • LIU DAN
  • Lv yue
  • QI PENG
  • KANG LISHAN
  • WANG SIQIN
  • JIN LEI

Assignees

  • 上海赛增医疗科技有限公司

Dates

Publication Date
20260512
Application Date
20251111
Priority Date
20241112

Claims (20)

  1. 1. A polypeptide construct comprising an antigen binding domain capable of specifically binding RANKL, a PTH peptide, and an Fc domain, wherein the Fc domain comprises a first Fc domain monomer and a second Fc domain monomer; The antigen binding domain and the PTH peptide are each linked to one of the first and second Fc domain monomers.
  2. 2. The polypeptide construct of claim 1 wherein the PTH peptide is selected from a wild-type PTH peptide or variant thereof; preferably, the wild-type PTH peptide is an active fragment of a wild-type PTH protein; Preferably, the wild-type PTH peptide possesses the biological activity (e.g., PTH1R binding activity and/or PTH1R activating activity) of the wild-type PTH protein from which it is derived; Preferably, the wild-type PTH peptide comprises amino acid residues in the wild-type PTH protein at positions corresponding to positions 1-16 of SEQ ID NO. 31; Preferably, the wild-type PTH peptide comprises an amino acid residue in the wild-type PTH protein at a position corresponding to positions 1-16 (or positions 1-17, or positions 1-27, or positions 1-33, or positions 1-34, or positions 1-35, or positions 1-40, or positions 1-50, or positions 1-60, or positions 1-70, or positions 1-80, or positions 1-84) of SEQ ID NO. 31; Preferably, the wild-type PTH peptide comprises amino acid residues in the wild-type PTH protein at positions corresponding to positions 1-33, or positions 1-34, or positions 1-40 of SEQ ID NO. 31.
  3. 3. The polypeptide construct of claim 2 wherein the PTH peptide variant has one or more characteristics selected from the group consisting of: (i) In comparison to the wild-type PTH peptide, the PTH peptide variant has an amino acid residue (e.g., an asparagine residue) at a position corresponding to position 16 of SEQ ID No. 31 replaced with an amino acid residue other than an asparagine residue, preferably with a glutamic acid residue, a glycine residue, or a serine residue; (ii) In comparison to the wild-type PTH peptide, the PTH peptide variant has an amino acid residue (e.g., serine residue) at a position corresponding to position 17 of SEQ ID NO. 31 replaced with an amino acid residue other than serine, glycine and proline residues, preferably with a glutamic acid residue; (iii) Preferably, the PTH peptide variant comprises a characteristic sequence N-X-S (or T) wherein N represents asparagine, X represents any one of the amino acids other than proline, S represents serine, and T represents threonine, compared to the wild-type PTH peptide, preferably, the PTH peptide variant is capable of undergoing an N-glycosylation modification at an amino acid residue (e.g., an asparagine residue) corresponding to position 33 of SEQ ID NO: 31, preferably, the PTH peptide variant is substituted with a serine residue or a threonine residue at an amino acid residue (e.g., a valine residue) corresponding to position 35 of SEQ ID NO: 31, compared to the wild-type PTH peptide, preferably, the PTH peptide variant is substituted with an amino acid residue (e.g., a valine residue) corresponding to position 35 of SEQ ID NO: 31, and an alanine residue (e.g., a phenylalanine residue) corresponding to position 34 of SEQ ID NO: 31.
  4. 4. The polypeptide construct of any one of claims 1-3, wherein the PTH polypeptide (e.g., the wild-type PTH peptide or the PTH polypeptide variant) has an amino acid sequence as set forth in any one of SEQ ID NOs 7-9, 11-16, 31.
  5. 5. The polypeptide construct of any one of claims 1 to 4 wherein the antigen binding domain is linked to the first Fc domain monomer and the PTH peptide is linked to the second Fc domain monomer, or wherein the antigen binding domain is linked to the second Fc domain monomer and the PTH peptide is linked to the first Fc domain monomer.
  6. 6. The polypeptide construct of claim 5 wherein the first and second Fc domain monomers each independently comprise one or more amino acid modifications capable of promoting heterodimerization of the first and second Fc domain monomers.
  7. 7. The polypeptide construct of claim 6 wherein the first Fc domain monomer comprises an amino acid modification capable of forming a hole structure and the second Fc domain monomer comprises an amino acid modification capable of forming a knob structure that can pair with the knob structure to form a heterodimeric Fc domain.
  8. 8. The polypeptide construct of any one of claims 5-7, wherein the first Fc domain monomer and/or the second Fc domain monomer is derived from a human immunoglobulin (e.g., igG1, igG2, igG3, or IgG 4); Preferably, the first Fc domain monomer and/or the second Fc domain monomer each independently has an amino acid modification capable of forming a hole or knob structure, altered (e.g., enhanced or reduced) effector function, prolonged half-life (e.g., enhanced FcRn binding activity), and/or altered (e.g., enhanced or reduced or eliminated) protein a (protein a) binding activity compared to the wild-type Fc domain monomer from which it originates; Preferably, the first Fc domain monomer and/or the second Fc domain monomer is derived from human immunoglobulin IgG4, and the first Fc domain monomer comprises the substitution mutation T366S, L A, and/or Y407V, and/or the second Fc domain monomer comprises the substitution mutation T366W; Preferably, the first Fc domain monomer and/or the second Fc domain monomer is derived from human immunoglobulin IgG4, and the first Fc domain monomer comprises a substitution mutation (i) F234A, L A, and/or (ii) T366S, L368A, Y407V, and/or the second Fc domain monomer comprises a substitution mutation (i) F234A, L A, and/or (ii) T366W; Preferably, one of the first Fc domain monomer and the second Fc domain monomer further comprises the substitution mutation H435R and/or Y436F.
