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KR-20260066142-A - Anti-PD1-IL10 Fusion Protein and Its Applications

KR20260066142AKR 20260066142 AKR20260066142 AKR 20260066142AKR-20260066142-A

Abstract

Belonging to the field of biotechnology, the invention provides an anti-PD1-IL10 fusion protein and its applications. Specifically, the invention provides a fusion protein comprising a PD1 binding unit and an IL10 unit, wherein the fusion protein possesses excellent targetability, antitumor effects, and safety, and can treat tumors or inflammatory diseases.

Inventors

  • 구 쩌
  • 예 린후이
  • 루 디
  • 우 샤오동
  • 오 잉예
  • 런 바이광
  • 궈 지엔
  • 한 저
  • 딩 자오자오
  • 덩 웬타오

Assignees

  • 광동 파폰 바이오파마 인크.

Dates

Publication Date
20260512
Application Date
20240529
Priority Date
20231008

Claims (17)

  1. As a fusion protein comprising a PD1 binding unit and an IL10 unit, A fusion protein comprising: the PD1 binding unit portion being an anti-PD1 antibody and the IL10 unit portion being an IL10 polypeptide; wherein the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 variable regions having at least 90% sequence identity with SEQ ID NO. 1, 3, 5, or 7, and/or the light chain variable region comprises LCDR1, LCDR2, and LCDR3 variable regions having at least 90% sequence identity with SEQ ID NO. 2, 4, 6, or 8.
  2. In paragraph 1, The above anti-PD1 antibody is, A. An anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 2; B. An anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 4; C. An anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 5, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 6; or D. An anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 8; selected from, Optionally, the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat numbering system, defined by the IMGT numbering system, defined by the Chothia numbering system, defined by the Contact numbering system, or defined by the AbM numbering system; Optionally, the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat numbering system; Optionally, HCDR1 of the heavy chain variable region of the anti-PD1 antibody comprises the amino acid sequence of SEQ ID NO. 9, HCDR2 comprises the amino acid sequence of SEQ ID NO. 10, 15, or 16, and HCDR3 comprises the amino acid sequence of SEQ ID NO. 11; LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO. 12, LCDR2 comprises the amino acid sequence of SEQ ID NO. 13, and LCDR3 comprises the amino acid sequence of SEQ ID NO. 14; Optionally, the anti-PD1 antibody comprises HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, respectively, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively, fusion proteins.
  3. In paragraph 1 or 2, The above anti-PD1 antibody is a humanized antibody, a murine antibody, or a chimeric antibody; Optionally, the antibody is a full-length antibody or a fusion protein that is an antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.
  4. In any one of paragraphs 1 through 3, The heavy chain variable region of the above anti-PD1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NOs 1, 3, 5, or 7, and/or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NOs 2, 4, 6, or 8; Optionally, (a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 2; (b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4; (c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 6; or (d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 8; Optionally, the anti-PD1 antibody is a fusion protein comprising a heavy chain variable region represented by SEQ ID NO. 1 and a light chain variable region represented by SEQ ID NO. 2.
  5. As a fusion protein comprising a PD1 binding unit and an IL10 unit, The above PD1 binding unit is an anti-PD1 antibody, and the above IL10 unit is an IL10 polypeptide; (a) The anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 95% sequence identity with SEQ ID NO. 1 and the light chain variable region has at least 95% sequence identity with SEQ ID NO. 2; (b) The anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 95% sequence identity with SEQ ID NO. 3 and the light chain variable region has at least 95% sequence identity with SEQ ID NO. 4; (c) The anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 95% sequence identity with SEQ ID NO. 5 and the light chain variable region has at least 95% sequence identity with SEQ ID NO. 6; (d) The anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 95% sequence identity with SEQ ID NO. 7 and the light chain variable region has at least 95% sequence identity with SEQ ID NO. 8; or (e) The anti-PD1 antibody is a PD1 antigen that competitively binds to the anti-PD1 antibody according to (a), (b), (c) or (d), or an antibody that binds to the same epitope; Optionally, the antibody is a fusion protein comprising a heavy chain variable region represented by SEQ ID NO. 1 and a light chain variable region represented by SEQ ID NO. 2.
  6. In any one of paragraphs 1 through 5, The above anti-PD1 antibody includes an invariant region; Optionally, the heavy chain constant region of the antibody is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, and/or the light chain constant region of the antibody is selected from the κ or λ chain constant regions; Optionally, the species origin of the above invariant region is murine or human; Optionally, the heavy chain invariant region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 19, and/or the light chain invariant region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 20; Optionally, the heavy chain of the anti-PD1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 17, and the light chain of the anti-PD1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 18; Optionally, the anti-PD1 antibody is a fusion protein comprising a heavy chain represented by SEQ ID NO. 17 and a light chain represented by SEQ ID NO. 18.
