CN115746124A

CN115746124A - TGF beta RII mutant and application thereof

Info

Publication number: CN115746124A
Application number: CN202211066160.7A
Authority: CN
Inventors: 张轶博; 芦迪; 路力生; 霍永庭
Original assignee: Guangdong Fapon Biopharma Inc
Current assignee: Guangdong Fapon Biopharma Inc
Priority date: 2021-09-02
Filing date: 2022-08-31
Publication date: 2023-03-07
Also published as: TW202328173A; WO2023030408A1

Abstract

The invention provides a TGF beta RII mutant fragment and application thereof, wherein the TGF beta RII mutant fragment comprises at most 122 amino acids at the C-terminal end of a TGF beta RII extracellular domain, a flexible fragment and an N-terminal fragment of the TGF beta RII extracellular domain, the N end of the flexible fragment is connected with the C end of the N-terminal fragment of the TGF beta RII extracellular domain, and the C end of the flexible fragment is connected with the N end of the C-terminal fragment of the TGF beta RII extracellular domain. The TGF beta RII mutant containing the fragment has consistent biological activity with wild TGF beta RII, the fragment content is obviously reduced compared with wild TGF beta RII in the preparation process, and the TGF beta RII mutant is combined with the TGF beta on the surface of a tumor cell to achieve the purpose of controlling the TGF beta content in the tumor microenvironment, so that the tumor is effectively prevented and treated.

Description

TGF beta RII mutant and application thereof

The present application claims priority from a chinese application (application No. 202111026893.3, entitled TGF β RII variants and uses thereof) filed on 3/9/2/2021, the entire contents of which are incorporated herein by reference in their entirety.

Technical Field

The invention relates to the field of biomedicine, in particular to a TGF (transforming growth factor) RII mutant and application thereof, and more particularly relates to the TGF RII mutant, a nucleic acid molecule for coding the TGF RII mutant, an expression vector, a recombinant cell, a pharmaceutical composition and application thereof in preparing medicines.

Background

Transforming growth factor beta (TGF β) is a TGF β superfamily that regulates cell growth and differentiation. In addition to TGF β, this family has activin, inhibin, mullerian inhibitor substance, and bone morphogenetic proteins. The body secretes TGF β in an inactive state in a variety of cells. TGF β in the inactive state, also known as the Latent Associated Peptide (LAP), can be activated in vitro by acid cleavage. In vivo, an acidic environment may be present near the fracture and in the healing wound. Cleavage of the protein itself may cause the TGF-beta complex to become activated TGF-beta.

TGF plays an important regulatory role in cell growth, differentiation and immune function. TGF β plays a tumor-inhibiting or tumor-promoting role in tumors in a cell-background dependent manner. TGF beta can inhibit the expression of the proto-oncogene c-myc, but during tumor development, following the introduction of mutations or changes in epigenetic modifications, cancer cells gradually tolerate the inhibitory effects of TGF beta signaling, ultimately leading to tumor development. Recent studies have found that increased TGF β in the tumor microenvironment is associated with immune escape, and that increased TGF β increases T cell rejection, blocking infiltration of TH1 effector T cells. The TGF beta-targeting protein can control the content of free TGF beta on the surface of a tumor, but a large amount of fragments and high polymers are generated in the current process of preparing the extracellular domain of the TGF beta receptor, and the fragments and the high polymers are difficult to remove through purification, so that the preparation efficiency of the TGF beta receptor is low, the yield is low, the drug property is seriously influenced, and the method for preparing the protein and the operation need to be improved.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following problems:

in the invention, through a large amount of experimental researches, the inventor adopts a method of truncating the amino acid sequence of the TGF beta RII extracellular domain to reduce the fragment content in the TGF beta RII preparation process, obtains TGF beta RII mutant fragments and TGF beta RII mutants, prepares fusion protein by utilizing the TGF beta RII mutants, and unexpectedly obtains the fusion protein with low fragment content in the TGF beta RII preparation process, wherein the fusion protein and TGF beta have high in-vitro binding activity, the drug effect is obviously improved, and tumors can be prevented or treated by controlling the content of TGF beta with up-regulated expression around tumor cells.

In a first aspect of the invention, the invention provides a TGF β RII mutant fragment. According to embodiments of the invention, the TGF β RII mutant fragment comprises up to 122 amino acids of the C-terminus of the TGF β RII extracellular domain; the TGF-beta RII mutant fragments further include flexible fragments and N-terminal fragments of the TGF-beta RII extracellular domain. In view of the fact that the TGF beta RII fragment prepared by the prior art has very high content and influences the drug effect, the inventor creatively discovers that after the N-terminal of the TGF beta RII extracellular domain is truncated according to the mutation mode of the embodiment of the invention, the obtained TGF beta RII mutant fragment has the biological activity of binding with TGF beta, in the process of preparing the TGF beta RII mutant, the amino acid sequence of the TGF beta RII mutant fragment is not easy to dissociate, the fragment content is obviously reduced, the TGF beta RII mutant containing the fragment also has the biological activity consistent with the wild-type TGF beta RII, in addition, the amino acid sequence is not easy to dissociate in the process of producing the TGF beta RII mutant, the fragment content is obviously reduced, and the drug property is improved.

In a second aspect of the invention, the invention provides a TGF β RII mutant. According to an embodiment of the present invention, comprises an extracellular region, a transmembrane region and an intracellular region; wherein the extracellular region comprises a TGF beta RII mutant fragment of the first aspect. In view of the fact that the wild-type TGF beta RII fragment produced by the prior art has very high content and influences the drug effect, the inventor creatively discovers that the TGF beta RII mutant containing the fragment of the first aspect of the invention has the biological activity consistent with that of the wild-type TGF beta RII, and in the process of producing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the drug property is improved.

In a third aspect of the invention, the invention provides a method of making a TGF-beta RII mutant fragment according to the first aspect. According to an embodiment of the present invention, the amino acid sequence of the tgfbetarii mutant is truncated by at least 14 amino acids from the N-terminus of the tgfbetarii extracellular domain, with no more than 20 amino acids inserted. In view of the fact that the TGF beta RII fragment prepared by the prior art has very high content and is difficult to remove in the purification process and influence the drug effect, the inventor conducts a great deal of experimental exploration, after the N-terminal of the TGF beta RII extracellular domain is truncated according to the mutation mode of the embodiment of the invention, the obtained TGF beta RII mutant fragment has the biological activity of combining with TGF beta, the fragment is not easy to dissociate and is not easy to break, the TGF beta RII mutant containing the fragment also has the biological activity consistent with the wild-type TGF beta RII, in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the drug effect is improved.

In a fourth aspect of the invention, the invention provides a TGF β RII mutant fragment. According to an embodiment of the present invention, the material is obtained by the method of the third aspect. According to the embodiment of the invention, the TGF beta RII mutant with the fragment has the biological activity consistent with that of a wild TGF beta RII, and in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the drug property is improved.

In a fifth aspect of the invention, the invention provides a method of making a tgfbetarii mutant as described in the second aspect. According to an embodiment of the invention, the tgfbetarii mutant comprises an extracellular region, a transmembrane region, and an intracellular region; the method comprises the following steps: truncation is by at least 14 amino acids from the N-terminus of the TGF β RII extracellular domain, with no more than 20 amino acids inserted. In view of the fact that the TGF beta RII fragment prepared by the prior art has very high content and is difficult to remove in the purification process, and the drug effect is influenced, the inventor conducts a great deal of experimental exploration, the TGF beta RII mutant obtained by the method provided by the invention has the biological activity consistent with that of the wild type TGF beta RII, and in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the drug effect is improved.

In a sixth aspect of the invention, the invention provides a TGF β RII mutant. According to an embodiment of the present invention, the method is prepared by the method of the fifth aspect.

In a seventh aspect of the invention, a nucleic acid molecule is presented. According to an embodiment of the invention, the nucleic acid molecule encodes a TGF β RII mutant fragment of the first or fourth aspect, or a TGF β RII mutant of the second or sixth aspect. According to the embodiment of the invention, the TGF beta RII mutant fragment coded by the nucleic acid molecule has the biological activity of combining with TGF beta, and in the process of preparing the TGF beta RII mutant, the amino acid sequence of the TGF beta RII mutant fragment is not easy to dissociate, and the fragment content is obviously reduced; the TGF beta RII mutant containing the fragment also has the biological activity consistent with that of the wild TGF beta RII, and in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, so that the fragment content is obviously reduced, and the druggability of the TGF beta RII mutant is improved.

In an eighth aspect of the invention, a fusion protein is provided. According to an embodiment of the invention, comprising: 1) A TGF β RII mutant fragment of the first or fourth aspect; and 2) an immunoglobulin Fc fragment, to which the TGF-beta RII mutant fragment is linked via a linker peptide. In view of the fact that the TGF beta RII fragment prepared by the prior art has very high content, influences the drug effect and has high administration frequency, the inventor carries out the truncation mutation on the N-terminal amino acid sequence of the TGF beta RII extracellular domain to obtain the TGF beta RII mutant with the bioactivity consistent with that of the wild TGF beta RII, adds the immunoglobulin Fc fragment on the TGF beta RII mutant to obtain the fusion protein with the bioactivity consistent with that of the wild TGF beta RII, and the amino acid sequence is not easy to dissociate in the preparation process of the fusion protein, so that the fragment content is obviously reduced, the drug forming property and the in vivo half-life are improved, and the fusion protein can control the content of the TGF beta with the up-regulated expression around tumor cells, thereby preventing or treating tumors for a long time and effectively.

In a ninth aspect, the present invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the fusion protein according to the eighth aspect. According to the embodiment of the invention, the fusion protein coded by the nucleic acid molecule has the biological activity consistent with that of wild TGF beta RII, the amino acid sequence of the fusion protein is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, the drug forming property and the in vivo half life are improved, and the fusion protein can control the content of TGF beta with up-regulated expression around tumor cells, so that the tumor can be effectively prevented or treated for a long time.

In a tenth aspect of the invention, the invention features an expression vector. According to an embodiment of the invention, the expression vector comprises the nucleic acid molecule of the seventh or ninth aspect.

In an eleventh aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the nucleic acid molecule of the seventh aspect or the ninth aspect, or the expression vector of the tenth aspect. According to the embodiment of the invention, the recombinant cell can express the fusion protein, the amino acid sequence of the recombinant cell is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, the drug forming property and the in vivo half-life period of the recombinant cell are improved, and the fusion protein can control the content of TGF beta with up-regulated expression around tumor cells, so that the tumor can be effectively prevented or treated for a long time.

In a twelfth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises a tgfbetarii mutant fragment of the first or fourth aspect, or a tgfbetarii mutant of the second or sixth aspect, or a nucleic acid molecule of the seventh aspect, or a fusion protein of the eighth aspect, or a nucleic acid molecule of the ninth aspect, or an expression vector of the tenth aspect, or a recombinant cell of the eleventh aspect. The pharmaceutical composition may include: pharmaceutically acceptable adjuvants including at least one of stabilizers, wetting agents, emulsifiers, binders, isotonicity agents; the pharmaceutical composition is at least one of tablets, granules, powder, capsules, solutions, suspensions and freeze-dried preparations. The pharmaceutical composition can control the content of TGF beta with up-regulated expression around tumor cells, thereby preventing or treating tumors with long-acting and effective effects.

In a thirteenth aspect, the present invention provides the use of a TGF β RII mutant fragment of the first or fourth aspect, a TGF β RII mutant of the second or sixth aspect, a nucleic acid molecule of the seventh aspect, a fusion protein of the eighth aspect, a nucleic acid molecule of the ninth aspect, an expression vector of the tenth aspect, a recombinant cell of the eleventh aspect, or a pharmaceutical composition of the twelfth aspect, in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the prevention or treatment of a tumor. The medicine provided by the invention has the long-acting function of combining with TGF beta, and achieves the purpose of treating or preventing tumors.

In a fourteenth aspect of the present invention, the present invention provides a method for preparing the fusion protein of the eighth aspect. According to an embodiment of the invention, the method comprises the steps of: 1) Constructing an expression vector according to the tenth aspect; 2) Introducing the expression vector into a host cell to obtain a recombinant cell so as to express the fusion protein. The method can efficiently obtain the fusion protein, the fusion protein has the biological activity consistent with that of wild TGF beta RII, the amino acid sequence of the fusion protein is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, and the drugginess and the half-life period in vivo are improved.

In a fifteenth aspect of the invention, there is provided a method of reducing the amount of debris in the process of producing the fusion protein of the eighth aspect. According to an embodiment of the invention, the method comprises the steps of: 1) Constructing an expression vector according to the tenth aspect; 2) Introducing the expression vector into a host cell. According to the method, the amino acid sequence of the fusion protein is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, and the drug effect is obviously improved.

In a sixteenth aspect of the invention, a method of preventing or treating a tumor is presented. According to an embodiment of the invention, the method comprises administering to the subject at least one of:

1) A TGF β RII mutant fragment of the first or fourth aspect; 2) The tgfbetarii mutant of the second or sixth aspect; 3) The fusion protein of the eighth aspect; 3) The nucleic acid molecule of the seventh or 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 TGF beta with up-regulated expression around tumor cells.

In a seventeenth aspect of the invention, a medicament is presented. According to an embodiment of the invention, the medicament comprises at least one of a tgfbetarii mutant fragment of the first or fourth aspect, a tgfbetarii mutant of the second or sixth aspect, a nucleic acid molecule of the seventh aspect, a fusion protein of the eighth aspect, a nucleic acid molecule of the ninth aspect, an expression vector of the tenth aspect, a recombinant cell of the eleventh aspect and a pharmaceutical composition of the twelfth aspect. The medicament according to the embodiment of the invention can effectively treat or prevent tumors.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic structural diagram of a TGF-beta RII mutant according to an embodiment of the present invention, wherein insertion represents the inserted amino acid sequence, i.e.: truncating the 1 st to the X th amino acids at the N-terminal of the TGF beta RII extracellular domain, and then inserting a flexible fragment and an N-terminal fragment of the TGF beta RII extracellular domain;

FIG. 2 is a graph showing the results of the contents of fragments and polymers generated during purification of a fusion protein formed by a TGF-beta RII mutant obtained by truncating the N-terminus of the TGF-beta RII extracellular domain, or inserting a flexible fragment and an N-terminal fragment of the TGF-beta RII extracellular domain after truncation, according to an embodiment of the present invention, wherein: the fusion proteins shown by the abscissa are WT, trunc #1-Trunc #19, trunc #21 and Trunc #22 in sequence; <xnotran> R0805: WT 1 WT, R0749:7-136 1 Trunc #1, R0776:8-136 1 Trunc #2, R0777:9-136 1 Trunc #3, R0778:10-136 1 Trunc #4, R0779:11-136 1 Trunc #5, R0780:12-136 1 Trunc #6, R0781:13-136 1 Trunc #7, R0782:14-136 1 Trunc #8, R0783:1-6+15-136 1 Trunc #9, R0784:16-136 1 Trunc #10, R0785:17-136 1 Trunc #11, R0786:18-136 1 Trunc #12, R0787:19-136 1 Trunc #13, R0788:1-6+20-136 1 Trunc #14, R0789:21-136 1 Trunc #15, R0790:1-6+22-136 1 Trunc #16, R0791:23-136 1 Trunc #17, R0792:24-136 1 Trunc #18, R0793: G4S5G +8-136 1 Trunc #19, R0795: G4S5G +8-136mut 1 Trunc #21, </xnotran> R0796, IP6+ GSGSGSGSG +20-136 represents fusion protein Trunc #22 in Table 1 of the present invention; furthermore, each fusion protein shown on the abscissa also shows the mutation pattern of the TGF-beta RII mutant fragment, for example, R0749:7-136 shows that the TGF-beta RII mutant fragment in the fusion protein is obtained by truncating amino acids 1-6 of the N-terminal of the TGF-beta RII extracellular domain; r0796:1-6+ GSGSGSGSGSG +20-136 shows that the TGF beta RII mutant fragment in the fusion protein is formed by truncating the 1 st to 19 th amino acids at the N-terminal of the TGF beta RII extracellular domain, and then inserting the 1 st to 6 th amino acids (IPPHVQ (SEQ ID NO: 1)) and a flexible fragment (GSGSGSGSG (SEQ ID NO: 2)) at the N-terminal of the TGF beta RII extracellular domain;

FIG. 3 is a graph showing the results of the contents of fragments and aggregates generated during purification of fusion proteins formed by a TGF-beta RII mutant obtained by truncating the N-terminus of the TGF-beta RII extracellular domain, inserting a flexible fragment and an N-terminal fragment of the TGF-beta RII extracellular domain, according to an embodiment of the present invention, wherein the specific mutation pattern of the TGF-beta RII mutant in each fusion protein shown in the abscissa is shown in Table 1;

FIG. 4 is a graph showing the results of ELISA binding activity detection of fusion proteins with human TGF-. Beta.1 according to an embodiment of the present invention;

FIG. 5 is a graph showing the results of the detection of the binding activity of a fusion protein blocking human TGF-beta 1 and TGF-beta RII according to an embodiment of the present invention;

FIG. 6 is a graph showing the experimental results of the effect of fusion proteins formed from TGF-beta RII mutants obtained after truncation of the N-terminus of the TGF-beta RII extracellular domain on T-cell proliferation according to an embodiment of the present invention;

FIG. 7 is a graph showing the experimental results of the effect on T cell proliferation of a fusion protein formed by a TGF-beta RII mutant obtained by truncating the N-terminus of the TGF-beta RII extracellular domain, inserting a flexible fragment and an N-terminal fragment of the TGF-beta RII extracellular domain according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative and intended to explain the present invention and should not be construed as limiting the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

"extracellular domain of TGF β RII" refers to a 136 amino acid residue long peptide segment from the N-terminus outside of a wild-type TGF β RII cell having the amino acid sequence set forth in SEQ ID NO:10, or a pharmaceutically acceptable salt thereof. The invention is also called wild type TGF beta RII.

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO：10)。

The present invention provides a TGF β RII mutant fragment, said TGF β RII mutant fragment comprising up to 122 amino acids of the C-terminus of a TGF β RII extracellular domain; the TGF-beta RII mutant fragments further include flexible fragments and N-terminal fragments of the TGF-beta RII extracellular domain. In view of the fact that the fragment content of TGF beta RII prepared by the prior art is very high and influences the drug effect, the inventor creatively discovers that after the N-terminal of the TGF beta RII extracellular domain is truncated according to the mutation mode of the embodiment of the invention, the obtained TGF beta RII mutant fragment has the biological activity of TGF beta binding, the amino acid sequence of the TGF beta RII mutant fragment is not easy to dissociate in the process of preparing the TGF beta RII mutant, the fragment content is obviously reduced, the TGF beta RII mutant containing the fragment also has the biological activity consistent with the wild-type TGF beta RII, the amino acid sequence is not easy to dissociate in the process of preparing the TGF beta RII mutant, the fragment content is obviously reduced, and the drug property of the TGF beta RII mutant is improved.

According to a specific embodiment of the invention, the N-terminus of the flexible fragment is linked to the C-terminus of the N-terminal fragment of the TGF-beta RII extracellular domain, and the C-terminus of the flexible fragment is linked to the N-terminus of up to 122 amino acids of the C-terminus of the TGF-beta RII extracellular domain.

According to some embodiments of the invention, the TGF β RII mutant fragment has an amino acid sequence in the extracellular domain of TGF β RII as set forth in SEQ ID NO:10, the C-terminal fragment of the TGF beta RII extracellular domain is an N-terminal truncated polypeptide of the TGF beta RII extracellular domain; in some embodiments, the C-terminal fragment of the TGF β RII extracellular domain is a polypeptide truncated N-terminal of the TGF β RII extracellular domain by 14-23 consecutive amino acid residues; in some embodiments, the C-terminal fragment of a tgfbetarii extracellular domain is a polypeptide truncated N-terminal of the tgfbetarii extracellular domain by between 16 and 23 consecutive amino acid residues; in some embodiments, the C-terminal fragment of the tgfbetarii extracellular domain is a tgfbetatrap (15-136), a tgfbetatrap (16-136), a tgfbetatrap (17-136), a tgfbetatrap (18-136), a tgfbetatrap (19-136), a tgfbetatrap (20-136), a tgfbetatrap (21-136), a tgfbetatrap (22-136), a tgfbetatrap (23-136), a tgfbetatrap (24-136), or a mutated sequence thereof having a substitution of one or more amino acids on the C-terminal fragment of the tgfbetarii extracellular domain, e.g., a tgfbetarii extracellular domain having an N18A and/or N19A amino acid substitution; in some embodiments, the C-terminal fragment of the tgfbetarii extracellular domain is SEQ ID NO: 41. SEQ ID NO:42 or SEQ ID NO: 43.

According to a specific embodiment of the invention, the TGF β RII mutant fragment comprises 0 to 6 amino acids of the N-terminus of the TGF β RII extracellular domain. In some embodiments, the TGF β RII mutant fragment comprises 1, 2, 3, 4, 5, or 6 amino acids of the N-terminus of the TGF β RII extracellular domain; in some embodiments, the TGF β RII mutant fragment comprises 6 amino acid residues N-terminal to the TGF β RII extracellular domain.

According to a particular embodiment of the invention, the N-terminal fragment of the TGF β RII extracellular domain has the amino acid sequence of SEQ ID NO: 1.

IPPHVQ(SEQ ID NO：1)。

According to a specific embodiment of the invention, the flexible fragment is a short peptide containing G, S amino acids. In some embodiments, the flexible segment is (G) ₄ S) _X G polypeptide, wherein X is preferably any integer from 1 to 6; in some embodiments, the flexible segment comprises ammoniaThe amino acid sequence is shown as SEQ ID NO: 2. SEQ ID NO:12 or SEQ ID NO: shown at 39.

According to a preferred embodiment of the invention, the short peptide comprises 5 to 15 amino acids.

According to a specific embodiment of the invention, the flexible fragment has the sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

GSGSGSGSG(SEQ ID NO：2)。

According to a specific embodiment of the invention, the TGF β RII mutant fragment comprises at most 115, at most 117 amino acids of the C-terminus of the TGF β RII extracellular domain.

According to a specific embodiment of the present invention, the TGF-beta RII mutant fragment includes 112 to 117 amino acids from the C-terminus of the TGF-beta RII extracellular domain.

According to a particular embodiment of the invention, the TGF-beta RII mutant fragment comprises the C-terminal 112, 115 or 117 amino acids of the TGF-beta RII extracellular domain.

According to a particular embodiment of the invention, the TGF β RII mutant fragment has the amino acid sequence of SEQ ID NO:3 to 5 or a pharmaceutically acceptable salt thereof.

IPPHVQGSGSGSGSGGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO：3)。

IPPHVQGSGSGSGSGVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO：4)。

IPPHVQGSGSGSGSGVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO：5)。

The invention provides a TGF beta RII mutant, which comprises an extracellular region, a transmembrane region and an intracellular region; wherein the extracellular region comprises a TGF-beta RII mutant fragment according to the first aspect. In view of the fact that the wild-type TGF beta RII fragment produced by the prior art has very high content and influences the drug effect, the inventor creatively discovers that the TGF beta RII mutant containing the fragment has the biological activity consistent with that of the wild-type TGF beta RII, the amino acid sequence of the TGF beta RII mutant is not easy to dissociate in the process of producing the TGF beta RII mutant, the content of the fragment is obviously reduced, and the drug property of the TGF beta RII mutant is improved.

The present invention provides a method for producing the above-described TGF-beta RII mutant fragment, wherein the amino acid sequence of the TGF-beta RII mutant fragment is obtained by truncating at least 14 amino acids from the N-terminus of the TGF-beta RII extracellular domain and inserting no more than 20 amino acids. In view of the fact that the TGF beta RII fragment prepared by the prior art has very high content and is difficult to remove in the purification process and influence the drug effect, the inventor conducts a great deal of experimental exploration, after the N-terminal of the TGF beta RII extracellular domain is truncated according to the mutation mode of the embodiment of the invention, the obtained TGF beta RII mutant fragment has the biological activity of combining with TGF beta, the fragment is not easy to dissociate and is not easy to break, the TGF beta RII mutant containing the fragment also has the biological activity consistent with the wild-type TGF beta RII, in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the drug effect is improved.

According to a specific embodiment of the invention, the amino acid sequence of the TGF-beta RII mutant fragment is obtained by truncating the N-terminus of the TGF-beta RII extracellular domain by 14-23 amino acids. The mutated site has a certain relation with the content of fragments generated in the process of preparing TGF beta RII, therefore, after 14-23 amino acids are truncated, the fragments still have the biological activity of combining with TGF beta, and in the process of preparing the TGF beta RII mutant fragments, the amino acid sequence is not easy to dissociate, so that the content of the fragments is obviously reduced, and the pharmaceutical property is improved; the TGF beta RII mutant containing the fragment also has the biological activity consistent with that of the wild TGF beta RII, and in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the druggability is improved.

According to a specific embodiment of the invention, the amino acid sequence of the TGF-beta RII mutant fragment is obtained by truncating the N-terminus of the TGF-beta RII extracellular domain by 14-21 amino acids. When the TGF beta RII mutant fragment is truncated by 14-21 amino acids, the obtained TGF beta RII mutant fragment still has the biological activity of combining with TGF beta, is not easy to dissociate, the fragment content is obviously reduced, the TGF beta RII mutant containing the fragment also has the biological activity consistent with wild type TGF beta RII, the amino acid sequence is not easy to dissociate in the process of preparing the TGF beta RII mutant, the fragment content is obviously reduced, and the druggability is improved.

According to a specific embodiment of the invention, the amino acid sequence of the TGF-beta RII mutant fragment is truncated by 14, 19 or 21 amino acids from the N-terminus of the TGF-beta RII extracellular domain. When the TGF beta RII mutant fragment is truncated by 14, 19 or 21 amino acids, the obtained TGF beta RII mutant fragment still has the biological activity of binding with TGF beta, the fragment is not easy to dissociate, the fragment content is obviously reduced, the TGF beta RII mutant containing the fragment has the biological activity consistent with that of a wild type TGF beta RII, the amino acid sequence is not easy to dissociate in the process of preparing the TGF beta RII mutant, the fragment content and the high polymer content are both obviously reduced, and the druggability is improved.

According to a specific embodiment of the invention, the amino acid sequence of the TGF-beta RII mutant fragment is obtained by inserting no more than 15 amino acids at the N-terminus of the TGF-beta RII extracellular domain. When no more than 15 amino acids are inserted, the amino acid sequence of the TGF beta RII mutant is not easy to dissociate in the preparation process, the fragment content is obviously reduced, the pharmaceutical property is improved, and the in vivo half-life period is prolonged.

According to a specific embodiment of the invention, the amino acid sequence of the TGF-beta RII mutant fragment is obtained by inserting 15 amino acids into the N-terminus of the TGF-beta RII extracellular domain. When 15 amino acids are inserted, the TGF beta RII mutant fragment still has the biological activity of combining with TGF beta, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, the TGF beta RII mutant containing the fragment is not easy to dissociate in the preparation process, the fragment content is obviously reduced, the pharmaceutical property is improved, and the half-life period in vivo is prolonged.

According to a specific embodiment of the invention, the inserted amino acid fragment comprises an N-terminal fragment of the extracellular domain of TGF-beta RII.

IPPHVQ(SEQ ID NO：1)。

According to a specific embodiment of the invention, the inserted amino acid fragment further comprises a flexible fragment. According to a specific embodiment of the present invention, the flexible segment is not particularly limited, and a conventional flexible segment may be used.

"Flexible fragment" refers to a peptide containing 2 or more amino acid residues joined by peptide bonds and providing greater rotational freedom for the 2 polypeptides to which it is attached (e.g., an N-terminal fragment of the TGF-beta RII extracellular domain, a C-terminal fragment of the TGF-beta RII extracellular domain).

According to a specific embodiment of the invention, the flexible fragment has the sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. In some embodiments, the flexible fragment is a polypeptide as set forth in SEQ ID NO:12 or SEQ ID NO: 39.

GSGSGSGSG(SEQ ID NO：2)。

According to a specific embodiment of the invention, the N-terminus of the flexible fragment is linked to the C-terminus of the N-terminal fragment of the extracellular domain of TGF-beta RII, and the C-terminus of the flexible fragment is linked to the N-terminus of the extracellular domain of TGF-beta RII remaining after truncation.

According to a specific embodiment of the invention, the sequence of SEQ ID NO:2 (e.g., IPPHVQGSGSGSGSG (SEQ ID NO: 11)) is arranged after the N-terminal fragment of the TGF-beta RII extracellular domain (e.g., when the N-terminal amino acid sequence of the TGF-beta RII extracellular domain is shown in SEQ ID NO: 1). In the preparation process, the amino acid sequence of the TGF beta RII mutant is not easy to dissociate, the fragment content is obviously reduced, the pharmaceutical property is improved, and the half-life period in vivo is prolonged.

The present invention provides a TGF-beta RII mutant fragment prepared by the method for preparing a TGF-beta RII mutant described above.

According to the embodiments of the present invention, since fragments are easily generated when TGF-beta RII is prepared and some sites in the amino acid sequence of TGF-beta RII are easily broken and closely linked, by the method provided by the embodiments of the present invention, TGF-beta RII mutant fragments having a low content of fragments and aggregates during production can be obtained by truncating and/or inserting a site where the N-terminus of the TGF-beta RII extracellular domain is easily broken or dissociated, and TGF-beta RII mutant fragments containing the TGF-beta RII mutant fragments are produced with a low content of aggregates and fragments.

The present invention provides a method for preparing the above-described TGF-beta RII mutant, which comprises an extracellular region, a transmembrane region and an intracellular region; the method comprises the following steps: truncation is by at least 14 amino acids from the N-terminus of the TGF β RII extracellular domain, with no more than 20 amino acids inserted. In view of the fact that the TGF beta RII fragment prepared by the prior art has very high content and is difficult to remove in the purification process and influence the drug effect, the inventor conducts a great deal of experimental exploration, after the N-terminal of the TGF beta RII extracellular domain is truncated according to the mutation mode of the embodiment of the invention, the obtained TGF beta RII mutant has the biological activity consistent with that of the wild type TGF beta RII, in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the drug property is improved.

According to a particular embodiment of the invention, the mutant is obtained by truncation of 14-23 amino acids from the N-terminus of the extracellular domain of TGF-beta RII. The mutated site has a certain relation with the content of fragments generated in the process of preparing TGF beta RII, therefore, after 14-23 amino acids are truncated, the obtained TGF beta RII mutant has the biological activity consistent with that of wild TGF beta RII, and in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the content of the fragments is obviously reduced, and the druggability is improved.

According to a specific embodiment of the invention, the TGF-beta RII mutant is obtained by truncating 14-21 amino acids from the N-terminus of the TGF-beta RII extracellular domain. After 14-21 amino acids are truncated, the obtained TGF beta RII mutant has the biological activity consistent with that of wild TGF beta RII, and in the process of preparing the TGF beta RII mutant, the amino acid sequence is not easy to dissociate, the fragment content is obviously reduced, and the drug property is improved.

According to a specific embodiment of the invention, the amino acid sequence of the TGF-beta RII mutant is obtained by truncating the N-terminus of the TGF-beta RII extracellular domain by 14, 19 or 21 amino acids. When the TGF beta RII mutant is truncated by 14, 19 or 21 amino acids, the obtained TGF beta RII mutant has the biological activity consistent with that of the wild TGF beta RII, the amino acid sequence is not easy to dissociate in the process of preparing the TGF beta RII mutant, the content of fragments and high polymers is greatly reduced, and the drug property of the TGF beta RII mutant is improved.

According to a specific embodiment of the present invention, the amino acid sequence of the tgfbetarii mutant is obtained by inserting no more than 15 amino acids at the N-terminus of the tgfbetarii extracellular domain.

According to a specific embodiment of the present invention, the amino acid sequence of the TGF-beta RII mutant is obtained by inserting 15 amino acids into the N-terminus of the TGF-beta RII extracellular domain. When 15 amino acids are inserted, the amino acid sequence of the TGF beta RII mutant is not easy to dissociate in the preparation process, the fragment content is obviously reduced, the pharmaceutical property is improved, and the half-life period in vivo is prolonged.

According to a particular embodiment of the invention, the inserted amino acid fragment further comprises a flexible fragment. According to a specific embodiment of the present invention, the flexible segment is not particularly limited, and a conventional flexible segment may be used.

GSGSGSGSG(SEQ ID NO：2)。

According to a specific embodiment of the invention, the sequence of SEQ ID NO:2 (e.g., IPPHVQGSGSGSGSG (SEQ ID NO: 11) when the N-terminal amino acid sequence of the TGF β RII extracellular domain is as set forth in SEQ ID NO: 1). In the preparation process, the amino acid sequence of the TGF beta RII mutant is not easy to dissociate, the fragment content is obviously reduced, the pharmaceutical property is improved, and the half-life period in vivo is prolonged.

The present invention provides a TGF beta RII mutant prepared by the above-described method.

According to the embodiments of the present invention, since fragments are easily generated when TGF-beta RII is prepared and some sites in the amino acid sequence of TGF-beta RII are easily broken and closely linked, a TGF-beta RII mutant having a low content of fragments and polymers during production can be obtained by truncating and/or inserting a site where the N-terminal of the extracellular domain of TGF-beta RII is easily broken or dissociated, by the method provided by the embodiments of the present invention.

The present invention provides a nucleic acid molecule encoding a TGF-beta RII mutant fragment or a TGF-beta RII mutant as hereinbefore described. The TGF beta RII mutant fragment or the TGF beta RII mutant coded by the nucleic acid molecule has the biological activity of combining with TGF beta, and the amino acid sequence is not easy to dissociate, so the fragment content is obviously reduced in the process of producing the TGF beta RII mutant fragment or the TGF beta RII mutant, and the drug property is improved.

The present invention provides a fusion protein, and a fusion protein according to an embodiment of the present invention includes: 1) A TGF β RII mutant fragment as described above; and 2) an immunoglobulin Fc fragment, to which the TGF-beta RII mutant fragment is linked via a linker peptide. In view of the fact that the fragment content of TGF beta RII prepared by the prior art is very high, the drug effect is influenced, and the administration frequency is high, the inventor carries out the truncation mutation on the N-terminal amino acid sequence of the extracellular domain of TGF beta RII, the obtained TGF beta RII mutant has the biological activity consistent with the wild TGF beta RII, after the immunoglobulin Fc fragment is added to the TGF beta RII mutant fragment, the obtained fusion protein also has the biological activity consistent with the wild TGF beta RII, the amino acid sequence is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, the drug forming property and the in vivo half-life are improved, and the fusion protein can control the content of TGF beta which is up-regulated in the periphery of tumor cells, so that the tumor can be effectively prevented or treated for a long time.

According to a specific embodiment of the present invention, the N-terminus of the linker peptide is linked to the C-terminus of the immunoglobulin Fc fragment, and the C-terminus of the linker peptide is linked to the N-terminus of the TGF β RII mutant fragment.

According to a particular embodiment of the invention, the immunoglobulin Fc fragment is derived from a human IgG antibody molecule.

According to a specific embodiment of the invention, the immunoglobulin Fc fragment comprises an Fc heavy chain fragment of an hIgG1 antibody.

According to a specific embodiment of the present invention, the C-terminal lysine of the immunoglobulin Fc fragment is mutated to alanine. The lysine is mutated into alanine, so that the cleavage hydrolysis of the fusion protein can be reduced, and the content of fragments in the process of preparing the fusion protein is reduced.

According to a particular embodiment of the invention, the immunoglobulin Fc fragment has the amino acid sequence as set forth in SEQ ID NO: 6.

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO：6)。

According to a specific embodiment of the invention, the linker peptide is a flexible linker peptide. The linker peptide is not particularly limited, and flexible fragments conventional in the art may be used.

According to a particular embodiment of the invention, the linker peptide comprises (G) ₄ S) _X G amino acid sequence, wherein X is an integer greater than 0. In some embodiments, the linker peptide is (G) ₄ S) _X G polypeptide, wherein X is preferably any integer from 1 to 6. In some embodiments, the linker peptide is as set forth in SEQ ID NO:12 or SEQ ID NO: 39.

According to a particular embodiment of the invention, the linker peptide comprises (G) ₄ S) ₄ G (SEQ ID NO: 12) sequence.

According to a particular embodiment of the invention, the fusion protein has the amino acid sequence as shown in SEQ ID NO:7 to 9.

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQGSGSGSGSGGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO：7)。

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQGSGSGSGSGVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO：8)。

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQGSGSGSGSGVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO：9)。

The present invention provides a nucleic acid molecule encoding a fusion protein as described above. The fusion protein coded by the nucleic acid molecule according to the specific embodiment of the invention has the biological activity consistent with that of wild TGF beta RII, the amino acid sequence of the fusion protein is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, the drug forming property and the in vivo half life of the fusion protein are improved, and the fusion protein can control the content of TGF beta with the up-regulated expression around tumor cells, so that the tumor can be effectively prevented or treated for a long time.

The present invention provides an expression vector. Expression vectors according to embodiments of the invention comprise a nucleic acid molecule encoding a TGF-beta RII mutant fragment or TGF-beta RII mutant, or a fusion protein, as described above. When the above-mentioned nucleic acid molecule is ligated to a vector, the nucleic acid molecule may be directly or indirectly ligated to control elements on the vector so long as the control elements can control translation, expression, and the like of the nucleic acid molecule. Of course, these control elements may be derived directly from the vector itself, or may be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule may be operably linked to a control element. "operably linked" herein refers to the attachment of a foreign gene to a vector such that control elements within the vector, such as transcriptional and translational control sequences and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene.

According to a particular embodiment of the invention, the expression vector is a eukaryotic expression vector.

The present invention provides a recombinant cell carrying a nucleic acid molecule encoding a TGF-beta RII mutant fragment or a TGF-beta RII mutant, or a fusion protein, or an expression vector as described above. The recombinant cell according to the embodiment of the invention can express the fusion protein, the amino acid sequence of the recombinant cell is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, the drug forming property and the in vivo half-life period of the recombinant cell are improved, and the fusion protein can control the content of TGF beta with up-regulated expression around tumor cells, so that the tumor can be effectively prevented or treated for a long time.

According to a particular embodiment of the invention, the recombinant cell is a mammalian cell, such as: human HEK-293F cells or CHO-K1 cells.

According to a specific embodiment of the invention, the recombinant cell does not comprise an animal germ cell, fertilized egg or embryonic stem cell.

The present invention provides a pharmaceutical composition comprising a TGF-beta RII mutant fragment, a TGF-beta RII mutant, a nucleic acid molecule encoding a TGF-beta RII mutant fragment or a TGF-beta RII mutant, a fusion protein, a nucleic acid molecule encoding a fusion protein, an expression vector, and a recombinant cell as described above according to an embodiment of the present invention. The pharmaceutical composition may include: pharmaceutically acceptable adjuvants including at least one of stabilizers, wetting agents, emulsifiers, binders, isotonicity agents; the pharmaceutical composition is at least one of tablets, granules, powder, capsules, solutions, suspensions and freeze-dried preparations. The pharmaceutical composition can control the content of TGF beta with up-regulated expression around tumor cells, thereby preventing or treating tumors for a long time and effectively.

The present invention provides the use of the above-described TGF-beta RII mutant fragment, TGF-beta RII mutant, nucleic acid molecule encoding TGF-beta RII mutant fragment or TGF-beta RII mutant, fusion protein, nucleic acid molecule encoding fusion protein, expression vector, recombinant cell in the preparation of a medicament. The medicament according to the embodiment of the present invention is used for preventing or treating tumors. The medicine provided by the invention has the long-acting function of combining with TGF beta, thereby effectively treating or preventing tumors.

The present invention provides a method for preparing the aforementioned fusion protein, comprising the steps of: 1) Constructing the expression vector as described above; 2) Introducing the expression vector into a host cell to obtain a recombinant cell so as to express the fusion protein. The fusion protein can be efficiently obtained by the method according to the specific embodiment of the invention, has the biological activity consistent with that of wild TGF beta RII, has an amino acid sequence which is not easy to dissociate in the preparation process of the fusion protein, has a remarkably reduced fragment content, and improves the drug property and the in vivo half-life period.

According to a specific embodiment of the present invention, the recombinant cell is a mammalian cell, such as human, monkey, rabbit, dog, cow, etc.; mammalian cells such as: such as: human HEK-293F cells or CHO-K1 cells.

The present invention provides a method for reducing the amount of debris in the process of preparing the aforementioned fusion protein, comprising the steps of: 1) Constructing the expression vector as described above; 2) Introducing the expression vector into a host cell. According to the method provided by the embodiment of the invention, the amino acid sequence of the fusion protein is not easy to dissociate in the preparation process of the fusion protein, the fragment content is obviously reduced, and the drug effect is obviously improved.

The present invention provides a method of preventing or treating a tumor, comprising administering to a subject at least one of: 1) A TGF β RII mutant fragment as described above; 2) The TGF β RII mutants described above; 3) The fusion protein as described above; 4) Nucleic acid molecules encoding said TGF β RII mutant fragment or TGF β RII mutant or fusion protein as described above; 5) The expression vector as described above; 6) Recombinant cells as described above; and 7) the pharmaceutical composition as described above. The method according to the embodiment of the present invention can effectively prevent or treat tumors by controlling the content of TGF β up-regulated in the periphery of tumor cells.

These tumors can be any unregulated cell growth. Specifically, it may be non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, colorectal cancer, melanoma, cholangiocarcinoma or sarcoma, acute myelogenous leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate carcinoma, neuroblastoma, pancreatic carcinoma, melanoma, head and neck squamous cell carcinoma or gastric carcinoma, and the like.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of TGF-. Beta.RII trap fusion proteins

In this example, the inventors used homologous recombination techniques to generate (G) ₄ S) ₄ G (SEQ ID NO: 12) as a linker peptide, the C-terminus of the Fc heavy chain fragment (SEQ ID NO: 6) of hIgG1 antibody was ligated with a variant fragment of TGF-. Beta.RII (Table 1) to obtain a TGF-. Beta.RII trap fusion protein. At the fusion junction, the C-terminal lysine residue (K) of the Fc heavy chain fragment of the hIgG1 antibody was mutated to alanine (A) (resulting in the sequence shown in SEQ ID NO: 6) to reduce cleavage hydrolysis of the fusion protein.

For the TGF β RII trap fusion proteins, the TGF β RII trap fusion proteins are prepared by transient transfection of human HEK-293F cells, TGF β RII trap fusion proteins prepared by transient transfection of mammalian cells with DNA encoding the Fc-TGF β RII receptor in the same expression vector or in a separate expression vector, and TGF β RII trap fusion proteins prepared by stable transfection of CHO-K1 cells using standard protocols for transient or stable transfection.

A schematic of the structure of the TGF-. Beta.RII mutant is shown in FIG. 1. The TGF β RII trap fusion proteins formed by the different mutant forms of the TGF β RII mutants are shown in table 1.

TABLE 1

Remarking: in Table 1, fc represents an 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 beta Trap (WT) represents the amino acid sequence of the extracellular domain of wild-type TGF beta RII (SEQ ID NO: 10); TGF-beta 0trap (X-136) represents a polypeptide of amino acid residues from position X to 136 of the extracellular domain of TGF-beta 1RII (the positions are numbered relative to the natural sequence of SEQ ID NO:10, the same applies below), e.g., TGF-beta trap (20-136) represents a polypeptide of amino acid residues 1-19 of the N-terminus of a truncated TGF-beta RII extracellular domain (i.e., residues 20-136 of the extracellular domain of TGF-beta RII are only retained), TGF-beta trap (8-136, N10G/N11G/N18A/N19A) represents amino acid residues 1-7 of the N-terminus of a truncated TGF-beta RII extracellular domain (i.e., residues 8-136 of the extracellular domain of TGF-beta RII are only retained), while post-mutating 3763 of amino acids 5236 z3236 zft 3762 zft 3718 x19 of the polypeptide at positions 10, 11, 18 and 19 (the mutating positions are numbered relative to the natural sequence of SEQ ID NO: 10); IP (XXX) represents the former XXX position of the N-terminal end of the TGF beta RII extracellular domain, for example, IP0, IP3, IP6, IP9, IP12 sequentially represent the former 0, 3, 6, 9, 12 amino acids of the N-terminal end of the TGF beta RII extracellular domain; GS9 represents a linker peptide (SEQ ID NO: 2); SA6 represents the sequence GGGGSA (SEQ ID NO: 32); EG14 represents the sequence EGKSSGSGSESKST (SEQ ID NO: 36); AE17 represents the sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 37); QF18 represents the sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 38);

in addition, the "number of truncated/inserted amino acids" in Table 1 indicates the difference between the number of amino acids in IP (XXX) + TGF β Trap (X-136) + linker peptide (polypeptide linking IP (XXX) and TGF β Trap (X-136)) and the number of amino acids in the wild-type TGF β RII extracellular domain (SEQ ID NO: 10) in the corresponding fusion protein, for example, the number of amino acids in IP6-GS9-TGF β Trap (20-136) in the Fc- (G4S) 4G-IP6-GS9-TGF β Trap (20-136) fusion protein is 132, which is 4 amino acids smaller than the wild-type TGF β RII extracellular domain, and thus the corresponding "number of truncated/inserted amino acids" is-4.

TGF beta trap (X-136) represents the amino acid sequence from position X to 136 of the extracellular domain of TGF beta RII, illustratively,

the amino acid sequence of TGF beta trap (20-136) is (SEQ ID NO: 41):

GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD；

the amino acid sequence of TGF beta trap (15-136) is (SEQ ID NO: 42):

VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD；

the amino acid sequence of TGF β trap (22-136) is (SEQ ID NO: 43):

VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

example 2 purification of TGF-. Beta.RII trap fusion proteins

The culture medium of human HEK-293F cells for producing TGF- β RII trap fusion protein obtained by transiently transfecting human HEK-293F cells and the culture medium of CHO-K1 cells capable of producing TGF- β RII trap fusion protein obtained by stably transfecting CHO-K1 cells were centrifuged at high speed, and the supernatants were collected.

Subjecting the collected supernatant to a first purification step by affinity chromatography, wherein the chromatography medium is Protein A or derivatized filler such as Mabselect from GE, which interacts with Fc; and (3) balancing 5 times of column volume by using 1 XPBS as the balancing buffer solution, combining the cell supernatant and a chromatography medium, and controlling the flow rate to be that the retention time of the sample on the affinity chromatography column is not less than 1min. After the loading was complete, the column was washed with 1 XPBS (pH 7.4) and the absorbance at 280nm was recorded until the A280 UV absorbance decreased to baseline. The column was then washed with 0.1M glycine (pH 3.0) elution buffer, and the elution peak was collected from the A280 UV absorbance peak and the collected eluate was neutralized with 1M Tris (pH 8.5).

Concentrating the neutralized elution sample by using an ultrafiltration device, and performing size exclusion chromatography, wherein the buffer solution for the size exclusion chromatography is 1 XPBS, the chromatography column is XK26/60Superdex200 (GE), the flow rate is controlled to be 4mL/min, the sample loading volume is less than 5mL, the target protein peaks are combined according to A280 ultraviolet absorption, and the purity of the collected TGF beta RII trap fusion protein is identified by SEC-HPLC.

Example 3 TGF-. Beta.RII trap fusion protein truncation assay

The inventors analyzed the high polymers and fragments during purification of different TGF- β RII trap fusion proteins as shown by SEC-HPLC results.

The results of the contents of fragments and polymers generated during purification of fusion proteins formed by a TGF-. Beta.RII mutant obtained by truncating the N-terminus of the TGF-. Beta.RII extracellular domain, or inserting a flexible fragment and an N-terminal fragment of the TGF-. Beta.RII extracellular domain after truncation are shown in FIG. 2, and can be seen: during the process that the truncation amount is increased from 6 amino acids to 13 amino acids, the high polymer content is increased by 7 percent; when the truncation number is increased to 15 amino acids, the high polymer content is increased by 13%; the truncation number is increased to 20 amino acids, and the high polymer content is increased to more than 20%; meanwhile, the inventors observed that when 6 to 15 amino acids were truncated, the fragment content did not change significantly; but when truncated by 16 to 23 amino acids, the fragment content is significantly reduced. Therefore, a truncation of 16 to 23 amino acids is effective in reducing the fragment content.

Example 4 Injectionengineering analysis of TGF-. Beta.RII trap fusion proteins

In FIG. 2, a notable observation is that, compared with WT, by truncating amino acids 1 to 19 at the N-terminus of the extracellular domain of TGF- β RII, and then inserting amino acids 1 to 6 at the N-terminus of the extracellular domain of TGF- β RII (IPPHVQ (SEQ ID NO: 1)) and a flexible fragment (GSGSGSGSG (SEQ ID NO: 2)), the fusion protein of TGF- β RII trap (Trunc # 22) was obtained with NO fragments and much reduced multimers. Therefore, the inventors further studied the structure of Trunc #22, and optimized the combination of the reserved portion, the inserted portion, and the truncated portion, respectively.

First, the inventors tried to retain the first 1 to 12 amino acids of the N-terminus of a TGF-. Beta.RII Trap for comparison in search of the N-terminus retention portion of the TGF-. Beta.RII Trap (Trunc #22, trunc #23, trunc #25, trunc #26, trunc # 27).

The results of the contents of fragments and polymers generated during purification of fusion proteins formed by a TGF-. Beta.RII mutant obtained by truncating the N-terminus of the TGF-. Beta.RII extracellular domain followed by insertion of a flexible fragment and an N-terminal fragment of the TGF-. Beta.RII extracellular domain are shown in FIG. 3, and it can be seen that: compared with the truncated control group of Trunc #31, trunc #22, trunc #23, trunc #25, trunc #26 and Trunc #27, the 5 recombinant protein molecules have obviously reduced high polymer; and as the number of amino acids retained increases, the fragments gradually increase from 2.8% to over 10%. The fragment content of the Trunc #22 is low, the high polymer content is effectively controlled, and a small amount of high polymer can be effectively removed in the subsequent purification process.

Furthermore, in order to verify whether the retained sequence must be the N-terminal amino acid sequence of the extracellular domain of TGF-. Beta.RII, the inventors tried other sequences, such as GGGSAG (Trunc # 24).

The results are shown in FIG. 3, where it can be seen that: compared with the Trunc #22 and the

Trunc #

24, 5 percent of fragments appear, and the high polymer is obviously increased; it was shown that the N-terminal fragment (IPPHVQ (SEQ ID NO: 1)) of the TGF-. Beta.RII extracellular domain selected by the retention sequence was more effective.

Secondly, for the exploration of the insertion part, the inventors tried various sequences including flexible linkers, rigid linkers and random sequences of different lengths, wherein the insertion sequence in Trunc #32 is a flexible linker, the insertion sequence in Trunc #33 is a rigid linker, and the insertion sequence in Trunc #34 is a random sequence.

As a result, as shown in fig. 3, the insertion of Trunc #32 and Trunc #33 resulted in a large increase in both the fragment content and the macromer content, and the insertion of Trunc #34 resulted in a smaller macromer content, but the fragment content was not significantly improved compared to the wild-type TGF β RII. Indicating that the insert is only a flexible fragment and that the length of the insert is less than 14 amino acids.

Finally, in order to verify the optimal truncation combination in the case where the reserved segment and the inserted segment are the same as those of Trunc #22, the inventors constructed Trunc #35 and Trunc #36.

The results are shown in FIG. 3, where it can be seen that: both Trunc #35 and Trunc #36 have reduced debris content and Trunc #36 has less debris content compared to wild-type TGF β RII; compared with Trunc #28 and Trunc #31, the content of the high polymer of Trunc #35 and Trunc #36 is obviously reduced, and the content of the high polymer of Trunc #35 is lower than that of Trunc #36. Thus, trunc #35, trunc #36 and Trunc #22 all solve the problem of fragmentation and high aggregation during expression, with Trunc #22 being the best.

Example 5 ELISA binding assay for TGF-beta RII trap fusion proteins

The protein used for the TGF beta RII Trap fusion protein Trap end binding assay is human TGF beta 1 (CA 59, purchased from Novoprotein), and the assay procedure is as follows:

a. TGF beta 1 is diluted to 0.5 mu g/mL by 1 Xphosphate buffer solution (PBS), 100 mu L/hole is coated on a 96-hole enzyme label plate, and the temperature is kept overnight at 4 ℃;

b.250 μ L1 XPBST (PBS +0.5% Tweenen20) 3 washes, add 200 μ L2% Bovine Serum Albumin (BSA) in PBS and block for 1 hour at room temperature;

c.250 μ L1 XPBST 3 washes, add gradient diluted TGF β RII trap, incubate for 2 hours at room temperature;

d.250 μ L1 × PBST wash 3 times, 100 μ L of diluted coat-anti-human Fc-HRP conjugate antibody (Sigma, 1;

e.250. Mu.L of 1 XPBST washed 3 times, 100. Mu.L of TMB developing solution was added to each well, incubated for 10 minutes at room temperature in the dark, 50. Mu.L of 2N H was added ₂ SO ₄ Terminating the reaction;

f. the absorbance at 450nM was read using an iX3 microplate reader (Molecular Device Co.) and analyzed for mapping.

The result of the detection of the ELISA binding activity of the fusion protein and the human TGF β 1 is shown in fig. 4, and it can be seen that: all TGF β RII traps were able to bind TGF β 1 coated on the plate, and no truncation modification affected the binding of TGF β RII to TGF β 1.

Example 6 detection of blocking Activity of TGF-beta RII trap fusion proteins

The protein used for the TGF-beta RII Trap fusion protein Trap end binding assay was human TGF-beta 1 (CA 59, from Novoprotein), and the assay protocol was as follows:

CHO cells (CHO-hTGF beta RII) which stably transfect and express human TGF beta RII are constructed, and a single clone is selected for establishing a line. The blocking activity against TGF β RII trap was tested using the following method:

a. CHO-hTGF. Beta. RII cells were counted at 2X 10 ⁵ Paving the density of each hole on a U-shaped bottom plate with 96 holes;

b. the plated CHO-hTGF. Beta. RII cells were centrifuged at 300g at 4 ℃ for 5 minutes, and the supernatant was discarded.

c. The precipitate containing the TGF-. Beta.RII trap fusion protein obtained in step b was diluted with 1 XPBS containing 1% BSA at an initial concentration of 50nM by a 4-fold dilution, with a total of 8 gradients, and TGF-. Beta.1-biotin (from Acrobiosystem) was also diluted to a concentration of 1. Mu.g/mL.

d. And mixing the fusion protein diluent and the TGF beta 1-botin diluent uniformly according to the volume proportion of 1:1, and standing for 30min at room temperature.

e. And d, adding the mixed sample obtained in the step d into CHO-TGF beta RII cells at a concentration of 100 mu L/hole, standing at 4 ℃ for 30min, centrifuging at room temperature under the condition of 300g for 5min, and throwing off the supernatant.

f. Adding SA-PE (400-fold dilution, jackson Immunoresearch, 016-110-084) into the product obtained in the step e, adding the mixture into the product with the volume of 100 mu l/hole, slightly mixing the mixture, placing the mixture at the temperature of 4 ℃ for 1 hour, centrifuging the mixture for 5min at the room temperature under the condition of 300g, and throwing off the supernatant; the obtained cells were added to 1 XPBS containing 1% BSA at 100. Mu.l/well, resuspended, and examined by flow cytometry.

The results of the fusion protein blocking the binding activity of human TGF beta 1 and TGF beta RII are shown in FIG. 5, and the truncated TGF beta RII Trap fusion protein can inhibit TGF beta 1 from binding to TGF beta RII, and the blocking capability is basically not greatly different from that of the wild type TGF beta RII Trap in terms of EC50 value.

Example 7T cell proliferation inhibition assay

Studies have shown that TGF beta 1 can significantly inhibit T cell proliferation, and to examine the function of the Trap end of the different modified TGF beta RII Trap fusion proteins at the cellular level, we examined the effect of the different modified TGF beta RII Trap fusion proteins on T cell proliferation in the presence of TGF beta. The following exemplary method was used for the experiments:

a. separating Peripheral Blood Mononuclear Cells (PBMCs) from whole blood of healthy volunteers using human lymphocyte separation liquid density gradient centrifugation, separating T cells from PBMCs using a human T cell enrichment kit (STEMCELL, 10951) according to instructions, staining T cells using CFSE (eBioscience, 85-65-0850-84) at a concentration of 1M according to the specific procedures with reference to the reagent instructions;

b. the TGF beta RII Trap fusion protein, CD3/CD28 beads (life, 40203D) and CFSE stained T cells were co-incubated at 5X 10 ⁴ Culturing in an incubator, wherein the volume ratio of CD3/CD28 beads to T cells is 1:5;

c. culturing the T cells to the fifth day, performing flow detection on the CFSE value of the T cells, calculating the proportion of the T cells in each generation, and mainly observing the leftmost peak.

The experimental results of the effect of fusion proteins formed by TGF-beta RII mutants obtained by shortening the N-terminus of the TGF-beta RII extracellular domain on T-cell proliferation are shown in FIG. 6, wherein the proliferation rate is about 15.4% after the stimulation without the addition of the antibody group (No TGF & No Ab group), and the proliferation rate is about 2.44% after the addition of TGF-beta 1, resulting in significant proliferation inhibition. After the fusion protein corresponding to the TGF beta RII mutant obtained by truncating the amino acid sequence at the N-terminal of the TGF beta RII extracellular domain is added, the proliferation proportion is obviously increased from 2.44 percent to 5.98 to 17.56 percent, and a concentration dependence relationship is presented. Wherein the Trunc #1, trunc #2, trunc #6, trunc #9, trunc #14, and Trunc #22 groups exhibited better T cell proliferation promoting effects than the WT group; meanwhile, the T cell proliferation promoting effect was weaker in the groups Trunc #3, trunc #7, trunc #8, trunc #11, trunc #13, and Trunc #15 to Trunc #18 than in the WT group.

The inventors further evaluated the activity of TGF-beta RII Trap fusion proteins corresponding to TGF-beta RII mutants obtained after insertion of the flexible fragment and the N-terminal fragment of the TGF-beta RII extracellular domain.

The experimental results of the T cell proliferation effect of the fusion protein formed by the tgfbetarii mutant obtained by truncating the N-terminus of the tgfbetarii extracellular domain and inserting the flexible fragment and the N-terminus fragment of the tgfbetarii extracellular domain are shown in fig. 7, wherein Trunc #25, trunc #32, trunc #35 and Trunc #36 exhibit better T cell proliferation promoting effects than the WT group; as a control, trunc #14 and Trunc #22 groups were used, and the results shown in fig. 7 were consistent with those shown in fig. 6, and Trunc #14 and Trunc #22 exhibited better T cell proliferation promoting effects than the WT group.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Moreover, various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A TGF β RII mutant fragment, comprising a C-terminal fragment, a flexible fragment, and an N-terminal fragment of a TGF β RII extracellular domain, wherein the C-terminal fragment comprises up to 122 amino acids of the C-terminus of the TGF β RII extracellular domain; the N end of the flexible segment is connected with the C end of the N-terminal segment of the TGF beta RII extracellular domain, and the C end of the flexible segment is connected with the N end of the C-terminal segment of the TGF beta RII extracellular domain.

2. The TGF β RII mutant fragment of claim 1, wherein the TGF β RII mutant fragment comprises 112 to 117 amino acids from the C-terminus of the TGF β RII extracellular domain; optionally, the amino acid sequence of the TGF β RII extracellular domain is as set forth in SEQ ID NO:10, the C-terminal fragment of the TGF beta RII extracellular domain is an N-terminal truncated polypeptide of the TGF beta RII extracellular domain; optionally, the C-terminal fragment of the TGF β RII extracellular domain is a polypeptide truncated at the N-terminus of the TGF β RII extracellular domain by 14-23 consecutive amino acid residues, preferably by 16-23 consecutive amino acid residues; optionally, the C-terminal fragment of the TGF β RII extracellular domain is SEQ ID NO: 41. SEQ ID NO:42 or SEQ ID NO: 43.

3. The TGF β RII mutant fragment according to claim 1 or 2, wherein the TGF β RII mutant fragment comprises 0 to 6 amino acids from the N-terminus of the TGF β RII extracellular domain; optionally, the TGF β RII mutant fragment comprises 1, 2, 3, 4, 5 or 6 amino acids of the N-terminus of the TGF β RII extracellular domain; alternatively, the amino acid sequence of the N-terminal fragment of the TGF β RII extracellular domain is as set forth in SEQ ID NO:1 is shown.

4. The TGF β RII mutant fragment according to any one of claims 1 to 3, wherein the flexible fragment is a short peptide comprising G and/or S amino acids; preferably, the short peptide contains 5 to 15 amino acids; optionally, the flexible segment is (G) ₄ S) _X G polypeptide, wherein X is preferably any integer from 1 to 6; optionally, the amino acid sequence of the flexible fragment is as shown in SEQ ID NO: 2. SEQ ID NO:12 or SEQ ID NO: shown at 39.

5. The TGF β RII mutant fragment according to any one of claims 1 to 4, wherein the N-terminal fragment of the TGF β RII extracellular domain has the amino acid sequence of SEQ ID NO:1, and the flexible fragment has an amino acid sequence shown in SEQ ID NO: 2. SEQ ID NO:12 or SEQ ID NO:39, and the C-terminal fragment of the TGF beta RII extracellular domain is SEQ ID NO: 41. SEQ ID NO:42 or SEQ ID NO: 43; alternatively, the tgfbetarii mutant fragment has the amino acid sequence of SEQ ID NO:3 to 5 or a pharmaceutically acceptable salt thereof.

6. The TGF β RII mutant fragment according to any one of claims 1 to 5, wherein the TGF β RII mutant fragment further comprises an immunoglobulin Fc fragment; optionally, the immunoglobulin Fc fragment is linked to the N-terminus of the TGF β RII mutant fragment by a linker peptide; alternatively, the immunoglobulin Fc fragment has the amino acid sequence as set forth in SEQ ID NO: 6; optionally, the linker peptide is (G) ₄ S) _X G polypeptide, wherein X is preferably any integer from 1 to 6, more preferably as set forth in SEQ ID NO:12 or SEQ ID NO: 39.

7. A fusion protein comprising a TGF-beta RII mutant fragment according to any one of claims 1 to 5;

preferably, the fusion protein comprises an immunoglobulin Fc fragment; optionally, the C end of the immunoglobulin Fc fragment is connected with the N end of the TGF beta RII mutant fragment through a connecting peptide; alternatively, the immunoglobulin Fc fragment has the amino acid sequence set forth in SEQ ID NO: 6; optionally, the linker peptide is (G) ₄ S) _X G polypeptide, wherein X is preferably any integer from 1 to 6, more preferably as set forth in SEQ ID NO:12 or SEQ ID NO: 39; alternatively, the fusion protein has the amino acid sequence of SEQ ID NO: 7-9.

8. A nucleic acid molecule encoding a TGF-beta RII mutant fragment according to any one of claims 1 to 6 or a fusion protein according to claim 7.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A pharmaceutical composition comprising a TGF-beta RII mutant fragment according to any one of claims 1 to 6, or a nucleic acid molecule according to claim 8, or an expression vector according to claim 9, or a fusion protein according to claim 7; preferably, it further comprises pharmaceutically acceptable adjuvants.

11. A TGF β RII mutant comprising an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region is the TGF β RII mutant fragment according to any one of claims 1 to 6.

12. A host cell transfected with the nucleic acid molecule of claim 8, or the expression vector of claim 9.

13. Use of a TGF-beta RII mutant fragment according to any one of claims 1 to 6, or a nucleic acid molecule according to claim 8, or an expression vector according to claim 9, or a pharmaceutical composition according to claim 10, or a TGF-beta RII mutant according to claim 11, or a host cell according to claim 12, or a fusion protein according to claim 7, for the preparation of a medicament for the prevention or treatment of a tumor.