  9. 9. The polypeptide construct of any one of claims 5 to 8, wherein the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID No. 20 and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID No. 32.
  10. 10. The polypeptide construct of any one of claims 1-9 wherein the antigen binding domain and the PTH peptide are each linked to the Fc domain monomer (e.g., the first Fc domain monomer or the second Fc domain monomer) by a hinge region; Preferably, the hinge region is derived from a human immunoglobulin (e.g., igG1, igG2, igG3, or IgG 4); Preferably, the hinge region is selected from the group consisting of wild-type human immunoglobulin hinge regions and variants thereof; Preferably, the hinge region is derived from human immunoglobulin IgG4 and comprises a substitution mutation S228P; preferably, the hinge region comprises the amino acid sequence shown as SEQ ID NO. 10.
  11. 11. The polypeptide construct of any one of claims 1 to 10 wherein the antigen binding domain is selected from the group consisting of Fab, fab ', F (ab') 2 , scFab, VHH, fv, disulfide-linked Fv, and scFv. Preferably, the antigen binding domain is a Fab.
  12. 12. The polypeptide construct of any one of claims 1 to 10, wherein the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), which VH and VL together form an antigen binding domain that specifically binds RANKL.
  13. 13. The polypeptide construct of claim 12 wherein the VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO. 1 and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO. 3; Preferably, the CDRs are defined by Kabat, chothia, abm or IMGT numbering system.
  14. 14. The polypeptide construct of claim 12 or 13 wherein the VH comprises HCDR1 as set forth in SEQ ID NO. 34, HCDR2 as set forth in SEQ ID NO. 35, and HCDR3 as set forth in SEQ ID NO. 36, and/or the VL comprises LCDR1 as set forth in SEQ ID NO. 37, LCDR2 as set forth in SEQ ID NO. 38, and LCDR3 as set forth in SEQ ID NO. 39; wherein the CDRs are defined by the Kabat numbering system; preferably, the VH comprises the amino acid sequence shown as SEQ ID NO. 1 and/or the VL comprises the amino acid sequence shown as SEQ ID NO. 3.
  15. 15. The polypeptide construct of any one of claims 12-14 comprising a peptide chain I-a comprising the VL and light chain constant regions, a peptide chain I-B comprising the VH, heavy chain CH1 region, hinge region, and the first Fc domain monomer (or the second Fc domain monomer), and a peptide chain I-C comprising the PTH peptide, hinge region, the second Fc domain monomer (or the first Fc domain monomer); Preferably, the peptide chain I-A comprises the VL and light chain constant regions from the N-terminus to the C-terminus, the peptide chain I-B comprises the VH, heavy chain CH1 region, hinge region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and/or the peptide chain I-C comprises the PTH peptide, hinge region, the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus.
  16. 16. The polypeptide construct of claim 15 wherein the adjacent domains of the peptide chain I-a are optionally connected by or without a linker, the adjacent domains of the peptide chain I-B are optionally connected by or without a linker, and/or the adjacent domains of the peptide chain I-C are optionally connected by or without a linker; Preferably, the linkers are each independently identical or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), preferably the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S), e.g., peptide linkers having the structure shown as G m S) n , wherein m, n are each independently integers not less than 0, e.g., each independently 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, preferably the peptide linkers each independently comprise an amino acid sequence as shown in any one of SEQ ID NOs 17-19.
  17. 17. The polypeptide construct of claim 15 or 16 wherein said peptide chain I-C comprises from N-terminus to C-terminus said PTH peptide, a peptide linker comprising an amino acid sequence as set forth in any one of SEQ ID NOs 17-19, a hinge region, said second Fc domain monomer (or said first Fc domain monomer).
  18. 18. The polypeptide construct of any one of claims 15 to 17 wherein the light chain constant region is a constant region of a human immunoglobulin kappa or lambda light chain; Preferably, the light chain constant region comprises the amino acid sequence shown as SEQ ID NO. 4.
  19. 19. The polypeptide construct of any one of claims 15 to 18 wherein the heavy chain CH1 region is the heavy chain CH1 region of a human immunoglobulin (e.g., igG1, igG2, igG3 or IgG 4); Preferably, the heavy chain CH1 region comprises the amino acid sequence shown as SEQ ID NO. 2.
  20. 20. The polypeptide construct of any one of claims 15 to 19, wherein the polypeptide construct comprises: a peptide chain I-A comprising an amino acid sequence as shown in SEQ ID NO. 6, a peptide chain I-B comprising an amino acid sequence as shown in SEQ ID NO. 5, a peptide chain I-C comprising an amino acid sequence as shown in any one of SEQ ID NOs 21-30.

