WO2023030408A1 - TGFβRII突变体及其应用 - Google Patents

TGFβRII突变体及其应用 Download PDF

Info

Publication number
WO2023030408A1
WO2023030408A1 PCT/CN2022/116320 CN2022116320W WO2023030408A1 WO 2023030408 A1 WO2023030408 A1 WO 2023030408A1 CN 2022116320 W CN2022116320 W CN 2022116320W WO 2023030408 A1 WO2023030408 A1 WO 2023030408A1
Authority
WO
WIPO (PCT)
Prior art keywords
tgfβrii
fragment
mutant
terminal
seq
Prior art date
Application number
PCT/CN2022/116320
Other languages
English (en)
French (fr)
Inventor
张轶博
芦迪
路力生
霍永庭
Original Assignee
广东菲鹏制药股份有限公司
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 广东菲鹏制药股份有限公司 filed Critical 广东菲鹏制药股份有限公司
Publication of WO2023030408A1 publication Critical patent/WO2023030408A1/zh

Links

Images

Classifications

    • 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
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • 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
    • 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
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • 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
    • 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

Definitions

  • the present invention relates to the field of biomedicine, in particular, to TGF ⁇ RII mutants and applications thereof, more specifically, to TGF ⁇ RII mutants, nucleic acid molecules encoding the TGF ⁇ RII mutants, expression vectors, recombinant cells, pharmaceutical compositions, and their use in Use in the preparation of medicines.
  • Transforming growth factor ⁇ belongs to the TGF ⁇ superfamily that regulates cell growth and differentiation. In addition to TGF ⁇ , this family also includes activin, inhibin, Müllerian inhibitory substance and bone morphogenic protein. Various cells in the body can secrete inactive TGF ⁇ . In vitro, the inactive state of TGF ⁇ , also known as latency associated peptide (LAP), can be activated by acid cleavage. In the body, an acidic environment can exist near fractures and in healing wounds. Cleavage of the protein itself turns the TGF ⁇ complex into activated TGF ⁇ .
  • LAP latency associated peptide
  • TGF ⁇ plays an important regulatory role in cell growth, differentiation and immune function. TGF ⁇ exerts tumor suppressor or tumor promoter role in tumors in a cell-context-dependent manner. TGF ⁇ can inhibit the expression of the proto-oncogene c-myc, but during tumor development, with the introduction of mutations or changes in epigenetic modifications, cancer cells gradually tolerate the inhibition of TGF ⁇ signaling, which eventually leads to tumor development. Recent studies have found that the increase of TGF ⁇ in the tumor microenvironment is related to immune escape, and the increase of TGF ⁇ will increase T cell rejection and block the infiltration of TH1 effector T cells.
  • Proteins targeting TGF ⁇ can control the content of free TGF ⁇ on the tumor surface, but the current process of preparing the extracellular domain of TGF ⁇ receptors will produce a large number of fragments and high polymers, which are difficult to remove through purification, resulting in the production efficiency of TGF ⁇ receptors Low, low yield, seriously affecting druggability, the method and operation of protein preparation still need to be improved.
  • the inventors used the method of truncating the amino acid sequence of the extracellular domain of TGF ⁇ RII to reduce the fragment content in the process of preparing TGF ⁇ RII, obtained TGF ⁇ RII mutant fragments and TGF ⁇ RII mutants, and utilized TGF ⁇ RII mutation
  • the fusion protein was prepared from the body, and the fusion protein with low fragment content in the process of preparing TGF ⁇ RII was unexpectedly obtained.
  • the fusion protein has high in vitro binding activity with TGF ⁇ , and the drug-making effect has been significantly improved. It can control the expression of up-regulated TGF ⁇ around tumor cells content to prevent or treat tumors.
  • the present invention provides a TGF ⁇ RII mutant fragment.
  • the TGF ⁇ RII mutant fragment includes at most 122 amino acids of 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 inventor found creatively that after truncating the N-terminal of the extracellular domain of TGF ⁇ RII according to the mutation method of the embodiment of the present invention, the obtained TGF ⁇ RII mutant
  • the fragment has the biological activity of binding to TGF ⁇ , 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, and the TGF ⁇ RII mutant containing the fragment also has the same biological activity, and its amino acid sequence is not easy to dissociate during the production of TGF ⁇ RII mutants, the fragment content is significantly reduced, and its druggability is improved.
  • the present invention provides a TGF ⁇ RII mutant.
  • it includes an extracellular region, a transmembrane region and an intracellular region; wherein, the extracellular region includes the TGF ⁇ RII mutant fragment described in the first aspect.
  • the inventors found creatively that the TGF ⁇ RII mutant comprising the fragment described in the first aspect of the present invention has the same biological activity as wild-type TGF ⁇ RII, and in In the process of producing the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its druggability is improved.
  • the present invention proposes a method for preparing the TGF ⁇ RII mutant fragment described in the first aspect.
  • the amino acid sequence of the TGF ⁇ RII mutant is obtained by truncating at least 14 amino acids and inserting no more than 20 amino acids starting from the N-terminal of the extracellular domain of TGF ⁇ RII.
  • the inventors explored through a large number of experiments, and the N- After the terminal is truncated, the obtained TGF ⁇ RII mutant fragment has the biological activity of binding to TGF ⁇ , and the fragment is not easy to dissociate, so it is not easy to be broken, and the TGF ⁇ RII mutant containing the fragment also has the same biological activity as the wild-type TGF ⁇ RII.
  • Biological activity, 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 druggability is improved.
  • the present invention provides a TGF ⁇ RII mutant fragment. According to the embodiment of the present invention, it is prepared by the method described in the third aspect. According to an embodiment of the present invention, 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 Medicinal.
  • the present invention proposes a method for preparing the TGF ⁇ RII mutant described in the second aspect.
  • the TGF ⁇ RII mutant includes an extracellular region, a transmembrane region and an intracellular region; the method comprises: truncating at least 14 amino acids from the N-terminal of the TGF ⁇ RII extracellular domain, inserting no more than 20 amino acids.
  • the inventors After a large number of experimental explorations, the inventors have found that the TGF ⁇ RII mutant obtained according to the method of the embodiment of the present invention has the same Consistent biological activity, 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 druggability is improved.
  • the present invention provides a TGF ⁇ RII mutant. According to the embodiment of the present invention, it is prepared by the method described in the fifth aspect.
  • the present invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the TGF ⁇ RII mutant fragment described in the first aspect or the fourth aspect, or the TGF ⁇ RII mutant described in the second aspect or the sixth aspect.
  • the TGF ⁇ RII mutant fragment encoded by the nucleic acid molecule has the biological activity of binding to TGF ⁇ , 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 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, so the fragment content is significantly reduced, which improves its druggability sex.
  • the present invention provides a fusion protein.
  • a fusion protein including: 1) the TGF ⁇ RII mutant fragment described in the first aspect or the fourth aspect; and 2) an immunoglobulin Fc fragment, the TGF ⁇ RII mutant fragment is connected to the immunoglobulin through a linking peptide The Fc fragments are connected.
  • the obtained TGF ⁇ RII mutation The body has the same biological activity as the wild-type TGF ⁇ RII. After adding the immunoglobulin Fc fragment to the TGF ⁇ RII mutant, the obtained fusion protein also has the same biological activity as the wild-type TGF ⁇ RII, and its amino acid sequence is different in the preparation process of the fusion protein.
  • the fusion protein can control the content of TGF ⁇ expressed up-regulated around tumor cells, so as to prevent or treat tumors with long-term effect.
  • the present invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the fusion protein described in the eighth aspect.
  • the fusion protein encoded by the nucleic acid molecule has the same biological activity as wild-type TGF ⁇ RII, and its amino acid sequence is not easy to dissociate during the preparation process of the fusion protein, and the fragment content is significantly reduced and increased. With its druggability and half-life in vivo, the fusion protein can control the content of TGF ⁇ expressed up-regulated around tumor cells, so as to prevent or treat tumors with long-term effect.
  • the present invention provides an expression vector.
  • the expression vector comprises the nucleic acid molecule described in the seventh aspect or the ninth aspect.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the nucleic acid molecule described in the seventh aspect or the ninth aspect, or the expression vector described in the tenth aspect.
  • the recombinant cell can express the above-mentioned fusion protein, and its amino acid sequence is not easily dissociated during the preparation process of the fusion protein, the fragment content is significantly reduced, and its druggability and half-life in vivo are improved.
  • the fusion protein can control the content of the up-regulated TGF ⁇ around tumor cells, so as to prevent or treat tumors with long-term effect.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the TGF ⁇ RII mutant fragment described in the first aspect or the fourth aspect, or the TGF ⁇ RII mutant described in the second aspect or the sixth aspect, or the nucleic acid molecule described in the seventh aspect , or the fusion protein described in the eighth aspect, or the nucleic acid molecule described in the ninth aspect, or the expression vector described in the tenth aspect, or the recombinant cell described in the eleventh aspect.
  • the present invention provides the TGF ⁇ RII mutant fragment described in the first aspect or the fourth aspect, the TGF ⁇ RII mutant described in the second aspect or the sixth aspect, the nucleic acid molecule described in the seventh aspect, the The fusion protein of the eighth aspect, the nucleic acid molecule of the ninth aspect, the expression vector of the tenth aspect, the recombinant cell of the eleventh aspect or the pharmaceutical composition of the twelfth aspect in the preparation of medicine the use of.
  • the drug is used for preventing or treating tumors.
  • the medicine provided by the invention has the function of long-acting combination with TGF ⁇ , so as to achieve the purpose of treating or preventing tumors.
  • the present invention provides a method for preparing the fusion protein described in the eighth aspect.
  • the method includes the following steps: 1) constructing the expression vector described in the tenth aspect; 2) introducing the expression vector into a host cell to obtain a recombinant cell to express the fusion protein.
  • the method can efficiently obtain the fusion protein, and the fusion protein has the same biological activity as the wild-type TGF ⁇ RII.
  • its amino acid sequence is not easy to dissociate, and the fragment content is significantly reduced, which improves its drug production. sex and half-life in vivo.
  • the present invention proposes a method for reducing the fragment content in the process of preparing the fusion protein described in the eighth aspect.
  • the method includes the following steps: 1) constructing the expression vector described in the tenth aspect; 2) introducing the expression vector into a host cell.
  • the amino acid sequence of the fusion protein is not easy to dissociate, the fragment content is significantly reduced, and the drug effect thereof is significantly improved.
  • the present invention provides a method for preventing or treating tumors.
  • the method comprises administering to the subject at least one of the following:
  • the TGF ⁇ RII mutant fragment described in the first aspect or the fourth aspect 2) The TGF ⁇ RII mutant described in the second aspect or the sixth aspect; 3) The fusion protein described in the eighth aspect; 3) The seventh aspect or the ninth aspect 4) the expression vector of the tenth aspect; 5) the recombinant cell of the eleventh aspect; and 6) the pharmaceutical composition of the twelfth aspect.
  • the method can effectively prevent or treat tumors by controlling the content of the upregulated TGF ⁇ around tumor cells.
  • the present invention provides a medicine.
  • the drug includes the TGF ⁇ RII mutant fragment described in the first aspect or the fourth aspect, the TGF ⁇ RII mutant described in the second aspect or the sixth aspect, the nucleic acid molecule described in the seventh aspect, the nucleic acid molecule described in the eighth aspect At least one of the fusion protein described in the ninth aspect, the nucleic acid molecule described in the ninth aspect, the expression vector described in the tenth aspect, and the pharmaceutical composition described in the eleventh aspect and the twelfth aspect.
  • the medicines according to the embodiments of the present invention can effectively treat or prevent tumors.
  • FIG. 1 is a schematic structural diagram of a TGF ⁇ RII mutant according to an embodiment of the present invention, wherein insertion represents the inserted amino acid sequence, that is, after truncating the first to X amino acids at the N-terminal of the extracellular domain of TGF ⁇ RII, Then insert the flexible fragment and the N-terminal fragment of the extracellular domain of TGF ⁇ RII;
  • Figure 2 is a fusion protein formed by TGF ⁇ RII mutants obtained after truncating the N-terminal of the TGF ⁇ RII extracellular domain, or inserting the flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain after truncation according to an embodiment of the present invention
  • the result diagram of the content of fragments and high polymers produced during the purification process wherein: the fusion proteins shown on the abscissa are WT, Trunc#1-Trunc#19, Trunc#21, Trunc#22; that is R0805: WT represents the fusion protein WT in Table 1 of the present invention, R0749: 7-136 represents the fusion protein Trunc#1 in Table 1 of the present invention, R0776: 8-136 represents the fusion protein Trunc#2 in Table 1 of the present invention, R0777:9-136 represents the fusion protein Trunc#3 in Table 1 of the present invention, R0778:10-136 represents the fusion protein Trun
  • R0788: 1-6+20-136 represents the fusion protein Trunc#14 in Table 1 of the present invention
  • R0789: 21-136 represents the fusion protein Trunc#15 in Table 1 of the present invention
  • R0790: 1-6+22- 136 represents the fusion protein Trunc#16 in Table 1 of the present invention
  • R0791:23-136 represents the fusion protein Trunc#17 in Table 1 of the present invention
  • R0792:24-136 represents the fusion protein Trunc#18 in Table 1 of the present invention
  • R0793: G4S5G+8-136 represents the fusion protein Trunc#19 in Table 1 of the present invention
  • R0795: G4S5G+8-136mut represents the fusion protein Trunc#21 in Table 1 of the present invention
  • R0796: IP6+GSGSGSGSG+20-136 represents The fusion protein Trunc#22 in Table 1 of the present invention
  • Each fusion protein shown on the abscissa 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 the N-terminal 1-6 position of the TGF ⁇ RII extracellular domain Amino acids were truncated; R0796: 1-6+GSGSGSGSG+20-136 indicated that the TGF ⁇ RII mutant in the fusion protein truncated the 1-19th amino acids of the N-terminal of the TGF ⁇ RII extracellular domain, and then inserted into the TGF ⁇ RII cell
  • the 1-6 amino acids IPPHVQ (SEQ ID NO: 1)
  • the flexible fragment GSGSGSGSG (SEQ ID NO: 2)
  • Figure 3 is the fusion protein formed by the TGF ⁇ RII mutant obtained by truncating the N-terminal 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 an embodiment of the present invention in the purification process
  • the result graph of the content of the generated fragments and high polymers, wherein, the specific mutation mode of the TGF ⁇ RII mutant in each fusion protein shown on the abscissa is shown in Table 1;
  • Fig. 4 is a graph showing the detection results of the ELISA binding activity between the fusion protein and human TGF ⁇ 1 according to an embodiment of the present invention
  • Fig. 5 is a diagram showing the detection results of the fusion protein blocking the binding activity of human TGF ⁇ 1 and TGF ⁇ RII according to an embodiment of the present invention
  • Fig. 6 is a graph showing the experimental results of the fusion protein formed by the TGF ⁇ RII mutant obtained after truncating the N-terminus of the extracellular domain of TGF ⁇ RII according to an embodiment of the present invention on the proliferation of T cells;
  • Fig. 7 shows that the TGF ⁇ RII mutant formed by truncating the N-terminus of the TGF ⁇ RII extracellular domain according to an embodiment of the present invention and then inserting the flexible fragment and the N-terminal fragment of the TGF ⁇ RII extracellular domain has an effect on T cell proliferation Effect of the experimental results plot.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • TGF ⁇ RII extracellular domain refers to a 136-amino acid residue peptide segment starting from the N-terminal outside the wild-type TGF ⁇ RII cell, which has the amino acid sequence shown in SEQ ID NO: 10. In the present invention, it is also called "wild-type TGF ⁇ RII".
  • the present invention provides a TGF ⁇ RII mutant fragment, the TGF ⁇ RII mutant fragment includes at most 122 amino acids of the C-terminus of the TGF ⁇ RII extracellular domain; the TGF ⁇ RII mutant fragment further includes a flexible fragment and the N of the TGF ⁇ RII extracellular domain - terminal fragments.
  • the inventor creatively found that after truncating the N-terminal of the extracellular domain of TGF ⁇ RII according to the mutation method of the embodiment of the present invention, the obtained TGF ⁇ RII mutation
  • the body 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 also has the same biological activity, and its amino acid sequence is not easy to dissociate in the process of preparing the TGF ⁇ RII mutant, the fragment content is significantly reduced, and its druggability is improved.
  • the N-terminal of the flexible fragment is connected to the C-terminal of the N-terminal fragment of the TGF ⁇ RII extracellular domain, and the C-terminal of the flexible fragment is connected to the TGF ⁇ RII extracellular domain
  • the N-terminus of at most 122 amino acids of the C-terminus is linked.
  • the amino acid sequence of the TGF ⁇ RII extracellular domain is shown in SEQ ID NO: 10, and the C-terminal fragment of the TGF ⁇ RII extracellular domain is TGF ⁇ RII cellular An N-terminal truncated polypeptide of the extracellular domain; in some embodiments, the C-terminal fragment of the TGF ⁇ RII extracellular domain is an N-terminal truncated continuous 14-23 amino acid residues of the TGF ⁇ RII extracellular domain Polypeptide; in some embodiments, the C-terminal fragment of the extracellular domain of TGF ⁇ RII is a polypeptide of N-terminal truncated consecutive 16-23 amino acid residues of the extracellular domain of TGF ⁇ RII; in some embodiments, the The C-terminal fragments of the extracellular domain of TGF ⁇ RII are TGF ⁇ Trap (15-136), TGF ⁇ Trap (16-136), TGF ⁇ Trap (17-136), TGF ⁇ Trap (13-136), TGF ⁇ Trap
  • the TGF ⁇ RII mutant fragment includes 0-6 amino acids at the N-terminal of the extracellular domain of TGF ⁇ RII.
  • the TGF ⁇ RII mutant fragment comprises 1, 2, 3, 4, 5 or 6 amino acids of the N-terminus of the extracellular domain of TGF ⁇ RII; in some embodiments, the TGF ⁇ RII The mutant fragment includes the 6 amino acid residues N-terminal to the extracellular domain of TGF ⁇ RII.
  • the N-terminal fragment of the extracellular domain of TGF ⁇ RII has the amino acid sequence shown in SEQ ID NO:1.
  • IPPHVQ (SEQ ID NO: 1).
  • the flexible fragment is a short peptide containing G and S amino acids.
  • the flexible fragment is a (G 4 S) X G polypeptide, wherein X is preferably any integer from 1 to 6; in some embodiments, the amino acid sequence of the flexible fragment is as shown in SEQ ID NO: 2. As shown in SEQ ID NO: 12 or SEQ ID NO: 39.
  • the short peptide contains 5-15 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, at most 117 amino acids at the C-terminus of the extracellular domain of TGF ⁇ RII.
  • the TGF ⁇ RII mutant fragment includes 112-117 amino acids at the C-terminal of the extracellular domain of TGF ⁇ RII.
  • the TGF ⁇ RII mutant fragment includes 112, 115 or 117 amino acids at the C-terminal of the extracellular domain of TGF ⁇ RII.
  • the TGF ⁇ RII mutant fragment has the amino acid sequence shown in any one of SEQ ID NO: 3-5.
  • the present invention provides a TGF ⁇ RII mutant, including an extracellular region, a transmembrane region and an intracellular region; wherein, the extracellular region includes the fragment of the TGF ⁇ RII mutant described in the first aspect.
  • the inventors creatively found that the TGF ⁇ RII mutant containing the aforementioned fragments has the same biological activity as the wild-type TGF ⁇ RII, and the production of TGF ⁇ RII mutants During the process, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its druggability is improved.
  • the present invention provides a method for preparing the aforementioned TGF ⁇ RII mutant fragment, the amino acid sequence of the TGF ⁇ RII mutant fragment is truncated from the N-terminal of the extracellular domain of TGF ⁇ RII by at least 14 amino acids, and inserted no more than 20 amino acids And get.
  • the inventors explored through a large number of experiments, and the N- After the terminal is truncated, the obtained TGF ⁇ RII mutant fragment has the biological activity of binding to TGF ⁇ , and the fragment is not easy to dissociate, so it is not easy to be broken, and the TGF ⁇ RII mutant containing the fragment also has the same biological activity as the wild-type TGF ⁇ RII.
  • Biological activity, 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 druggability is improved.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by truncating the N-terminal of the extracellular domain of TGF ⁇ RII by 14-23 amino acids.
  • the mutated site has a certain relationship with the content of fragments produced during the preparation of TGF ⁇ RII.
  • the fragment still has the biological activity of binding to TGF ⁇ , and the preparation of the During the process of TGF ⁇ RII mutant fragments, its amino acid sequence is not easy to dissociate, so the fragment content is significantly reduced, which improves its druggability; the TGF ⁇ RII mutants containing the fragments also have the same biological activity as wild-type TGF ⁇ RII, and in the preparation During the process of the TGF ⁇ RII mutant, its amino acid sequence is not easy to dissociate, the fragment content is significantly reduced, and its druggability is improved.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by truncating the N-terminal of the extracellular domain of TGF ⁇ RII by 14-21 amino acids. 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 fragment content is significantly reduced. Consistent biological activity, 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 druggability is improved.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by truncating the N-terminal of the extracellular domain of TGF ⁇ RII by 14, 19, or 21 amino acids. 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 fragment content is significantly reduced.
  • the TGF ⁇ RII comprising the fragment The mutant has the same biological activity as the wild-type TGF ⁇ RII, and its amino acid sequence is not easily dissociated during the preparation of the TGF ⁇ RII mutant, and the content of fragments and high polymers is significantly reduced, improving its druggability.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by inserting no more than 15 amino acids at the N-terminal of the extracellular domain of TGF ⁇ RII.
  • the amino acid sequence of the TGF ⁇ RII mutant is not easily dissociated during the preparation process, the fragment content is significantly reduced, druggability is improved, and the half-life in vivo is prolonged.
  • the amino acid sequence of the TGF ⁇ RII mutant fragment is obtained by inserting 15 amino acids at the N-terminal of the extracellular domain of TGF ⁇ RII.
  • 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 fragment content is significantly reduced. It is not easy to dissociate, the content of fragments is significantly reduced, its druggability is improved, and the half-life in vivo is prolonged.
  • the inserted amino acid fragment comprises the N-terminal fragment of the extracellular domain of TGF ⁇ RII.
  • the N-terminal fragment of the extracellular domain of TGF ⁇ RII has the amino acid sequence shown in SEQ ID NO: 1.
  • IPPHVQ (SEQ ID NO: 1).
  • the inserted amino acid fragment also includes a flexible fragment.
  • the flexible segment is not particularly limited, and conventional flexible segments can be used.
  • “Flexible fragment” refers to two polypeptides that contain two or more amino acid residues connected by peptide bonds and are linked thereto (for example, the N-terminal fragment of the TGF ⁇ RII extracellular domain, the C -terminal fragments) provide peptides with greater rotational freedom.
  • the flexible fragment has the amino acid sequence shown in SEQ ID NO: 2.
  • the flexible fragment is a polypeptide as shown in SEQ ID NO: 12 or SEQ ID NO: 39.
  • GSGSGSGSG (SEQ ID NO: 2).
  • the N-terminal of the flexible fragment is connected to the C-terminal of the N-terminal fragment of the extracellular domain of TGF ⁇ RII, and the C-terminal of the flexible fragment is connected to the remaining extracellular structure of TGF ⁇ RII after truncation.
  • the N-termini of the N-terminal domains are linked.
  • 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 of the TGF ⁇ RII extracellular domain
  • the inserted fragment is: IPPHVQGSGSGSGSG (SEQ ID NO: 11)
  • the amino acid sequence of the TGF ⁇ RII mutant is not easily dissociated during the preparation process, the fragment content is significantly reduced, its druggability is improved, and its half-life in vivo is prolonged.
  • the present invention provides a TGF ⁇ RII mutant fragment, which is prepared by the above-mentioned method for preparing TGF ⁇ RII mutant.
  • the TGF ⁇ RII mutant fragments with low fragment and high polymer content in the production process can be obtained by truncating and/or inserting the N-terminal site of the extracellular domain of TGF ⁇ RII that is easily broken or dissociated, and producing TGF ⁇ RII mutant fragments containing all In the TGF ⁇ RII mutant process of the above TGF ⁇ RII mutant fragments, the content of high polymers and fragments is still low.
  • the present invention provides a method for preparing the TGF ⁇ RII mutant described above, the TGF ⁇ RII mutant includes an extracellular region, a transmembrane region and an intracellular region; Short by at least 14 amino acids, with insertions of no more than 20 amino acids.
  • the inventors explored through a large number of experiments, and the N- After the terminal is truncated, 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 druggability is improved.
  • the mutant is obtained by truncating 14-23 amino acids from the N-terminal of the extracellular domain of TGF ⁇ RII.
  • the mutated site has a certain relationship with the content of fragments produced during the preparation of TGF ⁇ RII. Therefore, after truncating 14-23 amino acids, the obtained TGF ⁇ RII mutant has the same biological activity as 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 druggability is improved.
  • the TGF ⁇ RII mutant is obtained by truncating 14-21 amino acids from the N-terminal of the extracellular domain of TGF ⁇ RII. 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, and the fragment content is significantly reduced and increased. Its medicinal properties.
  • the amino acid sequence of the TGF ⁇ RII mutant is obtained by truncating the N-terminal of the extracellular domain of TGF ⁇ RII by 14, 19, or 21 amino acids. 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, fragments and high The polymer content is extremely significantly reduced, and its druggability is improved.
  • the amino acid sequence of the TGF ⁇ RII mutant is obtained by inserting no more than 15 amino acids at the N-terminal of the extracellular domain of TGF ⁇ RII.
  • the amino acid sequence of the TGF ⁇ RII mutant is obtained by inserting 15 amino acids at the N-terminal of the extracellular domain of TGF ⁇ RII.
  • 15 amino acids are inserted, the amino acid sequence of the TGF ⁇ RII mutant is not easily dissociated during the preparation process, the fragment content is significantly reduced, the druggability is improved, and the half-life in vivo is prolonged.
  • the inserted amino acid fragment comprises the N-terminal fragment of the extracellular domain of TGF ⁇ RII.
  • the N-terminal fragment of the extracellular domain of TGF ⁇ RII has the amino acid sequence shown in SEQ ID NO:1.
  • the inserted amino acid fragment also includes a flexible fragment.
  • the flexible segment is not particularly limited, and conventional flexible segments can be used.
  • the flexible fragment has the amino acid sequence 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-terminal of the flexible fragment is connected to the C-terminal of the N-terminal fragment of the extracellular domain of TGF ⁇ RII, and the C-terminal of the flexible fragment is connected to the remaining extracellular structure of TGF ⁇ RII after truncation.
  • the N-termini of the N-terminal domains are linked.
  • 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 of the TGF ⁇ RII extracellular domain
  • the inserted fragment is: IPPHVQGSGSGSGSG (SEQ ID NO: 11)
  • the amino acid sequence of the TGF ⁇ RII mutant is not easily dissociated during the preparation process, the fragment content is significantly reduced, its druggability is improved, and its half-life in vivo is prolonged.
  • the present invention provides a TGF ⁇ RII mutant, which is prepared by the aforementioned method.
  • the TGF ⁇ RII mutant with low content of fragments and high polymers in the production process can be obtained by truncating and/or inserting the N-terminal site of the extracellular domain of TGF ⁇ RII that is easily broken or dissociated.
  • the present invention provides a nucleic acid molecule, which encodes the aforementioned TGF ⁇ RII mutant fragment or TGF ⁇ RII mutant.
  • the TGF ⁇ RII mutant fragment or TGF ⁇ RII mutant encoded by the nucleic acid molecule according to the specific embodiment of the present invention has the biological activity of binding to TGF ⁇ , and the amino acid sequence is not easy to dissociate, so in the process of producing TGF ⁇ RII mutant fragment or TGF ⁇ RII mutant Fragment content is significantly reduced, improving its druggability.
  • the present invention provides a fusion protein.
  • the fusion protein according to a specific embodiment of the present invention includes: 1) the above-mentioned TGF ⁇ RII mutant fragment; linked to the Fc fragment of the immunoglobulin.
  • TGF ⁇ RII mutant fragment prepared by the prior art, which affects the efficacy of the drug and the frequency of administration is high
  • after the inventors performed the above-mentioned truncation mutation on the N-terminal amino acid sequence of the extracellular domain of TGF ⁇ RII after the inventors performed the above-mentioned truncation mutation on the N-terminal amino acid sequence of the extracellular domain of TGF ⁇ RII, the obtained TGF ⁇ RII mutation
  • the body has the same biological activity as the wild-type TGF ⁇ RII.
  • the obtained fusion protein After adding the immunoglobulin Fc fragment to the TGF ⁇ RII mutant fragment, the obtained fusion protein also has the same biological activity as the wild-type TGF ⁇ RII.
  • the sequence is not easy to dissociate, the content of fragments is significantly reduced, and its druggability and half-life in vivo are improved.
  • the fusion protein can control the content of TGF ⁇ expressed up-regulated around tumor cells, so as to prevent or treat tumors in a long-term and effective manner.
  • the N-terminal of the connecting peptide is connected to the C-terminal of the immunoglobulin Fc fragment, and the C-terminal of the connecting peptide is connected to the N-terminal of the TGF ⁇ RII mutant fragment.
  • the immunoglobulin Fc fragment is derived from a human IgG antibody molecule.
  • the immunoglobulin Fc fragment comprises an Fc heavy chain fragment of hIgG1 antibody.
  • the lysine at the C-terminus of the immunoglobulin Fc fragment is mutated to alanine. After mutating lysine to alanine, the cleavage and hydrolysis of the fusion protein can be reduced, thereby reducing the content of fragments during the preparation of the fusion protein.
  • the immunoglobulin Fc fragment has the amino acid sequence shown in SEQ ID NO:6.
  • the connecting peptide is a flexible connecting peptide.
  • the connecting peptide is not particularly limited, and conventional flexible fragments in the art can be used.
  • the connecting peptide comprises (G 4 S) X G amino acid sequence, wherein X is an integer greater than 0.
  • the connecting peptide is (G 4 S) X G polypeptide, wherein X is preferably any integer from 1 to 6.
  • the connecting peptide is the polypeptide 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 NO: 7-9.
  • the present invention provides a nucleic acid molecule, which encodes the aforementioned fusion protein.
  • the fusion protein encoded by the nucleic acid molecule according to the specific embodiment of the present invention has the same biological activity as wild-type TGF ⁇ RII, and its amino acid sequence is not easy to dissociate during the preparation process of the fusion protein, and the fragment content is significantly reduced, which improves its drug production. Sex and half-life in vivo, the fusion protein can control the content of TGF ⁇ expressed up-regulated around tumor cells, so as to prevent or treat tumors with long-term effect.
  • the invention provides an expression vector.
  • the expression vector according to the specific embodiment of the present invention comprises the aforementioned nucleic acid molecules encoding TGF ⁇ RII mutant fragments or TGF ⁇ RII mutants, or fusion proteins.
  • the nucleic acid molecule can be directly or indirectly linked to the control elements on the carrier, 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.
  • it is sufficient that the nucleic acid molecule is operably linked to the control element.
  • “Operably linked” herein refers to linking 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 role in regulating the transcription and translation of the exogenous gene function.
  • the expression vector is a eukaryotic expression vector.
  • the present invention provides a recombinant cell.
  • the recombinant cell according to the specific embodiment of the present invention carries the aforementioned nucleic acid molecule encoding TGF ⁇ RII mutant fragment or TGF ⁇ RII mutant, or fusion protein nucleic acid molecule, or expression vector.
  • the recombinant cells according to specific embodiments of the present invention can express the above-mentioned fusion protein, and its amino acid sequence is not easy to dissociate during the preparation process of the fusion protein, the fragment content is significantly reduced, and its druggability and half-life in vivo are improved, so
  • the fusion protein can control the content of TGF ⁇ expressed up-regulated around the tumor cells, so as to prevent or treat tumors with long-term effect.
  • the recombinant cells are mammalian cells, such as: human HEK-293F cells or CHO-K1 cells.
  • the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention provides a pharmaceutical composition
  • the pharmaceutical composition according to the specific embodiment of the present invention comprises the aforementioned TGF ⁇ RII mutant fragments, TGF ⁇ RII mutants, nucleic acid molecules encoding TGF ⁇ RII mutant fragments or TGF ⁇ RII mutants, fusion proteins, encoding Nucleic acid molecule of fusion protein, expression vector, recombinant cell.
  • the pharmaceutical composition may include: a pharmaceutically acceptable adjuvant, which includes at least one of a stabilizer, a wetting agent, an emulsifier, a binding agent, and an isotonic agent;
  • the composition is at least one of tablet, granule, powder, capsule, solution, suspension and freeze-dried preparation.
  • the pharmaceutical composition according to the present invention can control the content of the up-regulated TGF ⁇ around tumor cells, so as to prevent or treat tumors with long-term effect.
  • the present invention provides the aforementioned TGF ⁇ RII mutant fragments, TGF ⁇ RII mutants, nucleic acid molecules encoding TGF ⁇ RII mutant fragments or TGF ⁇ RII mutants, fusion proteins, nucleic acid molecules encoding fusion proteins, expression vectors, and recombinant cells used in the preparation of medicines .
  • the medicaments according to specific embodiments of the present invention are used for preventing or treating tumors.
  • the medicine provided by the invention has the function of long-acting combination with TGF ⁇ , so as to effectively treat or prevent tumors.
  • the present invention provides a method for preparing the aforementioned fusion protein, comprising the following steps: 1) constructing the aforementioned expression vector; 2) introducing the expression vector into host cells to obtain recombinant cells to express the fusion protein protein.
  • the method according to the specific embodiment of the present invention can efficiently obtain the fusion protein, and the fusion protein has the same biological activity as wild-type TGF ⁇ RII, and its amino acid sequence is not easy to dissociate during the preparation process of the fusion protein, and the fragment content is significant Reduce and improve its druggability and half-life in vivo.
  • the recombinant cells are mammalian cells, such as human, monkey, rabbit, dog, bovine, etc.; mammalian cells such as: human HEK-293F cells or CHO-K1 cells .
  • the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention 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 aforementioned expression vector into host cells.
  • the amino acid sequence of the fusion protein is not easy to dissociate during the preparation process of the fusion protein, the fragment content is significantly reduced, and its drug effect is significantly improved.
  • the present invention provides a method for preventing or treating tumors, comprising administering to a subject at least one of the following: 1) the aforementioned TGF ⁇ RII mutant fragment; 2) the aforementioned TGF ⁇ RII mutant; 3) the aforementioned TGF ⁇ RII mutant 4) the aforementioned nucleic acid molecule encoding the TGF ⁇ RII mutant fragment or TGF ⁇ RII mutant or fusion protein; 5) the aforementioned expression vector; 6) the aforementioned recombinant cell; and 7) The aforementioned pharmaceutical composition.
  • the methods according to specific embodiments of the present invention can effectively prevent or treat tumors by controlling the content of up-regulated TGF ⁇ around tumor cells.
  • tumors can be any unregulated cell growth.
  • non-small cell lung cancer papillary thyroid cancer, glioblastoma multiforme, colorectal cancer, melanoma, cholangiocarcinoma or sarcoma, acute myelogenous leukemia, large cell neuroendocrine carcinoma, neuroblastoma , prostate cancer, neuroblastoma, pancreatic cancer, melanoma, squamous cell carcinoma of the head and neck, or gastric cancer, among others.
  • the inventors used homologous recombination technology, using (G 4 S) 4 G (SEQ ID NO: 12) as the linker peptide, to combine the C-terminal of the Fc heavy chain fragment (SEQ ID NO: 6) of hIgG1 antibody with TGF ⁇ RII mutant fragments of different mutant forms (Table 1) were ligated 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) (giving the sequence shown in SEQ ID NO: 6) to reduce fusion Protein cleavage and hydrolysis.
  • TGF ⁇ RII trap fusion protein transfect mammalian cells with DNA encoding the Fc-TGF ⁇ RII receptor in the same expression vector or in a separate expression vector, using standard protocols for transient transfection or stable transfection, transiently transfecting human HEK TGF ⁇ RII trap fusion protein was prepared from -293F cells, TGF ⁇ RII trap fusion protein was stably transfected into CHO-K1 cells, and TGF ⁇ RII trap fusion protein was obtained.
  • the schematic diagram of the structure of the TGF ⁇ RII mutant is shown in FIG. 1 .
  • the TGF ⁇ RII trap fusion proteins formed by different mutant forms of TGF ⁇ RII mutants are shown in Table 1.
  • Fc represents the immunoglobulin Fc fragment (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 wild-type TGF ⁇ RII extracellular domain (SEQ ID NO: 10);
  • TGF ⁇ trap (X-136) represents the X to 136th amino acid of TGF ⁇ RII extracellular domain Residues of the polypeptide (the positions are numbered relative to the natural sequence of SEQ ID NO: 10, the same below), for example, TGF ⁇ trap (20-136) represents the 1st- 19 amino acids (that is, only retain the 20th-136th residues of the TGF ⁇ RII extracellular domain),
  • TGF ⁇ trap (8-136, N10G/N11G/N18A/N19A) represents the N- The
  • TGF ⁇ trap (X-136) represents the X to 136th amino acid sequence of the extracellular domain of TGF ⁇ RII, exemplarily,
  • 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 transient transfection of human HEK-293F cells obtained in Example 1 and the CHO-K1 cell capable of preparing TGF ⁇ RII trap fusion protein obtained by stably transfecting CHO-K1 cells The culture solution was subjected to high-speed centrifugation and the supernatant was collected.
  • the collected supernatant was purified by affinity chromatography in the first step, in which the chromatographic medium was Protein A or a derivative filler that could interact with Fc, such as GE’s Mabselect; the equilibration buffer was 1 ⁇ PBS, equilibrated After 5 times the column volume, the cell supernatant was loaded and combined with the chromatography medium, and the flow rate was controlled so that the retention time of the sample on the affinity chromatography column was ⁇ 1 min. After loading the sample, wash the column with 1 ⁇ PBS (pH7.4), and record the light absorbance value when the wavelength of ultraviolet light is 280nm, until the ultraviolet absorption of A280 drops to the baseline.
  • the chromatographic medium was Protein A or a derivative filler that could interact with Fc, such as GE’s Mabselect
  • the equilibration buffer was 1 ⁇ PBS
  • the inventors analyzed the high polymers and fragments in the purification process of different TGF ⁇ RII trap fusion proteins shown by SEC-HPLC results.
  • Trunc#32 is a flexible linker
  • Trunc#33 is a rigid linker
  • Trunc#34 insertion sequence is a random sequence.
  • the protein used in the TGF ⁇ RIITrap fusion protein Trap binding detection is humanTGF ⁇ 1 (CA59, purchased from Novoprotein), and the detection process is as follows:
  • TGF ⁇ 1 Dilute TGF ⁇ 1 to 0.5 ⁇ g/mL with 1 ⁇ phosphate buffered saline (PBS), coat 100 ⁇ L/well on a 96-well microtiter plate, and overnight at 4°C;
  • PBS phosphate buffered saline
  • TGF ⁇ RIITrap fusion protein Trap end binding detection protein is humanTGF ⁇ 1 (CA59, purchased from Novoprotein), the detection process is as follows:
  • TGF ⁇ RII trap was detected by the following method:
  • step c Dilute the precipitate containing the TGF ⁇ RII trap fusion protein obtained in step b with 1 ⁇ PBS containing 1% BSA, the initial concentration is 50nM, and the dilution factor is 4 times, and a total of 8 gradients are set, and TGF ⁇ 1-biotin ( Purchased from Acrobiosystem) for dilution, the diluted concentration was 1 ⁇ g/mL.
  • step d Add the mixed sample obtained in step d to CHO-TGF ⁇ RII cells, 100 ⁇ L/well, place at 4°C for 30 minutes, centrifuge at room temperature and 300 g for 5 minutes, and discard the supernatant.
  • step f Add SA-PE (400-fold dilution, Jackson immunoresearch, 016-110-084) to the product obtained in step e, add a volume of 100 ⁇ l/well, mix slightly, and place it at 4°C for 1 hour, then Centrifuge at room temperature and 300 g for 5 min, shake off the supernatant; add 1 ⁇ PBS containing 1% BSA to the obtained cells, 100 ⁇ l/well, resuspend the cells, and perform flow cytometry detection.
  • SA-PE 400-fold dilution, Jackson immunoresearch, 016-110-084
  • the fusion protein blocks the binding activity of human TGF ⁇ 1 and TGF ⁇ R II. As shown in Figure 5, the truncated TGF ⁇ RII Trap fusion protein can inhibit the binding of TGF ⁇ 1 to TGF ⁇ R II. From the perspective of EC50, the blocking ability is similar to that of wild-type TGF ⁇ RII Trap Basically there is not much difference.
  • 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 tested the effects of different modified TGF ⁇ RII Trap fusion proteins on the proliferation of T cells in the presence of TGF ⁇ . .
  • the experiment adopts the following exemplary method:
  • PBMC peripheral blood mononuclear cells
  • STMCELL human T cell enrichment kit
  • CFSE eBioscience, 85-65-0850-844
  • the CFSE value of the T cells is detected by flow cytometry, and the proportion of T cells in each generation is calculated, and the leftmost peak is mainly observed.
  • Trunc#1, Trunc#2, Trunc#6, Trunc#9, Trunc#14, Trunc#22 group showed a better effect of promoting T cell proliferation compared with the WT group; at the same time, Trunc#3, Trunc# 7.
  • Trunc#8, Trunc#11, Trunc#13, Trunc#15 ⁇ Trunc#18 groups were weaker than WT group in promoting T cell proliferation.
  • 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.