  7. In any one of paragraphs 1 through 6, The above IL10 polypeptide is a natural IL10 polypeptide or a modified IL10 polypeptide; Optionally, the modified IL10 polypeptide is in monomeric form, and compared to the natural IL10 polypeptide, a spacer peptide is inserted between any two adjacent amino acids, and the spacer peptide is a flexible peptide; Optionally, the position of one of the two adjacent amino acids is at position 115, position 116, position 117, position 118, and position 119; Optionally, the spacer peptide is located between positions 116 and 117; Optionally, the spacer peptide is located between positions 115 and 116; Optionally, the spacer peptide is located between positions 117 and 118; Optionally, the spacer peptide is at least one selected from GGGSGG, GGGSG, (GS)n, (GGGS)n, (GSGGS)n, (GGGGS)n, and n is any integer between 1 and 10; Optionally, the amino acid sequence of the spacer peptide is represented by SEQ ID NO. 21; Optionally, the modified IL10 polypeptide has n amino acids deleted at the N-terminus compared to the natural IL10 polypeptide, where n is any integer between 1 and 12; Optionally, the modified IL10 polypeptide has two amino acids deleted at the N-terminus compared to the natural IL10 polypeptide; Optionally, the IL10 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 22 or 23; Optionally, the amino acid sequence of the IL10 polypeptide is a fusion protein represented by SEQ ID NO. 22.
  8. In any one of paragraphs 1 through 7, The above IL10 polypeptide is directly linked to the heavy or light chain of the above anti-PD1 antibody or linked via a linker peptide; Optionally, the N-terminus of the IL10 polypeptide is connected to the C-terminus of the light chain of the anti-PD1 antibody via a linker peptide; Optionally, the fusion protein comprises two identical first chains represented by formula P1 and two identical second chains represented by formula P2; P1: [light chain of anti-PD1 antibody]-[linker peptide]-[IL10 polypeptide], P2: Heavy chain of anti-PD1 antibody; Optionally, in the above formula P1, the linker peptide is a flexible linker peptide; Optionally, the linker peptide is at least one selected from (GGGGS)nG, (GGGGS)n, (GS)n, (GGGS)n, (GSGGS)n, (GGGGSG)n, and n is any integer between 1 and 10; Optionally, the amino acid sequence of the linker peptide is denoted by SEQ ID NO. 24; Optionally, the fusion protein comprises a first chain of amino acid sequences having at least 90% sequence identity with SEQ ID NO. 25, and a second chain of amino acid sequences having at least 90% sequence identity with SEQ ID NO. 17; Optionally, the fusion protein comprises a first chain of two amino acid sequences represented by SEQ ID NO. 25 and a second chain of two amino acid sequences represented by SEQ ID NO. 17.
  9. In any one of paragraphs 1 through 8, The above fusion protein is, A. Can specifically bind to human PD1; optionally, said fusion protein can bind to human PD1 protein with an EC50 value of ≤10 nM, said EC50 value being a characteristic measured by FACS analysis; B. Characteristics of having low or no hematological toxicity; C. Characteristics capable of enhancing the cytotoxic activity of CD8+ T cells; D. Properties capable of inhibiting tumor growth; E. A fusion protein having at least one of the following characteristics: excellent stability.
  10. A nucleic acid encoding a fusion protein according to any one of claims 1 to 9.
  11. A vector containing nucleic acid according to paragraph 10.
  12. A cell containing a nucleic acid according to paragraph 10 or a vector according to paragraph 11.
  13. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 9, a nucleic acid according to claim 10, a vector according to claim 11, or a cell according to claim 12; optionally further comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.
  14. In the manufacture of a drug for the treatment or prevention of tumors or inflammatory diseases, as an application of a fusion protein according to any one of claims 1 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a cell according to claim 12, or a pharmaceutical composition according to claim 13, Optionally, the tumor is a tumor that highly expresses PDL1; Optionally, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematological cancer; Optionally, the tumor is an application selected from non-small cell lung cancer, renal cell carcinoma, and gastric cancer.
  15. A method for treating a disease comprising the step of administering a therapeutically effective amount of a fusion protein according to any one of claims 1 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a cell according to claim 12, or a pharmaceutical composition according to claim 13 to a subject in need thereof, Optionally, the disease is a tumor or an inflammatory disease; Optionally, the tumor or inflammatory disease is a disease that highly expresses human PD1 and/or PDL1; Optionally, the tumor is a tumor that highly expresses PDL1; Optionally, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematological cancer; Optionally, the above tumor is a disease treatment method selected from non-small cell lung cancer, renal cell carcinoma, and gastric cancer.
  16. A kit comprising a fusion protein according to any one of claims 1 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a cell according to claim 12, or a pharmaceutical composition according to claim 13, Optionally, the above kit is a kit used to detect or measure human PD1.
  17. A method for manufacturing a fusion protein according to any one of claims 1 to 9, A manufacturing method comprising the step of obtaining a fusion protein by culturing cells according to claim 12, followed by isolation and purification.

Description

Anti-PD1-IL10 Fusion Protein and Its Applications [Cross-reference of related applications] This application claims priority to a Chinese invention patent with application number 202311297567.5, filing date October 8, 2023, titled “Anti-PD1-IL10 fusion protein and applications thereof,” the entire contents of which are incorporated herein by reference. [Technology Field] The present disclosure relates to the field of biotechnology, specifically to anti-PD1-IL10 fusion proteins and their applications. Programmed cell death protein-1 (also referred to as PD1 or PD-1) is a CD28 component and is expressed in activated B cells, T cells, and myeloid cells. Human PD1 is encoded by the gene Pdcd1, is located at 2q37.3, has a total length of 9.6 kb, consists of 5 exons and 4 introns, and contains a 663 bp promoter upstream. PD1 is a 55 kDa type I transmembrane protein, and its molecular structure consists of an extracellular domain, a transmembrane domain, and an intracellular domain; the extracellular domain contains a single immunoglobulin variable domain (IgV) domain, and the intracellular domain contains an immunoreceptor tyrosine inhibition motif (ITIM) and an immunoreceptor tyrosine switching module (ITSM). The amino acid sequence of the extracellular domain of PD-1 is 24% homologous to CTLA-4 and 28% homologous to CD28. When T cells are activated, PD-1 assembles into the tyrosine phospholipase SHP2, primarily via ITIM, to induce dephosphorylation of downstream effector molecules. PD-1 has two ligands: PD-L1 and PD-L2. Both PD-L1 and PD-L2 are B7 homologues; the PD-L1 gene is located at the human chromosome 9P24.2 region, measures 42kb, and their molecular structures both include one immunoglobulin-like variable domain, one constant domain-like domain, one transmembrane domain, and one short cytoplasmic tail. PD-1 can bind to PD-L1 and PD-L2 to downregulate T cell activation. PD-L1 is expressed on the surface of various tumor cells, including those of lung, gastric, liver, esophageal, renal, ovarian, cervical, breast, skin, colon, bladder, glioma, head and neck, and oral squamous cell carcinoma. Additionally, a large number of PD1-expressing CD8+ T cells were found surrounding these tumors, and clinical statistical results indicate that high expression levels of PD-L1 in tumor cells are associated with a poor prognosis in cancer patients. Exhausted CD8+ T cells play a significant role in resistance to immune checkpoint therapy. T cells transition from an activated state to an exhausted state under the action of continuous antigen stimulation and various inhibitory molecules. Exhausted CD8+ T cells exhibit significantly reduced proliferative capacity, cytokine secretion ability, and tumor-killing capacity, making them unable to provide effective anti-tumor immunity. Even if anti-PD-1 antibodies are administered to block the PD-1/PD-L1 pathway while CD8+ T cells are in an exhausted state, they cannot be restored to an activated state. The present disclosure provides a fusion protein comprising a PD1 binding unit portion and an IL10 unit portion. In some embodiments, the present disclosure discloses a fusion protein comprising a PD1 binding unit portion and an IL10 unit portion, wherein the PD1 binding unit portion is an anti-PD1 antibody and the IL10 unit portion is an IL10 polypeptide; Herein, the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 variable regions having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO. 1, 3, 5, or 7, and/or the light chain variable region of the antibody comprises LCDR1, LCDR2, and LCDR3 variable regions having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO. 2, 4, 6, or 8; In some embodiments, in a fusion protein according to any one of the aforementioned claims, the anti-PD1 antibody is, A. An anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 2; B. An anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 4; C. An anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 5, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 6; or D. Selected from an anti-PD1 antibody in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 8. In some embodiments, in a fusion protein according to any one of the aforementioned claims, in the anti-PD1 antibody, HCDR1, HCDR2, HCDR3,