Description

Bifunctional fusion proteins and uses thereof Technical Field The present invention relates to the field of biological medicine, more specifically to polypeptide constructs comprising an antigen binding domain capable of specifically binding RANKL, and a PTH peptide, and related uses thereof for the treatment of diseases. Background Osteoporosis (osteoporosis) is a systemic bone disease characterized by low bone mass, damaged bone tissue microstructure, increased bone fragility, and susceptibility to fracture. Osteoporosis can occur at any age, but is common in postmenopausal women and older men. Osteoporosis is classified into two major categories, primary and secondary, depending on the etiology. Primary osteoporosis includes postmenopausal osteoporosis (type I), senile osteoporosis (type II), and idiopathic osteoporosis (juvenile type). Postmenopausal osteoporosis generally occurs within 5-10 years after menopause in women, senile osteoporosis generally refers to osteoporosis occurring after 70 years old, and idiopathic osteoporosis mainly occurs in teenagers, and the etiology is unknown. Secondary osteoporosis refers to osteoporosis caused by a disease or drug affecting bone metabolism or other well-defined etiology. An osteoporotic fracture (or brittle fracture) refers to a fracture that occurs when it is subjected to a minor trauma (equivalent to falling from a standing height or less), and is a serious consequence of osteoporosis. Common sites of osteoporotic fracture include vertebral body, distal forearm, hip, proximal humerus, pelvis, etc., with vertebral body fractures being most common. Hip fractures are the most severe osteoporotic fractures. Osteoporosis therapeutic agents are mainly classified into four categories according to their biological mechanisms, namely, basic modulators, bone resorption inhibitors, bone formation promoters, and bone metabolism modulators. Wherein, denosumab (DMAB) and teriparatide (TERIPARATIDE) are used clinically for many years as main anti-osteoporosis biological medicines, and the advantages and disadvantages are clear. Deshumab is a neutralizing antibody to RANKL (receptor activator of NF- κB ligand) and can inhibit bone resorption and significantly reduce the risk of postmenopausal women's vertebral, non-vertebral and hip bone fracture in clinical use. But limited by biological mechanisms, desulzumab is less effective in patients with severe osteoporosis, and increased Bone resorption occurs following de-activation of Desulzumab, and BMD (Bone MINERAL DENSITY) falls back to pre-treatment levels. And there is an increased risk of multiple vertebral fractures. Teriparatide is recombinant human PTH 1-34, and acts mainly on PTH1R (Parathyroid hormone receptor 1), regulating bone metabolism and promoting bone remodeling. PTH can promote differentiation of osteoblasts, promote cell maturation and bone formation, and can also up regulate RANKL expression on osteoblasts, promote differentiation of osteoclasts and promote bone resorption. Teriparatide can significantly reduce the risk of vertebral and non-vertebral fracture in postmenopausal women in clinical use, but the effect of reducing the risk of hip fracture has not been demonstrated. In addition, teriparatide needs to be administered daily due to its short half-life, and is expensive and has poor patient compliance. In addition, because the expression spectrum of PTH1R is wider, the expression of the PTH1R in tissues such as kidneys, intestinal tracts and the like is higher, the targeting of bone tissues is poorer, the high activation activity of teriparatide leads to the risk of high blood calcium in the use process, and potential renal toxicity can be caused. The biological mechanism is complementary to that of the Deshu monoclonal antibody and the teriparatide, and the Deshu monoclonal antibody and the teriparatide are combined in some retrospective researches, so that the density of vertebrae, thighbone neck and hip bone can be remarkably improved, and the increase of Yu Teli Pa peptide or Deshu monoclonal antibody is achieved. However, the combined drug still cannot solve the problems of short half-life period, high administration frequency and poor targeting of bone tissues of teriparatide. Disclosure of Invention The present invention provides a polypeptide construct (also referred to herein as a bifunctional fusion protein) comprising an antigen binding domain capable of specifically binding RANKL and a PTH peptide, said polypeptide construct having significantly superior stability, significantly increased half-life at the PTH terminus, and improved pharmacodynamic activity in modulating bone metabolism. Furthermore, the polypeptide construct can reduce potential toxic and side effects by reducing the activation activity of PTH peptide ends on PTH1R on single-expression PTH1R cells, and simultaneously realize the conditional activation activity of PTH1R in double-expression PTH1R and RANKL cells by util