DR5 domain variants and their applications

Abstract

The present application relates to a death receptor 5 (DR5) domain variant. The present application further relates to a fusion protein containing the DR5 domain variant, which has one or more of the following properties: (1) the ability to bind to TRAIL, (2) blocking apoptosis induced by the TRAIL / DR5 pathway, (3) reducing the size of myocardial infarction in cardiac ischemia and / or ischemia-reperfusion, and (4) having a protective effect against cerebral infarction. The present application further provides a method for producing the fusion protein and uses thereof.

Description

[Technical Field]

[0001] The present application relates to the biomedicine field, and specifically to a DR5 domain variant, a fusion protein comprising the DR5 domain variant, and applications thereof. [Background technology]

[0002] Acute myocardial infarction (AMI) is a serious cardiovascular disease. In China, the incidence rate of myocardial infarction is approximately 50 / 100,000, with more than 600,000 new cases per year and more than 1 million deaths due to myocardial infarction and its complications. As the population ages, the incidence rate of AMI is increasing year by year, and there is also a trend toward younger patients.

[0003] After myocardial infarction, cardiomyocytes die at a rate of 20% per hour. Therefore, time saves myocardium, and time saves lives. With advances in medical technology, patients with acute myocardial infarction can undergo thrombolytic therapy or percutaneous coronary intervention (PCI, i.e., stent placement) early on to restore blood supply and reduce the size of the infarct. However, the "golden time" for PCI is only 120 minutes (the mortality rate for PCI completed within 120 minutes is approximately 5%, while the mortality rate for PCI completed within 240 minutes reaches 10%), and less than 5% of patients both in Japan and abroad are able to complete PCI within this "golden time." Furthermore, upon resumption of blood flow, cardiomyocytes suffer secondary damage, known as "reperfusion injury," which leads to increased cell death and is life-threatening. Reperfusion injury also leads to late ventricular remodeling and heart failure, severely impairing patients' quality of life. Currently, there are no specific therapeutic agents for ischemia and ischemia-reperfusion injury of cardiomyocytes in clinical settings both in Japan and overseas.

[0004] Cell death includes necrosis and apoptosis, and necrosis has been considered the primary cause of myocardial infarction. In recent years, the role of apoptosis in myocardial infarction has been attracting increasing attention. The death receptor pathway, activated by binding of death receptor 5 (DR5) to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), recruits related proteins to form the death-inducing signaling complex (DISC), which then activates caspase-8, caspase-3, and other proteins in a cascade, leading to apoptosis. Soluble death receptor 5 (sDR5) is expressed at low levels in the peripheral blood of healthy individuals. Because sDR5 possesses an intact extracellular segment that binds to the TRAIL ligand, it can block TRAIL-induced apoptosis by competing with the death receptor on the cell membrane to bind to TRAIL molecules.

[0005] Current DR5 fusion proteins still have problems in the treatment of myocardial infarction, such as high doses required and limited administration routes, so there is an urgent need to develop new DR5 fusion proteins that will solve these problems. Summary of the Invention

[0006] The present application provides death receptor 5 (DR5) domain variants and fusion proteins containing the DR5 domain variants, which have one or more of the following properties: (1) binding to TRAIL with high affinity, (2) blocking apoptosis induced by the TRAIL / DR5 pathway, (3) reducing myocardial infarction size in cardiac ischemia and / or ischemia-reperfusion, and (4) having a protective effect against cerebral infarction. The fusion proteins described in the present application can exert specific protective effects against myocardial infarction and / or cerebral infarction at low doses. The fusion proteins described in the present application can also effectively extend the in vivo half-life of drugs, solving the problem of the need for frequent administration. The fusion proteins described in the present application can exert protective effects at low doses and can be administered intracranially or by intramuscular injection.

[0007] In one aspect, the present application provides a Death Receptor 5 (DR5) domain variant.

[0008] In some embodiments, the DR5 domain comprises the extracellular domain of DR5.

[0009] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more of amino acid positions 86-155 compared to the extracellular domain of human DR5 variant 1 (SEQ ID NO: 94);

[0010] In some embodiments, the death receptor 5 (DR5) comprises soluble death receptor 5 (sDR5).

[0011] In some embodiments, the DR5 is human DR5.

[0012] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more of amino acid positions 86-92.

[0013] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more of amino acid positions 105-112.

[0014] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more of amino acid positions 114-155.

[0015] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more of amino acid positions 114-115.

[0016] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more of amino acid positions 147-152.

[0017] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more of amino acid positions 154-155.

[0018] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations at one or more amino acid positions selected from the group consisting of I87, E89, D90, T105, N108, D109, L114, R115, E147, D148, E151, R154 and K155 compared to the amino acid sequence set forth in SEQ ID NO: 94.

[0019] In some embodiments, the DR5 domain variant has a sequence similar to that of SEQ ID NO: 94: (1) L114 and R115, (2) R154 and K155, (3) I87, D90, T105, D109 and D148, (4)I87, D90, T105, D109, L114, E147, (5)I87, E89, D90, D109, E147, (6)I87, E89, D90, T105, D109, L114, D148, (7)D90, T105, D109, L114, D148, R154, (8) D90, D109 and D148, (9) I87, D90 and R154, (10) T105, N108 and F112, (11) L114, E147 and E151, and (12)R115 The mutation is at an amino acid site selected from any one of the following groups:

[0020] In some embodiments, in the amino acid mutation, the mutated amino acid is a polar amino acid.

[0021] In some embodiments, in the amino acid mutation, the mutated amino acid is a neutral amino acid.

[0022] In some embodiments, in the amino acid mutation, the mutated amino acid is an acidic amino acid.

[0023] In some embodiments, in the amino acid mutation, the mutated amino acid is a basic amino acid.

[0024] In some embodiments, in the amino acid mutation, the mutated amino acid is a non-polar amino acid.

[0025] In some embodiments, in the amino acid mutation, the mutated amino acid is an amino acid with a relatively small side chain volume.

[0026] In some embodiments, the DR5 domain variant comprises one or more amino acid mutations selected from the group consisting of I87A / V / L, E89N / Q, D90N / S, T105N / S, N108Q, D109H / S / Q / E, F112Y, L114A / G / V / S / W / Y, R115A / G / V / L / S / W / Y, E147S / Y, D148N / K / E, E151S, R154A / G / V / L / S / W / Y and K155A / G / V / L / S / W / Y compared to the amino acid sequence set forth in SEQ ID NO: 94.

[0027] In some embodiments, the DR5 domain variant has a sequence similar to that of SEQ ID NO: 94: (1) L114A and R115A, (2) R154A and K155A, (3) I87L, D90S, T105N, D109H and D148N, (4) I87A, D90S, T105N, D109S, L114V and E147S, (5) I87A, E89N, D90N, D109Q and E147Y, (6) I87A, E89Q, D90S, T105S, D109S, L114V and D148K, (7) D90S, T105N, D109E, L114I, D148E and R154Y, (8) D90S, D109N and D148E, (9) I87V, D90N and R154Y, (10) T105S, N108Q and F112Y, (11) L114V, E147Q and E151S, (12) L114G and R115G, (13) L114V and R115V, (14)R115L, (15) L114S and R115S, (16) L114W and R115W, (17) L114Y and R115Y, (18) R154G and K155G, (19) R154V and K155V, (20) R154L and K155L, (21) R154S and K155S, (22) R154W and K155W, and (23) R154Y and K155Y The amino acid mutations include those selected from any one of the following groups:

[0028] In some embodiments, the DR5 domain variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-23.

[0029] In another aspect, the present application provides a fusion protein comprising said DR5 domain variant.

[0030] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region.

[0031] In some embodiments, the immunoglobulin Fc region comprises an IgG Fc region.

[0032] In some embodiments, the IgG is selected from the group consisting of IgG1 and IgG4.

[0033] In some embodiments, the Fc region of the immunoglobulin comprises the amino acid sequence set forth in any one of SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99.

[0034] In some embodiments, in the fusion protein, the DR5 domain variant is located at the N-terminus of the immunoglobulin Fc region.

[0035] In some embodiments, in the fusion protein, the DR5 domain variant is located at the C-terminus of the immunoglobulin Fc region.

[0036] In some embodiments, the fusion protein further comprises a protein capable of extending half-life.

[0037] In some embodiments, the protein capable of extending half-life comprises human serum albumin.

[0038] In some embodiments, in the fusion protein, the protein capable of extending half-life is located N-terminal to the DR5 domain variant.

[0039] In some embodiments, in the fusion protein, the protein capable of extending half-life is located C-terminal to the DR5 domain variant.

[0040] In some embodiments, the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 24-92.

[0041] In another aspect, the present application further provides one or more isolated nucleic acid molecules encoding said DR5 domain variant or said fusion protein.

[0042] In another aspect, the present application further provides a vector comprising the nucleic acid molecule.

[0043] In another aspect, the present application further provides a cell comprising said nucleic acid molecule or said vector.

[0044] In another aspect, the present application further provides a method for producing said DR5 domain variant or said fusion protein, said method comprising culturing said cell under conditions allowing expression of said DR5 domain variant or fusion protein.

[0045] In another aspect, the present application further provides a pharmaceutical composition comprising said DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector, and / or said cell, and optionally a pharmaceutically acceptable carrier.

[0046] In some embodiments, the pharmaceutical composition further comprises another active ingredient.

[0047] In another aspect, the present application further provides a method for preventing and / or treating a disease and / or condition, comprising administering to a subject in need thereof said DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector, said cell, and / or said pharmaceutical composition.

[0048] In some embodiments, the disease and / or condition comprises myocardial infarction and / or myocardial infarction-reperfusion injury.

[0049] In some embodiments, the disease and / or condition comprises cerebral infarction.

[0050] In another aspect, the present application further provides the use of said DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition.

[0051] In some embodiments, the disease and / or condition comprises myocardial infarction and / or myocardial infarction-reperfusion injury.

[0052] In some embodiments, the disease and / or condition comprises cerebral infarction.

[0053] In another aspect, the present application further provides said DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector, said cell, and / or said pharmaceutical composition for preventing and / or treating a disease and / or condition.

[0054] In some embodiments, the disease and / or condition comprises myocardial infarction and / or myocardial infarction-reperfusion injury.

[0055] In some embodiments, the disease and / or condition comprises cerebral infarction.

[0056] In another aspect, the present application further provides a method for blocking the TRAIL / DR5 pathway, the method comprising administering the fusion protein or the pharmaceutical composition.

[0057] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be apparent to those skilled in the art, the contents of this application will enable those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this disclosure pertains. Correspondingly, the drawings and description in this application are illustrative only and not limiting. [Brief explanation of the drawings]

[0058] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below, which are generally described as follows:

[0059] [Figure 1] FIG. 1 is a schematic diagram of the physical structure of vector pTM. [Figure 2] FIG. 1 is a schematic diagram of a method for detecting the interaction between DR5 truncated domains and their variants with TRAIL. [Figure 3] FIG. 1 shows the results of a flow cytometry enrichment screen of DR5 truncated domain variants. [Figure 4] FIG. 1 shows the sequence alignment of DR5 truncated domains and their variants. [Figure 5]FIG. 1 shows the situation of the recognition of TRAIL protein by the fusion protein of the present application. [Figure 6] FIG. 1 shows the activity of the fusion protein of the present application in blocking TRAIL-induced apoptosis. [Figure 7] FIG. 1 shows the activity of the fusion protein of the present application in reducing cardiac ischemia / reperfusion injury in rats. [Figure 8] FIG. 1 shows the activity of the fusion protein of the present application in reducing cardiac ischemia / reperfusion injury in rhesus monkeys. [Figure 9] FIG. 1 shows the activity of the fusion protein of the present application in blocking TRAIL-induced apoptosis. [Figure 10] FIG. 1 shows the activity of the fusion protein of the present application in reducing cardiac ischemia / reperfusion injury in rats. [Figure 11] FIG. 1 shows the activity of the fusion protein of the present application in alleviating cerebral ischemia / reperfusion injury in rats. [Figure 12] FIG. 1 shows the protective activity of the fusion protein of the present application against myocardial infarction / reperfusion injury by intramuscular injection. DETAILED DESCRIPTION OF THE INVENTION

[0060] Hereinafter, embodiments of the present application will be described through specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Definition of Terms

[0061] In this application, the term "fusion protein" generally refers to a protein consisting of two or more polypeptides, which are not normally linked in the natural state but are joined via a peptide bond at their respective amino and carboxyl termini to form a continuous polypeptide. It should be understood that the two or more polypeptide components may be directly linked or indirectly linked via a peptide linker / spacer. Fusion proteins can generally be artificially produced by recombinant nucleic acid methods or chemical synthesis.

[0062] In the present application, the term "DR5" is also referred to as death receptor 5 (DR5), and the death receptor pathway activated by DR5 binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis by recruiting related proteins to form a death-inducing signaling complex (DISC), which then activates caspase-8, caspase-3, and the like in a cascade manner. In the present application, DR5 may be human DR5. In the present application, DR5 may be soluble DR5 (sDR5). The term further encompasses full-length DR5, its functionally active fragments, its variants, its homologs, or its analogs.

[0063] In the present application, the term "DR5 domain" generally refers to wild-type DR5, a fragment or a functional variant thereof. For example, the DR5 domain may comprise the ectodomain of DR5. For example, the human DR5 variant 1 ectodomain may comprise the amino acid sequence set forth in SEQ ID NO: 94. For example, the DR5 domain may comprise one or more amino acid mutations compared to the corresponding human DR5 variant 1 or a fragment thereof.

[0064] In the present application, the term "immunoglobulin Fc region" generally refers to the base region of the Y-shaped antibody structure, also known as the fragment crystallizable region (Fc region). In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, consisting of the second and third constant domains of the antibody's two heavy chains. The Fc regions of IgM and IgE contain three heavy chain constant domains in each polypeptide chain. For example, an immunoglobulin Fc region described in the present application may comprise an IgG, IgA, IgD, IgM, or IgE Fc region. In some embodiments, the immunoglobulin Fc region may comprise an IgG Fc region. For example, the immunoglobulin Fc region may comprise an IgG1 Fc region. For example, the immunoglobulin Fc region may comprise an IgG4 Fc region.

[0065] In the present application, the term "IgG" generally refers to immunoglobulin G. IgG is one of the human immunoglobulins, the others being IgA, IgM, IgD, and IgE. Depending on the antigenicity of the γ chain in the IgG molecule, human IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4. In the present application, the term "IgG1" generally refers to the subtype that accounts for the highest proportion of IgG and has high affinity for Fc receptors. For example, the IgG may be human IgG. Also, for example, the IgG may be IgG1.

[0066] In the present application, the DR5 domain may comprise a domain of human DRR5, a fragment or a variant thereof. For example, the DR5 domain may comprise the DR5 ectodomain, a fragment or a variant thereof.

[0067] In the present application, the term "extracellular domain" can be used interchangeably with "ectodomain" and generally refers to a functional structural region of a protein that is located outside the cell membrane. In some embodiments, the ectodomain refers to the ectodomain of the DR5 domain, a fragment thereof, or a variant thereof. For example, the human DR5 variant 1 ectodomain may comprise the amino acid sequence set forth in SEQ ID NO: 94.

[0068] The DR5 domain variants described herein may comprise an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-23.

[0069] The fusion protein described in the present application may comprise an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 24 to 92.

[0070] In this application, the term "sequence homology" generally refers to sequence similarity, compatibility, resemblance, or relatedness between two or more polynucleotide sequences or two or more polypeptide sequences. When using a computer program or software (e.g., Emboss Needle or BestFit) to determine sequence identity, similarity, or homology between different amino acid sequences, default parameter settings can be used. An appropriate scoring matrix, e.g., blosum45 or blosum80, can also be selected to optimize identity, similarity, or homology scores. In some embodiments, a homologous polynucleotide includes a polynucleotide that can hybridize to a reference polynucleotide sequence under stringent conditions and has at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even at least 100% sequence identity relative to the reference polynucleotide sequence. A homologous polypeptide may be a polypeptide that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even at least 100% sequence identity with a reference polypeptide sequence when sequence alignment is performed under appropriate conditions.

[0071] In the present application, "percent sequence identity" can be calculated as follows: two sequences to be aligned are compared within a comparison window, and the number of positions in the two sequences at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present is determined to obtain the number of matching positions, and the number of matching positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to calculate the percent sequence identity. Alignment performed to determine percent sequence identity can be achieved by various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment within the full-length sequences being compared or within a sequence region of interest. Homology can also be measured using the FASTA and BLAST methods. For a description of the FASTA algorithm, see W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988, and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Search for Protein Similarity," Science, 227:1435-1441, 1989. For a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "Basic Search Tools for Local Alignments," Journal of Molecular Biology, 215:403-410, 1990.

[0072] In the present application, the amino acid sequence (e.g., a protein domain or protein fragment, a DR5 domain variant described herein, or a fusion protein described herein) may comprise a substitution, deletion, or addition of one or more amino acid residues. For example, in the present application, the DR5 domain variant may comprise an amino acid mutation at one or more residues selected from the group consisting of I87, E89, D90, T105, N108, D109, L114, R115, E147, D148, E151, R154, and K155.

[0073] In the present application, the positions of the amino acid residues in the amino acid mutations are determined based on the amino acid sequence shown in SEQ ID NO: 96 (human DR5 variant 1 extracellular domain (including signal peptide)).

[0074] In this application, "amino acid site Xn" generally refers to residue X corresponding to position n in the amino acid sequence set forth in SEQ ID NO:96, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "residue I87" refers to amino acid residue I corresponding to position 87 in the amino acid sequence set forth in SEQ ID NO:96.

[0075] In the present application, the amino acid sequence shown in SEQ ID NO: 94 corresponds to amino acid positions 57 to 182 of the amino acid sequence shown in SEQ ID NO: 96. The amino acid sequence positions in the present application are determined by SEQ ID NO: 96, i.e., the numbers of the amino acid sequence shown in SEQ ID NO: 94 are I57 to E182, and the first amino acid of the amino acid sequence shown in SEQ ID NO: 94 is numbered I57.

[0076] In this application, a residue in one amino acid sequence "corresponds to" a residue in another amino acid sequence generally refers to the residue correspondence obtained when the amino acid sequences are aligned under optimized conditions. The sequence alignment can be performed by methods known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms necessary to achieve optimal alignment of the full-length sequences being compared.

[0077] The amino acid substitutions described herein may be non-conservative substitutions, which may involve non-conservatively altering an amino acid residue in a protein or polypeptide of interest, for example, changing an amino acid residue having a certain side chain size or certain properties (e.g., hydrophilicity) to an amino acid residue having a different side chain size or properties (e.g., hydrophobicity).

[0078] The amino acid substitution may also be a conservative substitution. The conservative substitution involves conservatively modifying an amino acid residue in a target protein or polypeptide, for example, changing an amino acid residue having a certain side chain size or certain properties (e.g., hydrophilicity) to an amino acid residue having the same or similar side chain size or the same or similar properties (e.g., still hydrophilic). Such conservative substitutions usually do not significantly affect the structure or function of the produced protein. In the present application, the amino acid sequence variant of the fusion protein or a fragment thereof may include a conservative amino acid substitution that does not significantly alter the protein structure or its function (e.g., the ability to block the TRAIL / DR5 pathway).

[0079] In the present application, the amino acid mutation "XnY / Z" refers to the substitution of amino acid residue Y or Z for residue X corresponding to position n in the amino acid sequence set forth in SEQ ID NO:96, where n is a positive integer, X, Y, and Z are each independently an abbreviation for any amino acid residue, and X is different from Y or Z. For example, the amino acid substitution "L114A / G / V / S / W / Y" refers to the substitution of amino acid residue A, G, V, S, W, or Y for residue L corresponding to position 114 in the amino acid sequence set forth in SEQ ID NO:96.

[0080] In this application, the term "amino acid mutation" generally encompasses amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be used to achieve the final construct, as long as the final construct possesses the desired attributes. Deletions and insertions in the amino acid sequence include deletions of the amino and / or carboxyl termini and insertions of amino acids. A specific amino acid mutation is an amino acid substitution. For example, one amino acid is replaced with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with unnatural amino acids or replacement with natural amino acid derivatives of the 20 common amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering, such as chemical modification to alter amino acid side groups, are also available.

[0081] In this application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides, or ribonucleotides, or analogs thereof, of any length, isolated from their natural environment or artificially synthesized. A nucleic acid molecule described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) in vitro amplification, e.g., amplification by polymerase chain reaction (PCR); (ii) cloning recombination; (iii) purification, e.g., enzymatic digestion and fractionation by gel electrophoresis; or (iv) synthesis, e.g., chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA technology. In the present application, nucleic acids encoding the antibodies or antigen-binding fragments thereof can be produced by various methods known in the art, including, but not limited to, restriction fragment manipulation or overlap-extension PCR using synthetic oligonucleotides. For specific procedures, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0082] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating within a suitable host and transporting an inserted nucleic acid molecule within and / or between host cells. Such vectors may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for replicating DNA or RNA, and expression vectors primarily used for transcribing and / or translating DNA or RNA. Such vectors may also include vectors having multiple of the above functions. Such vectors may be polynucleotides that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector. In the present application, such vectors may contain one or more of the above nucleic acid molecules. The vector may also contain other genes, such as marker genes, that allow for selection of the vector in a suitable host cell under suitable conditions. The vector may further contain expression control elements that allow for precise expression of the coding region in a suitable host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatable element. The specific structure of the expression control sequence can vary depending on the species or cell type, but typically includes a 5' non-transcribed sequence and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, capping sequence, or CAAT sequence. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for controlling the transcription of an operably linked nucleic acid. The vector may also include a replication origin. The vector may further include components that aid in its entry into cells, such as, but not limited to, a viral particle, liposome, or capsid.

[0083] In the present application, the terms "host cell," "cell," and "host" can be used interchangeably and generally refer to a single cell, cell line, or cell culture that can contain or contains a plasmid or vector comprising a nucleic acid molecule described herein, or that can express a fusion protein, fragment, or variant thereof described herein. The host cell may include progeny of a single host cell. Due to natural, unintended, or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but they may express the antibodies or antigen-binding fragments thereof described herein. The host cell may be obtained by transfecting cells in vitro with a vector described herein. The host cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cell, e.g., COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell).

[0084] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and, optionally, a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise an appropriate formulation of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Preferably, acceptable components of the composition are non-toxic to recipients at the dosages and concentrations used. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0085] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, preservatives, low molecular weight (fewer than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, e.g., sodium, and / or non-ionic surfactants.

[0086] In this application, the term "subject" generally refers to any human or non-human animal. The term "non-human animal" may include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, goats, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0087] In this application, the term "about" generally refers to a variation within 0.5% to 10% above or below the specified numerical value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0088] In this application, the term "comprise" generally refers to the meaning of comprising, including, containing or including. In some cases, it also means "is" or "consists of." Detailed Description of the Invention

[0089] In one aspect, the present application provides a death receptor 5 (DR5) domain variant, wherein the DR5 domain variant is capable of binding to TRAIL.

[0090] In the present application, the DR5 domain variant can block the TRAIL / DR5 signaling pathway, thereby blocking apoptosis induced by the TRAIL / DR5 pathway. In the present application, the DR5 domain variant can form a fusion protein with other fragments, and the fusion protein can reduce the size of myocardial infarction in cardiac ischemia. In the present application, the fusion protein can reduce the size of myocardial infarction in cardiac ischemia-reperfusion. In the present application, the fusion protein can play a protective role against cerebral infarction.

[0091] In the present application, the DR5 binding domain may comprise a functionally active fragment of DR5 or a variant thereof. For example, the DR5 domain may comprise the extracellular domain of DR5. For example, the DR5 extracellular domain may comprise a variant of the DR5 extracellular domain, which may be obtained by amino acid mutation based on the extracellular domain of human DR5 variant 1. For example, the extracellular domain of human DR5 variant 1 may comprise the amino acid sequence set forth in SEQ ID NO: 94. For example, the variant of the DR5 ectodomain may have at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence homology to the extracellular domain of human DR5 variant 1. In the present application, the DR5 may comprise human DR5. For example, the DR5 ectodomain variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more amino acid mutations compared to the wild-type DR5 ectodomain. In the present application, the DR5 domain may have one or more amino acid mutations based on the extracellular domain of human DR5 variant 1.

[0092] In the present application, the DR5 domain variant may include one or more amino acid mutations at one or more of amino acid positions 86-92.

[0093] In the present application, the DR5 domain variant may include one or more amino acid mutations at one or more of the amino acid positions 105-1122.

[0094] In the present application, the DR5 domain variant may include one or more amino acid mutations at one or more of amino acid positions 114-155.

[0095] In the present application, the DR5 domain variant may include one or more amino acid mutations at one or more of amino acid positions 114-115.

[0096] In the present application, the DR5 domain variant may include one or more amino acid mutations at one or more of amino acid positions 147-152.

[0097] In the present application, the DR5 domain variant may include one or more amino acid mutations at one or more of amino acid positions 154-155.

[0098] For example, the DR5 domain in the fusion protein may comprise one or more amino acid mutations at one or more amino acid positions selected from the group consisting of I87, E89, D90, T105, N108, D109, L114, R115, E147, D148, E151, R154 and K155.

[0099] For example, the DR5 domain variant may include mutations at the L114 and R115 amino acid positions. For example, the DR5 domain variant may include mutations at the R154 and K155 amino acid positions. For example, the DR5 domain variant may include mutations at the I87, D90, T105, D109, and D148 amino acid positions. For example, the DR5 domain variant may include mutations at the I87, D90, T105, D109, L114, and E147 amino acid positions. For example, the DR5 domain variant may include mutations at the I87, E89, D90, D109, and E147 amino acid positions. For example, the DR5 domain variant may include mutations at the I87, E89, D90, T105, D109, L114, and D148 amino acid positions. For example, the DR5 domain variant may include mutations at the D90, T105, D109, L114, D148, and R154 amino acid positions. For example, the DR5 domain variant may include mutations at the D90, D109, and D148 amino acid positions. For example, the DR5 domain variant may include mutations at the I87, D90, and R154 amino acid positions. For example, the DR5 domain variant may include mutations at the T105, N108, and F112 amino acid positions. For example, the DR5 domain variant may include mutations at the L114, E147, and E151 amino acid positions. For example, the DR5 domain variant may include a mutation at the R115 amino acid position.

[0100] In the present application, the mutated amino acid at the site of the amino acid mutation may be a nonpolar amino acid. In the present application, the mutated amino acid at the site of the amino acid mutation may be a polar amino acid. In the present application, the mutated amino acid at the site of the amino acid mutation may be a neutral amino acid.

[0101] In the present application, the mutated amino acid at the site having the amino acid mutation may be a hydrophilic amino acid. In the present application, the mutated amino acid at the site having the amino acid mutation may be a hydrophobic amino acid.

[0102] In the present application, the mutated amino acid at the site of the amino acid mutation may be an amino acid with a relatively small side chain volume, for example, an amino acid with a relatively small side chain volume characterized by a side chain that is a small-volume group such as a hydrogen atom, a methyl group, or an ethyl group.

[0103] For example, the group of amino acids having non-polar side chains may include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0104] For example, the group of amino acids having polar side chains may include glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0105] In the present application, at the amino acid mutation site, the mutated amino acid may be independently selected from alanine (A), glycine (G), valine (V), leucine (L), serine (S), tryptophan (W), and tyrosine (Y).

[0106] In the present application, the DR5 domain variant may comprise one or more amino acid mutations selected from the group consisting of I87A / V / L, E89N / Q, D90N / S, T105N / S, N108Q, D109H / S / Q / E, F112Y, L114A / G / V / S / W / Y, R115A / G / V / L / S / W / Y, E147S / Y, D148N / K / E, E151S, R154A / G / V / L / S / W / Y and K155A / G / V / L / S / W / Y.

[0107] In the present application, the DR5 domain variants are: (1) L114A and R115A (SEQ ID NO: 1); (2) R154A and K155A (SEQ ID NO: 2); (3) I87L, D90S, T105N, D109H, and D148N (SEQ ID NO: 3); (4) I87A, D90S, T105N, D109S, L114V, and E147S (SEQ ID NO: 4); (5) I87A, E89N, D90N, D109Q, and E147Y (SEQ ID NO: 5); and (6) I87A, E89Q, D90S, T105S, D109S, L114V, and D148K (SEQ ID NO: 6). 6), (7) D90S, T105N, D109E, L114I, D148E, and R154Y (SEQ ID NO: 7), (8) D90S, D109N, and D148E (SEQ ID NO: 8), (9) I87V, D90N, and R154Y (SEQ ID NO: 9), (10) T105S, N108Q, and F112Y (SEQ ID NO: 10), (11) L114V, E147Q, and E151S (SEQ ID NO: 11), (12) L114G and R115G (SEQ ID NO: 12), (13) L114V and R115V (SEQ ID NO: 13), (14) R115L (SEQ ID NO: 14), (15) L114S and R115S (SEQ ID NO: (15), (16) L114W and R115W (SEQ ID NO: 16), (17) L114Y and R115Y (SEQ ID NO: 17), (18) R154G and K155G (SEQ ID NO: 18), (19) R154V and K155V (SEQ ID NO: 19), (20) R154L and K155L (SEQ ID NO: 20), (21) R154S and K155S (SEQ ID NO: 21), (22) R154W and K155W (SEQ ID NO: 22), and (23) R154Y and K155Y (SEQ ID NO: 23).

[0108] In the present application, the DR5 domain variant may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23.

[0109] In another aspect, the present application further provided a fusion protein comprising a DR5 domain variant described in the present application.

[0110] In the present application, the fusion protein may further comprise other functional proteins.

[0111] For example, the functional protein may include an immunoglobulin Fc region. For example, the fusion protein may include a DR5 domain and an immunoglobulin Fc region. For example, the immunoglobulin Fc region may include an IgG Fc region. For example, the IgG may be selected from IgG1, IgG2, IgG3, and IgG4. For example, the IgG may be IgG1. For example, the IgG may be IgG4.

[0112] In the present application, the Fc region of the immunoglobulin may further comprise one or more amino acid mutations. For example, the amino acid mutation may be any amino acid mutation known to those skilled in the art. For example, the IgG1 Fc region may comprise an amino acid mutation at position 265 (SEQ ID NO: 99). For example, the IgG4 Fc region comprises an amino acid mutation of S228P.

[0113] For example, the DR5 domain variant and the Fc region of the immunoglobulin of the fusion protein may be directly linked. In the present application, the Fc region of the immunoglobulin of the fusion protein and the DR5 domain variant may be indirectly linked. For example, the N-terminus of the Fc region of the immunoglobulin of the fusion protein may be directly or indirectly linked to the C-terminus of the DR5 domain variant. For example, the C-terminus of the Fc region of the immunoglobulin of the fusion protein may be directly or indirectly linked to the N-terminus of the DR5 domain variant. For example, the indirect link may be via a linker. For example, the linker may include a peptide linker. For example, the fusion protein may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 24 to 92.

[0114] For example, the functional protein may include a protein capable of extending the in vivo half-life of a drug. The protein capable of extending the in vivo half-life of a drug and the DR5 domain may be directly linked. The protein capable of extending the in vivo half-life of a drug and the DR5 domain may also be indirectly linked.

[0115] For example, the functional protein may comprise human serum albumin. For example, the fusion protein may comprise a DR5 domain variant and human serum albumin. For example, the human serum albumin and the DR5 domain variant of the fusion protein may be directly linked. In the present application, the human serum albumin and the DR5 domain variant of the fusion protein may be indirectly linked. For example, the N-terminus of the human serum albumin of the fusion protein may be directly or indirectly linked to the C-terminus of the DR5 domain variant. For example, the C-terminus of the human serum albumin of the fusion protein may be directly or indirectly linked to the N-terminus of the DR5 domain variant. For example, the indirect link may be via a linker. For example, the linker may include a peptide linker.

[0116] Nucleic acid molecules, vectors, cells, pharmaceutical compositions, manufacturing methods and applications

[0117] In another aspect, the present application further provides one or more isolated nucleic acid molecules capable of encoding the DR5 domain variants or fusion proteins described herein. In another aspect, the present application provides one or more vectors that may comprise one or more nucleic acid molecules described herein. In another aspect, the present application provides cells (e.g., host cells) that may comprise the nucleic acid molecules described herein or the vectors described herein.

[0118] The nucleic acid molecules described herein may be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification, e.g., polymerase chain reaction (PCR); (ii) cloning or recombination; (iii) purification, e.g., enzymatic digestion and gel electrophoretic fractionation; or (iv) synthesis, e.g., chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA technology. In the present application, nucleic acids encoding the antibodies or antigen-binding fragments thereof can be produced by various methods known in the art, including, but not limited to, restriction fragment manipulation or overlap-extension PCR using synthetic oligonucleotides. For specific procedures, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0119] In the present application, the vector may include a vector primarily used for inserting DNA or RNA into a cell, a vector primarily used for replicating DNA or RNA, and an expression vector primarily used for transcribing and / or translating DNA or RNA. The vector further includes a vector having multiple types of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate host cell. Typically, the vector can produce a desired expression product by culturing an appropriate host cell containing the vector. In the present application, the vector may include one or more of the nucleic acid molecules. The vector may also include other genes, such as marker genes, that allow the vector to be selected in an appropriate host cell under appropriate conditions. The vector may further include expression control elements that allow the accurate expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatable element. The specific structure of the expression control sequence may vary depending on the species or cell type, but typically includes a 5' non-transcribed sequence and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for controlling the transcription of an operably linked nucleic acid.

[0120] In the present application, the host cell may include progeny of a single host cell. Due to spontaneous, unintended, or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but they may express the antibody or antigen-binding fragment thereof described in the present invention. The host cell may be obtained by in vitro transfecting cells with a vector described in the present application. The host cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cell, e.g., COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell). The vector described in the present application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, or lipofectamine transfection.

[0121] In another aspect, the present application further provides a method for producing the DR5 domain variant or the fusion protein, which may comprise culturing the cells under conditions that allow expression of the fusion protein, for example, using an appropriate medium, an appropriate temperature and incubation time, and these methods are known to those skilled in the art.

[0122] In another aspect, the present application further provided a pharmaceutical composition which may comprise said DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector and / or said cell, and optionally a pharmaceutically acceptable carrier.

[0123] For example, the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex, and / or a non-ionic surfactant.

[0124] In the present application, the pharmaceutical compositions can be formulated with pharmaceutically acceptable vectors or diluents and any other known adjuvants and excipients according to conventional techniques in the art, for example, according to the techniques disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995.

[0125] In some embodiments, the pharmaceutical compositions may further contain two or more active compounds, active compounds with complementary activities that typically do not adversely affect each other.

[0126] In some embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intracavitary, intraarterially, intrathecally, and / or intranasally, or by direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered by various methods, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved through a miniature pump system worn by the patient, which meters the therapeutic agent into the patient's body, as described in WO 2015 / 036583.

[0127] In another aspect, the present application further provides a method for preventing and / or treating a disease and / or condition, comprising administering to a subject in need thereof the DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition.

[0128] In the present application, said diseases and / or conditions may include diseases and / or conditions associated with DR5 / TRAIL pathway-induced apoptosis.

[0129] In the present application, the disease and / or condition may include myocardial infarction and / or myocardial infarction-reperfusion injury. In the present application, the disease and / or condition may include cerebral infarction.

[0130] In another aspect, the present application further provides the use of a DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition.

[0131] In the present application, the disease and / or condition may include a disease and / or condition associated with apoptosis induced by the DR5 / TRAIL pathway. In the present application, the disease and / or condition may include cerebral infarction.

[0132] In the present application, the disease and / or condition may include myocardial infarction and / or myocardial infarction-reperfusion injury.

[0133] In another aspect, the present application further provides a DR5 domain variant, said fusion protein, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition for preventing and / or treating a disease and / or condition.

[0134] In the present application, said diseases and / or conditions may include diseases and / or conditions associated with DR5 / TRAIL pathway-induced apoptosis.

[0135] In the present application, the disease and / or condition may include myocardial infarction and / or myocardial infarction-reperfusion injury. In the present application, the disease and / or condition may include cerebral infarction.

[0136] In another aspect, the present application further provides a method for interrupting and / or blocking the TRAIL / DR5 pathway, said method comprising administering said fusion protein or said pharmaceutical composition.

[0137] In some cases, the method may be an in vivo method.

[0138] In some cases, the method may be an in vitro method.

[0139] In some cases, the method may be an ex vivo method.

[0140] In some cases, the method may be for non-disease diagnostic or therapeutic purposes.

[0141] Without wishing to be bound by any theory, the following examples are merely intended to illustrate the variants, fusion proteins, production methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0142] Example

[0143] Example 1 Screening for DR5 variants

[0144] The truncated domain (SEQ ID NO: 94) of human DR5 variant 1 (NP_003833.4) was obtained, and a structure of the truncated domain interacting with TRAIL (NP_003801.1) was constructed using software. The sites involved in the interaction between the two proteins and the interaction mode were theoretically analyzed. The amino acid sites directly or indirectly involved in the interaction with TRAIL in the truncated domain were determined to be I87, E89, D90, T105, N108, D109, L114, R115, E147, D148, E151, R154, and K155. The positions of these amino acid residues were numbered based on the amino acid sequence shown in SEQ ID NO: 96. Random mutations were performed on these active sites to construct a mutant library. The mutant library was then cloned into the pTM vector. The pTM vector contains a signal peptide and a transmembrane domain sequence, which allows genes cloned into the vector to be displayed on the cell surface.

[0145] The constructed expression vector for the variant library was transfected into CHO cells (ATCC), and the variant library was displayed and expressed on the cell surface. TRAIL protein was then fluorescently labeled with APC to obtain APC-TRAIL. Based on the strength of the binding activity between APC-TRAIL and the truncated domain variants on the CHO cell surface, variants capable of binding to APC-TRAIL were enriched and screened by flow cytometry. The specific screening principle is shown in Figure 2, where the truncated domain and variants bind to TRAIL protein bearing a fluorescent molecule, and the binding results are reflected by the level of the fluorescent molecule.

[0146] After three rounds of screening and enrichment, cells that strongly bound to TRAIL-APC were collected (Figure 3). Their mRNA was extracted and reverse-transcribed to obtain cDNA, and the truncated domain variant genes were subjected to sequencing analysis (Figure 4). The sequencing results showed that different mutation combinations existed at the aforementioned sites: I87, E89, D90, T105, N108, D109, L114, R115, E147, D148, E151, R154, and K155.

[0147] As can be seen from the results, different mutation combinations at residues I87, E89, D90, T105, N108, D109, L114, R115, E147, D148, E151, R154 and K155 can yield new truncated domain variants that can specifically recognize DR5.

[0148] Further analysis of the mutation sites revealed that the mutated amino acid residues at each site were I87A / V / L, E89N / Q, D90N / S, T105N / S, N108Q, D109H / S / Q / E, F112Y, L114A / G / V / S / W / Y, R115A / G / V / L / S / W / Y, E147S / Y, D148N / K / E, E151S, R154A / G / V / L / S / W / Y and K155A / G / V / L / S / W / Y.

[0149] Mutations were made to the amino acid sequence shown in SEQ ID NO: 94, and these DR5 domain variants were designated M3 (SEQ ID NO: 1), M5 (SEQ ID NO: 2), M14 (SEQ ID NO: 3), M16 (SEQ ID NO: 4), M29 (SEQ ID NO: 5), M36 (SEQ ID NO: 6), M42 (SEQ ID NO: 7), M47 (SEQ ID NO: 8), M51 (SEQ ID NO: 9), M52 (SEQ ID NO: 10), M54 (SEQ ID NO: 11), M62 (SEQ ID NO: 12), M63 (SEQ ID NO: 13), M64 (SEQ ID NO: 14), M65 (SEQ ID NO: 15), M66 (SEQ ID NO: 16), M67 (SEQ ID NO: 17), M82 (SEQ ID NO: 18), M83 (SEQ ID NO: 19), M84 (SEQ ID NO: 20), M85 (SEQ ID NO: 21), M86 (SEQ ID NO: 22), and M87 (SEQ ID NO: 23), respectively.

[0150] Example 2 Measurement of binding activity of fusion protein (ELISA)

[0151] The ectodomain of human DR5 variant 1 in Example 1 (also referred to as wild-type DR5 truncated domain) and the variants of the DR5 ectodomain obtained in Example 1 were each fused with human IgG1-Fc (the amino acid sequence of which is shown in SEQ ID NO: 97) and expressed to obtain the corresponding DR5 ectodomain-human Fc fusion proteins (abbreviated as fusion proteins). These fusion proteins were designated as YF01M3 (SEQ ID NO: 24), YF01M5 (SEQ ID NO: 25), YF01M14 (SEQ ID NO: 26), YF01M16 (SEQ ID NO: 27), YF01M29 (SEQ ID NO: 28), YF01M36 (SEQ ID NO: 29), YF01M42 (SEQ ID NO: 30), YF01M47 (SEQ ID NO: 31), YF01M51 (SEQ ID NO: 32), YF01M52 (SEQ ID NO: 33), YF01M54 (SEQ ID NO: 34), YF01M55 (SEQ ID NO: 35), YF01M56 (SEQ ID NO: 36), YF01M57 (SEQ ID NO: 37), YF01M58 (SEQ ID NO: 38), YF01M59 (SEQ ID NO: 39), YF01M60 (SEQ ID NO: 40), YF01M61 (SEQ ID NO: 41), YF01M62 (SEQ ID NO: 42), YF01M63 (SEQ ID NO: 43), YF01M64 (SEQ ID NO: 44), YF01M65 (SEQ ID NO: 45), YF01M66 (SEQ ID NO: 46), YF 34), YF01M62 (SEQ ID NO: 35), YF01M63 (SEQ ID NO: 36), YF01M64 (SEQ ID NO: 37), YF01M65 (SEQ ID NO: 38), YF01M66 (SEQ ID NO: 39), YF01M67 (SEQ ID NO: 40), YF01M82 (SEQ ID NO: 41), YF01M83 (SEQ ID NO: 42), YF01M84 (SEQ ID NO: 43), YF01M85 (SEQ ID NO: 44), YF01M86 (SEQ ID NO: 45), and YF01M87 (SEQ ID NO: 46).

[0152] As an example, YF01M3, YF01M14, YF01M16, YF01M36, YF01M42, and YF01M47 were selected for biological activity analysis. An ELISA plate was coated with 10 μg / ml of the target antigen TRAIL-His and incubated overnight at 4°C. After washing with PBST, 10% fetal bovine serum was added and blocked at 37°C for 1 hour. Then, YF01M3, YF01M14, YF01M16, YF01M36, YF01M42, and YF01M47 (at concentrations of 1 μg / ml, 2 μg / ml, 10 μg / ml, and 50 μg / ml) were added and incubated at 37°C for 1 hour. The plates were then washed with PBST, and horseradish peroxidase-conjugated goat anti-human IgG secondary antibody (Goat Anti-human IgG HRP, Thermo Fisher Scientific) was added and incubated at room temperature for 30 minutes. The plates were then washed five times with PBST, and the remaining liquid was gently patted dry with absorbent paper. 100 ml of TMB (eBioscience) was added to each well, and the plates were left at room temperature (20 ± 5°C) for 1–5 minutes in the dark. The substrate reaction was stopped by adding 100 ml of 2N H2SO4 to each well. The OD values ​​were read at 450 nm using a microplate reader, and the affinity of each fusion protein for the TRAIL molecule was analyzed (Figure 5).

[0153] The results in Figure 5 indicate that YF01M3, YF01M14, YF01M16, YF01M36, YF01M42, and YF01M47 can all bind to TRAIL with a certain degree of binding affinity, and that their effects are comparable to those of WT.

[0154] Example 3 Affinity Analysis

[0155] For example, the affinity of each fusion protein such as YF01M3 and YF01M5 to the TRAIL molecule was measured using the Biacore method, and the results are shown in Table 1 below.

[0156] [Table 1]

[0157] The results in Table 1 demonstrate that each of the fusion proteins, such as YF01M3 and YF01M5, can recognize TRAIL molecules with high affinity.

[0158] Example 4 Specific Blockade of TRAIL-Induced Cell Killing by Fusion Proteins

[0159] TRAIL can induce apoptosis of Jurkat cells in vitro by binding to DR5 on the cell surface, and we used flow cytometry to detect the blocking effect of the fusion protein on this process.

[0160] As examples, YF01M63, YF01M65, YF01M14, YF01M16, YF01M29, YF01M36, YF01M42, and YF01M47 were selected for TRAIL killing blocking activity analysis, and YF01 protein was used as a control.

[0161] Harvest 5 x 10 Jurkat cells in logarithmic growth phase. 5 The cells were seeded at 100 cells / well into a 24-well cell culture plate, and 200 ng / ml of TRAIL (Novoprotein) and 0.25 mg / ml of the fusion protein were added to 0.5 ml of the culture medium. After shaking evenly, the cells were further cultured in an incubator for 6 hours.

[0162] After staining with AnnexinV-FITC and 7AAD apoptosis detection kits (Biyuntian), AnnexinV was detected by flow cytometry (BD FACSCalibur). + 7AAD - and Annexin V + 7AAD + The total apoptotic cell ratio was measured (results are shown in Figure 6).

[0163] The results in Figure 6 show that at a concentration of 0.25 μg / ml, YF01M63, YF01M65, YF01M14, YF01M16, and YF01M36 could effectively block 200 ng / ml TRAIL-induced apoptosis, and their apoptosis inhibitory effects were superior to those of YF01 protein.

[0164] Example 5: Detection of the protective effect of the fusion protein against myocardial ischemia-reperfusion injury

[0165] Taking YF01M5 as an example, the in vivo protective effect of the fusion protein against cardiac ischemia-reperfusion injury in rats was detected.

[0166] Male clean-grade Wistar rats (200-250 g) were anesthetized with an intraperitoneal injection of 10% chloral hydrate. The trachea was intubated and a ventilator was connected before opening the intercostal space. Parameters were adjusted to achieve an IE ratio of 1:2, a respiratory rate of 90-120 breaths per minute, and a tidal volume of 2-4 ml. A transverse incision was made between the third and fourth intercostal spaces on the left sternal border, and the pericardium was incised to expose the heart. Using the left coronary vein at the junction of the pulmonary artery and the left atrial appendage as a marker, a needle was inserted 0.1 cm from the midpoint of the lower border of the left atrial appendage and ligated. After a set time, the ligature was loosened, and the myocardium turned red, indicating successful reperfusion. After surgery, penicillin was typically injected at 400,000 U / rat. The fusion protein (concentration calibrated) was administered 10 min before reperfusion. 24 h later, left ventricular myocardial tissue was removed and sectioned. The area of ​​myocardial infarction in each treatment group was observed by Evans Blue / TTC staining and photographed with a digital camera. The size of the planar infarct lesion area was measured using Image-Pro Plus 6.0 software. The area unstained with Evans Blue was the risk area, and the area stained white with TTC was the infarct area. The degree of cardiac damage was determined by the relative infarction rate, which was calculated using the following formula:

[0167]

number

[0168] Each group contained 5 or more animals, and the results are shown in FIG.

[0169] The results shown in Figure 7 indicate that YF01M5 protein protects rats from cardiac ischemia-reperfusion injury in a dose-dependent manner, and its myocardial infarction protective effect at a dose of 3 mg / kg (26%) is equivalent to that of YF01 (WT) protein at 15 mg / kg (24%), indicating that it has a clear dose advantage.

[0170] Taking YF01M3 as an example, the in vivo protective effect of the fusion protein against cardiac ischemia-reperfusion injury in rhesus monkeys was detected.

[0171] Animal anesthesia and preoperative preparation: The monkey was immobilized and anesthetized with an intramuscular injection of 0.15 ml / kg Luminning (manufactured by Kamoku Co., Ltd., drug concentration 100 mg / ml, diluted 10-fold before use). Its ear number was recorded. After weighing, the monkey was secured to the operating table. Cardiac function was measured using color Doppler ultrasound. An intravenous line was opened, saline was administered intravenously, and sufentanil, etomidate, and vecuronium bromide were administered intravenously in bolus doses. The trachea was intubated. Anticoagulated blood was collected from the groin before surgery. A veterinary hematology analyzer was used to measure blood counts, cardiac function (CK-MB), renal function (creatinine and urea nitrogen), and liver function (alanine aminotransferase and aspartate aminotransferase). Electrocautery electrode pads were attached. Electrocardiograms, blood oxygen saturation, and blood pressure were recorded. The surgical site was disinfected with iodine complex.

[0172] Drug preparation: Drugs of appropriate concentrations were prepared according to the weight of the rhesus monkeys and randomly numbered. The injection dose of YF01M3 was 2 mg / kg, and the injection dose of YF01(WT) was 6 mg / kg, with an injection volume of 5 mL. After preparation, the drugs were temporarily stored at 4°C, protected from light.

[0173] Animal surgery: After the rhesus monkey's heart rate and other indicators had stabilized, the skin and muscle were incised at the first to second intercostal spaces under the left chest (the fifth to sixth intercostal spaces on the left side), exposing the heart. The pericardium was incised and suspended to expose the left anterior descending coronary artery. After the heart rate and other indicators had stabilized, the blood vessel of the left anterior descending coronary artery was ligated at one-third of the apex (below the diagonal branch) using sutures and a urinary catheter. Five minutes later, the electrocardiogram, blood oxygen saturation, and blood pressure were recorded. (5) After 50 min, blood was collected and blood counts, cardiac function, renal function, and liver function were measured. After 55 min, 5 mL of drug was slowly administered intravenously over 2-3 min (the drug number was recorded). At 60 min, the ligature was released and the blood vessels were reopened. At 120 min, 240 min, and 360 min, blood was collected and blood counts, cardiac function, renal function, and liver function were measured. Six hours after the reperfusion, the rhesus monkeys were sacrificed by administering a large dose of anesthetic and injecting air.

[0174] Tissue acquisition and cardiac TTC staining: The heart was acquired and washed with saline, then placed in a -20°C freezer overnight. A certain amount of TTC powder was weighed and dissolved in 1x PBS (protected from light) to prepare a 1% working solution. The prepared TTC staining solution was placed in a 37°C incubator and preheated for 15-30 minutes in the dark. The monkey heart tissue was removed from the -20°C freezer and, once its surface had softened, sectioned and stained. 3-4 mm thick slices were uniformly cut from the apex toward the ligature line. The sliced ​​hearts were placed in the preheated TTC staining solution and incubated in a 37°C incubator for 15 minutes in the dark. After 15 minutes, the heart slices were inverted once to ensure uniform staining. (7) After staining was complete, the stained heart tissue was photographed.

[0175] Photographing and weighing after TTC staining: Each cardiac slice of each monkey was photographed, the right ventricle was removed, and the weight of the cardiac slice was recorded. The white infarcted area was collected from the slice and weighed. The degree of cardiac damage was determined by the relative infarction rate. The cardiac infarction rate was calculated using the following formula:

[0176]

number

[0177] Each group had 3 or more animals, and the results are shown in FIG.

[0178] The results in Figure 8 showed that the YF01M3 protein significantly protected rhesus monkeys from cardiac ischemia-reperfusion injury (78%) at a dose of 2 mg / kg, which was equivalent to the YF01(WT) protein at a dose of 6 mg / kg (75%), indicating that it has a dose advantage.

[0179] Example 6. Effect of different subtypes of IgG Fc fusion on fusion protein activity

[0180] According to the method for constructing fusion proteins in Example 2, the DR5 domain truncated variants M3, M5, M14, M16, M29, M36, M42, M47, M51, M52, M54, M62, M63, M64, M65, M66, M67, M82, M83, M84, M85, M86, and M87 obtained in Example 1 were each fused with human IgG4-Fc (the amino acid sequence of which is shown in SEQ ID NO: 98) and expressed to obtain the corresponding DR5 variant truncated domain-human Fc fusion proteins (abbreviated as fusion proteins). These fusion proteins were designated YF01M3G4 (SEQ ID NO: 47), YF01M5G4 (SEQ ID NO: 48), YF01M14G4 (SEQ ID NO: 49), YF01M16G4 (SEQ ID NO: 50), and YF01M29G4 (SEQ ID NO: 51), respectively. 51), YF01M36G4 (SEQ ID NO: 52), YF01M42G4 (SEQ ID NO: 53), YF01M47G4 (SEQ ID NO: 54), YF01M51G4 (SEQ ID NO: 55), YF01M52G4 (SEQ ID NO: 56), YF01M54G4 (SEQ ID NO: 57), YF01M62G4 (SEQ ID NO: 58), YF01M63G4 (SEQ ID NO: 59), YF01M64G4 (SEQ ID NO: 60), YF01M65G4 (SEQ ID NO: 61), YF01M66G4 (SEQ ID NO: 62), YF01M67G4 (SEQ ID NO: 63), YF01M82G4 (SEQ ID NO: 64), YF01M83G4 (SEQ ID NO: 65), YF01M84G4 (SEQ ID NO: 66), YF01M85G4 (SEQ ID NO: 67), YF01M86G4 (SEQ ID NO: 68), and YF01M87G4 (SEQ ID NO: 69).

[0181] The DR5 domain variants M3, M5, M14, M16, M29, M36, M42, M47, M62, M63, M64, M65, M66, M67, M82, M83, M84, M85, M86, and M87 obtained in Example 1 were each fused with a mutated human IgG1-Fc (the amino acid sequence of which is shown in SEQ ID NO: 99) and expressed to obtain the corresponding DR5 variant truncated domain-human Fc fusion proteins (abbreviated as fusion proteins). These fusion proteins were designated YF01M3G1m (SEQ ID NO: 70), YF01M5G1m (SEQ ID NO: 71), YF01M14G1m (SEQ ID NO: 72), YF01M16G1m (SEQ ID NO: 73), YF01M29G1m (SEQ ID NO: 74), and YF01M36G1m (SEQ ID NO: 75), respectively. 75), YF01M42G1m (SEQ ID NO: 76), YF01M47G1m (SEQ ID NO: 77), YF01M51G1m (SEQ ID NO: 78), YF01M52G1m (SEQ ID NO: 79), YF01M54G1m (SEQ ID NO: 80), YF01M62G1m (SEQ ID NO: 81), YF01M63G1m (SEQ ID NO: 82), YF01M64G1m (SEQ ID NO: 83), YF01M65G1m (SEQ ID NO: 84), YF01M66G1m (SEQ ID NO: 85), YF01M67G1m (SEQ ID NO: 86), YF01M82G1m (SEQ ID NO: 87), YF01M83G1m (SEQ ID NO: 88), YF01M84G1m (SEQ ID NO: 89), YF01M85G1m (SEQ ID NO: 90), YF01M86G1m (SEQ ID NO: 91), and YF01M87G1m (SEQ ID NO: 92).

[0182] As an example, YF01M3G4 and YF01M3G1m were selected for biological activity analysis.

[0183] According to the method analysis for specifically blocking TRAIL-induced cell killing in Example 4, the blocking activity of YF01M3G4 and YF01M3G1m on TRAIL-induced Jurkat apoptosis was analyzed, and the results are shown in Figure 9. The results in Figure 9 indicate that both YF01M3G4 and YF01M3G1m at a dose of 0.125 mg / ml were able to block apoptosis induced by 200 ng / ml of TRAIL, an effect comparable to that of M3-1 and superior to that of YF01 (WT).

[0184] According to the method for protecting rats from myocardial ischemia-reperfusion injury in Example 5, the protective effects of YF01M3G4 and YF01M3G1m against myocardial ischemia-reperfusion injury in vivo were analyzed, and the results are shown in Figure 10. The results in Figure 10 indicate that YF01M3G4 and YF01M3G1m can both protect rats from myocardial ischemia-reperfusion injury at a dose of 3 mg / kg, and the effect is equivalent to that of 3 mg / kg M3 and 15 mg / kg WT.

[0185] These results demonstrate that different subtypes of IgG Fc fusion proteins do not significantly affect the activity of the fusion proteins constructed in this application.

[0186] Example 7 Protective effect of fusion protein against cerebral infarction / reperfusion injury

[0187] Using YF01M3 as an example, we detected a protective effect against brain damage caused by middle cerebral artery occlusion and reperfusion.

[0188] Rats were housed in a clean-grade environment with an ambient temperature of 24 ± 2°C, humidity of 45 ± 5%, and a 12-hour light-dark cycle. They were fed a balanced diet. They were randomly divided into a PBS control group and an M3-1 treatment group, with at least five rats in each group. They were fasted for 12 hours before the experiment and allowed free access to water. After anesthetizing with sodium pentobarbital, rats were placed in a supine position, and the hair was roughly removed and disinfected (hair removal can also be performed with a depilatory). The neck skin was moistened with iodophor and disinfected. An incision was made along the right side of the middle portion of the neck below the mandible, and each layer of tissue was bluntly dissected. The membrane surrounding the vagus nerve and common carotid artery was cut with ophthalmic scissors. The vagus nerve, which was in close contact with the common carotid artery, was bluntly dissected with a glass needle to expose the right common carotid artery (CCA). The external carotid artery (ECA) and internal carotid artery (ICA) were isolated at the distal end of the common carotid artery. A 4-0 suture was passed through the proximal end of the common carotid artery (CCA) and below the internal carotid artery (ICA) to control arterial blood flow during embolization (hemostasis could also be achieved by clamping with an arterial clamp). Two 5-0 sutures were passed through the inferior portion of the external carotid artery (ECA) and tied twice at the distal end. After ligation, the vessel was transected from the center. The sutures on the ICA and CCA were gently tightened to prevent bleeding during insertion of the suture obturator. The ECA stump was aligned with the ICA, and the ECA bifurcation near the CCA was incised and a suture was inserted toward the intracranial cavity. The suture was made of 4-0 nylon thread, 28 μm in diameter, and had a certain hardness. The tip that was inserted into the blood vessel was slightly bulged and spherical. The suture was inserted to a depth of approximately 18.5 ± 0.5 mm. When resistance was felt, the suture was stopped and a ligature was placed below the incision to prevent blood leakage. The hemostat was removed, the dissected tissue was returned to its original state, and a heating pad was placed on top to keep it warm. After surgery, rats were warmed using a heat lamp or a heating pad, maintaining a rectal temperature of approximately 37°C.

[0189] Thirty minutes after embolization, a 10 μL intracranial injection of 60 μg of M3-1 was administered to the ischemic side of the brain using a stereotaxic apparatus. The injection volume was then sutured and disinfected. Two hours after embolization, the rats were again anesthetized and the thread was removed. The thin thread below the external carotid artery was tightly tied. An appropriate amount of antibiotic was sprayed into the skin incision, which was then sutured using standard methods. The rats were then returned to their cages, kept warm, and fed a 5% glucose solution.

[0190] Infarct volume was quantified using 2,3,5-triphenyltetrazolium chloride (TTC) staining. 24 hours after MCAO surgery, rats were deeply anesthetized, and brain tissue was removed. Using a brain section template, the brain tissue was uniformly cut into six 2-mm-thick coronal sections along the coronal plane. Using tweezers, the sections were gently pressed into pre-prepared 1% TTC staining solution (in a 6-well cell culture plate), ensuring that the same coronal orientation of the sections was attached to the bottom of the 6-well plate, ensuring that the staining solution covered the brain tissue sections. The 6-well plate was covered with a light shield and placed in a 37°C water bath for 20 minutes. After inverting with tweezers, the sections were continued to stain for 5–10 minutes, after which they were removed and placed in 4% paraformaldehyde for overnight fixation. The coronal sections of the TTC-stained brain sections were uniformly placed from the cerebellum to the olfactory bulb and photographed with a digital camera. The size of the planar infarct lesion area was measured using Image-Pro Plus 6.0 software, and the calculation formula for the relative infarct volume was as follows (the results are shown in Figure 11):

[0191]

number

[0192] The results in Figure 11 demonstrate that YF01M3 can significantly protect against tissue damage caused by cerebral infarction / reperfusion.

[0193] Example 8 Protective effect of intramuscular injection of fusion protein against myocardial infarction / reperfusion injury

[0194] Taking YF01M5 as an example, the protective effect of intramuscular injection of the fusion protein against cardiac ischemia-reperfusion injury in rats was detected.

[0195] The rat cardiac ischemia-reperfusion injury model was established and evaluated as described in Example 5. The administration method was as follows: calibrated drugs were administered by intramuscular injection into the hind limbs 10 minutes before reperfusion, with a drug volume of 200 μl.

[0196] As shown in the results of Figure 12, YF01M5, when injected intramuscularly, can significantly protect myocardial tissue from damage caused by cerebral infarction / reperfusion.

Claims

1. A Death Receptor 5 (DR5) domain variant comprising: (1) Ability to bind to TRAIL; (2) blocking TRAIL / DR5 pathway-induced apoptosis; (3) reducing myocardial infarction size in cardiac ischemia and / or ischemia-reperfusion; and (4) having a protective effect against cerebral infarction; having one or more of the following characteristics: Death receptor 5 domain variant.

2. The DR5 domain variant of claim 1 , wherein the DR5 domain comprises the extracellular domain of DR5.

3. The DR5 domain variant of claim 1 or 2, wherein the DR5 domain variant comprises one or more amino acid mutations at one or more amino acid positions 86 to 155 compared to the extracellular domain of human DR5 variant 1 (SEQ ID NO: 94).

4. The DR5 domain variant according to any one of claims 1 to 3, wherein the death receptor 5 (DR5) comprises soluble death receptor 5 (sDR5).

5. The DR5 domain variant according to any one of claims 1 to 4, wherein the DR5 is human DR5.

6. The DR5 domain variant according to any one of claims 3 to 5, wherein the extracellular domain of human DR5 variant 1 comprises the amino acid sequence shown in SEQ ID NO:

94.

7. The DR5 domain variant according to any one of claims 1 to 6, comprising one or more amino acid mutations at one or more of amino acid positions 86 to 92.

8. The DR5 domain variant according to any one of claims 1 to 7, comprising one or more amino acid mutations at one or more amino acid positions 105 to 112.

9. The DR5 domain variant according to any one of claims 1 to 8, comprising one or more amino acid mutations at one or more amino acid positions 114 to 155.

10. The DR5 domain variant according to any one of claims 1 to 9, comprising one or more amino acid mutations at one or more of amino acid positions 114 to 115.

11. The DR5 domain variant of any one of claims 1 to 10, comprising one or more amino acid mutations at one or more of amino acid positions 147 to 152.

12. The DR5 domain variant according to any one of claims 1 to 11, comprising one or more amino acid mutations at one or more of amino acid positions 154 to 155.

13. The DR5 domain variant of any one of claims 1 to 12, comprising one or more amino acid mutations at one or more amino acid positions selected from the group consisting of I87, E89, D90, T105, N108, D109, L114, R115, E147, D148, E151, R154 and K155, compared to the amino acid sequence set forth in SEQ ID NO:

94.

14. Compared to the amino acid sequence shown in SEQ ID NO: 94, (1) L114 and R115, (2) R154 and K155, (3) I87, D90, T105, D109 and D148, (4)I87, D90, T105, D109, L114, E147, (5) I87, E89, D90, D109, E147, (6) I87, E89, D90, T105, D109, L114, D148, (7)D90, T105, D109, L114, D148, R154, (8) D90, D109 and D148, (9) I87, D90 and R154, (10) T105, N108 and F112, (11) L114, E147 and E151, and (12) R115 The DR5 domain variant according to any one of claims 1 to 13, comprising a mutation at an amino acid site selected from the group consisting of:

15. The DR5 domain variant according to any one of claims 1 to 14, wherein the mutated amino acid is a polar amino acid.

16. The DR5 domain variant according to any one of claims 1 to 14, wherein the mutated amino acid is a neutral amino acid.

17. The DR5 domain variant according to any one of claims 1 to 14, wherein the mutated amino acid is an acidic amino acid.

18. The DR5 domain variant according to any one of claims 1 to 14, wherein the mutated amino acid is a basic amino acid.

19. The DR5 domain variant according to any one of claims 1 to 14, wherein the mutated amino acid is a non-polar amino acid.

20. The DR5 domain variant according to any one of claims 1 to 14, wherein the mutated amino acid in the amino acid mutation is an amino acid with a relatively small side chain volume.

21. 21. The DR5 domain variant of any one of claims 1 to 20, comprising one or more amino acid mutations selected from the group consisting of I87A / V / L, E89N / Q, D90N / S, T105N / S, N108Q, D109H / S / Q / E, F112Y, L114A / G / V / S / W / Y, R115A / G / V / L / S / W / Y, E147S / Y, D148N / K / E, E151S, R154A / G / V / L / S / W / Y and K155A / G / V / L / S / W / Y, compared to the amino acid sequence set forth in SEQ ID NO:

94.

22. Compared to the amino acid sequence shown in SEQ ID NO: 94, (1) L114A and R115A, (2) R154A and K155A, (3) I87L, D90S, T105N, D109H and D148N, (4) I87A, D90S, T105N, D109S, L114V and E147S, (5) I87A, E89N, D90N, D109Q and E147Y, (6) I87A, E89Q, D90S, T105S, D109S, L114V and D148K, (7) D90S, T105N, D109E, L114I, D148E and R154Y, (8) D90S, D109N and D148E, (9) I87V, D90N and R154Y, (10) T105S, N108Q and F112Y, (11) L114V, E147Q and E151S, (12) L114G and R115G, (13) L114V and R115V, (14) R115L, (15) L114S and R115S, (16) L114W and R115W, (17) L114Y and R115Y, (18) R154G and K155G, (19) R154V and K155V, (20) R154L and K155L, (21) R154S and K155S, (22) R154W and K155W, and (23) R154Y and K155Y 22. The DR5 domain variant of claim 1, comprising an amino acid mutation selected from the group consisting of:

23. A DR5 domain variant according to any one of claims 1 to 22, comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 23.

24. A fusion protein comprising the DR5 domain variant of any one of claims 1 to 23.

25. 25. The fusion protein of claim 24, further comprising an immunoglobulin Fc region.

26. 26. The fusion protein of claim 25, wherein the immunoglobulin Fc region comprises an IgG Fc region.

27. 27. The fusion protein of claim 26, wherein the IgG is selected from the group consisting of IgG1 and IgG4.

28. The fusion protein of any one of claims 25 to 27, wherein the Fc region of the immunoglobulin comprises an amino acid sequence set forth in any one of SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO:

99.

29. The fusion protein of any one of claims 25 to 28, wherein the Fc region of the immunoglobulin is directly or indirectly linked to the DR5 domain.

30. The fusion protein of any one of claims 25 to 29, wherein the DR5 domain variant is located at the N-terminus of the immunoglobulin Fc region.

31. The fusion protein of any one of claims 25 to 29, wherein the DR5 domain variant is located at the C-terminus of the immunoglobulin Fc region.

32. The fusion protein of any one of claims 24 to 31, further comprising a protein capable of extending its half-life.

33. 33. The fusion protein of claim 32, wherein the protein capable of extending half-life comprises human serum albumin.

34. 34. The fusion protein of claim 32 or 33, wherein the protein capable of extending half-life is directly or indirectly linked to the DR5 domain variant.

35. The fusion protein according to any one of claims 32 to 34, wherein the protein capable of extending half-life is located at the N-terminus of the DR5 domain variant.

36. The fusion protein according to any one of claims 32 to 34, wherein the protein capable of extending half-life is located at the C-terminus of the DR5 domain variant.

37. The fusion protein according to any one of claims 24 to 36, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 24 to 92.

38. 38. An isolated nucleic acid molecule or molecules encoding a DR5 domain variant according to any one of claims 1 to 23, or a fusion protein according to any one of claims 24 to 37.

39. A vector comprising the nucleic acid molecule of claim 38.

40. 40. A cell comprising the nucleic acid molecule of claim 38 or the vector of claim 39.

41. 41. A method for producing a DR5 domain variant of any one of claims 1 to 23 or a fusion protein of any one of claims 24 to 37, said method comprising culturing a cell of claim 40 under conditions allowing expression of the DR5 domain variant or fusion protein.

42. 41. A pharmaceutical composition comprising a DR5 domain variant according to any one of claims 1 to 23, a fusion protein according to any one of claims 24 to 37, a nucleic acid molecule according to claim 38, a vector according to claim 39, and / or a cell according to claim 40, and optionally a pharmaceutically acceptable carrier.

43. 43. The pharmaceutical composition of claim 42, further comprising another active ingredient.

44. 10. A method for preventing and / or treating a disease and / or condition, comprising administering to a subject in need thereof a DR5 domain variant according to any one of claims 1 to 23, a fusion protein according to any one of claims 24 to 37, a nucleic acid molecule according to claim 38, a vector according to claim 39, a cell according to claim 40, and / or a pharmaceutical composition according to claim 42 or 43.

45. 45. The method of claim 44, wherein the disease and / or condition comprises myocardial infarction and / or myocardial infarction-reperfusion injury.

46. 46. ​​The method of claim 44 or 45, wherein the disease and / or condition comprises cerebral infarction.

47. Use of a DR5 domain variant according to any one of claims 1 to 23, a fusion protein according to any one of claims 24 to 37, a nucleic acid molecule according to claim 38, a vector according to claim 39, a cell according to claim 40, and / or a pharmaceutical composition according to claim 42 or 43 in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition.

48. 48. The use according to claim 47, wherein the disease and / or condition comprises myocardial infarction and / or myocardial infarction-reperfusion injury.

49. 49. The use according to claim 47 or 48, wherein the disease and / or condition comprises cerebral infarction.

50. A DR5 domain variant according to any one of claims 1 to 23, a fusion protein according to any one of claims 24 to 37, a nucleic acid molecule according to claim 38, a vector according to claim 39, a cell according to claim 40, and / or a pharmaceutical composition according to claim 42 or 43 for the prevention and / or treatment of a disease and / or condition.

51. The DR5 domain variant, fusion protein, nucleic acid molecule, vector, cell and / or pharmaceutical composition of claim 50, wherein the disease and / or condition comprises myocardial infarction and / or myocardial infarction-reperfusion injury.

52. 52. The fusion protein, nucleic acid molecule, vector, cell and / or pharmaceutical composition of claim 50 or 51, wherein the disease and / or condition comprises cerebral infarction.

53. A method for blocking the TRAIL / DR5 pathway, said method comprising administering a DR5 domain variant according to any one of claims 1 to 23, a fusion protein according to any one of claims 24 to 37, or a pharmaceutical composition according to claim 42 or 43.

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