Landscapes

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

Abstract

提供了TGFβRII突变体片段及其应用，该TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端的至多122个氨基酸、柔性片段和TGFβRII胞外结构域的N-末端片段，所述柔性片段的N端与所述TGFβRII胞外结构域的N-末端片段的C端相连，所述柔性片段的C端与所述TGFβRII胞外结构域的C-末端片段的N端相连。包含所述片段的TGFβRII突变体与野生型TGFβRII具有一致的生物活性，且其制备过程中碎片含量相较于野生型TGFβRII显著降低，所述TGFβRII突变体通过与肿瘤细胞表面TGFβ结合以达到控制肿瘤微环境中TGFβ含量的目的，从而有效预防和治疗肿瘤。

Description

TGFβRII突变体及其应用
本申请要求2021年9月2日提交的中国申请(申请号：202111026893.3，发明名称：TGFβRII变体及其应用)的优先权，该中国申请的全部内容通过引用整体结合到本申请中。
技术领域
本发明涉及生物医药领域，具体地，涉及TGFβRII突变体及其应用，更具体地，涉及TGFβRII突变体、编码所述TGFβRII突变体的核酸分子、表达载体、重组细胞、药物组合物、及其在制备药物中的用途。
背景技术
转化生长因子β(transforming growth factor β，TGFβ)是属于调节细胞生长和分化的TGFβ超家族。这一家族除TGFβ外，还有活化素、抑制素、缪勒氏管抑制质和骨形成蛋白。机体多种细胞均可分泌非活性状态的TGFβ。在体外，非活性状态的TGFβ又称为latency associated peptide(LAP)，通过酸裂解可被活化。在体内，酸性环境可存在于骨折附近和正在愈合的伤口。蛋白本身的裂解作用可使TGFβ复合体变为活化TGFβ。
TGFβ在细胞的生长、分化和免疫功能中发挥重要的调节作用。TGFβ在肿瘤中以细胞背景依赖的方式发挥肿瘤抑制或肿瘤促进的作用。TGFβ能够抑制原癌基因c-myc的表达，但在肿瘤发展过程中，随着突变的引入或表观遗传修饰的变化后，癌细胞逐渐耐受TGFβ信号的抑制作用，最终导致肿瘤的发展。近年的研究发现，肿瘤微环境中TGFβ增加与免疫逃逸相关，TGFβ的增加会增加T细胞排斥，阻断TH1效应T细胞的浸润。靶向TGFβ的蛋白可控制肿瘤表面游离TGFβ的含量，但目前制备TGFβ受体胞外结构域的过程中会产生大量碎片和高聚体，且通过纯化也难以去除，导致TGFβ受体的制备效率低、产量低，严重影响成药性，制备蛋白的方法及操作仍需改进。
发明内容
本申请是基于发明人对以下问题的发现和认识作出的：
本发明中，发明人通过大量实验研究，采用将TGFβRII胞外结构域的氨基酸序列进行截短的方法降低制备TGFβRII过程中的碎片含量，获得了TGFβRII突变体片段以及TGFβRII突变体，并利用TGFβRII突变体制备了融合蛋白，意外地获得了制备TGFβRII过程中碎片含量低的融合蛋白，该融合蛋白与TGFβ具有较高的体外结合活性，成药效果得到显著提升，可通过控制肿瘤细胞周围表达上调的TGFβ的含量来预防或治疗肿瘤。
在本发明的第一方面，本发明提出了一种TGFβRII突变体片段。根据本发明的实施例，所述TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端的至多122个氨基酸；所述TGFβRII突变体片段进一步 包括柔性片段和TGFβRII胞外结构域的N-末端片段。鉴于现有技术制备的TGFβRII碎片含量非常高，影响药效，发明人创造性的发现，根据本发明实施例的突变方式对TGFβRII胞外结构域的N-末端进行截短后，获得的TGFβRII突变体片段具有与TGFβ结合的生物活性，且在制备TGFβRII突变体的过程中，TGFβRII突变体片段氨基酸序列不容易解离，碎片含量显著降低，包含所述片段的TGFβRII突变体同样具有与野生型TGFβRII一致的生物活性，且在生产TGFβRII突变体的过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
在本发明的第二方面，本发明提出了一种TGFβRII突变体。根据本发明的实施例，包括胞外区、跨膜区和胞内区；其中，所述胞外区包括第一方面所述的TGFβRII突变体片段。鉴于现有技术生产的野生型TGFβRII碎片含量非常高，影响药效，发明人创造性的发现，包含本发明第一方面所述的片段的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在生产TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
在本发明的第三方面，本发明提出了一种制备第一方面所述的TGFβRII突变体片段的方法。根据本发明的实施例，所述TGFβRII突变体的氨基酸序列是从TGFβRII胞外结构域的N-末端开始，截短至少14个氨基酸，插入不超过20个氨基酸而得到。鉴于现有技术制备的TGFβRII碎片含量非常高，且在纯化过程中很难去除，影响药效，发明人经过大量的实验探索，根据本发明实施例的突变方式对TGFβRII胞外结构域的N-末端进行截短后，获得的TGFβRII突变体片段具有与TGFβ结合的生物活性，且所述片段不易解离，因此不容易被折断，包含所述片段的TGFβRII突变体同样具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
在本发明的第四方面，本发明提出了一种TGFβRII突变体片段。根据本发明的实施例，是通过第三方面所述的方法制备获得的。根据本发明实施例，具有所述片段的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
在本发明的第五方面，本发明提出了一种制备第二方面所述的TGFβRII突变体的方法。根据本发明的实施例，所述TGFβRII突变体包括胞外区、跨膜区和胞内区；所述方法包括：从TGFβRII胞外结构域的N-末端截短至少14个氨基酸，插入不超过20个氨基酸。鉴于现有技术制备的TGFβRII碎片含量非常高，且在纯化过程中很难去除，影响药效，发明人经过大量的实验探索，根据本发明实施例的方法获得的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
在本发明的第六方面，本发明提出了一种TGFβRII突变体。根据本发明的实施例，是通过第五方面所述的方法制备获得的。
在本发明的第七方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码第一 方面或第四方面所述的TGFβRII突变体片段、或第二方面或第六方面所述的TGFβRII突变体。根据本发明的实施例，所述核酸分子编码的所述TGFβRII突变体片段具有与TGFβ结合的生物活性，且在制备TGFβRII突变体的过程中，TGFβRII突变体片段氨基酸序列不容易解离，碎片含量显著降低；包含所述片段的TGFβRII突变体同样具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，因此碎片含量显著降低，提高了其成药性。
在本发明的第八方面，本发明提出了一种融合蛋白。根据本发明的实施例，包括：1)第一方面或第四方面所述的TGFβRII突变体片段；以及2)免疫球蛋白Fc片段，所述TGFβRII突变体片段通过连接肽与所述免疫球蛋白Fc片段相连。鉴于现有技术制备的TGFβRII碎片含量非常高，影响药效以及给药频率较高，发明人对TGFβRII胞外结构域的N-末端氨基酸序列进行前面所述的截短突变后，获得的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，在TGFβRII突变体上加入免疫球蛋白Fc片段后，获得融合蛋白同样具有与野生型TGFβRII一致的生物活性，且在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期，所述融合蛋白能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
在本发明的第九方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码第八方面所述的融合蛋白。根据本发明的实施例，所述核酸分子编码的所述融合蛋白具有与野生型TGFβRII一致的生物活性，且在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期，所述融合蛋白能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
在本发明的第十方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体包含第七方面或第九方面所述的核酸分子。
在本发明的第十一方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带第七方面或第九方面所述的核酸分子、或第十方面所述的表达载体。根据本发明的实施例，所述重组细胞能够表达前面所述的融合蛋白，且在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期，所述融合蛋白能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
在本发明的第十二方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包含第一方面或第四方面所述TGFβRII突变体片段、或第二方面或第六方面所述TGFβRII突变体、或第七方面所述的核酸分子、或第八方面所述的融合蛋白、或第九方面所述的核酸分子、或第十方面所述的表达载体、或第十一方面所述的重组细胞。所述药物组合物可包括：药学上可接受的辅剂，所述药学上可接受的辅剂包括稳定剂、湿润剂、乳化剂、粘合剂、等渗剂的至少之一；所述药物组合物呈片剂、颗粒剂、散剂、胶囊剂、溶液剂、悬浮剂、冻干制剂的至少一种。所述药物组合物能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
在本发明的第十三方面，本发明提出了第一方面或第四方面所述TGFβRII突变体片段、第二方面或第六方面所述TGFβRII突变体、第七方面所述的核酸分子、第八方面所述的融合蛋白、第九方面所述的核酸分子、第十方面所述的表达载体、第十一方面所述的重组细胞或第十二方面所述的药物组合物在制备药物中的用途。根据本发明的实施例，所述药物用于预防或治疗肿瘤。本发明提供的药物具有长效与TGFβ结合的功能，达到治疗或预防肿瘤的目的。
在本发明的第十四方面，本发明提出了一种制备第八方面所述的融合蛋白的方法。根据本发明的实施例，所述方法包括以下步骤：1)构建第十方面所述的表达载体；2)将所述表达载体导入宿主细胞中，获得重组细胞，以表达所述融合蛋白。所述方法可高效获得所述融合蛋白，且所述融合蛋白具有与野生型TGFβRII一致的生物活性，在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期。
在本发明的第十五方面，本发明提出了一种降低制备第八方面所述的融合蛋白过程中碎片含量的方法。根据本发明的实施例，所述方法包括以下步骤：1)构建第十方面所述的表达载体；2)将所述表达载体导入宿主细胞中。所述方法在融合蛋白的制备过程中融合蛋白的氨基酸序列不容易解离，碎片含量显著降低，显著提高了其药效。
在本发明的第十六方面，本发明提出了一种预防或治疗肿瘤的方法。根据本发明的实施例，所述方法包括向受试者施用以下中的至少之一：
1)第一方面或第四方面所述TGFβRII突变体片段；2)第二方面或第六方面所述TGFβRII突变体；3)第八方面所述的融合蛋白；3)第七方面或第九方面所述的核酸分子；4)第十方面所述的表达载体；5)第十一方面所述的重组细胞；以及6)第十二方面所述的药物组合物。所述方法能够通过控制肿瘤细胞周围表达上调的TGFβ的含量，从而有效预防或治疗肿瘤。
本发明的第十七方面，本发明提出了一种药物。根据本发明的实施例，所述药物包括第一方面或第四方面所述TGFβRII突变体片段、第二方面或第六方面所述TGFβRII突变体、第七方面所述的核酸分子、第八方面所述的融合蛋白、第九方面所述的核酸分子、第十方面所述的表达载体、第十一方面和第十二方面所述的药物组合物中的至少之一。根据本发明实施例的药物能够有效治疗或预防肿瘤。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的TGFβRII突变体的结构示意图，其中，insertion表示插入的氨基酸序列，即：将TGFβRII胞外结构域的N-末端的第1至第X个氨基酸进行截短后、再插入柔性片段和TGFβRII胞外结构域的N-末端片段；
图2是根据本发明实施例的截短TGFβRII胞外结构域的N-末端、或截短后再插入柔性片段和TGFβRII胞外结构域的N-末端片段后得到的TGFβRII突变体形成的融合蛋白在纯化过程中所产生的碎片及高聚体的含量的结果图，其中：横坐标所示的各融合蛋白依次为WT、Trunc#1-Trunc#19、Trunc#21、Trunc#22；也即R0805：WT表示本发明表1中的融合蛋白WT、R0749：7-136表示本发明表1中的融合蛋白Trunc#1、R0776：8-136表示本发明表1中的融合蛋白Trunc#2、R0777：9-136表示本发明表1中的融合蛋白Trunc#3、R0778：10-136表示本发明表1中的融合蛋白Trunc#4、R0779：11-136表示本发明表1中的融合蛋白Trunc#5、R0780：12-136表示本发明表1中的融合蛋白Trunc#6、R0781：13-136表示本发明表1中的融合蛋白Trunc#7、R0782：14-136表示本发明表1中的融合蛋白Trunc#8、R0783：1-6+15-136表示本发明表1中的融合蛋白Trunc#9、R0784：16-136表示本发明表1中的融合蛋白Trunc#10、R0785：17-136表示本发明表1中的融合蛋白Trunc#11、R0786：18-136表示本发明表1中的融合蛋白Trunc#12、R0787：19-136表示本发明表1中的融合蛋白Trunc#13、R0788：1-6+20-136表示本发明表1中的融合蛋白Trunc#14、R0789：21-136表示本发明表1中的融合蛋白Trunc#15、R0790：1-6+22-136表示本发明表1中的融合蛋白Trunc#16、R0791：23-136表示本发明表1中的融合蛋白Trunc#17、R0792：24-136表示本发明表1中的融合蛋白Trunc#18、R0793：G4S5G+8-136表示本发明表1中的融合蛋白Trunc#19、R0795：G4S5G+8-136mut表示本发明表1中的融合蛋白Trunc#21、R0796：IP6+GSGSGSGSG+20-136表示本发明表1中的融合蛋白Trunc#22；
横坐标所示的各融合蛋白只表示了TGFβRII突变体的突变方式，如，R0749：7-136表示该融合蛋白中TGFβRII突变体是将TGFβRII胞外结构域的N-末端的第1-6位氨基酸进行截短；R0796：1-6+GSGSGSGSG+20-136表示该融合蛋白中TGFβRII突变体是将TGFβRII胞外结构域的N-末端的第1-19位氨基酸进行截短，然后插入TGFβRII胞外结构域的N-末端的第1-6位氨基酸(IPPHVQ(SEQ ID NO：1))和柔性片段(GSGSGSGSG(SEQ ID NO：2))；
图3是根据本发明实施例的截短TGFβRII胞外结构域的N-末端后、再插入柔性片段和TGFβRII胞外结构域的N-末端片段得到的TGFβRII突变体形成的融合蛋白在纯化过程中所产生的碎片及高聚体的含量的结果图，其中，横坐标所示的各融合蛋白中TGFβRII突变体的具体突变方式见表1；
图4是根据本发明实施例的融合蛋白与人源TGFβ1的ELISA结合活性检测结果图；
图5是根据本发明实施例的融合蛋白阻断人源TGFβ1与TGFβRII的结合活性检测结果图；
图6是根据本发明实施例的截短TGFβRII胞外结构域的N-末端后得到的TGFβRII突变体形成的融合蛋白对T细胞增殖影响的实验结果图；
图7是根据本发明实施例的截短TGFβRII胞外结构域的N-末端后、再插入柔性片段和TGFβRII胞外结构域的N-末端片段得到的TGFβRII突变体形成的融合蛋白对T细胞增殖影响的实验结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例 是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
“TGFβRII胞外结构域”是指野生型TGFβRII细胞外自N端开始的一段长136个氨基酸残基的肽段，具有如SEQ ID NO：10所示的氨基酸序列。本发明中又称“野生型TGFβRII”。
Figure PCTCN2022116320-appb-000001
本发明提供一种TGFβRII突变体片段，所述TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端的至多122个氨基酸；所述TGFβRII突变体片段进一步包括柔性片段和TGFβRII胞外结构域的N-末端片段。鉴于现有技术制备的TGFβRII的碎片含量非常高，影响药效，发明人创造性的发现，根据本发明实施例的突变方式对TGFβRII胞外结构域的N-末端进行截短后，获得的TGFβRII突变体片段具有TGFβ结合的生物活性，且在制备TGFβRII突变体的过程中，TGFβRII突变体片段氨基酸序列不容易解离，碎片含量显著降低，包含所述片段的TGFβRII突变体同样具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述柔性片段的N端与所述TGFβRII胞外结构域的N-末端片段的C端相连，所述柔性片段的C端与所述TGFβRII胞外结构域的C-末端的至多122个氨基酸的N端相连。
根据本发明的一些实施方式，所述TGFβRII突变体片段中，所述TGFβRII胞外结构域的氨基酸序列如SEQ ID NO：10所示，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短多肽；在一些实施方式中，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短连续14-23个氨基酸残基的多肽；在一些实施方式中，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短连续16-23个氨基酸残基的多肽；在一些实施方式中，所述TGFβRII胞外结构域的C-末端片段为TGFβTrap(15-136)、TGFβTrap(16-136)、TGFβTrap(17-136)、TGFβTrap(18-136)、TGFβTrap(19-136)、TGFβTrap(20-136)、TGFβTrap(21-136)、TGFβTrap(22-136)、TGFβTrap(23-136)、TGFβTrap(24-136)、或其突变序列，所述突变序列为在TGFβRII胞外结构域的C-末端片段上具有一个或多个氨基酸的取代，例如TGFβRII胞外结构域具有N18A和/或/N19A氨基酸取代；在一些实施方式中，所述TGFβRII胞外结构域的C-末端片段为SEQ ID NO：41、SEQ ID NO：42或SEQ ID NO：43所示多肽。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的0～6个氨基酸。在一些实施方式中，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的1 个、2个、3个、4个、5个或6个氨基酸；在一些实施方式中，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的6个氨基酸残基。
根据本发明的一个具体的实施例，所述TGFβRII胞外结构域的N-末端片段具有SEQ ID NO：1所示的氨基酸序列。
IPPHVQ(SEQ ID NO：1)。
根据本发明的一个具体的实施例，所述柔性片段为含有G、S氨基酸的短肽。在一些实施方式中，所述柔性片段为(G 4S) XG多肽，其中，X优选为1至6的任意整数；在一些实施方式中，所述柔性片段的氨基酸序列如SEQ ID NO：2、SEQ ID NO：12或SEQ ID NO：39所示。
根据本发明的一个具优选的实施例，所述短肽含有5～15个氨基酸。
根据本发明的一个具体的实施例，所述柔性片段具有SEQ ID NO：2所示的氨基酸序列。
GSGSGSGSG(SEQ ID NO：2)。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端的至多115个、至多117个氨基酸。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端的112～117个氨基酸。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端的112个、115个或117个氨基酸。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段具有SEQ ID NO：3～5任一所示的氨基酸序列。
Figure PCTCN2022116320-appb-000002
Figure PCTCN2022116320-appb-000003
Figure PCTCN2022116320-appb-000004
本发明提供一种TGFβRII突变体，包括胞外区、跨膜区和胞内区；其中，所述胞外区包括第一方面所述的TGFβRII突变体片段。鉴于现有技术生产的野生型TGFβRII碎片含量非常高，影响药效，发明人创造性的发现，包含前面所述的片段的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在生产TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
本发明提供一种制备前面所述的TGFβRII突变体片段的方法，所述TGFβRII突变体片段的氨基酸序列是从TGFβRII胞外结构域的N-末端截短至少14个氨基酸，插入不超过20个氨基酸而得到。鉴于现有技术制备的TGFβRII碎片含量非常高，且在纯化过程中很难去除，影响药效，发明人经过大量的实验探索，根据本发明实施例的突变方式对TGFβRII胞外结构域的N-末端进行截短后，获得的TGFβRII突变体片段具有与TGFβ结合的生物活性，且所述片段不易解离，因此不容易被折断，包含所述片段的TGFβRII突变体同样具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段的氨基酸序列是对TGFβRII胞外结构域的N-末端截短14-23个氨基酸而得到。发生突变的所述位点与制备TGFβRII过程中产生的碎片的含量具有一定的联系，因此，当截短14-23个氨基酸后，所述片段仍具有与TGFβ结合的生物活性，且制备所述TGFβRII突变体片段的过程中，其氨基酸序列不容易解离，因此碎片含量显著降低，提高了其成药性；包含所述片段的TGFβRII突变体同样具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段的氨基酸序列是对TGFβRII胞外结构域的N-末端截短14-21个氨基酸而得到。当截短14-21个氨基酸后，获得的TGFβRII突变体片段仍具有与TGFβ结合的生物活性，且不容易解离，碎片含量显著降低，包含所述片段的TGFβRII突变体同样具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段的氨基酸序列是对TGFβRII胞外结构域的N-末端截短14个、19个、或21个氨基酸而得到。当截短14个、19个、或21个氨基酸后，获得的TGFβRII突变体片段仍具有与TGFβ结合的生物活性，且所述片段不容易解离，碎片含量显著降低，包含所述片段的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中其氨基酸序列不容易解离，碎片和高聚体含量均极显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段的氨基酸序列是在TGFβRII胞外结构域的N-末端插入不超过15个氨基酸而得到。当插入不超过15个氨基酸时，所述TGFβRII突变体在制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性，且体内半衰期延长。
根据本发明的一个具体的实施例，所述TGFβRII突变体片段的氨基酸序列是在TGFβRII胞外结构域的N-末端插入15个氨基酸而得到。当插入15个氨基酸时，所述TGFβRII突变体片段仍然具备与TGFβ结合的生物活性，其氨基酸序列不容易解离，碎片含量显著降低，包含所述片段的TGFβRII突变体在制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性，且体内半衰期延长。
根据本发明的一个具体的实施例，所述插入的氨基酸片段包含TGFβRII胞外结构域的N-末端片段。
根据本发明的一个具体的实施例，所述TGFβRII胞外结构域的N-末端片段具有SEQ ID NO：1所 示的氨基酸序列。
IPPHVQ(SEQ ID NO：1)。
根据本发明的一个具体的实施例，所述插入的氨基酸片段还包含柔性片段。根据本发明的具体实施例，所述柔性片段不受特别限制，常规的柔性片段均可使用。
″柔性片段″是指含有由肽键连接的2个或更多个氨基酸残基、并且为其连接的2个多肽(例如TGFβRII胞外结构域的N-末端片段、TGFβRII胞外结构域的C-末端片段)提供更大转动自由度的肽。
根据本发明的一个具体的实施例，所述柔性片段具有SEQ ID NO：2所示的氨基酸序列。在一些实施方式中，所述柔性片段为如SEQ ID NO：12或SEQ ID NO：39所示的多肽。
GSGSGSGSG(SEQ ID NO：2)。
根据本发明的一个具体的实施例，所述柔性片段的N端与TGFβRII胞外结构域的N-末端片段的C端相连，所述柔性片段的C端与截短后剩余的TGFβRII胞外结构域N-末端的N端相连。
根据本发明的一个具体实施例，所述SEQ ID NO：2所示的氨基酸序列排列在所述TGFβRII胞外结构域的N-末端片段之后(例如，当TGFβRII胞外结构域的N-末端氨基酸序列如SEQ ID NO：10所示时，所述插入的片段为：IPPHVQGSGSGSGSG(SEQ ID NO：11))。此时，所述TGFβRII突变体在制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性，且体内半衰期延长。
本发明提供一种TGFβRII突变体片段，是通过前面所述的制备TGFβRII突变体的方法制备获得的。
根据本发明的具体实施例，制备TGFβRII时易产生碎片和TGFβRII的氨基酸序列中某些位点易被折断具有紧密的联系，因此，采用本发明具体实施例所提供的方法，在制备TGFβRII突变体片段时可以采用将TGFβRII胞外结构域的N-末端易被折断或解离的位点截短和/插入的方式获得生产过程中碎片和高聚体含量低的TGFβRII突变体片段，生产包含所述TGFβRII突变体片段的TGFβRII突变体过程中，其高聚体以及碎片含量依然较低。
本发明提供一种制备前面所述的TGFβRII突变体的方法，所述TGFβRII突变体包括胞外区、跨膜区和胞内区；所述方法包括：从TGFβRII胞外结构域的N-末端截短至少14个氨基酸，插入不超过20个氨基酸。鉴于现有技术制备的TGFβRII碎片含量非常高，且在纯化过程中很难去除，影响药效，发明人经过大量的实验探索，根据本发明实施例的突变方式对TGFβRII胞外结构域的N-末端进行截短后，获得的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述突变体从TGFβRII胞外结构域的N-末端截短14-23个氨基酸而得到。发生突变的所述位点与制备TGFβRII过程中产生的碎片的含量具有一定的联系，因此，当截短14-23个氨基酸后，获得的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述TGFβRII突变体是从TGFβRII胞外结构域的N-末端截短 14-21个氨基酸而得到。当截短14-21个氨基酸后，获得的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中，其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述TGFβRII突变体的氨基酸序列是对TGFβRII胞外结构域的N-末端截短14个、19个、或21个氨基酸而得到。当截短14个、19个、或21个氨基酸后，获得的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，且在制备TGFβRII突变体的过程中其氨基酸序列不容易解离，碎片和高聚体含量均极显著降低、提高了其成药性。
根据本发明的一个具体的实施例，所述TGFβRII突变体的氨基酸序列是在TGFβRII胞外结构域的N-末端插入不超过15个氨基酸而得到。
根据本发明的一个具体的实施例，所述TGFβRII突变体的氨基酸序列是在TGFβRII胞外结构域的N-末端插入15个氨基酸而得到。当插入15个氨基酸时，所述TGFβRII突变体在制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性，且体内半衰期延长。
根据本发明的一个具体的实施例，所述插入的氨基酸片段包含TGFβRII胞外结构域的N-末端片段。
根据本发明的一个具体的实施例，所述TGFβRII胞外结构域的N-末端片段具有SEQ ID NO：1所示的氨基酸序列。
根据本发明的一个具体的实施例，所述插入的氨基酸片段还包含柔性片段。根据本发明的具体实施例，所述柔性片段不受特别限制，常规的柔性片段均可使用。
根据本发明的一个具体的实施例，所述柔性片段具有SEQ ID NO：2所示的氨基酸序列。在一些实施方案中，所述柔性片段为如SEQ ID NO：12或SEQ ID NO：39所示的多肽。
Figure PCTCN2022116320-appb-000005
根据本发明的一个具体的实施例，所述柔性片段的N端与TGFβRII胞外结构域的N-末端片段的C端相连，所述柔性片段的C端与截短后剩余的TGFβRII胞外结构域N-末端的N端相连。
根据本发明的一个具体实施例，所述SEQ ID NO：2所示的氨基酸序列排列在所述TGFβRII胞外结构域的N-末端片段之后(例如，当TGFβRII胞外结构域的N-末端氨基酸序列如SEQ ID NO：10所示时，所述插入的片段为：IPPHVQGSGSGSGSG(SEQ ID NO：11))。此时，所述TGFβRII突变体在制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性，且体内半衰期延长。
本发明提供一种TGFβRII突变体，是通过前面所述的方法制备获得的。
根据本发明的具体实施例，制备TGFβRII时易产生碎片和TGFβRII的氨基酸序列中某些位点易被折断具有紧密的联系，因此，采用本发明具体实施例所提供的方法，在制备TGFβRII突变体时可以采用将TGFβRII胞外结构域的N-末端易被折断或解离的位点截短和/插入的方式获得生产过程中碎片和高聚体含量低的TGFβRII突变体。
本发明提供一种核酸分子，所述核酸分子编码前面所述的TGFβRII突变体片段或TGFβRII突变体。 根据本发明具体实施例的核酸分子编码的所述TGFβRII突变体片段或TGFβRII突变体具有与TGFβ结合的生物活性，且氨基酸序列不容易解离，因此在生产TGFβRII突变体片段或TGFβRII突变体过程中碎片含量显著降低，提高了其成药性。
本发明提供一种融合蛋白，根据本发明具体实施例的融合蛋白包括：1)前面所述的TGFβRII突变体片段；以及2)免疫球蛋白Fc片段，所述TGFβRII突变体片段通过连接肽与所述免疫球蛋白Fc片段相连。鉴于现有技术制备的TGFβRII碎片含量非常高，影响药效以及给药频率较高，发明人对TGFβRII胞外结构域的N-末端氨基酸序列进行前面所述的截短突变后，获得的TGFβRII突变体具有与野生型TGFβRII一致的生物活性，在TGFβRII突变体片段上加入免疫球蛋白Fc片段后，获得的融合蛋白同样具有与野生型TGFβRII一致的生物活性，且在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期，所述融合蛋白能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
根据本发明的一个具体的实施例，所述连接肽的N端与所述免疫球蛋白Fc片段的C端相连，所述连接肽的C端与所述的TGFβRII突变体片段的N端相连。
根据本发明的一个具体的实施例，所述免疫球蛋白Fc片段来源于人源IgG抗体分子。
根据本发明的一个具体的实施例，所述免疫球蛋白Fc片段包含hIgG1抗体的Fc重链片段。
根据本发明的一个具体的实施例，所述免疫球蛋白Fc片段的C末端的赖氨酸突变为丙氨酸。将赖氨酸突变为丙氨酸后，能够减少融合蛋白的切割水解，从而降低制备融合蛋白过程中碎片的含量。
根据本发明的一个具体的实施例，所述免疫球蛋白Fc片段具有如SEQ ID NO：6所示氨基酸序列。
Figure PCTCN2022116320-appb-000006
根据本发明的一个具体的实施例，所述连接肽为柔性连接肽。所述连接肽不受特别限制，本领域常规的柔性片段均可使用。
根据本发明的一个具体的实施例，所述连接肽包含(G 4S) XG氨基酸序列，其中，X为大于0的整数。在一些实施方式中，所述连接肽为(G 4S) XG多肽，其中，X优选为1至6的任意整数。在一些实施方式中，所述连接肽为如SEQ ID NO：12或SEQ ID NO：39所示的多肽。
根据本发明的一个具体的实施例，所述连接肽包含(G 4S) 4G(SEQ ID NO：12)序列。
根据本发明的一个具体的实施例，所述融合蛋白具有如SEQ ID NO：7～9任一所示的氨基酸序列。
Figure PCTCN2022116320-appb-000007
Figure PCTCN2022116320-appb-000008
Figure PCTCN2022116320-appb-000009
Figure PCTCN2022116320-appb-000010
本发明供一种核酸分子，所述核酸分子编码前面所述的融合蛋白。根据本发明具体实施例的核酸分子编码的所述融合蛋白具有与野生型TGFβRII一致的生物活性，且在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期，所述融合蛋白能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
本发明提供一种表达载体。根据本发明具体实施例的表达载体包含前面所述的编码TGFβRII突变体片段或TGFβRII突变体的核酸分子、或融合蛋白的核酸分子。在将上述核酸分子连接到载体上时，可以将核酸分子与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。当然，核酸分子与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。
根据本发明的一个具体的实施例，所述表达载体为真核表达载体。
本发明提供一种重组细胞，根据本发明具体实施例的重组细胞携带前面所述的编码TGFβRII突变体片段或TGFβRII突变体的核酸分子、或融合蛋白的核酸分子、或表达载体。根据本发明的具体实施例的重组细胞能够表达前面所述的融合蛋白，且在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期，所述融合蛋白能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
根据本发明的一个具体的实施例，所述重组细胞为哺乳动物细胞，如：人HEK-293F细胞或CHO-K1细胞。
根据本发明的一个具体实施例，所述重组细胞不包括动物生殖细胞、受精卵或胚胎干细胞。
本发明提供一种药物组合物，根据本发明具体实施例的药物组合物包含前面所述的TGFβRII突变体片段、TGFβRII突变体、编码TGFβRII突变体片段或TGFβRII突变体的核酸分子、融合蛋白、编码融合蛋白的核酸分子、表达载体、重组细胞。所述药物组合物可包括：药学上可接受的辅剂，所述药学上可接受的辅剂包括稳定剂、湿润剂、乳化剂、粘合剂、等渗剂的至少之一；所述药物组合物呈片剂、颗粒剂、散剂、胶囊剂、溶液剂、悬浮剂、冻干制剂的至少一种。根据本发明的药物组合物能够控制肿瘤细胞周围表达上调的TGFβ的含量，从而长效、有效的预防或治疗肿瘤。
本发明提供前面所述的TGFβRII突变体片段、TGFβRII突变体、编码TGFβRII突变体片段或TGFβRII突变体的核酸分子、融合蛋白、编码融合蛋白的核酸分子、表达载体、重组细胞在制备药物中的用途。根据本发明具体实施例的药物用于预防或治疗肿瘤。本发明提供的药物具有长效与TGFβ结合的功能，从而有效治疗或预防肿瘤。
本发明提供一种制备前面所述的融合蛋白的方法，包括以下步骤：1)构建前面所述的表达载体；2)将所述表达载体导入宿主细胞中，获得重组细胞，以表达所述融合蛋白。根据本发明的具体实施例的方法可高效获得所述融合蛋白，且所述融合蛋白具有与野生型TGFβRII一致的生物活性，在融合蛋白的制备过程中其氨基酸序列不容易解离，碎片含量显著降低、提高了其成药性及体内半衰期。
根据本发明的一个具体的实施例，所述重组细胞为哺乳动物细胞，哺乳动物例如人、猴、兔、犬、牛等；哺乳动物细胞如：如：人HEK-293F细胞或CHO-K1细胞。
根据本发明的一个具体实施例，所述重组细胞不包括动物生殖细胞、受精卵或胚胎干细胞。
本发明提供一种降低制备前面所述的融合蛋白过程中碎片含量的方法，包括以下步骤：1)构建前面所述的表达载体；2)将所述表达载体导入宿主细胞中。根据本发明具体实施例的方法在融合蛋白的制备过程中融合蛋白的氨基酸序列不容易解离，碎片含量显著降低，显著提高了其药效。
本发明提供一种预防或治疗肿瘤的方法，包括向受试者施用以下中的至少之一：1)前面所述的TGFβRII突变体片段；2)前面所述的TGFβRII突变体；3)前面所述的融合蛋白；4)前面所述的编码所述TGFβRII突变体片段或TGFβRII突变体或融合蛋白的核酸分子；5)前面所述的表达载体；6)前面所述的重组细胞；以及7)前面所述的药物组合物。根据本发明具体实施例的方法能够通过控制肿瘤细胞周围表达上调的TGFβ的含量，从而有效预防或治疗肿瘤。
这些肿瘤可以是任何不受调控的细胞生长。具体地，可以是非小细胞肺癌、乳头状甲状腺癌、多形性成胶质细胞瘤、结肠直肠癌、黑色素瘤、胆管癌或肉瘤、急性骨髓性白血病、大细胞神经内分泌癌、成神经细胞瘤、前列腺癌、成神经细胞瘤、胰腺癌、黑色素瘤、头颈鳞状细胞癌或胃癌等等。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任 何方式限制本发明。
实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明制备厂商者，均为可以通过市购获得的常规产品。
实施例1制备TGFβRII trap融合蛋白
本实施例中发明人利用同源重组技术，以(G 4S) 4G(SEQ ID NO：12)作为连接肽，将hIgG1抗体的Fc重链片段(SEQ ID NO：6)的C末端与不同突变形式(表1)的TGFβRII突变体片段连接，得到TGFβRII trap融合蛋白。在融合结合处，将hIgG1抗体的Fc重链片段的C末端的赖氨酸残基(K)突变成丙氨酸(A)(得到SEQ ID NO：6所示的序列)，以减少融合蛋白的切割水解。
对于TGFβRII trap融合蛋白，采用瞬时转染或稳定转染的标准方案，用位于相同表达载体或分开的表达载体中的编码Fc-TGFβRII受体的DNA来转染哺乳动物细胞，瞬时转染人HEK-293F细胞制备TGFβRII trap融合蛋白，稳定转染CHO-K1细胞制备TGFβRII trap融合蛋白，得到TGFβRII trap融合蛋白。
TGFβRII突变体的结构示意图如图1所示。不同突变形式的TGFβRII突变体形成的TGFβRII trap融合蛋白如表1所示。
表1
Figure PCTCN2022116320-appb-000011
Figure PCTCN2022116320-appb-000012
Figure PCTCN2022116320-appb-000013
备注：表1中，Fc代表免疫球蛋白Fc片段(SEQ ID NO：6)；(G 4S) 4G代表序列GGGGSGGGGSGGGGSGGGGSG(SEQ ID NO：12)、(G 4S) 5G代表序列GGGGSGGGGSGGGGSGGGGSGGGGSG(SEQ ID NO：39)；TGFβTrap(WT)代表野生型TGFβRII胞外结构域的氨基酸序列(SEQ ID NO：10)；TGFβtrap(X-136)代表TGFβRII胞外结构域的第X至第136位氨基酸残基的多肽(所述位点为相对于SEQ ID NO：10的自然顺序编号，下同)，例如，TGFβtrap(20-136)代表截短TGFβRII胞外结构域的N-末端的第1-19位氨基酸(也即只保留TGFβRII胞外结构域的第20-136位残基)的多肽，TGFβtrap(8-136，N10G/N11G/N18A/N19A)代表截短TGFβRII胞外结构域的N-末端的第1-7位氨基酸(也即只保留TGFβRII胞外结构域的第8-136位残基)，同时对第10位、11位、18位和19位(所述突变位点为相对于SEQ ID NO：10的自然顺序编号)进行N10G、N11G、N18A、N19A氨基酸突变后的多肽；IP(XXX)表示TGFβRII胞外结构域的N-末端的前XXX位，例如IP0、IP3、IP6、IP9、IP12依次代表TGFβRII胞外结构域的N-末端的前0位、前3位、前6位、前9位、前12位氨基酸；GS9代表连接肽(SEQ ID NO：2)；SA6代表序列GGGGSA(SEQ ID NO：32)；EG14代表序列EGKSSGSGSESKST(SEQ ID NO：36)；AE17代表序列AEAAAKEAAAKEAAAKA(SEQ ID NO：37)；QF18代表序列KESGSVSSEQLAQFRSLD(SEQ ID NO：38)；另外，表1中的“截短/插入氨基酸个数”，该列的数值为对应的融合蛋白中，IP(XXX)+TGFβtrap(X-136)+连接肽(连接IP(XXX)和TGFβtrap(X-136)的多肽)的氨基酸个数与野生型TGFβRII胞外结构域(SEQ ID NO：10)的氨基酸个数的差值，例如Fc-(G4S)4G-IP6-GS9-TGFβTrap(20-136)融合蛋白中IP6-GS9-TGFβTrap(20-136)的氨基酸个数为132，其比野生型TGFβRII胞外结构域少4个氨基酸，因此，其对应的“截短/插入氨基酸个数”为-4。
TGFβtrap(X-136)代表TGFβRII胞外结构域的第X至第136位氨基酸序列，示例性地，
TGFβtrap(20-136)的氨基酸序列为(SEQ ID NO：41)：
Figure PCTCN2022116320-appb-000014
TGFβtrap(15-136)的氨基酸序列为(SEQ ID NO：42)：
Figure PCTCN2022116320-appb-000015
TGFβtrap(22-136)的氨基酸序列为(SEQ ID NO：43)：
Figure PCTCN2022116320-appb-000016
实施例2 TGFβRII trap融合蛋白纯化
将实施例1瞬时转染人HEK-293F细胞所获得的制备TGFβRII trap融合蛋白的人HEK-293F细胞培养液、以及稳定转染CHO-K1细胞获得的能够制备TGFβRII trap融合蛋白的CHO-K1细胞培养液分别进行高速离心后收集上清液。
将收集到的上清液利用亲合层析进行第一步纯化，其中，层析介质为可与Fc相互作用的Protein A或者衍生填料，如GE的Mabselect；平衡缓冲液为1×PBS，平衡5倍管柱体积后，将细胞上清液上样与层析介质进行结合，流速控制为样品在亲和层析管柱上的保留时间≥1min。上样结束后，用1×PBS(pH7.4)冲洗管柱，记录紫外光波长为280nm时的光吸收值，直至A280紫外吸收降至基线。然后用0.1M甘胺酸(pH为3.0)的洗脱缓冲液冲洗层析管柱，根据A280紫外吸收峰收集洗脱峰，收集的洗脱样品用1MTris(pH 8.5)进行中和。
将上述中和后的洗脱样品超滤装置浓缩后进行体积排阻层析，其中，体积排阻层析的缓冲液为1×PBS，层析管柱为XK26/60Superdex200(GE)，流速控制在4mL/min，上样体积小于5mL，根据A280紫外吸收合并目的蛋白峰，收集的TGFβRII trap融合蛋白经SEC-HPLC鉴定纯度。
实施例3 TGFβRII trap融合蛋白截短分析
发明人对SEC-HPLC结果显示的不同TGFβRII trap融合蛋白纯化过程中的高聚体和碎片进行了分析。
截短TGFβRII胞外结构域的N-末端、或截短后再插入柔性片段和TGFβRII胞外结构域的N-末端片段后得到的TGFβRII突变体形成的融合蛋白在纯化过程中所产生的碎片及高聚体的含量的结果如图2所示，可以看到：截短数量从6个氨基酸增加到13个氨基酸过程中，高聚体含量增加了7％；截短数量增加到15个氨基酸时，高聚体含量增加了13％；截短数量增加到20个氨基酸，高聚体含量增加到20％以上；同时，发明人观察到当截短6～15个氨基酸时，碎片含量变化并不明显；但是当截短16～23个氨基酸时，碎片含量明显减少。因此，截短范围为16～23个氨基酸时，能够有效减少碎片含量。
实施例4 TGFβRII trap融合蛋白的插入改造分析
在图2中，一个值得注意的现象是，与WT相比，将TGFβRII胞外结构域的N-末端的第1-19位氨基酸进行截短，然后插入TGFβRII胞外域的N-末端的第1-6位氨基酸(IPPHVQ(SEQ ID NO：1))和柔性片段(GSGSGSGSG(SEQ ID NO：2))后，得到的TGFβRIItrap融合蛋白(Trunc#22)，不但没有碎片，高聚体也降低很多。因此，发明人针对Trunc#22的结构进行了进一步研究，分别对保留部分、插入部分和截短部分的组合进行了优化。
首先，对于TGFβRII Trap的N-末端保留部分的探索，发明人尝试保留TGFβRII Trap的N-末端的前1～12个氨基酸进行比较(Trunc#22、Trunc#23、Trunc#25、Trunc#26、Trunc#27)。
截短TGFβRII胞外结构域的N-末端后、再插入柔性片段和TGFβRII胞外结构域的N-末端片段得到的TGFβRII突变体形成的融合蛋白在纯化过程中所产生的碎片及高聚体的含量的结果如图3所示，可以看到：相较于只截短的对照组Trunc#31，Trunc#22、Trunc#23、Trunc#25、Trunc#26、Trunc#27这5个重组蛋白分子高聚体明显减少；且随着保留的氨基酸数量增加，碎片逐渐增多，从2.8％增加到10％以上。其中，Trunc#22的碎片含量较少，同时高聚体含量也得到有效控制，少量高聚体在后续纯化过程中可以有效去除。
此外，为了验证保留序列是否必须为TGFβRII胞外结构域的N-末端氨基酸序列，发明人尝试了其他序列，如GGGSAG(Trunc#24)。
结果如图3所示，可以看到：相较于Trunc#22，Trunc#24又出现了5％的碎片，同时高聚体也明显增加；说明保留序列选择TGFβRII胞外结构域的N-末端片段(IPPHVQ(SEQ ID NO：1))效果较佳。
其次，对于插入部分的探索，发明人尝试了多种序列，包括不同长度的柔性linker、刚性linker和随机序列，其中，Trunc#32中插入序列为柔性linker、Trunc#33中插入序列为刚性linker、Trunc#34插入序列为随机序列。
结果如图3所示，Trunc#32和Trunc#33的插入使得碎片含量和高聚体含量都大幅度增加，Trunc#34的插入高聚体含量较少，但是碎片含量相较于野生型TGFβRII没有明显改善。说明插入部分只能是柔性片段，并且插入部分的长度要小于14个氨基酸。
最后，为了验证在保留段、插入段与Trunc#22相同情况下，最优的截短组合，发明人构建了Trunc#35和Trunc#36。
结果如图3所示，可以看到：相较于野生型TGFβRII，Trunc#35和Trunc#36的碎片含量都有所降低，并且Trunc#36的碎片含量更少；相较于Trunc#28和Trunc#31，Trunc#35和Trunc#36的高聚体含量明显减少，并且Trunc#35的高聚体含量较Trunc#36更少。因此，Trunc#35、Trunc#36和Trunc#22都能解决表达过程中碎片和高聚体的问题，其中以Trunc#22效果最佳。
实施例5 TGFβRII trap融合蛋白的ELISA结合检测
TGFβRIITrap融合蛋白Trap端结合检测所用蛋白为humanTGFβ1(CA59，购自Novoprotein)，检 测流程如下：
a.用1×磷酸盐缓冲液(PBS)稀释TGFβ1至0.5μg/mL，100μL/孔包被96孔酶标板，4℃过夜；
b.250μL 1×PBST(PBS+0.5％Tween20)洗涤3次，加入200μL含2％牛血清白蛋白(BSA)的PBS室温封闭1小时；
c.250μL 1×PBST洗涤3次，加入梯度稀释的TGFβRII trap，室温孵育2小时；
d.250μL 1×PBST洗涤3次，每孔加入100μL稀释好的Goat-anti-human Fc-HRP藕联抗体(Sigma，1∶15k)，室温孵育1小时；
e.250μL 1×PBST洗涤3次，每孔加入100μLTMB显色液，室温避光孵育10分钟，加入50μL2N H2SO4终止反应；
f.用iX3酶标仪(Molecular Device公司)读取450nM处的吸收值，分析作图。
融合蛋白与人源TGFβ1的ELISA结合活性检测结果如图4所示，可以看出：所有TGFβRII Trap都能够结合包被在板上的TGFβ1，并且截短改造均不影响TGFβRII与TGFβ1的结合。
实施例6 TGFβRII trap融合蛋白的阻断活性检测
TGFβRIITrap融合蛋白Trap端结合检测所用蛋白为humanTGFβ1(CA59，购自Novoprotein)，检测流程如下：
构建稳定转染表达人TGFβRII的CHO细胞(CHO-hTGFβRII)，挑取单克隆建系。采用如下方法对抗TGFβRII trap的阻断活性进行检测：
a.对CHO-hTGFβRII细胞进行计数，将细胞以2×10 5每孔的密度铺至96孔U底板；
b.将铺好的CHO-hTGFβRII细胞于300g、4℃条件下离心处理5分钟，弃上清液。
c.用含1％BSA的1×PBS稀释步骤b中获得的含有TGFβRII trap融合蛋白的沉淀，起始浓度为50nM，稀释倍数为4倍，共设置8个梯度，同时也对TGFβ1-biotin(购自Acrobiosystem)进行稀释，稀释后的浓度为1μg/mL。
d.将融合蛋白稀释液和TGFβ1-botin稀释液按1∶1的体积比例混匀，室温下放置30min。
e.将步骤d获得的混合样品加入CHO-TGFβRII细胞中，100μL/孔，于4℃放置30min后，在室温、300g的条件下离心处理5min，甩去上清液。
f.向步骤e获得的产物中加入SA-PE(400倍稀释，Jackson immunoresearch，016-110-084)，添加体积为100μl/孔，轻微混匀，于4℃条件下放置1小时后，于室温、300g的条件下离心处理5min，甩去上清液；将获得的细胞中加入含1％BSA的1×PBS，100μl/孔，重悬细胞，流式上机检测。
融合蛋白阻断人源TGFβ1与TGFβR II的结合活性检测结果如图5所示，截短TGFβRII Trap融合蛋白均能够抑制TGFβ1结合到TGFβR II，从EC50值来看，阻断能力与野生型TGFβRII Trap基本没有太大的差异。
实施例7 T细胞增殖抑制试验
研究表明TGFβ1会明显抑制T细胞的增殖，为了检测不同改造的TGFβRII Trap融合蛋白Trap端在细胞水平的功能，我们检测了在TGFβ存在情况下，不同改造的TGFβRII Trap融合蛋白对T细胞增殖的影响。实验采用如下示例性方法：
a.使用人淋巴细胞分离液密度梯度离心从健康志愿者的全血中，分离外周血单个核细胞(PBMC)，使用人T细胞富集试剂盒(STEMCELL，10951)，根据说明从PBMC中分离T细胞，使用CFSE(eBioscience，85-65-0850-84)对T细胞进行染色，具体操作参考试剂说明书，CFSE的使用浓度为1M；
b.将TGFβRII Trap融合蛋白、CD3/CD28beads(life，40203D)和经CFSE染色后的T细胞进行共孵育，T细胞的数量为5×104个/孔，CD3/CD28beads与T细胞的体积比例为1∶5，在培养箱进行培养；
c.将步骤所述的T细胞培养至第五天后，流式检测T细胞的CFSE值，算出T细胞在每一代中所占的比例，主要观测最左侧的峰。
截短TGFβRII胞外结构域的N-末端后得到的TGFβRII突变体形成的融合蛋白对T细胞增殖影响的实验结果如图6所示，不加抗体组(No TGF&No Ab组)刺激后，增殖比例约为15.4％，加入TGFβ1后，增殖比例约为2.44％，产生了明显的增殖抑制。截短TGFβRII胞外结构域的N-末端的氨基酸序列后得到的TGFβRII突变体对应的融合蛋白加入后，增殖比例从2.44％明显增加，增加至5.98％～17.56％，并呈现出浓度依赖关系。其中，Trunc#1、Trunc#2、Trunc#6、Trunc#9、Trunc#14、Trunc#22组相较于WT组展现出更好促进T细胞增殖的效果；同时，Trunc#3、Trunc#7、Trunc#8、Trunc#11、Trunc#13、Trunc#15～Trunc#18组促进T细胞增殖的效果弱于WT组。
发明人进一步评估了插入柔性片段和TGFβRII胞外结构域的N-末端片段后得到的TGFβRII突变体对应的TGFβRII Trap融合蛋白的活性。
截短TGFβRII胞外结构域的N-末端后、再插入柔性片段和TGFβRII胞外结构域的N-末端片段得到的TGFβRII突变体形成的融合蛋白对T细胞增殖影响的实验结果如图7所示，其中，Trunc#25、Trunc#32、Trunc#35、Trunc#36相较于WT组展现出更好促进T细胞增殖的效果；Trunc#14、Trunc#22组作为对照，图7所示结果与图6所示结果一致，Trunc#14和Trunc#22相较于WT组展现出更好的促进T细胞增殖的效果。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为 对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (15)

  1. 一种TGFβRII突变体片段，其特征在于，所述TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端片段、柔性片段和TGFβRII胞外结构域的N-末端片段，其中，所述C-末端片段包括TGFβRII胞外结构域的C-末端的至多122个氨基酸；所述柔性片段的N端与所述TGFβRII胞外结构域的N-末端片段的C端相连，所述柔性片段的C端与所述TGFβRII胞外结构域的C-末端片段的N端相连。
  2. 根据权利要求1所述TGFβRII突变体片段，其特征在于，所述TGFβRII突变体片段包括TGFβRII胞外结构域的C-末端的112～117个氨基酸；可选的，所述TGFβRII胞外结构域的氨基酸序列如SEQ ID NO：10所示，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短多肽；可选的，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短连续14-23个氨基酸残基的多肽，优选截短连续16-23个氨基酸残基的多肽；可选的，所述TGFβRII胞外结构域的C-末端片段为SEQ ID NO：41、SEQ ID NO：42或SEQ ID NO：43所示多肽。
  3. 根据权利要求1或2所述TGFβRII突变体片段，其特征在于，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的0～6个氨基酸；可选的，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的1个、2个、3个、4个、5个或6个氨基酸；可选的，所述TGFβRII胞外结构域的N-末端片段的氨基酸序列如SEQ ID NO：1所示。
  4. 根据权利要求1至3任一项所述TGFβRII突变体片段，其特征在于，所述柔性片段为含有G和/或S氨基酸的短肽，优选地，所述短肽含有5～15个氨基酸；可选的，所述柔性片段为(G 4S) XG多肽，其中，X优选为1至6的任意整数；可选的，所述柔性片段的氨基酸序列如SEQ ID NO：2、SEQ ID NO：12或SEQ ID NO：39所示。
  5. 根据权利要求1至4任一项所述TGFβRII突变体片段，其特征在于，所述TGFβRII胞外结构域的N-末端片段具有SEQ ID NO：1所示的氨基酸序列，所述柔性片段具有SEQ ID NO：2、SEQ ID NO：12或SEQ ID NO：39所示的氨基酸序列，且所述TGFβRII胞外结构域的C-末端片段为SEQ ID NO：41、SEQ ID NO：42或SEQ ID NO：43所示多肽；可选的，所述TGFβRII突变体片段具有SEQ ID NO：3～5任一所示的氨基酸序列。
  6. 根据权利要求1至5任一项所述TGFβRII突变体片段，其特征在于，所述TGFβRII突变体片段还连接有免疫球蛋白Fc片段；可选的，所述免疫球蛋白Fc片段通过连接肽与所述TGFβRII突变体片段的N端相连；可选的，所述免疫球蛋白Fc片段具有如SEQ ID NO：6所示氨基酸序列；可选的，所述连接肽为(G 4S) XG多肽，其中，X优选为1至6的任意整数，更优选如SEQ ID NO：12或SEQ ID NO：39所示的多肽。
  7. 一种融合蛋白，其特征在于，包含权利要求1至5任一项所述TGFβRII突变体片段；优选的，所述融合蛋白包含免疫球蛋白Fc片段；可选的，所述免疫球蛋白Fc片段C端通过连接肽与所述TGFβRII 突变体片段的N端连接；可选的，所述免疫球蛋白Fc片段具有如SEQ ID NO：6所示氨基酸序列；可选的，所述连接肽为(G 4S) XG多肽，其中，X优选为1至6的任意整数，优选如SEQ ID NO：12或SEQ ID NO：39所示的多肽；可选的，所述融合蛋白具有SEQ ID NO：7-9任一所示的氨基酸序列。
  8. 一种核酸分子，其特征在于，编码权利要求1～6任一项所述的TGFβRII突变体片段或权利要求7所述融合蛋白。
  9. 一种表达载体，其特征在于，包含权利要求8所述的核酸分子。
  10. 一种药物组合物，其特征在于，包含权利要求1～6任一项所述的TGFβRII突变体片段、或权利要求8所述的核酸分子、或权利要求9所述的表达载体、或权利要求7所述的融合蛋白。
  11. 一种TGFβRII突变体，包括胞外区、跨膜区和胞内区，其特征在于，所述胞外区为权利要求1～6任一项所述的TGFβRII突变体片段。
  12. 一种宿主细胞，其特征在于，所述宿主细胞转染有权利要求8所述的核酸分子，或权利要求9所述的表达载体。
  13. 权利要求1～6任一项所述的TGFβRII突变体片段、或权利要求8所述的核酸分子、或权利要求9所述的表达载体、或权利要求10所述的药物组合物、或权利要求11所述的TGFβRII突变体、或权利要求12所述的宿主细胞、或权利要求7所述的融合蛋白在制备用于预防或治疗肿瘤的药物中的用途。
  14. 一种预防或治疗肿瘤的方法，其特征在于，所述方法包括：向受试者施用预防或治疗有效量的以下至少之一：权利要求1～6任一项所述的TGFβRII突变体片段、或权利要求8所述的核酸分子、或权利要求9所述的表达载体、或权利要求10所述的药物组合物、或权利要求11所述的TGFβRII突变体、或权利要求12所述的宿主细胞、或权利要求7所述的融合蛋白。
  15. 一种用于预防或治疗肿瘤的药物，其特征在于，所述药物为权利要求1～6任一项所述的TGFβRII突变体片段、或权利要求8所述的核酸分子、或权利要求9所述的表达载体、或权利要求10所述的药物组合物、或权利要求11所述的TGFβRII突变体、或权利要求12所述的宿主细胞、或权利要求7所述的融合蛋白。
PCT/CN2022/116320 2021-09-02 2022-08-31 TGFβRII突变体及其应用 WO2023030408A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111026893 2021-09-02
CN202111026893.3 2021-09-02

Publications (1)

Publication Number Publication Date
WO2023030408A1 true WO2023030408A1 (zh) 2023-03-09

Family

ID=85349528

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/116320 WO2023030408A1 (zh) 2021-09-02 2022-08-31 TGFβRII突变体及其应用

Country Status (3)

Country Link
CN (1) CN115746124A (zh)
TW (1) TW202328173A (zh)
WO (1) WO2023030408A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105658672A (zh) * 2013-08-22 2016-06-08 阿塞勒隆制药公司 TGF-β受体II型变体及其用途
CN110050000A (zh) * 2017-05-12 2019-07-23 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途
CN110072885A (zh) * 2016-11-17 2019-07-30 蓝鸟生物公司 TGFβ信号转换器
WO2021115456A1 (en) * 2019-12-11 2021-06-17 Wuxi Biologics (Shanghai) Co., Ltd. BI-FUNCTIONAL ANTIBODY AGAINST PD-L1 AND TGFβ

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105658672A (zh) * 2013-08-22 2016-06-08 阿塞勒隆制药公司 TGF-β受体II型变体及其用途
CN110072885A (zh) * 2016-11-17 2019-07-30 蓝鸟生物公司 TGFβ信号转换器
CN110050000A (zh) * 2017-05-12 2019-07-23 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途
WO2021115456A1 (en) * 2019-12-11 2021-06-17 Wuxi Biologics (Shanghai) Co., Ltd. BI-FUNCTIONAL ANTIBODY AGAINST PD-L1 AND TGFβ

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAKER K, RAUT P, JASS JR: "Microsatellite unstable colorectal cancer cell lines with truncating TGFβRII mutations remain sensitive to endogenous TGFβ", THE JOURNAL OF PATHOLOGY, vol. 213, no. 3, 1 November 2007 (2007-11-01), Hoboken, USA, pages 257 - 265, XP093041537, ISSN: 0022-3417, DOI: 10.1002/path.2235 *
DI JINMING, ZHANG YICHU, GU QINLONG, CHEN LEI, JIANG BAOCHENG, DONG QIAN: "Study on the mutation of TGF-βRⅡ gene in colorectal cancer with MSI", JOURNAL OF PRACTICAL ONCOLOGY, vol. 15, no. 2, 15 May 2001 (2001-05-15), pages 88 - 91, XP093041544 *

Also Published As

Publication number Publication date
CN115746124A (zh) 2023-03-07
TW202328173A (zh) 2023-07-16

Similar Documents

Publication Publication Date Title
RU2711979C2 (ru) Белковый комплекс интерлейкина 15 и его применение
EP3453401A1 (en) Interleukin combination and use thereof
CN113444182B (zh) 一种靶向递送IgG类抗体的融合蛋白载体及其用途
JP2004203890A (ja) マクロファージ炎症蛋白変種
AU623589B2 (en) Platelet related growth regulator
KR20220012938A (ko) 펩타이드
CN113179631A (zh) 通过临近使能反应疗法开发的共价蛋白质药物
CN112111013A (zh) 一种靶向claudin 18.2的通用型嵌合抗原受体T细胞、构建方法及其用途
CN109867725B (zh) PD-1-Fc融合蛋白及其制备方法和用途
CN107936109B (zh) 衍生自sage1的肿瘤抗原短肽
CN115916251A (zh) 肽混合物
JPH03178999A (ja) 脳由来の膜蛋白質に対する抗体
WO2023030408A1 (zh) TGFβRII突变体及其应用
CN109157656B (zh) 一种双靶标肿瘤疫苗及其制备方法和应用
WO2008145013A1 (zh) 包含cd13靶向肽和力达霉素的融合蛋白
CN108218977B (zh) 源自于肿瘤抗原sage1的短肽
WO2022105922A1 (zh) 源自于ssx2抗原的短肽
US9663562B2 (en) Compositions and methods for treating cancer
CN115746123A (zh) TGFβRII突变体及其应用
CN106674352B (zh) 抗肿瘤蛋白内皮抑素的衍生物及应用
CN105985424B (zh) 一种干细胞因子功能性肽段及其应用
CN115724942A (zh) TGFβRII活性改造突变体及其应用
WO2022007885A1 (zh) 融合多肽和多肽二聚体及其用途
WO2022143938A1 (zh) 一种激活nk细胞的肿瘤免疫治疗方法
WO2022078518A1 (zh) 经修饰的il-2分子及其用途

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: 22863543

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE