[go: up one dir, main page]

WO2024179553A1 - PROTÉINE DE FUSION CIBLANT TGFβ ET SON UTILISATION - Google Patents

PROTÉINE DE FUSION CIBLANT TGFβ ET SON UTILISATION Download PDF

Info

Publication number
WO2024179553A1
WO2024179553A1 PCT/CN2024/079436 CN2024079436W WO2024179553A1 WO 2024179553 A1 WO2024179553 A1 WO 2024179553A1 CN 2024079436 W CN2024079436 W CN 2024079436W WO 2024179553 A1 WO2024179553 A1 WO 2024179553A1
Authority
WO
WIPO (PCT)
Prior art keywords
tgfβrii
fusion protein
antibody
tgf
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/079436
Other languages
English (en)
Chinese (zh)
Inventor
张轶博
霍永庭
周新新
张喆
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Fapon Biopharma Inc
Original Assignee
Guangdong Fapon Biopharma Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Fapon Biopharma Inc filed Critical Guangdong Fapon Biopharma Inc
Publication of WO2024179553A1 publication Critical patent/WO2024179553A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present disclosure relates to the field of biomedicine, and in particular, to a fusion protein targeting TGF ⁇ and applications thereof.
  • TGF- ⁇ Transforming growth factor- ⁇
  • TGF- ⁇ belongs to the TGF- ⁇ superfamily that regulates cell growth and differentiation. It has three forms, including TGF- ⁇ 1/2/3. In addition to TGF- ⁇ , this family also includes activins, inhibins, Mullerian inhibitor substance (MIS) and bone morpho-genetic proteins (BMPs). TGF- ⁇ was originally named because this cytokine can transform normal fibroblasts into non-anchorage-dependent growth cells. It plays an important regulatory role in cell growth, differentiation and immune function. TGF- ⁇ mediates TGF ⁇ signaling by binding to TGF- ⁇ receptors (TGF ⁇ type I receptor (TGF ⁇ RI, GenBank Accession No. of human TGF ⁇ RI: L11695) and TGF ⁇ type II receptor (TGF ⁇ RII, GenBank Accession No. of human TGF ⁇ RII: M85079)).
  • TGF ⁇ type I receptor TGF ⁇ RI, GenBank Accession No. of human TGF ⁇ RI: L11695
  • TGF ⁇ RII
  • inactive TGF- ⁇ is also called latency associated peptide (LAP), which can be activated by acid cleavage.
  • LAP latency associated peptide
  • acidic environment can exist near fractures and in healing wounds. The cleavage of the protein itself can convert the TGF- ⁇ complex into activated TGF- ⁇ .
  • TGF ⁇ can inhibit the proliferation of T cells stimulated by mitogens and alloantigens or the growth of IL-2-dependent T cells, and can also inhibit IFN- ⁇ -induced MHC class II antigen expression in melanoma cells.
  • TGF ⁇ can also inhibit the production of IFN- ⁇ and TNF- ⁇ in PBMCs and promote the expression of IL-6.
  • TGF- ⁇ is an effective immunosuppressant, and the disorder of TGF- ⁇ signaling can lead to autoimmunity, inflammation, and cancer.
  • TGF- ⁇ is produced by cancer cells and several other cell types present in the TME, including Treg. Fibroblasts, macrophages, and platelets are also important TGF- ⁇ producers in tumors. Elevated levels of TGF- ⁇ prevent naive T cells from differentiating into a Th1 effector phenotype, promote their transformation into a Treg subtype, and inhibit the antigen presentation function of dendritic cells.
  • the inventors have constructed a TGF- ⁇ receptor fusion protein through a large number of experimental studies, which includes a TGF- ⁇ receptor portion and an immunoglobulin portion, wherein the TGF- ⁇ receptor portion includes at least 2 TGF- ⁇ receptor structures.
  • the TGF- ⁇ receptor portion includes 2, 3, 4, 5 or 6 or more TGF- ⁇ receptor domains.
  • the TGF- ⁇ receptor portion comprises a first TGF- ⁇ receptor domain and a second TGF- ⁇ receptor domain in series; in some embodiments, the first TGF- ⁇ receptor domain is connected to the second TGF- ⁇ receptor domain directly or through a linker; in some embodiments, the first TGF- ⁇ receptor domain is directly connected to the second TGF- ⁇ receptor domain.
  • the linker is selected from a (G x S) y linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6; in some embodiments, the linker is as shown in SEQ ID NO: 12 or 39.
  • the immunoglobulin portion is an antibody or its Fc region, and the N-terminus of the TGF- ⁇ receptor portion is connected to the C-terminus of the immunoglobulin portion; or the C-terminus of the TGF- ⁇ receptor portion is connected to the N-terminus of the immunoglobulin portion;
  • the TGF- ⁇ receptor fusion protein described in any of the above items is a fusion protein selected from the following:
  • the fusion protein comprises two polypeptide chains of the structure shown in formula (a):
  • the fusion protein comprises two polypeptide chains of the structure shown in formula (b):
  • the fusion protein comprises two polypeptide chains of the structures shown in formula (c) and two polypeptide chains of the structures shown in formula (d):
  • TGF ⁇ R-1, TGF ⁇ R-2, and TGF ⁇ R-3 are TGF- ⁇ receptor domains
  • L1 and L2 are linkers
  • Fc is an antibody Fc region
  • VH is an antibody heavy chain variable region
  • VL is an antibody light chain variable region
  • CH1, CH2, and CH3 are antibody heavy chain constant regions
  • CL is an antibody light chain constant region
  • the L1 and L2 are independently selected from (G x S) y linkers, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6; optionally, the L1 and L2 linkers are as shown in SEQ ID NO: 12 or 39.
  • TGF ⁇ R-1, TGF ⁇ R-2, and TGF ⁇ R-3 may be the same TGF- ⁇ receptor domain or different TGF- ⁇ receptor domains; in some embodiments, the amino acid sequences of TGF ⁇ R-1, TGF ⁇ R-2, and TGF ⁇ R-3 are the same.
  • the TGF- ⁇ receptor domain is a TGF ⁇ RII polypeptide.
  • the TGF- ⁇ receptor domain is a TGF ⁇ RII mutant fragment
  • the TGF ⁇ RII mutant fragment includes a C-terminal fragment of the TGF ⁇ RII extracellular domain, a flexible fragment and an N-terminal fragment of the TGF ⁇ RII extracellular domain, wherein the C-terminal fragment includes up to 122 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acid residues at the C-terminus of the TGF ⁇ RII extracellular domain); the N-terminus of the flexible fragment is connected to the C-terminus of the N-terminal fragment of the TGF ⁇ RII extracellular domain, and the C-terminus of the flexible fragment is connected to the N-terminus of the C-terminal fragment of the TGF ⁇ RII extracellular domain.
  • the TGF ⁇ RII mutant fragment includes 112-122 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acid residues at the C-terminus of the TGF ⁇ RII extracellular domain), or in some embodiments, the TGF ⁇ RII mutant fragment includes 112 to 117 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the amino acid sequence of the TGF ⁇ RII extracellular domain is as shown in SEQ ID NO: 1 0, the C-terminal fragment of the TGF ⁇ RII extracellular domain is a truncated polypeptide of the N-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the C-terminal fragment of the TGF ⁇ RII extracellular domain is a polypeptide of the TGF
  • the TGF ⁇ RII mutant fragment in the TGF- ⁇ receptor fusion protein described in any of the preceding items, includes 0 to 6 amino acids at the N-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the TGF ⁇ RII mutant fragment includes 1, 2, 3, 4, 5 or 6 amino acids at the N-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the amino acid sequence of the N-terminal fragment of the TGF ⁇ RII extracellular domain is as shown in SEQ ID NO: 1.
  • the flexible segment is a connecting peptide containing G and/or S amino acids; in some embodiments, the flexible segment is a (GS) x G polypeptide, wherein x is preferably any integer from 1 to 6; or, the flexible segment is selected from a (G x S) y linker, wherein x is selected from an integer from 1 to 5, and y is selected from an integer from 0 to 6; in some embodiments, the amino acid sequence of the flexible segment is as shown in SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 39; in some embodiments, the TGF ⁇ RII mutant fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 5.
  • the immunoglobulin portion is an antibody or an Fc region thereof.
  • the TGF- ⁇ receptor fusion protein described in any of the preceding items the antibody is a murine, chimeric, humanized or human antibody; in some embodiments, in some embodiments, the antibody specifically binds to a tumor-associated antigen or an immune checkpoint-associated antigen, and the antigen may be selected from but is not limited to VEGF, GUCY2C, MSLN, Claudin18.2, GPC3, EGFR, HER2, CEA, GD2, EGFRvIII, MUC1, PRLR, CLCA1, MUC12, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS1IE, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISSIR, QRFPR, GPR119, CLDN6, UPK2, ADAM1 2.
  • VEGF GUCY2C
  • MSLN Claudin18.2
  • the antibody contains a heavy chain constant region and a light chain constant region
  • the heavy chain constant region sequence is selected from the constant region sequence of any one of IgG1, IgG2, IgG3, and IgG4, and the light chain constant region is a ⁇ or ⁇ chain
  • the immunoglobulin portion is an antibody Fc region; in some embodiments, the Fc region includes SEQ ID NO:6 or SEQ ID NO:54 sequence.
  • the TGF- ⁇ receptor fusion protein described in any of the preceding items wherein A) the immunoglobulin portion is an antibody Fc region, and the fusion protein comprises two identical polypeptide chains, optionally, the polypeptide chains are selected from: any one of SEQ ID NO: 44-49; B) the immunoglobulin portion is an antibody, and the fusion protein comprises two identical heavy chains and two identical light chains; in some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 50 or 52, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 51.
  • the present disclosure provides a method for preparing any of the above TGF ⁇ RII mutant fragments.
  • the amino acid sequence of the TGF ⁇ RII mutant is a sequence starting from the N-terminus of the TGF ⁇ RII extracellular domain, truncated by at least 14 amino acids, and inserted with no more than 20 amino acids.
  • the wild-type TGF ⁇ RII fragment content is very high, and it is difficult to remove it during the purification process, which affects the efficacy.
  • the inventors found that after truncating the N-terminus of the TGF ⁇ RII extracellular domain according to the mutation method of the embodiment of the present disclosure, the obtained TGF ⁇ RII mutant fragment has biological activity binding to TGF ⁇ , and the fragment is not easy to dissociate, so it is not easy to be broken.
