TGFβRII activity-modified mutants and their applications

Abstract

This invention proposes a TGFβRII activity-modified mutant and its applications. Compared to the amino acid sequence of the extracellular domain of TGFβRII, the TGFβRII mutant fragment has mutations at the following sites: at least one of amino acids 49 and 50, or at least one of amino acids 49 and 53, and at least one of amino acids 27, 30, 50, 51, 52, 55, and 77. The TGFβRII mutant fragment according to this invention can effectively bind to TGFβ1 but not to TGFβ3. Fusion proteins prepared from the extracellular region of the TGFβRII mutant containing this fragment also possess the biological characteristics of binding to TGFβ1 but not to TGFβ3. These fusion proteins can effectively prevent and treat tumors, and their drug toxicity is significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to TGFβRII activity-modified mutants and their applications, and more specifically to TGFβRII mutant fragments, nucleic acid molecules encoding TGFβRII mutants, fusion proteins, nucleic acid molecules encoding fusion proteins, expression vectors, recombinant cells, pharmaceutical compositions, the use of said TGFβRII mutants or fusion proteins or nucleic acid molecules or expression vectors or recombinant cells in the preparation of drugs, methods for preparing said fusion proteins, and methods for reducing the fragment content during the production of fusion proteins. Background Technology

[0002] Transforming growth factor-β (TGFβ) belongs to the TGFβ superfamily, which regulates cell growth and differentiation.

[0003] TGFβ1 is a polypeptide cell growth negative regulator discovered by Tuker et al. in 1984 that is associated with the growth of various epithelial tumors. It is widely involved in various pathophysiological processes in the body and is closely related to the occurrence and development of various diseases such as inflammation, trauma, and organ fibrosis. In particular, its regulatory role in the occurrence and development of tumors has profound significance for tumor research.

[0004] TGFβ2 plays an important role in cell proliferation, differentiation, embryonic development, extracellular matrix formation, bone formation and remodeling, as well as tumor suppression and metastasis. TGFβ2 has been shown to inhibit IL-2-dependent T cell growth and suppress immune surveillance during tumor development, thus promoting tumor growth in an autocrine manner. TGFβ2 can also affect the activity of killer cells and reduce the expression of cytokines such as IL-2, IL-6, IL-10, and IFN-γ.

[0005] TGFβ3 stimulates extracellular matrix synthesis and accelerates vascularization in target cells (fibroblasts, vascular endothelial cells, etc.) during tissue repair, and promotes wound healing and reduces scar formation. During mammalian embryonic development, TGFβ3 promotes morphogenesis and plays a crucial role in vertebral formation, acral budding, teething, facial bone formation, and heart valve formation. TGFβ3 can regulate bone formation and influence adult bone regeneration. Studies have found elevated serum TGFβ3 concentrations in osteoporosis patients, which may reflect decreased TGFβ3 activity in bone tissue, thus contributing to the pathological process of osteoporosis.

[0006] Recent studies have found that increased TGFβ in the tumor microenvironment is associated with immune escape. Increased TGFβ increases T cell rejection and blocks the infiltration of TH1 effector T cells. Studies have shown that TGFβ blockade makes mouse liver metastasis cancer models more sensitive to PD1 / PDL1 therapy. Furthermore, studies have reported that the combination of TGFβ blockade and PD-L1 antibody can downregulate the TGFβ signaling pathway in stromal cells, promote T cell infiltration into the tumor, and activate a strong anti-tumor immune response. Summary of the Invention

[0007] This application is based on the inventor's discoveries and understanding of the following problems:

[0008] TGFβRII can bind to both TGFβ1 and TGFβ3. TGFβ1 plays an important regulatory role in the occurrence and development of tumors, while TGFβ3 plays an important role in mammalian embryonic development and can regulate bone formation. However, studies have found that the serum TGFβ3 concentration is elevated in patients with osteoporosis. Therefore, after extensive experiments, the inventors unexpectedly obtained a TGFβRII mutant with high affinity for TGFβ1 but without binding to TGFβ3. When this mutant is developed into a drug, it can effectively prevent and treat tumors, and the drug toxicity is significantly reduced.

[0009] In a first aspect, the present invention provides a TGFβRII mutant fragment. According to embodiments of the invention, compared to the amino acid sequence of the extracellular domain of TGFβRII, the TGFβRII mutant fragment has mutations at the following sites: at least one of amino acids 49 and 53, or at least one of amino acids 49 and 53 and at least one of amino acids 27, 30, 50, 51, 52, 55, and 77. Given that TGFβRII produced in the prior art can bind to both TGFβ1 and TGFβ3, and that binding of TGFβRII to TGFβ3 can affect normal physiological functions and produce drug toxicity, the inventors have creatively discovered that the TGFβRII mutant fragment according to embodiments of the present invention can effectively bind to TGFβ1 and not to TGFβ3. The fusion protein containing said fragment can effectively prevent and treat tumors, and its drug toxicity and impact on normal bodily functions are significantly reduced.

[0010] In a second aspect, the present invention provides a TGFβRII mutant. According to embodiments of the invention, it includes an extracellular region, a transmembrane region, and an intracellular region, wherein the extracellular region includes the TGFβRII mutant fragment described in the first aspect. Given that existing TGFβRII production can bind to both TGFβ1 and TGFβ3, and that binding of TGFβRII to TGFβ3 can affect normal physiological functions and cause drug toxicity, the inventors have creatively discovered that the TGFβRII mutant according to embodiments of the present invention can effectively bind to TGFβ1 and not to TGFβ3. The fusion protein containing the extracellular region of the TGFβRII mutant can effectively prevent and treat tumors, and its drug toxicity and impact on normal bodily functions are significantly reduced.

[0011] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes either the TGFβRII mutant fragment described in the first aspect or the TGFβRII mutant described in the second aspect. The TGFβRII mutant fragment encoded by the nucleic acid molecule according to embodiments of the invention can effectively bind to TGFβ1 but not to TGFβ3. Similarly, the TGFβRII mutant encoded by the nucleic acid molecule according to embodiments of the invention also possesses the characteristic of binding to TGFβ1 but not to TGFβ3. Fusion proteins containing the TGFβRII mutant fragment or the extracellular region of the TGFβRII mutant can effectively prevent and treat tumors, and their drug toxicity and impact on normal bodily functions are significantly reduced.

[0012] In a fourth aspect, the present invention provides a fusion protein. According to embodiments of the invention, it comprises: 1) a TGFβRII mutant fragment as described in the first aspect; and 2) an immunoglobulin Fc fragment, wherein the TGFβRII mutant fragment is linked to the immunoglobulin Fc fragment via a linker peptide. After the inventors performed the aforementioned mutation on the amino acid sequence of the extracellular domain of TGFβRII, the obtained TGFβRII mutant exhibits a significantly enhanced characteristic of binding to TGFβ1 and not binding to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein are significantly reduced. Adding the immunoglobulin Fc fragment to the TGFβRII mutant fragment further enhances the fusion protein's characteristic of binding to TGFβ1 and not binding to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein are significantly reduced, its drug-likeness and in vivo half-life are improved, and the fusion protein can control the upregulated expression of TGFβ around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0013] In a fifth aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the fusion protein described in the fourth aspect. The fusion protein encoded by the nucleic acid molecule according to embodiments of the present invention has the characteristic of binding to TGFβ1 but not to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein are significantly reduced, its drug-likeness and in vivo half-life are improved, and the fusion protein can control the upregulated expression of TGFβ around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0014] In a sixth aspect, the present invention provides an expression vector comprising the nucleic acid molecules described in the third or fifth aspect, according to embodiments of the invention.

[0015] In a seventh aspect, the present invention provides a recombinant cell. According to embodiments of the invention, it carries the nucleic acid molecule described in the third or fifth aspect, or the expression vector described in the sixth aspect. The recombinant cell according to embodiments of the invention can express the aforementioned fusion protein, which has the property of binding to TGFβ1 but not to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein are significantly reduced, its drug-likeness and in vivo half-life are improved, and the fusion protein can control the upregulated TGFβ content around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0016] In an eighth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises the TGFβRII mutant fragment described in the first aspect, or the TGFβRII mutant described in the second aspect, or the nucleic acid molecule described in the third aspect, or the fusion protein described in the fourth aspect, or the nucleic acid molecule described in the fifth aspect, or the expression vector described in the sixth aspect, or the recombinant cell described in the seventh aspect. The pharmaceutical composition according to the invention has low drug toxicity and low impact on normal bodily functions, and can control the upregulated expression of TGFβ around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0017] In a ninth aspect of the invention, the invention proposes the use of the TGFβRII mutant fragment described in the first aspect, or the TGFβRII mutant described in the second aspect, or the nucleic acid molecule described in the third aspect, or the fusion protein described in the fourth aspect, the nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, and the recombinant cells described in the seventh aspect in the preparation of a drug. According to embodiments of the invention, the drug is used for the prevention or treatment of tumors. The drug provided by the present invention has the characteristic of long-lasting binding to TGFβ1 and not binding to TGFβ3, thereby achieving the purpose of treating or preventing tumors.

[0018] In a tenth aspect of the invention, a method for preparing the fusion protein described in the fourth aspect is provided. According to an embodiment of the invention, the method includes the following steps: 1) constructing the expression vector described in the sixth aspect; 2) introducing the expression vector into host cells to obtain recombinant cells to express the fusion protein. The method according to the embodiment of the invention can efficiently obtain the fusion protein, and the fusion protein has the characteristic of binding to TGFβ1 but not to TGFβ3. It has low drug toxicity and low impact on normal bodily functions, and can control the upregulated TGFβ content around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0019] In an eleventh aspect, the present invention provides a method for preventing or treating tumors. According to embodiments of the invention, the method includes administering to a subject at least one of the following: 1) a TGFβRII mutant fragment as described in the first aspect; 1) a TGFβRII mutant as described in the second aspect; 3) a fusion protein as described in the fourth aspect; 3) isolated nucleic acid molecules as described in the third and fifth aspects; 4) an expression vector as described in the sixth aspect; 5) recombinant cells as described in the seventh aspect; and 6) a pharmaceutical composition as described in the eighth aspect. The method according to embodiments of the invention can effectively prevent or treat tumors by controlling the upregulated expression of TGFβ1 around tumor cells.

[0020] 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. Attached Figure Description

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0022] Figure 1 This is a graph showing the ELISA binding activity detection results of the TGFβRII trap fusion protein with human TGFβ1 and human TGFβ3 according to an embodiment of the present invention.

[0023] Figure 2 This is a graph showing the effect of the TGFβRII trap fusion protein on T cell proliferation according to an embodiment of the present invention. Detailed Implementation

[0024] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0025] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0026] The “TGFβRII extracellular domain” refers to a 136-amino acid residue peptide segment outside the cells of wild-type TGFβRII, starting from the N-terminus, with the amino acid sequence shown in SEQ ID NO: 1.

[0027] This invention provides a TGFβRII mutant fragment. Compared to the amino acid sequence of the extracellular domain of TGFβRII, the TGFβRII mutant fragment has mutations at the following sites: at least one of amino acids 49 and 53, or at least one of amino acids 49 and 53 and at least one of amino acids 27, 30, 50, 51, 52, 55, and 77. Given that existing TGFβRII production technology can bind to both TGFβ1 and TGFβ3, and that the binding of TGFβRII to TGFβ3 can affect the normal physiological functions of the body and produce drug toxicity, the inventors have creatively discovered that, according to the mutation method of the present invention, after mutating the amino acid sequence (SEQ ID NO: 1) of the extracellular domain of TGFβRII, the obtained TGFβRII mutant fragment can effectively bind to TGFβ1 and not bind to TGFβ3. The TGFβRII mutant containing the fragment also has the characteristic of binding to TGFβ1 and not binding to TGFβ3. The fusion protein prepared using the extracellular region of the TGFβRII mutant can effectively prevent and treat tumors, and its drug toxicity and impact on the normal functions of the body are significantly reduced.

[0028] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 1).

[0029] According to a specific embodiment of the present invention, the TGFβRII mutant fragment has at least one of the following mutations compared to the amino acid sequence of the TGFβRII extracellular domain: 1) S is replaced with N at position 49, or 2) I is replaced with K at position 53, 3) S is replaced with N at position 49 and L is replaced with S at position 27, or 4) S is replaced with N at position 49 and F is replaced with E at position 30, or 5) S is replaced with N at position 49 and I is replaced with W at position 50, or 6) S is replaced with N at position 49 and L is replaced with S at position 27, or 7) S is replaced with N at position 49 and L is replaced with S at position 27, or 7) S is replaced with N at position 49 and L is replaced with E at position 30 ... Replace S with N, and replace T in the 51st position with I, or 7) replace S in the 49th position with N, and replace T in the 51st position with W, or 8) replace S in the 49th position with N, and replace T in the 51st position with R, or 9) replace S in the 49th position with N, and replace S in the 52nd position with D, or 10) replace S in the 49th position with N, and replace I in the 53rd position with K, or 11) replace S in the 49th position with N, and replace E in the 55th position with W, or 12) replace S in the 49th position with N, and replace E in the 55th position with R, or 1 3) Replace S in the 49th position with N, and V in the 77th position with W; or 14) Replace I in the 53rd position with K, and L in the 27th position with S; or 15) Replace I in the 53rd position with K, and F in the 30th position with E; or 16) Replace I in the 53rd position with K, and I in the 50th position with W; or 17) Replace I in the 53rd position with K, and T in the 51st position with I; or 18) Replace I in the 53rd position with K, and T in the 51st position with W; or 19) Replace I in the 53rd position with K, and L in the 51st position with W. Replace T in bit 50 with R, or 20) replace I in bit 53 with K, and S in bit 52 with D, or 21) replace I in bit 53 with K, and E in bit 55 with W, or 22) replace I in bit 53 with K, and E in bit 55 with R, or 23) replace I in bit 53 with K, and V in bit 77 with W, or 24) replace I in bit 50 with W, or 25) replace T in bit 51 with I, or 26) replace E in bit 55 with W, or 27) replace V in bit 77 with W. The TGFβRII mutant fragment obtained by the mutation method according to a specific embodiment of the present invention has the characteristic of binding to TGFβ1 but not to TGFβ3. The TGFβRII mutant containing the fragment also has the characteristic of binding to TGFβ1 but not to TGFβ3. The fusion protein prepared using the extracellular region of the TGFβRII mutant can effectively prevent and treat tumors, and its drug toxicity and impact on the normal function of the body are significantly reduced.

[0030] According to a specific embodiment of the present invention, the TGFβRII mutant fragment has any of the amino acid sequences shown in SEQ ID NO: 2 to 28.

[0031]

[0032]

[0033]

[0034] According to a preferred embodiment of the present invention, the TGFβRII mutant fragment has at least one of the following mutations compared to the amino acid sequence of the TGFβRII extracellular domain: 1) S is replaced with N at position 49, or 2) I is replaced with K at position 53, 3) S is replaced with N at position 49 and L is replaced with S at position 27, or 4) S is replaced with N at position 49 and F is replaced with E at position 30, or 5) S is replaced with N at position 49 and L is replaced with E at position 50. Replace I with W, or 6) replace S in the 49th position with N and T in the 51st position with I, or 7) replace S in the 49th position with N and T in the 51st position with W, or 8) replace S in the 49th position with N and T in the 51st position with R, or 9) replace S in the 49th position with N and S in the 52nd position with D, or 10) replace S in the 49th position with N and I in the 53rd position with K, or 11) replace S in the 49th position with N and E in the 55th position with W, or 12) replace S in the 49th position with N, and E in the 55th position with R, or 13) replace S in the 49th position with N, and V in the 77th position with W, or 14) replace I in the 53rd position with K, and L in the 27th position with S, or 15) replace I in the 53rd position with K, and F in the 30th position with E, or 16) replace I in the 53rd position with K, and I in the 50th position with W, or 17) replace I in the 53rd position with K, and T in the 51st position with I. , or 18) Replace the 53rd bit I with K, and the 51st bit T with W, or 19) Replace the 53rd bit I with K, and the 51st bit T with R, or 20) Replace the 53rd bit I with K, and the 52nd bit S with D, or 21) Replace the 53rd bit I with K, and the 55th bit E with W, or 22) Replace the 53rd bit I with K, and the 55th bit E with R, or 23) Replace the 53rd bit I with K, and the 77th bit V with W.

[0035] According to a preferred embodiment of the present invention, the TGFβRII mutant fragment has any of the amino acid sequences shown in SEQ ID NO: 2 to 24.

[0036] According to a preferred embodiment of the present invention, the TGFβRII mutant fragment has at least one of the following mutations compared to the amino acid sequence of the TGFβRII extracellular domain: 1) S at position 49 is replaced with N, or 2) I at position 53 is replaced with K.

[0037] According to a preferred embodiment of the present invention, the TGFβRII mutant fragment has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0038] This invention provides a nucleic acid molecule encoding the aforementioned TGFβRII mutant fragment or TGFβRII mutant. According to a specific embodiment of the invention, the TGFβRII mutant fragment encoded by the nucleic acid molecule has the characteristic of binding to TGFβ1 but not to TGFβ3. Similarly, the TGFβRII mutant encoded by the nucleic acid molecule according to a specific embodiment of the invention also possesses the characteristic of binding to TGFβ1 but not to TGFβ3. The fusion protein prepared using the extracellular region of the TGFβRII mutant exhibits significantly reduced drug toxicity and impact on normal bodily functions, effectively preventing and treating tumors.

[0039] This invention provides a fusion protein comprising: 1) the aforementioned TGFβRII mutant fragment; and 2) an immunoglobulin Fc fragment, wherein the TGFβRII mutant fragment is linked to the immunoglobulin Fc fragment via a linker peptide. After the inventors mutated the wild-type TGFβRII shown in SEQ ID NO: 1 as described above, the resulting TGFβRII mutant exhibited a significantly enhanced characteristic of binding to TGFβ1 but not to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein were significantly reduced. Adding the immunoglobulin Fc fragment to the TGFβRII mutant fragment further enhanced the fusion protein's characteristic of binding to TGFβ1 but not to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein were significantly reduced, while its drug-likeness and in vivo half-life were improved. This fusion protein can control the upregulated expression of TGFβ around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0040] 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 TGFβRII mutant fragment.

[0041] According to a specific embodiment of the present invention, the immunoglobulin Fc fragment is derived from human IgG antibody molecules.

[0042] According to a specific embodiment of the present invention, the immunoglobulin Fc fragment comprises an Fc heavy chain fragment of an hIgG1 antibody.

[0043] According to a specific embodiment of the present invention, the lysine residue at the C-terminus of the immunoglobulin Fc fragment is mutated to alanine, and the immunoglobulin Fc fragment has the amino acid sequence shown in SEQ ID NO: 25. Mutating the lysine residue at the C-terminus of the immunoglobulin Fc fragment to alanine can reduce the cleavage and hydrolysis of the fusion protein, thereby reducing the fragment content during the production of the fusion protein.

[0044] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO: 29).

[0045] According to a specific embodiment of the present invention, the linker peptide is a flexible linker peptide. The linker peptide is not particularly limited; any conventional flexible fragment in the art can be used.

[0046] According to a specific embodiment of the present invention, the linker peptide comprises (G4S). X The G amino acid sequence, where X is an integer greater than 0. According to a specific embodiment of the present invention, the linker peptide comprises a (G4S)4G amino acid sequence (SEQ ID NO: 30).

[0047] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 30).

[0048] According to a specific embodiment of the present invention, the fusion protein has an amino acid sequence as shown in any of SEQ ID NO: 31-36. In the amino acid sequences shown in SEQ ID NO: 31-36, the amino acid sequence marked with a dotted line below represents alanine residues mutated at the C-terminus of the immunoglobulin Fc fragment, such as A in SEQ ID NO: 31; the amino acid sequence marked with a single solid line below represents the amino acid sequence of the linker peptide, such as A in SEQ ID NO: 31. GGGGSGGGGSGGGGSGGGGSG The amino acids marked with double solid lines below are the mutated amino acids of the TGFβRII mutant, such as those in SEQ ID NO: 31.

[0049]

[0050]

[0051] According to a preferred embodiment of the present invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 31 or SEQ ID NO: 32.

[0052] This invention provides a nucleic acid molecule that encodes the aforementioned fusion protein. According to a specific embodiment of the invention, the fusion protein encoded by the nucleic acid molecule has the characteristic of binding to TGFβ1 but not to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein are significantly reduced, while its drug-likeness and in vivo half-life are improved. The fusion protein can control the upregulated expression of TGFβ around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0053] This invention provides an expression vector containing a nucleic acid molecule encoding a TGFβRII mutant fragment, a TGFβRII mutant, or a fusion protein, as described above. When ligating the nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the operative linking of the nucleic acid molecule to the control elements is sufficient. In this context, "operative linking" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0054] According to a specific embodiment of the present invention, the expression vector is a eukaryotic expression vector.

[0055] This invention provides a recombinant cell carrying a nucleic acid molecule encoding a TGFβRII mutant fragment or a TGFβRII mutant, or a nucleic acid molecule encoding a fusion protein, or an expression vector. According to a specific embodiment of the invention, the recombinant cell can express the aforementioned fusion protein, which has the characteristic of binding to TGFβ1 but not to TGFβ3. The drug toxicity and impact on normal bodily functions of the fusion protein are significantly reduced, while its drug-likeness and in vivo half-life are improved. The fusion protein can control the upregulated TGFβ expression level around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0056] According to a specific embodiment of the present invention, the recombinant cell is a mammalian cell, and the mammalian is such as a human, monkey, rabbit, dog, or cow; the mammalian cell is such as a human HEK-293F cell or a CHO-K1 cell.

[0057] According to a specific embodiment of the present invention, the recombinant cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0058] This invention provides a pharmaceutical composition comprising the aforementioned TGFβRII mutant fragment, or a TGFβRII mutant, or a nucleic acid molecule encoding a TGFβRII mutant fragment or a TGFβRII mutant, or a fusion protein, or a nucleic acid molecule encoding a fusion protein, or an expression vector, or recombinant cells. The pharmaceutical composition may include: pharmaceutically acceptable excipients, including at least one of stabilizers, wetting agents, emulsifiers, binders, and isotonic agents; the pharmaceutical composition is in at least one of tablets, granules, powders, capsules, solutions, suspensions, and lyophilized formulations. The pharmaceutical composition according to this invention has low drug toxicity and low impact on normal bodily functions, and can control the upregulated expression of TGFβ around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0059] This invention provides the use of the aforementioned TGFβRII mutant fragment, or TGFβRII mutant, or nucleic acid molecules encoding TGFβRII mutant fragments or TGFβRII mutants, fusion proteins, nucleic acid molecules encoding fusion proteins, expression vectors, and recombinant cells in the preparation of drugs. According to specific embodiments of the invention, the drug is used for the prevention or treatment of tumors. The drug provided by this invention has the characteristic of long-lasting binding to TGFβ1 and not binding to TGFβ3, thereby achieving the purpose of treating or preventing tumors.

[0060] This 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 for expressing the fusion protein. The method according to specific embodiments of this invention can efficiently obtain the fusion protein, which has the characteristic of binding to TGFβ1 but not to TGFβ3. It exhibits low drug toxicity and minimal impact on normal bodily functions, and can control the upregulated TGFβ expression around tumor cells, thereby providing long-term and effective prevention or treatment of tumors.

[0061] According to a specific embodiment of the present invention, the recombinant cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0062] This invention provides a method for preventing or treating tumors, comprising administering to a subject at least one of the following: 1) the aforementioned TGFβRII mutant fragment or TGFβRII mutant; 2) the aforementioned fusion protein; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; 5) the aforementioned recombinant cells; and 6) the aforementioned pharmaceutical composition. The method according to specific embodiments of the invention can effectively prevent or treat tumors by controlling the upregulated expression of TGFβ around tumor cells.

[0063] These tumors can be caused by any unregulated cell growth. Specifically, they can be non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, colorectal cancer, melanoma, cholangiocarcinoma or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, or gastric cancer, etc.

[0064] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0065] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0066] Example 1 Expression of TGFβRII trap fusion protein

[0067] Using homologous recombination technology, the inventors used (G4S)4G as the linker peptide (SEQ ID NO: 30) to link the C-terminal amino acid of the Fc heavy chain fragment of hIgG1 antibody (SEQ ID NO: 29) with a TGFβRII mutant fragment with different mutation sites (Table 1) to form a TGFβRII trap fusion protein.

[0068] At the fusion site, the C-terminal lysine residue (K) of the Fc heavy chain fragment of the hIgG1 antibody was mutated to alanine (A) to reduce the cleavage and hydrolysis of the fusion protein. For the TGFβRII trap fusion protein, mammalian cells were transfected using standard protocols of transient or stable transfection with DNA encoding the Fc-TGFβRII receptor located in the same or separate expression vectors. Transient transfection of human HEK-293F cells prepared the TGFβRII trap fusion protein, and stable transfection of CHO-K1 cells prepared the TGFβRII trap fusion protein.

[0069] The sequence descriptions of the TGFβRII trap fusion protein and the TGFβRII mutant fragments and their mutation sites are shown in Table 1.

[0070] Table 1

[0071]

[0072]

[0073] In Table 1, for example, L27S represents the L mutation to S at position 27 of the amino acid sequence of the extracellular domain of TGFβRII.

[0074] Example 2 Purification of TGFβRII trap fusion protein

[0075] After high-speed centrifugation of cell culture medium, the supernatant was collected and purified using affinity chromatography as the first step. The chromatography medium was Protein A or a derivative of Fc, such as GE's Mabselect. Equilibration buffer was 1×PBS. After equilibrating to 5 column volumes, the cell supernatant was loaded, with the flow rate controlled to ensure the sample retention time on the column was ≥1 min. After loading, the column was washed with 1×PBS (pH 7.4) until the A280 UV absorbance dropped to baseline. The column was then washed with 0.1 M glycine (pH 3.0) elution buffer, and the elution peak was collected based on the A280 UV absorption peak. The collected eluted sample was neutralized with 1 M Tris (pH 8.5).

[0076] The neutralized eluted sample was concentrated by ultrafiltration and then subjected to size exclusion chromatography. The buffer was 1×PBS, the chromatography column was an XK26 / 60 Superdex 200 (GE), the flow rate was controlled at 4 mL / min, and the sample loading volume was less than 5 mL. The target protein peak was combined based on A280 UV absorption. The purity of the collected TGFβRII trap fusion protein was identified by SEC-HPLC.

[0077] Example 3: ELISA detection of TGFβRII trap fusion protein

[0078] The proteins used for the Trap-terminal binding assay of the TGFβRII fusion protein were human TGFβ1 (CA59, purchased from Novoprotein) and human TGFβ3 (CJ44, purchased from Novoprotein). The assay procedure is as follows:

[0079] a. Dilute TGFβ to 0.5 μg / mL with 1× phosphate-buffered saline (PBS), coat 96-well microplates with 100 μL / well, and incubate overnight at 4°C;

[0080] b. Wash 3 times with 250 μL 1×PBST (PBS + 0.5% Tween 20), then add 200 μL of PBS containing 2% bovine serum albumin (BSA) and block at room temperature for 1 hour;

[0081] c. Wash 3 times with 250 μL 1×PBST, add serially diluted TGFβRII trap fusion protein, and incubate at room temperature for 2 hours;

[0082] d. Wash 3 times with 250 μL 1×PBST, add 100 μL of diluted Goat-anti-human Fc-HRP conjugate antibody (Sigma, 1:15k) to each well, and incubate at room temperature for 1 hour;

[0083] e. Wash 3 times with 250 μL 1×PBST, add 100 μL TMB colorimetric solution to each well, incubate at room temperature in the dark for 10 min, and add 50 μL 2NH2SO4 to terminate the reaction;

[0084] f. Use an iX3 microplate reader (Molecular Devices) to read the absorbance at 450 nM, analyze and plot the results.

[0085] The ELISA results of the binding activity assay of the TGFβRII trap fusion protein with human TGFβ1 and human TGFβ3 are as follows: Figure 1 As shown, compared to the wild-type WT group, the TGFβRII trap fusion proteins with mutations at L27S, F30E, T51W, T51R, and S52D significantly weaken the binding of both TGFβ1 and TGFβRII, indicating that changes at these five sites may have a significant impact on the structure of TGFβRII. Furthermore, we observed that the TGFβRII trap fusion proteins with mutations at S49N, I50W, T51I, I53K, E55W, and V77W significantly weaken the binding of TGFβ3 and TGFβRII. More notably, the EC50 of the TGFβ1-TGFβRII binding activity of S49N and I53K is close to that of the wild-type WT group, with I53K even achieving the goal of retaining the binding of TGFβ1 and TGFβRII while completely preventing TGFβ3 from binding to TGFβRII.

[0086] Example 4: Detection of the blocking activity of TGFβRII trap fusion protein

[0087] Stable CHO cells expressing human TGFβRII (CHO-hTGFβRII) were constructed, and single clones were selected for cell line establishment. The blocking activity against the TGFβRII trap was detected using the following method.

[0088] a. Count CHO-hTGFβRII cells, dividing the cells into groups of 2 × 10⁻⁶. 5 The density of each hole is laid on a 96-hole U-shaped base plate;

[0089] b. Centrifuge cells at 300g, 4℃, for 5 minutes, and discard the supernatant.

[0090] c. Dilute the TGFβRII trap fusion protein with 1×PBS containing 1% BSA, starting at a concentration of 50 nM, with 4-fold dilutions in 8 gradients. Simultaneously dilute TGFβ1-biotin (purchased from Acrobiosystem) to a concentration of 1 μg / mL.

[0091] d. Mix the fusion protein dilution and TGFβ1-botin dilution at a volume ratio of 1:1 and let stand at room temperature for 30 minutes.

[0092] e. Add the mixed sample to CHO-TGFβRII cells, 100 μL / well, and incubate at 4°C for 30 min. Centrifuge at room temperature, 300 g, for 5 min, and discard the supernatant.

[0093] f. Add SA-PE (400-fold dilution, Jackson immunoresearch, 016-110-084), 100 μL / well, mix gently, and incubate at 4°C for 1 hour.

[0094] g. Centrifuge at room temperature, 300g, for 5 min, and discard the supernatant. Add 100 μL of 1×PBS containing 1% BSA to each well to resuspend the cells, and then perform flow cytometry analysis.

[0095] The effect of the TGFβRII trap fusion protein on T cell proliferation is shown in the figure below. Figure 2 As shown, compared with the wild-type WT group, the TGFβRII trap fusion proteins with L27S, S49N, I50W, I53K, E55W, and V77W mutations exhibited blocking activity against TGFβ1. Among them, S49N and I53K showed better blocking activity. The EC50 value of S49N was 0.2544 nM, which was better than that of the wild-type WT group, while the EC50 value of I53K was 0.4037 nM, which was close to that of the wild-type WT group.

[0096] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention. SEQUENCE LISTING <110> Guangdong Feipeng Pharmaceutical Co., Ltd. <120> TGFβRII activity-modified mutants and their applications <130> SI4210199 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 1 <400> 1 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 2 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 2 <400> 2 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 3 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 3 <400> 3 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 4 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 4 <400> 4 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Ser Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 5 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 5 <400> 5 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Glu Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 6 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 6 <400> 6 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Trp Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 7 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 7 <400> 7 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Ile Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 8 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 8 <400> 8 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Trp Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 9 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 9 <400> 9 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Arg Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 10 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 10 <400> 10 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Asp Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 11 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 11 <400> 11 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 12 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 12 <400> 12 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Ser Ile Cys Arg Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 13 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 13 <400> 13 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Ser Ile Cys Trp Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 14 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 14 <400> 14 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Asn Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Trp Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 15 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 15 <400> 15 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Ser Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 16 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 16 <400> 16 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Glu Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 17 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 17 <400> 17 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Trp Thr Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 18 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 18 <400> 18 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Ile Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 19 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 19 <400> 19 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Trp Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 20 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 20 <400> 20 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Arg Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 21 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 21 <400> 21 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Asp Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 22 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 22 <400> 22 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Lys Cys Arg Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 23 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 23 <400> 23 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Lys Cys Trp Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 24 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 24 <400> 24 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Lys Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Trp Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 25 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 25 <400> 25 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Trp Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 26 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 26 <400> 26 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Ile Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 27 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 27 <400> 27 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Trp Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 28 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> 28 <400> 28 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Trp Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 29 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> 29 <400> 29 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala 225 <210> 30 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> 30 <400> 30 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly 20 <210> 31 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> 31 <400> 31 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 245 250 255 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 260 265 270 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 275 280 285 Gln Lys Ser Cys Met Ser Asn Cys Asn Ile Thr Ser Ile Cys Glu Lys 290 295 300 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 305 310 315 320 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 325 330 335 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 340 345 350 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 355 360 365 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 370 375 380 <210> 32 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> 32 <400> 32 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 245 250 255 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 260 265 270 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 275 280 285 Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Lys Cys Glu Lys 290 295 300 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 305 310 315 320 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 325 330 335 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 340 345 350 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 355 360 365 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 370 375 380 <210> 33 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> 33 <400> 33 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 245 250 255 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 260 265 270 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 275 280 285 Gln Lys Ser Cys Met Ser Asn Cys Ser Trp Thr Ser Ile Cys Glu Lys 290 295 300 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 305 310 315 320 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 325 330 335 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 340 345 350 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 355 360 365 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 370 375 380 <210> 34 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> 34 <400> 34 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 245 250 255 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 260 265 270 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 275 280 285 Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Ile Ser Ile Cys Glu Lys 290 295 300 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 305 310 315 320 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 325 330 335 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 340 345 350 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 355 360 365 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 370 375 380 <210> 35 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> 35 <400> 35 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 245 250 255 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 260 265 270 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 275 280 285 Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Trp Lys 290 295 300 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 305 310 315 320 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 325 330 335 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 340 345 350 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 355 360 365 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 370 375 380 <210> 36 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> 36 <400> 36 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 245 250 255 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 260 265 270 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 275 280 285 Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys 290 295 300 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 305 310 315 320 Thr Leu Glu Thr Trp Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 325 330 335 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 340 345 350 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 355 360 365 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 370 375 380

Claims

1. A TGFβRII mutant fragment, characterized in that, Compared to the amino acid sequence of the extracellular domain of TGFβRII shown in SEQ ID NO: 1, the mutations in the TGFβRII mutant fragment are as follows: 1) Replace the 49th 'S' with 'N', or 2) Replace the 'I' in the 53rd position with 'K', or 25) Replace the T in the 51st position with I.

2. The TGFβRII mutant fragment according to claim 1, characterized in that, The amino acid sequence of the TGFβRII mutant fragment is shown in SEQ ID NO: 2, 3 or 26.

3. A fusion protein, characterized in that, It consists of the following: 1) The TGFβRII mutant fragment according to any one of claims 1 to 2; and 2) Immunoglobulin Fc fragment; 3) Linking peptides; Wherein, the N-terminus of the linking peptide is linked to the C-terminus of the immunoglobulin Fc fragment, and the C-terminus of the linking peptide is linked to the N-terminus of the TGFβRII mutant fragment; The immunoglobulin Fc fragment is derived from human IgG antibody molecules; The linker peptide is a flexible linker peptide.

4. The fusion protein according to claim 3, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO: 31, 32 or 34.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the TGFβRII mutant fragment as described in any one of claims 1 to 2, or the fusion protein as described in any one of claims 3 to 4.

6. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 5.

7. A recombinant cell, characterized in that, Carrying the expression vector as described in claim 6.

8. A pharmaceutical composition, characterized in that, It comprises the TGFβRII mutant fragment as described in any one of claims 1 to 2 or the fusion protein as described in any one of claims 3 to 4.

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