  • the TGF ⁇ RII mutant containing the fragment also has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its drugability is improved.
  • the present disclosure proposes a TGF ⁇ RII mutant fragment. According to the embodiments of the present disclosure, it is prepared by the method described above. According to the embodiments of the present disclosure, the TGF ⁇ RII mutant having the fragment has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its drugability is improved.
  • the present disclosure proposes a TGF ⁇ RII mutant and a method for preparing the same, wherein the TGF ⁇ RII mutant includes an extracellular region, a transmembrane region, and an intracellular region; the method includes: truncating at least 14 amino acids from the N-terminus of the TGF ⁇ RII extracellular domain and inserting no more than 20 amino acids.
  • the inventors have conducted a large number of experimental explorations and found that the TGF ⁇ RII mutant obtained by the method of the embodiment of the present disclosure has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its drugability is improved.
  • the present disclosure provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the fusion protein described in any one of the above.
  • the present disclosure provides an expression vector comprising any of the above nucleic acid molecules.
  • the present disclosure provides a host cell comprising any of the above expression vectors or nucleic acid molecules.
  • the present disclosure provides a recombinant cell comprising any of the above nucleic acid molecules or expression vectors.
  • the present disclosure provides a pharmaceutical composition, which comprises the fusion protein, or nucleic acid molecule, or expression vector, or cell described in any of the above; in some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition comprises a pharmaceutically acceptable adjuvant, and the pharmaceutically acceptable adjuvant comprises at least one of a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent; the pharmaceutical composition is in the form of at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension, and a lyophilized preparation.
  • the pharmaceutical composition can control the content of upregulated TGF ⁇ expressed around tumor cells, thereby preventing or treating tumors in a long-term and effective manner.
  • the present disclosure provides use of any of the foregoing fusion proteins, or the nucleic acid molecules, or the expression vectors, or the cells, or the pharmaceutical compositions in the preparation of drugs for treating tumors.
  • the present disclosure provides a method for treating a tumor, comprising: administering a therapeutically effective amount of any of the foregoing fusion proteins, or the nucleic acid molecules, or the expression vectors, or the cells, or the pharmaceutical compositions to a subject in need thereof.
  • the present disclosure provides any of the above-mentioned fusion proteins, or the nucleic acid molecules, or the expression vectors, or the cells, or the pharmaceutical compositions for treating tumors.
  • the present disclosure provides a method for preparing any of the above fusion proteins, comprising the following steps: 1) constructing an expression vector as described in any of the above; 2) introducing the expression vector into a host cell to obtain a recombinant cell to express the fusion protein.
  • FIG. 1 is a schematic diagram of the structure of a TGF ⁇ RII mutant according to an embodiment of the present disclosure, wherein insertion represents an inserted amino acid sequence, i.e., the 1st to Xth amino acids of the N-terminus of the TGF ⁇ RII extracellular domain are truncated, and then a flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain are inserted.
  • insertion represents an inserted amino acid sequence, i.e., the 1st to Xth amino acids of the N-terminus of the TGF ⁇ RII extracellular domain are truncated, and then a flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain are inserted.
  • FIG2 is a result diagram of the content of fragments and polymers generated during the purification process of the fusion protein formed by truncating the N-terminus of the TGF ⁇ RII extracellular domain, or by inserting a flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain after truncation according to an embodiment of the present disclosure, wherein: the fusion proteins shown on the horizontal axis are WT, Trunc#1-Trunc#19, Trunc#21, and Trunc#22, respectively; that is, R0805: WT represents the fusion protein WT in Table 1 of the present disclosure, R0749: 7-136 represents the fusion protein Trunc#1 in Table 1 of the present disclosure, R0776: 8-136 represents the fusion protein Trunc#2 in Table 1 of the present disclosure, R0777: 9-136 represents the fusion protein Trunc#3 in Table 1 of the present disclosure, and R0778: 10-136 represents the fusion protein T in Table 1 of the present disclosure
  • R0779: 11-136 represents the fusion protein Trunc#5 in Table 1 of the present disclosure
  • R0780: 12-136 represents the fusion protein Trunc#6 in Table 1 of the present disclosure
  • R0781: 13-136 represents the fusion protein Trunc#7 in Table 1 of the present disclosure
  • R0782: 14-136 represents the fusion protein Trunc#8 in Table 1 of the present disclosure
  • R0783: 1-6+15-136 represents the fusion protein Trunc#9 in Table 1 of the present disclosure
  • R0784: 16-136 represents the fusion protein Trunc#10 in Table 1 of the present disclosure
  • R0785: 17-136 represents the fusion protein Trunc#11 in Table 1 of the present disclosure
  • R0786: 18-136 represents the fusion protein Trunc#12 in Table 1 of the present disclosure
  • R0787: 19-136 represents the fusion protein Trunc#13 in Table 1 of the present disclosure
  • R0788: 1-6+20-136 represents the fusion protein Trunc
  • Each fusion protein shown on the horizontal axis only represents the mutation mode of the TGF ⁇ RII mutant, for example, R0749:7-136 indicates that the TGF ⁇ RII mutant in the fusion protein is truncated at the 1st to 6th amino acids of the N-terminus of the TGF ⁇ RII extracellular domain; R0796:1-6+GSGSGSGSSG+20-136 indicates that the TGF ⁇ RII mutant in the fusion protein is truncated at the 1st to 19th amino acids of the N-terminus of the TGF ⁇ RII extracellular domain, and then the 1st to 6th amino acids (IPPHVQ (SEQ ID NO: 1)) and the flexible fragment (GSGSGSGSG (SEQ ID NO: 2)) of the N-terminus of the TGF ⁇ RII extracellular domain are inserted.
  • IPPHVQ SEQ ID NO: 1
  • GSGSGSGSG flexible fragment
  • Figure 3 is a result graph showing the content of fragments and polymers produced during the purification process of the fusion protein formed by the TGF ⁇ RII mutant obtained by truncating the N-terminus of the TGF ⁇ RII extracellular domain and then inserting the flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain according to the embodiments of the present disclosure, wherein the specific mutation mode of the TGF ⁇ RII mutant in each fusion protein shown on the horizontal axis is shown in Table 1.
  • FIG. 4 is a graph showing the results of an ELISA binding activity test between the fusion protein and human TGF ⁇ 1 according to an embodiment of the present disclosure.
  • FIG5 is a graph showing the results of detecting the binding activity of the fusion protein blocking human TGF ⁇ 1 and TGF ⁇ RII according to an embodiment of the present disclosure.
  • FIG. 6 is a graph showing the experimental results of the effect of the fusion protein formed by the TGF ⁇ RII mutant obtained by truncating the N-terminus of the TGF ⁇ RII extracellular domain on T cell proliferation according to an embodiment of the present disclosure.
  • FIG. 7 is a graph showing the experimental results of the effect of a fusion protein formed by truncating the N-terminus of the TGF ⁇ RII extracellular domain and then inserting a flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain to obtain a TGF ⁇ RII mutant on T cell proliferation according to an embodiment of the present disclosure.
  • FIG. 8 is a schematic diagram of the structure of a tandem hTGF ⁇ Trap fusion protein.
  • FIG. 9 is a graph showing the in vitro binding activity results of the tandem hTGF ⁇ Trap fusion protein and hTGF ⁇ 1, m/hTGF ⁇ 3.
  • Figure 10 shows the experimental results of blocking the binding of TGF ⁇ RII and TGF ⁇ on the cell surface by the tandem hTGF ⁇ Trap fusion protein.
  • FIG. 11 is a graph showing the results of a T cell proliferation inhibition test using tandem hTGF ⁇ Trap fusion proteins.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated.
  • a feature defined as “first” or “second” may explicitly or implicitly include at least one of the features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • amino acid refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs or amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids, such as hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine; common natural amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), thre
  • Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.
  • Amino acid mimetics are chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.
  • antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibody fusion proteins, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, humanized antibodies, single domain antibodies, full-length antibodies, or antigen-binding fragments thereof (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Natural antibodies refer to naturally occurring immunoglobulin molecules.
  • natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains. From N to C-terminus, each heavy chain has a heavy chain variable region (VH, also known as a variable heavy domain), followed by a heavy chain constant region (CH), and the natural IgG heavy chain constant region generally includes three constant domains (CH1, CH2, and CH3).
  • VH heavy chain variable region
  • CH heavy chain constant region
  • each light chain has a light chain variable region (VL, also known as a variable light domain), followed by a light chain constant region (CL, also known as a light chain constant domain).
  • full length antibody or “intact antibody” refers to an antibody that contains a structure substantially similar to a native antibody, the heavy chain of which contains an Fc region.
  • antibody “variable region” refers to the region of the antibody heavy chain or light chain involved in antibody binding to antigen.
  • the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved framework regions (FR) and three complementary determining regions (CDR).
  • "Antibody portion that specifically binds to TIGIT” is a portion of a TIGIT antibody that contains an anti-TIGIT antibody variable region; in some embodiments, the anti-TIGIT antibody variable region refers to the variable region of the anti-TIGIT antibody in International Patent Application PCT/CN2022/120984 (e.g., the light and heavy chain variable regions of 15H10L3), and the present disclosure incorporates PCT/CN2022/120984 in its entirety by reference.
  • CDR complementarity determining region
  • framework or “FR” refers to the variable domain residues other than the CDR residues.
  • VH contains three CDR regions: HCDR1, HCDR2, and HCDR3;
  • VL contains three CDR regions: LCDR1, LCDR2, and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus (also called the N terminus) to the carboxyl terminus (also called the C terminus) in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDRs refer to more than two CDRs in the heavy chain and light chain of the antibody.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: “Kabat” numbering rules (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), “Chothia” numbering rules, “ABM” numbering rules, “contact” numbering rules (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering rules (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc.; the correspondence between various numbering systems is well known to those skilled in the art.
  • antigen binding fragment and "antigen binding domain” refer to a portion of an intact antibody that is capable of specifically binding to an antigen to which the intact antibody binds.
  • antigen binding fragments include, but are not limited to, Fv (composed of VH and VL), Fab (composed of a light chain and a constant region 1 (CH1) of a heavy chain and a heavy chain variable region), Fab' (composed of a Fab region and a hinge region), Fab'-SH (the cysteine residue in the hinge region of the Fab' fragment carries a free thiol group), (Fab')2 (dimeric Fab'), scFv (single-chain antibody molecule, in which the light chain variable region is directly connected to the heavy chain variable region or connected by a linker), scFab (single-chain Fab), dsFv (disulfide-stabilized Fv fragment), (dsFv)2 (dimeric dsFv), VHH (
  • Fc region is used to define the C-terminal region of the heavy chain of an antibody, including a native Fc region and a remodeled Fc region.
  • the Fc region for antibodies described herein includes the Fc region of human IgG1, IgG2 (IgG2a, IgG2b), IgG3, and IgG4.
  • the Fc region is a human IgG1 comprising a DLE mutation.
  • the boundaries of the Fc region can also vary, such as the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine and lysine in the Fc region (residues 446 and 447 according to the EU numbering system) are missing.
  • the numbering convention of the Fc region is the EU numbering system, also known as the EU index.
  • the C-terminal lysine residue (K) in the Fc region is mutated into alanine (A) to reduce the cleavage and hydrolysis of the fusion protein.
  • chimeric antibody refers to an antibody in which a portion of the heavy chain and/or light chain is derived from one species, while the other portion of the heavy chain and/or light chain is derived from another species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, it can be achieved by retaining the non-human CDR region and replacing the rest with a human antibody counterpart (i.e., the constant region and the framework region portion of the variable region).
  • human antibody humanized antibody
  • fully human antibody fully human antibody
  • completely human antibody refers to antibodies whose variable and constant regions are human sequences.
  • linker refers to a connecting unit connecting two polypeptide fragments, which usually has a certain flexibility, and the use of the linker will not cause the loss of the original function of the protein domain.
  • the linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids.
  • the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 0-6.
  • the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 1-6.
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, in conjunction with “affinity” refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen).
  • affinity of a molecule X to its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art.
  • KD refers to a dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as a molar concentration (M).
  • M molar concentration
  • the KD value of an antibody can be measured using methods well known in the art. For example, a biosensor system such as a system measuring surface plasmon resonance (e.g., Biacore) is used, or the affinity in a solution is measured by a solution equilibrium titration method (SET).
  • SET solution equilibrium titration method
  • effector function refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an Fc region of amino acid sequence mutation).
  • antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
  • the term “monoclonal antibody” refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for possible natural mutations that are present in small amounts.
  • polyclonal antibody preparations are typically comprised of a variety of different antibodies having different amino acid sequences in their variable domains, which are typically specific for different epitopes.
  • “Monoclonal” represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring the production of antibodies by any particular method.
  • the antibody provided by the present disclosure is a monoclonal antibody.
  • antigen refers to a molecule that can be bound by an antigen binding protein (eg, an antibody) selective binding agent.
  • An antigen may have one or more epitopes that can interact with different antigen binding proteins (eg, antibodies).
  • epitope refers to an area or region on an antigen that can specifically bind to an antibody.
  • An epitope can be formed by a continuous string of amino acids (linear epitope) or contain non-continuous amino acids (conformational epitope), for example, due to the folding of the antigen (i.e., the tertiary folding of the antigen by protein properties) and become spatially close.
  • the difference between a conformational epitope and a linear epitope is that the binding of the antibody to the conformational epitope is lost in the presence of a denaturing solvent.
  • a conformational epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind to a specific epitope can be performed using routine methods in the art, such as alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.
  • An antibody that binds to the same epitope as a reference antibody or an antibody that competes for binding with a reference antibody means an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more.
  • test antibody binds to the same epitope as a reference antibody
  • the reference antibody is allowed to bind to the antigen under saturating conditions, and then the ability of the test antibody to bind to the antigen is assessed after removing excess reference antibody; if the test antibody is able to bind to the antigen after saturation binding of the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody; and if the test antibody is unable to bind to the antigen after saturation binding of the reference antibody, then the test antibody can bind to the same epitope as the reference antibody.
  • test antibody binds to the same epitope
  • routine experiments e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art
  • two antibodies are considered to bind to the same or overlapping epitope if a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).
  • KD equilibrium dissociation constant
  • the KD of the antibody binding to the antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein).
  • KD can be measured using known methods, including but not limited to Biacore assays, Octet methods, microthermophoresis, HPLC MS methods, and flow cytometry fluorescence sorting techniques.
  • TGF ⁇ RII is a TGF ⁇ type II receptor, including a wild-type TGF ⁇ type II receptor or a mutant fragment thereof, wherein the mutant fragment is capable of binding to TGF ⁇ .
  • TGF ⁇ RII mediates TGF ⁇ signal transduction by binding to TGF ⁇ .
  • a wild-type human TGF ⁇ RII cell has a 136-amino acid residue peptide starting from the N-terminus, having an amino acid sequence as shown in SEQ ID NO: 10, and a wild-type TGF ⁇ RII extracellular domain sequence:
  • the TGF ⁇ RII described in the present disclosure can be selected from Chinese patent applications 202111026893.3, 202111028163.7, 202111026881.0, or 202210476344.4 and PCT applications or Chinese patent applications with Chinese patent applications 202111026893.3, 202111028163.7, 202111026881.0, or 202210476344.4 as priority, and the entire texts of these patents are incorporated into the present disclosure by reference.
  • the present disclosure provides a TGF ⁇ RII mutant fragment, which includes up to 122 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acid residues at the C-terminus of the TGF ⁇ RII extracellular domain); the TGF ⁇ RII mutant fragment further includes a flexible fragment and an N-terminal fragment of the TGF ⁇ RII extracellular domain.
  • up to 122 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acid residues at the C-terminus of the TGF ⁇ RII extracellular domain
  • the TGF ⁇ RII mutant fragment further includes a flexible fragment and an N-terminal fragment of the TGF ⁇ RII extracellular domain.
  • the inventors creatively discovered that after truncating the N-terminus of the TGF ⁇ RII extracellular domain according to the mutation method of the embodiments of the present disclosure, the obtained TGF ⁇ RII mutant fragment has the biological activity of TGF ⁇ binding, and in the process of preparing the TGF ⁇ RII mutant, the amino acid sequence of the TGF ⁇ RII mutant fragment is not easy to dissociate, and the fragment content is significantly reduced.
  • the TGF ⁇ RII mutant containing the fragment has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its drugability is improved.
  • the N-terminus of the flexible fragment is connected to the C-terminus of the N-terminal fragment of the TGF ⁇ RII extracellular domain
  • the C-terminus of the flexible fragment is connected to the N-terminus of a polypeptide of up to 122 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain.
  • the amino acid sequence of the TGF ⁇ RII extracellular domain is as shown in SEQ ID NO: 10
  • the C-terminal fragment of the TGF ⁇ RII extracellular domain is an N-terminal truncated polypeptide of the TGF ⁇ RII extracellular domain
  • the C-terminal fragment of the TGF ⁇ RII extracellular domain is 14-23 consecutive (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75
  • the C-terminal fragment of the TGF ⁇ RII extracellular domain is 14-23 consecutive (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • the TGF ⁇ RII mutant fragment includes 0 to 6 amino acids at the N-terminus of the TGF ⁇ RII extracellular domain. In some embodiments, the TGF ⁇ RII mutant fragment includes 1, 2, 3, 4, 5 or 6 amino acids at the N-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the TGF ⁇ RII mutant fragment includes 6 amino acid residues at the N-terminus of the TGF ⁇ RII extracellular domain.
  • the N-terminal fragment of the TGF ⁇ RII extracellular domain has the amino acid sequence shown in SEQ ID NO: 1.
  • IPPHVQ SEQ ID NO: 1.
  • the flexible segment is a connecting peptide containing G and S amino acids.
  • the flexible segment is a (GS) x G polypeptide, wherein X is preferably any integer from 1 to 6; in some embodiments, the amino acid sequence of the flexible segment is shown in SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 39.
  • the flexible segment contains 5 to 15 amino acids.
  • the flexible fragment has the amino acid sequence shown in SEQ ID NO: 2.
  • GSGSGSGSG SEQ ID NO: 2.
  • the TGF ⁇ RII mutant fragment includes at most 115 or at most 117 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain.
  • the TGF ⁇ RII mutant fragment includes 112 to 117 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain.
  • the TGF ⁇ RII mutant fragment includes 112, 115 or 117 amino acids at the C-terminus of the TGF ⁇ RII extracellular domain.
  • the TGF ⁇ RII mutant fragment has an amino acid sequence shown in any one of SEQ ID NOs: 3-5.
  • the present disclosure provides a TGF ⁇ RII mutant, comprising an extracellular region, a transmembrane region and an intracellular region; wherein the extracellular region comprises the TGF ⁇ RII mutant fragment described in the first aspect.
  • the present disclosure provides a method for preparing the aforementioned TGF ⁇ RII mutant fragment.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by truncating at least 14 amino acids from the N-terminus of the TGF ⁇ RII extracellular domain and inserting no more than 20 amino acids.
  • the inventors in view of the fact that the TGF ⁇ RII fragments prepared by the prior art have a very high content and are difficult to remove during the purification process, which affects the efficacy, the inventors have conducted a large number of experimental explorations.
  • the obtained TGF ⁇ RII mutant fragment After truncating the N-terminus of the TGF ⁇ RII extracellular domain according to the mutation method of the embodiment of the present disclosure, the obtained TGF ⁇ RII mutant fragment has biological activity that binds to TGF ⁇ , and the fragment is not easy to dissociate, so it is not easy to be broken.
  • the TGF ⁇ RII mutant containing the fragment also has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its drugability is improved.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by truncating 14-23 (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) amino acids from the N-terminus of the TGF ⁇ RII extracellular domain.
  • 14-23 e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23
  • the site where the mutation occurs has a certain relationship with the content of fragments produced during the preparation of TGF ⁇ RII, so that after truncating 14-23 amino acids, the fragment still has biological activity binding to TGF ⁇ , and in the process of preparing the TGF ⁇ RII mutant fragment, its amino acid sequence is not easy to dissociate, so the content of fragments is significantly reduced, thereby improving its drugability.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by truncating 14-21 amino acids from the N-terminus of the TGF ⁇ RII extracellular domain. After truncating 14-21 amino acids, the obtained TGF ⁇ RII mutant fragment still has the biological activity of binding to TGF ⁇ , and is not easy to dissociate, and the content of fragments is significantly reduced.
  • the TGF ⁇ RII mutant containing the fragment also has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the content of fragments is significantly reduced, and its drugability is improved.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by truncating 14, 19, or 21 amino acids from the N-terminus of the TGF ⁇ RII extracellular domain. After truncating 14, 19, or 21 amino acids, the obtained TGF ⁇ RII mutant fragment still has the biological activity of binding to TGF ⁇ , and the fragment is not easy to dissociate, and the content of fragments is significantly reduced.
  • the TGF ⁇ RII mutant containing the fragment has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, and the content of fragments and polymers is significantly reduced, thereby improving its drugability.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by inserting no more than 15 amino acids at the N-terminus of the TGF ⁇ RII extracellular domain.
  • the amino acid sequence of the TGF ⁇ RII mutant is not easy to dissociate during the preparation process, the fragment content is significantly reduced, its drugability is improved, and the in vivo half-life is prolonged.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by inserting 15 amino acids into the N-terminus of the TGF ⁇ RII extracellular domain.
  • the TGF ⁇ RII mutant fragment still has the biological activity of binding to TGF ⁇ , its amino acid sequence is not easy to dissociate, and the content of fragments is significantly reduced.
  • the amino acid sequence of the TGF ⁇ RII mutant containing the fragment is not easy to dissociate during the preparation process, the content of fragments is significantly reduced, its drugability is improved, and the half-life in vivo is prolonged.
  • the inserted amino acid fragment comprises the N-terminal fragment of the TGF ⁇ RII extracellular domain.
  • the N-terminal fragment of the TGF ⁇ RII extracellular domain has the amino acid sequence shown in SEQ ID NO: 1.
  • IPPHVQ SEQ ID NO: 1.
  • the inserted amino acid fragment further comprises a flexible fragment.
  • the flexible fragment is not particularly limited, and any conventional flexible fragment can be used.
  • “Flexible fragment” refers to a peptide containing two or more amino acid residues connected by peptide bonds and providing greater rotational freedom to the two polypeptides it connects (e.g., the N-terminal fragment of the TGF ⁇ RII extracellular domain, the C-terminal fragment of the TGF ⁇ RII extracellular domain).
  • the soft segment has an amino acid sequence as shown in SEQ ID NO: 2.
  • the The flexible fragment is a polypeptide as shown in SEQ ID NO:12 or SEQ ID NO:39.
  • the N-terminus of the flexible fragment is connected to the C-terminus of the N-terminal fragment of the TGF ⁇ RII extracellular domain, and the C-terminus of the flexible fragment is connected to the N-terminus of the remaining TGF ⁇ RII extracellular domain after truncation.
  • the amino acid sequence shown in SEQ ID NO: 2 is arranged after the N-terminal fragment of the TGF ⁇ RII extracellular domain (for example, when the N-terminal amino acid sequence of the TGF ⁇ RII extracellular domain is shown in SEQ ID NO: 10, the inserted fragment is: IPPHVQGSGSGSGSG (SEQ ID NO: 11)).
  • the amino acid sequence of the TGF ⁇ RII mutant is not easy to dissociate during the preparation process, the fragment content is significantly reduced, the drugability is improved, and the in vivo half-life is prolonged.
  • the present disclosure provides a TGF ⁇ RII mutant fragment, which is prepared by the aforementioned method for preparing a TGF ⁇ RII mutant.
  • the present disclosure provides a method for preparing the aforementioned TGF ⁇ RII mutant, wherein the TGF ⁇ RII mutant comprises an extracellular region, a transmembrane region and an intracellular region; the method comprises: truncating at least 14 amino acids from the N-terminus of the TGF ⁇ RII extracellular domain and inserting no more than 20 amino acids.
  • the TGF ⁇ RII mutant can be obtained by truncating 14-23 (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) amino acids from the N-terminus of the TGF ⁇ RII extracellular domain.
  • 14-23 e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23
  • the site where the mutation occurs has a certain relationship with the content of fragments generated during the preparation of TGF ⁇ RII. Therefore, after truncating 14-23 amino acids, the obtained TGF ⁇ RII mutant has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the content of fragments is significantly reduced, and its drugability is improved.
  • the TGF ⁇ RII mutant is obtained by truncating 14-21 amino acids from the N-terminus of the TGF ⁇ RII extracellular domain. After truncating 14-21 amino acids, the obtained TGF ⁇ RII mutant has the same biological activity as the wild-type TGF ⁇ RII, and in the process of preparing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its drugability is improved.
  • the amino acid sequence of the TGF ⁇ RII mutant is obtained by truncating 14, 19, or 21 amino acids from the N-terminus of the TGF ⁇ RII extracellular domain. After truncating 14, 19, or 21 amino acids, the obtained TGF ⁇ RII mutant has the same biological activity as the wild-type TGF ⁇ RII, and its amino acid sequence is not easy to dissociate during the preparation of the TGF ⁇ RII mutant, and the content of fragments and polymers is significantly reduced, thereby improving its drugability.
  • the amino acid sequence of the TGF ⁇ RII mutant is obtained by inserting no more than 15 amino acids into the N-terminus of the TGF ⁇ RII extracellular domain.
  • the amino acid sequence of the TGF ⁇ RII mutant is obtained by inserting 15 amino acids into the N-terminus of the TGF ⁇ RII extracellular domain.
  • 15 amino acids are inserted, the amino acid sequence of the TGF ⁇ RII mutant is not easy to dissociate during the preparation process, the fragment content is significantly reduced, the drugability is improved, and the half-life in vivo is prolonged.
  • the inserted amino acid fragment comprises the N-terminal fragment of the TGF ⁇ RII extracellular domain.
  • the N-terminal fragment of the TGF ⁇ RII extracellular domain has the amino acid sequence shown in SEQ ID NO: 1.
  • the inserted amino acid fragment further comprises a flexible fragment.
  • the flexible fragment is not particularly limited, and any conventional flexible fragment can be used.
  • the flexible fragment has an amino acid sequence as shown in SEQ ID NO: 2.
  • the flexible fragment is a polypeptide as shown in SEQ ID NO: 12 or SEQ ID NO: 39.
  • the N-terminus of the flexible fragment is connected to the C-terminus of the N-terminal fragment of the TGF ⁇ RII extracellular domain, and the C-terminus of the flexible fragment is connected to the N-terminus of the remaining TGF ⁇ RII extracellular domain after truncation.
  • the amino acid sequence shown in SEQ ID NO: 2 is arranged after the N-terminal fragment of the TGF ⁇ RII extracellular domain (for example, when the N-terminal amino acid sequence of the TGF ⁇ RII extracellular domain is shown in SEQ ID NO: 10, the inserted fragment is: IPPHVQGSGSGSGSG (SEQ ID NO: 11)).
  • the amino acid sequence of the TGF ⁇ RII mutant is not easy to dissociate during the preparation process, the fragment content is significantly reduced, its drugability is improved, and the half-life in vivo is prolonged.
  • the present disclosure provides a TGF ⁇ RII mutant, which is prepared by the method described above.
  • the easy generation of fragments during the preparation of TGF ⁇ RII and the easy breakage of certain sites in the amino acid sequence of TGF ⁇ RII are closely related. Therefore, when preparing TGF ⁇ RII mutants, the sites at the N-terminus of the TGF ⁇ RII extracellular domain that are easy to break or dissociate can be shortened and/or inserted to obtain TGF ⁇ RII mutants with low fragment and polymer content during the production process.
  • the present disclosure provides a TGF- ⁇ receptor fusion protein, which includes a TGF- ⁇ receptor portion and an immunoglobulin portion, wherein the TGF- ⁇ receptor portion includes at least 2 TGF- ⁇ receptor structures; in some embodiments, the TGF- ⁇ receptor portion includes 2, 3, 4, 5 or 6 or more TGF- ⁇ receptor domains.
  • the TGF- ⁇ receptor portion includes a first TGF- ⁇ receptor domain and a second TGF- ⁇ receptor domain, and the first TGF- ⁇ receptor domain is directly or through a linker connected to the second TGF- ⁇ receptor domain; in some embodiments, the first TGF- ⁇ receptor domain is directly or through a linker connected to the second TGF- ⁇ receptor domain; The second TGF- ⁇ receptor domain is directly connected.
  • the first TGF- ⁇ receptor domain is connected to the second TGF- ⁇ receptor domain by a linker.
  • the linker is selected from a (G x S) y linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6; in some embodiments, the linker is as shown in SEQ ID NO: 12 or 39.
  • the immunoglobulin portion is an antibody or its Fc region, and the N-terminus of the TGF- ⁇ receptor portion is connected to the C-terminus of the immunoglobulin portion; or the C-terminus of the TGF- ⁇ receptor portion is connected to the N-terminus of the immunoglobulin portion; in some embodiments, any of the above TGF- ⁇ receptor fusion proteins is a fusion protein selected from the following:
  • the fusion protein comprises two polypeptide chains of the structure shown in formula (a):
  • the fusion protein comprises two polypeptide chains of the structure shown in formula (b):
  • the fusion protein comprises two polypeptide chains of the structures shown in formula (c) and two polypeptide chains of the structures shown in formula (d):
  • TGF ⁇ R-1, TGF ⁇ R-2, and TGF ⁇ R-3 are TGF- ⁇ receptor domains
  • L1 and L2 are linkers
  • Fc is an antibody Fc region
  • VH is an antibody heavy chain variable region
  • VL is an antibody light chain variable region
  • CH1, CH2, and CH3 are antibody heavy chain constant regions
  • CL is an antibody light chain constant region
  • L1 and L2 are independently selected from (G x S) y linkers, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6; optionally, the L1 and L2 linkers are as shown in SEQ ID NO: 12 or 39.
  • the TGF- ⁇ receptor domain is a TGF ⁇ RII polypeptide.
  • the TGF ⁇ RII can be selected from Chinese patent applications 202111026893.3, 202111028163.7, 202111026881.0, or 202210476344.4 and any TGF ⁇ RII polypeptide described in PCT applications (e.g., PCT/CN2022/116320) or Chinese patent applications with Chinese patent applications 202111026893.3, 202111028163.7, 202111026881.0, or 202210476344.4 as priority, the entire text of these patents is incorporated into the present disclosure by reference.
  • the TGF- ⁇ receptor is a TGF ⁇ RII mutant fragment as described in any of the foregoing.
  • the N-terminus of the connecting peptide is connected to the C-terminus of the antibody Fc region, and the C-terminus of the connecting peptide is connected to the N-terminus of the TGF ⁇ RII mutant fragment.
  • the antibody Fc region is derived from a human IgG antibody molecule.
  • the antibody Fc region comprises the Fc of the hIgG1 antibody.
  • the lysine at the C-terminus of the antibody Fc region is mutated to alanine.
  • the antibody Fc region has an amino acid sequence as shown in SEQ ID NO: 6:
  • the addition of arginine-glycine-aspartic acid (e.g., RGDRGD (SEQ ID NO: 53)) to the N-terminus of the antibody Fc region can improve the ability of the fusion protein to bind to cell surface integrins.
  • the antibody Fc region has an amino acid sequence as shown in SEQ ID NO: 54:
  • the connecting peptide is a flexible connecting peptide.
  • the connecting peptide is not particularly limited, and any conventional flexible fragment in the art can be used.
  • the connecting peptide comprises a (G 4 S) X G amino acid sequence, wherein X is an integer greater than 0.
  • the connecting peptide is a (G 4 S) X G polypeptide, wherein X is preferably any integer from 1 to 6.
  • the connecting peptide is a polypeptide as shown in SEQ ID NO: 12 or SEQ ID NO: 39.
  • the connecting peptide comprises a (G 4 S) 4 G (SEQ ID NO: 12) sequence.
  • the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 7-9.
  • antibody-dependent cellular cytotoxicity is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells with lytic activity (such as natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fc ⁇ receptors (Fc ⁇ Rs) expressed on effector cells.
  • lytic activity such as natural killer cells (NK), monocytes, macrophages, and neutrophils
  • Fc ⁇ Rs Fc ⁇ receptors
  • NK cells express Fc ⁇ RIIIa
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIIIa.
  • the ADCC activity of the antibodies provided herein can be assessed using in vitro assays using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on markers released from lysed cells (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins).
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to a mechanism of inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death.
  • Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.
  • complement receptors e.g., CR3
  • nucleic acid is used interchangeably with the term “polynucleotide” herein, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides.
  • Isolated nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which cells typically contain the nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or at a chromosomal position different from its natural chromosomal position.
  • the isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or a separate vector, and such one or more nucleic acid molecules present in one or more positions in a host cell.
  • a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • sequence identity refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned (introducing gaps, if necessary, to obtain the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity).
  • sequence identity can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 Megalign
  • vector means a polynucleotide molecule capable of transporting another polynucleotide connected thereto.
  • plasmid refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected.
  • viral vector such as an adeno-associated virus vector (AAV or AAV2), wherein additional DNA segments can be connected to the viral genome.
  • AAV adeno-associated virus vector
  • Some vectors can replicate autonomously in the host cell in which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins).
  • vectors can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome.
  • expression vector or "expression construct” refers to a vector that can transform a host cell, and contains a nucleic acid sequence for guiding and/or controlling (together with the host cell) the expression of one or more heterologous coding regions operably connected thereto.
  • the expression construct may include, but is not limited to, affecting or controlling transcription, translation, and affecting the sequence of RNA splicing of the coding region operably connected thereto when there are introns.
  • host cell refers to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Mutant offspring with the same function or biological activity as screened or selected in the initial transformed cells are included herein.
  • Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include but are not limited to mammalian cells, insect cell lines, plant cells and fungal cells.
  • Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells.
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • A549 cells e.g., 3T3 cells and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells, including For example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia cactus (Pichia puntiae), Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha, polymorpha), Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma re
  • Pichia any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.
  • progeny As used in this application, “cell”, “cell line” and “cell culture” can be used interchangeably, and all such names include progeny.
  • the words “transformant” and “converted cell” include the primary subject cell and the culture derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny have exactly the same DNA content. Included are mutant progeny that have the same function or biological activity as the original transformed cell from which it was screened.
  • the present disclosure provides a nucleic acid molecule encoding the fusion protein described above.
  • the fusion protein encoded by the nucleic acid molecule according to the specific embodiment of the present disclosure has the same biological activity as the wild-type TGF ⁇ RII, and its amino acid sequence is not easy to dissociate during the preparation of the fusion protein, the fragment content is significantly reduced, and its drugability and in vivo half-life are improved.
  • the fusion protein can control the content of upregulated TGF ⁇ expressed around tumor cells, thereby preventing or treating tumors in a long-term and effective manner.
  • the present disclosure provides an expression vector.
  • the expression vector according to the specific embodiment of the present disclosure comprises the aforementioned nucleic acid molecule encoding the fusion protein.
  • the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
  • the nucleic acid molecule can be operably connected to the control element.
  • "operably connected” means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended function of regulating the transcription and translation of the exogenous gene.
  • the expression vector is a eukaryotic expression vector.
  • the present disclosure provides a recombinant cell.
  • the recombinant cell according to a specific embodiment of the present disclosure carries the aforementioned nucleic acid molecule encoding the fusion protein or the expression vector.
  • the recombinant cell according to a specific embodiment of the present disclosure can express the aforementioned fusion protein, and its amino acid sequence is not easy to dissociate during the preparation of the fusion protein, the fragment content is significantly reduced, and its drugability and in vivo half-life are improved.
  • the fusion protein can control the content of upregulated TGF ⁇ around tumor cells, thereby preventing or treating tumors in a long-term and effective manner.
  • the recombinant cell is a mammalian cell, such as a human HEK-293F cell or a CHO-K1 cell.
  • the recombinant cell does not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the recombinant cell is a mammalian cell, such as a human, monkey, rabbit, dog, cow, etc.; a mammalian cell such as a human HEK-293F cell or a CHO-K1 cell.
  • the recombinant cell does not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present disclosure provides a method for reducing the content of fragments in the process of preparing the aforementioned fusion protein, comprising the following steps: 1) constructing the aforementioned expression vector; 2) introducing the expression vector into a host cell.
  • the amino acid sequence of the fusion protein is not easily dissociated during the preparation of the fusion protein, the content of fragments is significantly reduced, and its efficacy is significantly improved.
  • the tumor disclosed herein can be non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colorectal cancer, melanoma, bile duct cancer or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma or gastric cancer, etc.
  • composition refers to a mixture containing one or more active ingredients such as the antibodies described herein and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation other than the active ingredient.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein.
  • cynomolgus or “cynomolgus” refers to cynomolgus monkeys (Macaca fascicularis).
  • the individual or subject is a human.
  • administering refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.
  • sample refers to a collection of fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, secretory tissues, and mucosal secretions of organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, abdominal and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures or cell cultures.
  • biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, secretory tissues, and mucosal secretions of organ
  • Treatment refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology.
  • the desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis.
  • the antibodies of the present disclosure are used to delay the development of the disease or slow the progression of the disease.
  • an “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or potential causes, prevent the occurrence of symptoms and/or their potential causes, and/or improve or ameliorate damage (e.g., lung disease) caused by or associated with a disease state.
  • an effective amount is a therapeutically effective amount or a preventive effective amount.
  • a “therapeutically effective amount” is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay, or reverse the disease state or any other undesirable symptom associated with the disease in any way.
  • a complete treatment or preventive effect may not occur after administering a dose, but may occur after administering a series of doses.
  • a therapeutic or preventive effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “preventive effective amount” may vary depending on a variety of factors: such as the disease state, age, sex, and weight of an individual, and the ability of a therapeutic agent or combination of therapeutic agents to induce a desired response in an individual.
  • Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health conditions of the patient.
  • (G 4 S) 4 G (SEQ ID NO: 12) was used as a connecting peptide to connect the C-terminus of the Fc heavy chain fragment of the hIgG1 antibody (SEQ ID NO: 6) with TGF ⁇ RII mutant fragments of different mutation forms (Table 1) to obtain TGF ⁇ RII trap fusion protein.
  • the lysine residue (K) at the C-terminus of the Fc heavy chain fragment of the hIgG1 antibody was mutated to alanine (A) (obtaining the sequence shown in SEQ ID NO: 6) to reduce the cleavage and hydrolysis of the fusion protein.
  • TGF ⁇ RII trap fusion protein For TGF ⁇ RII trap fusion protein, standard transient transfection or stable transfection protocols were used to transfect mammalian cells with DNA encoding the Fc-TGF ⁇ RII receptor in the same expression vector or in a separate expression vector. Human HEK-293F cells were transiently transfected to prepare TGF ⁇ RII trap fusion protein, and CHO-K1 cells were stably transfected to prepare TGF ⁇ RII trap fusion protein to obtain TGF ⁇ RII trap fusion protein.
  • TGF ⁇ RII mutants The schematic diagram of the structure of TGF ⁇ RII mutants is shown in Figure 1.
  • the TGF ⁇ RII trap fusion proteins formed by different mutant forms of TGF ⁇ RII mutants are shown in Table 1.
  • Fc represents the Fc region of an antibody (SEQ ID NO: 6);
  • G 4 S) 4 G represents the sequence GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 12),
  • G 4 S) 5 G represents the sequence GGGGSGGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 39);
  • TGF ⁇ Trap (WT) represents the amino acid sequence of the wild-type TGF ⁇ RII extracellular domain (SEQ ID NO: 10);
  • TGF ⁇ trap (X-136) represents the polypeptide of amino acid residues X to 136 of the TGF ⁇ RII extracellular domain (the positions are numbered in the natural sequence relative to SEQ ID NO: 10, the same below), for example, TGF ⁇ trap(20-136) represents a polypeptide in which amino acids 1-19 of the N-terminus of the TGF ⁇ RII extracellular domain are truncated (i.e., only residues 20-136 of the TGF ⁇ RII extracellular domain are retained), TGF ⁇ trap(8-136,
  • the amino acid truncation described in the table refers to the deletion of amino acids in the wild-type human TGF ⁇ RII extracellular domain (TGF ⁇ Trap (WT), SEQ ID NO: 10), and the amino acid insertion refers to the insertion into the N-terminus of the TGF ⁇ RII polypeptide; for example, Trunc#22, the structure of the fusion protein Fc-(G 4 S) 4 G-IP6-GS9-TGF ⁇ Trap (20-136) refers to the following sequence from the N-terminus to the C-terminus: antibody constant region Fc, Linker (G 4 S) 4 G, IP6, linker GS9, and TGF ⁇ Trap (20-136), wherein the IP6-GS9-TGF ⁇ Trap (20-136) polypeptide can be obtained by deleting the IPPHVQKSVNNDMIVTDNN (SEQ ID NO: 25) amino acid at the N-terminus of the wild-type human TGF ⁇ RII extracellular domain (SEQ ID NO: 10), and then
  • TGF ⁇ trap represents the amino acid sequence from position X to position 136 of the TGF ⁇ RII extracellular domain.
  • TGF ⁇ trap (20-136) is (SEQ ID NO: 41):
  • TGF ⁇ trap 15-136) is (SEQ ID NO: 42):
  • TGF ⁇ trap 22-1336 is (SEQ ID NO: 43):
  • the human HEK-293F cell culture medium obtained by transiently transfecting human HEK-293F cells in Example 1 for preparing TGF ⁇ RII trap fusion protein and the CHO-K1 cell culture medium capable of preparing TGF ⁇ RII trap fusion protein obtained by stably transfecting CHO-K1 cells were respectively subjected to high-speed centrifugation and the supernatants were collected.
  • the collected supernatant is purified by affinity chromatography in the first step, wherein the chromatography medium is Protein A or a derivative filler that can interact with Fc, such as GE's Mabselect; the equilibration buffer is 1 ⁇ PBS, and after equilibration of 5 times the volume of the column, the cell supernatant is loaded and combined with the chromatography medium, and the flow rate is controlled so that the retention time of the sample on the affinity chromatography column is ⁇ 1min. After the loading is completed, the column is rinsed with 1 ⁇ PBS (pH7.4), and the light absorption value at a UV wavelength of 280nm is recorded until the A280 UV absorption drops to the baseline.
  • the chromatography medium is Protein A or a derivative filler that can interact with Fc, such as GE's Mabselect
  • the equilibration buffer is 1 ⁇ PBS
  • the chromatography column is rinsed with 0.1M glycine (pH 3.0) elution buffer, and the elution peak is collected according to the A280 UV absorption peak.
  • the collected elution sample is neutralized with 1M Tris (pH 8.5).
  • the neutralized eluted sample was concentrated by ultrafiltration device and then subjected to size exclusion chromatography, wherein the buffer for size exclusion chromatography was 1 ⁇ PBS, the chromatography column was XK26/60 Superdex200 (GE), the flow rate was controlled at 4 mL/min, the loading volume was less than 5 mL, and the target protein peaks were merged according to A280 UV absorption.
  • the collected TGF ⁇ RII trap fusion protein was identified for purity by SEC-HPLC.
  • the inventors analyzed the polymers and fragments during the purification process of different TGF ⁇ RII trap fusion proteins shown by SEC-HPLC results.
  • a noteworthy phenomenon is that compared with WT, after truncating the 1st to 19th amino acids of the N-terminus of the TGF ⁇ RII extracellular domain and then inserting the 1st to 6th amino acids (IPPHVQ (SEQ ID NO: 1)) and the flexible fragment (GSGSGSGSG (SEQ ID NO: 2)) of the N-terminus of the TGF ⁇ RII extracellular domain, the obtained TGF ⁇ RII trap fusion protein (Trunc#22) not only has no fragments, but also has much lower polymers. Therefore, the inventors further studied the structure of Trunc#22 and optimized the combination of the retained part, the inserted part and the truncated part.
  • the inventors tried a variety of sequences, including flexible linkers of different lengths, rigid linkers and random sequences, among which the insertion sequence in Trunc#32 was a flexible linker, the insertion sequence in Trunc#33 was a rigid linker, and the insertion sequence in Trunc#34 was a random sequence.
  • the protein used in the TGF ⁇ RII Trap fusion protein Trap end binding assay is human TGF ⁇ 1 (CA59, purchased from Novoprotein).
  • the detection process is as follows:
  • TGF ⁇ 1 Dilute TGF ⁇ 1 to 0.5 ⁇ g/mL with 1 ⁇ phosphate buffered saline (PBS), coat 96-well ELISA plate with 100 ⁇ L/well, and incubate at 4°C overnight;
  • PBS phosphate buffered saline
  • the protein used in the TGF ⁇ RII Trap fusion protein Trap end binding assay is human TGF ⁇ 1 (CA59, purchased from Novoprotein).
  • the assay process is as follows:
  • CHO-hTGF ⁇ RII Stably transfected CHO cells expressing human TGF ⁇ RII
  • TGF ⁇ RII trap fusion protein obtained in step b with 1 ⁇ PBS containing 1% BSA.
  • the starting concentration is 50 nM
  • the dilution factor is 4 times
  • a total of 8 gradients are set.
  • TGF ⁇ 1-biotin purchased from Acrobiosystem
  • the diluted concentration is 1 ⁇ g/mL.
  • step d The mixed sample obtained in step d was added to CHO-TGF ⁇ RII cells at 100 ⁇ L/well, placed at 4°C for 30 min, and then centrifuged at room temperature and 300g for 5 min, and the supernatant was discarded.
  • step f Add SA-PE (400-fold dilution, Jackson immunoresearch, 016-110-084) to the product obtained in step e at a volume of 100 ⁇ l/well, mix gently, place at 4°C for 1 hour, centrifuge at room temperature and 300 g for 5 minutes, and discard the supernatant; add 1 ⁇ PBS containing 1% BSA to the obtained cells at 100 ⁇ l/well, resuspend the cells, and detect on a flow cytometer.
  • SA-PE 400-fold dilution, Jackson immunoresearch, 016-110-084
  • the results of the fusion protein blocking the binding activity of human TGF ⁇ 1 and TGF ⁇ RII are shown in Figure 5.
  • the truncated TGF ⁇ RII Trap fusion protein can inhibit the binding of TGF ⁇ 1 to TGF ⁇ R II. From the EC50 value, the blocking ability is basically not much different from that of the wild-type TGF ⁇ RII Trap.
  • TGF ⁇ 1 can significantly inhibit the proliferation of T cells.
  • TGF ⁇ RII Trap fusion proteins In order to detect the function of the Trap end of different modified TGF ⁇ RII Trap fusion proteins at the cellular level, we detected the effects of different modified TGF ⁇ RII Trap fusion proteins on T cell proliferation in the presence of TGF ⁇ . The experiment uses the following exemplary method:
  • PBMC Peripheral blood mononuclear cells
  • T cells were isolated from PBMC using a human T cell enrichment kit (STEMCELL, 10951) according to the instructions.
  • T cells were stained with CFSE (eBioscience, 85-65-0850-84). For specific operations, refer to the reagent instructions. The concentration of CFSE used was 1 M.
  • TGF ⁇ RII Trap fusion protein CD3/CD28 beads (life, 40203D) and CFSE-stained T cells were co-incubated, the number of T cells was 5 ⁇ 10 4 /well, the volume ratio of CD3/CD28 beads to T cells was 1:5, and cultured in an incubator;
  • the CFSE value of the T cells was detected by flow cytometry to calculate the proportion of T cells in each generation, mainly observing the leftmost peak.
  • Trunc#1, Trunc#2, Trunc#6, Trunc#9, Trunc#14, and Trunc#22 groups showed better effects in promoting T cell proliferation than the WT group; at the same time, the effects of Trunc#3, Trunc#7, Trunc#8, Trunc#11, Trunc#13, Trunc#15 to Trunc#18 groups in promoting T cell proliferation were weaker than those of the WT group.
  • the inventors further evaluated the activity of the TGF ⁇ RII Trap fusion protein corresponding to the TGF ⁇ RII mutant obtained after inserting the flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain.
  • Example 1.1 The same method as in Example 1.1 was used to construct a fusion protein of hTGF ⁇ Trap in series.
  • the antibody or its Fc was connected to the N-terminus or C-terminus of the extracellular domain of the TGF ⁇ receptor in series through a linker (e.g., Gly 4 Ser) 4 G) to construct a fusion protein containing the extracellular region of hTGF ⁇ Trap in series.
  • a linker e.g., Gly 4 Ser
  • the mutation of the lysine residue (K) at the C-terminus of the Fc of the antibody to alanine (A) can reduce the cleavage and hydrolysis of the fusion protein, and the addition of arginine-glycine-aspartic acid (e.g., RGDRGD (SEQ ID NO: 53)) to the N-terminus of the Fc can improve the function of the fusion protein to bind to cell surface integrins;
  • arginine-glycine-aspartic acid e.g., RGDRGD (SEQ ID NO: 53)
  • Tandem hTGF ⁇ Trap Fusion Proteins Mammalian cells are transfected with DNA encoding the TGF ⁇ receptor fusion protein using standard protocols for transient or stable transfection.
  • TGF ⁇ Trap is selected from the TGF ⁇ Trap described in Table 1.
  • Table 1 For example, we constructed a variety of tandem hTGF ⁇ Trap fusion proteins:
  • a fusion protein comprising two polypeptide chains of the structure shown in formula (a):
  • a fusion protein comprising two polypeptide chains having the structure shown in formula (b):
  • a fusion protein comprising two polypeptide chains of the structures shown in formula (c) and two polypeptide chains of the structures shown in formula (d):
  • amino acid sequence of chain 2 of R1705 is the same as the amino acid sequence of chain 2 of R1704 (SEQ ID NO: 51)
  • the experimental method was the same as that in Example 1.2.
  • An expression plasmid was constructed and transiently transfected into human embryonic kidney HEK 293 cells.
  • the protein produced by the cells was isolated and purified.
  • the molecular weight of the band on SDS-PAGE under non-reducing conditions was approximately 120 kD, 150 Kd or 210 Kd, and the molecular weight of the band on SDS-PAGE under reducing conditions was approximately 60 kD, 75 kD, and 100 kD.
  • Example 4 ELISA to detect the in vitro binding activity of tandem hTGF ⁇ Trap fusion protein and hTGF ⁇ 1, m/hTGF ⁇ 3
  • Antigens used for antibody binding detection are human TGF ⁇ 1 (CA59, protein purchased from Novoprotein) and mouse/human TGF ⁇ 3 (CJ44) (protein purchased from Novoprotein).
  • the detection process is as follows:
  • CHO cells stably transfected with human TGF ⁇ RII (CHO-hTGF ⁇ RII) were constructed, and a single clone was selected to establish a line.
  • the blocking activity of Fc-hTGF ⁇ trap was detected using the following method.
  • CHO-TGF ⁇ RII cells were plated into 96-well plates at 2E5/well, 100 ⁇ l per well; the cells were centrifuged at 350G for 5 min at room temperature, the supernatant was discarded, and the mixture of hTGF ⁇ 1.biotin and antibody was added at 100ul/well, mixed, and incubated at 4°C for 1 hour;
  • the fusion protein of the present invention exhibited a similar blocking effect as the control antibody (R0796) on the binding of CHO-hTGF ⁇ RII cells to hTGF ⁇ 1.biotin.
  • the negative control had no blocking effect on the binding of TGF ⁇ RII to hTGF ⁇ 1.biotin.
  • TGF ⁇ 1 inhibits the proliferation of T cells.
  • PBMC peripheral blood mononuclear cells
  • STMCELL human T cell enrichment kit
  • T cell density 5E6/ml with DPBS, and use CFSE (eBioscience, 85-65-0850-84) to stain the T cells.
  • CFSE eBioscience, 85-65-0850-864
  • concentration of CFSE is 0.5 ⁇ M.
  • T cell density 2E6 and add 50ul/well into a 96-well U-shaped plate, i.e. the final number of cells is 1E5/well.
  • CD3/CD28 beads (life, 11161D)
  • the ratio of CD3/CD28 beads to T cells was 1:30.
  • the present disclosure provides a TGF- ⁇ receptor fusion protein, which includes a TGF- ⁇ receptor portion and an immunoglobulin portion.
  • a TGF- ⁇ receptor fusion protein which includes a TGF- ⁇ receptor portion and an immunoglobulin portion.
  • the fusion protein of the present disclosure can be used for tumor treatment. Therefore, the present disclosure has broad application prospects and good industrial applicability in the field of biomedicine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation relève du domaine de la biomédecine, et concerne une protéine de fusion ciblant TGFβ, et concerne en particulier une protéine de fusion du récepteur de TGF-β, comprenant une fraction de récepteur de TGF-β et une fraction d'immunoglobuline. La protéine de fusion peut être utilisée pour traiter des tumeurs.
PCT/CN2024/079436 2023-03-01 2024-02-29 PROTÉINE DE FUSION CIBLANT TGFβ ET SON UTILISATION Pending WO2024179553A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310195354 2023-03-01
CN202310195354.5 2023-03-01

Publications (1)

Publication Number Publication Date
WO2024179553A1 true WO2024179553A1 (fr) 2024-09-06

Family

ID=92537616

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/079436 Pending WO2024179553A1 (fr) 2023-03-01 2024-02-29 PROTÉINE DE FUSION CIBLANT TGFβ ET SON UTILISATION

Country Status (2)

Country Link
CN (1) CN118580372A (fr)
WO (1) WO2024179553A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104508133A (zh) * 2012-08-02 2015-04-08 弗·哈夫曼-拉罗切有限公司 用于产生单体和多聚体分子的方法及其用途
CN110546168A (zh) * 2016-09-27 2019-12-06 埃皮辛特瑞柯斯公司 免疫调节性融合蛋白
WO2020094122A1 (fr) * 2018-11-09 2020-05-14 江苏恒瑞医药股份有限公司 COMPOSITION PHARMACEUTIQUE À BASE D'UNE PROTÉINE DE FUSION DU RÉCEPTEUR TGF-β ET SON UTILISATION
CN113667016A (zh) * 2020-05-15 2021-11-19 普米斯生物技术(珠海)有限公司 一种构建冠状病毒抗体的平台

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104508133A (zh) * 2012-08-02 2015-04-08 弗·哈夫曼-拉罗切有限公司 用于产生单体和多聚体分子的方法及其用途
CN110546168A (zh) * 2016-09-27 2019-12-06 埃皮辛特瑞柯斯公司 免疫调节性融合蛋白
WO2020094122A1 (fr) * 2018-11-09 2020-05-14 江苏恒瑞医药股份有限公司 COMPOSITION PHARMACEUTIQUE À BASE D'UNE PROTÉINE DE FUSION DU RÉCEPTEUR TGF-β ET SON UTILISATION
CN113667016A (zh) * 2020-05-15 2021-11-19 普米斯生物技术(珠海)有限公司 一种构建冠状病毒抗体的平台

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KIYOSHI MASATO, TATEMATSU KEN-ICHIRO, TADA MINORU, SEZUTSU HIDEKI, SHIBATA HIROKO, ISHII-WATABE AKIKO: "Structural insight and stability of TNFR-Fc fusion protein (Etanercept) produced by using transgenic silkworms", JOURNAL OF BIOCHEMISTRY, OXFORD UNIVERSITY PRESS, GB, vol. 169, no. 1, 6 February 2021 (2021-02-06), GB , pages 25 - 33, XP093205513, ISSN: 0021-924X, DOI: 10.1093/jb/mvaa092 *
SANDALON ZIV, BRUCKHEIMER ELIZABETH M., LUSTIG KURT H., ROGERS LINDA C., PELUSO RICHARD W., BURSTEIN HAIM: "Secretion of a TNFR:Fc Fusion Protein following Pulmonary Administration of Pseudotyped Adeno-Associated Virus Vectors", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 78, no. 22, 15 November 2004 (2004-11-15), US , pages 12355 - 12365, XP093205515, ISSN: 0022-538X, DOI: 10.1128/JVI.78.22.12355-12365.2004 *

Also Published As

Publication number Publication date
CN118580372A (zh) 2024-09-03

Similar Documents

Publication Publication Date Title
JP7034950B2 (ja) 抗ｈｌａ－ｇ特異的抗体
CN113166246A (zh) 一种抗体及其用途
EP4582449A1 (fr) Anticorps monoclonal humanisé recombinant ciblant gprc5d humain et son utilisation
CN112154157A (zh) 抗ror1抗体及其用途
CN111349163B (zh) 针对cd123的单克隆抗体
CN113527493B (zh) 一种b7-h3抗体及其应用
CN116731188A (zh) 抗pd-l1和vegf双特异性抗体及其应用
WO2021143914A1 (fr) Anticorps anti-ox40, son procédé de production et son application
CN114656566B (zh) 一种靶向cd47的抗体及其应用
JP2022550121A (ja) Ｌｉｆに特異的な結合分子及びその使用
JP7734856B2 (ja) 二重特異性抗体及びその使用
WO2024120418A1 (fr) Anticorps anti-ccr8 et utilisation associée
WO2024179553A1 (fr) PROTÉINE DE FUSION CIBLANT TGFβ ET SON UTILISATION
WO2023143484A1 (fr) Protéine de liaison à l'antigène et son utilisation
CN118580373A (zh) 一种TGF-β和TIGIT双特异性融合蛋白及其用途
TWI835166B (zh) 靶向pd-1和/或ox40的特異性結合蛋白及其應用
EP4446341A1 (fr) Anticorps se liant au bcma et son utilisation
CN118240092A (zh) 一种抗pd-l1和tigit双特异性抗体融合蛋白及其应用
CN111704668B (zh) 抗ccr4抗体及其在治疗癌症中的应用
CN118240091A (zh) 一种抗pd-l1和tigit双特异性抗体及其应用
WO2025051083A1 (fr) Anticorps spécifique anti-bdca-2 et son utilisation
WO2024164807A1 (fr) Anticorps multispécifique et son utilisation
CN120230222A (zh) 一种靶向CD39和TGFβ的融合蛋白及其应用
KR20230169942A (ko) 항-pd-l1 항체 및 이의 용도
WO2024199294A1 (fr) Anticorps ou fragment de liaison à l'antigène de celui-ci ciblant cd3 et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24763233

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE