LIGHT mutein and its use

Abstract

Provided are a LIGHT mutein and an LTβR-binding LIGHT mutein, and also provided are related polynucleotides, isolated vectors, host cells, and pharmaceutical compositions. Further provided are the use of the LIGHT mutein, or the isolated polynucleotide, isolated vector, host cell, or pharmaceutical composition, in the manufacture of a medicament for preventing or treating a disease, and a method of doing so in a subject in need of prevention or treatment of a disease. For the above purposes, novel LIGHT muteins are provided herein. In some embodiments, the LIGHT mutein has a sequence set forth in SEQ ID NO: 87, or SEQ ID NO: 88, or SEQ ID NOs: 1-85, or SEQ ID NOs: 89-93.

Description

【Technical Field】 【0001】 (Cross - reference to related applications) This application claims the priority of PCT Application No. PCT / CN2022 / 097735, filed on June 8, 2022. The content of the previous PCT application is regarded as part of this disclosure and is incorporated herein by reference in its entirety. 【0002】 (Reference to sequence listing) The sequence listing titled DCF230337WO - Sequence listing.xml, created on June 6, 2023, with a size of 95,646 bytes, is incorporated herein by reference in its entirety. 【0003】 (Field of the invention) This disclosure relates to a LIGHT mutein (or "LIGHT variant"), an isolated vector containing a polynucleotide encoding the LIGHT mutein, a host cell containing an isolated polynucleotide or an isolated vector encoding the LIGHT mutein, a pharmaceutical composition containing the LIGHT mutein, the use of the LIGHT mutein, an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition in the manufacture of a drug for preventing or treating a disease, and a method of performing the prevention or treatment in a subject in need thereof, including administering a therapeutically effective amount of the LIGHT mutein, or an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition to the subject. 【Background Art】 【0004】 The documents in this section are only intended to provide background information related to this disclosure, and the information existing below this section does not necessarily constitute prior art. 【0005】 LIGHT (which is like lymphotoxin, shows inducible expression, and competes with herpes simplex virus glycoprotein D for the herpes virus entry mediator receptor expressed by T cells) is known as tumor necrosis factor superfamily member 14 (TNFSF14) and is also referred to as HVEM-Ligand (HVEM-L). LIGHT is a membrane protein composed of 240 amino acids (AA) (SEQ ID NO: 86), of which 37 AAs form the cytoplasmic domain, 22 AAs form the transmembrane domain, and 181 AAs form the extracellular domain. LIGHT is transiently induced on immune cells, particularly immature dendritic cells (DCs) and activated T cells. The membrane-bound form of LIGHT can be cleaved by proteases, resulting in a soluble functional structure (Yu et al., 2004). 【0006】 LIGHT has three receptors: herpes virus entry mediator (HVEM), lymphotoxin beta receptor (LTβR), and decoy receptor 3 (DcR3). HVEM is expressed on T cells, NK cells, and dendritic cells. The interaction between LIGHT and HVEM stimulates T cell activation, proliferation, and survival. Another receptor, LTβR, is found on the surface of epithelial cells, stromal cells, immature DCs, and myeloid cells but not on lymphocytes. The LIGHT-LTβR interaction results in the expression of chemokines and adhesion molecules involved in lymph node formation and dendritic cell migration. The third binding partner, DcR3, is a soluble protein that attenuates the activation signal initiated by LIGHT (Liu et al., 2021). The introduction of LIGHT into tumors or the tumor microenvironment could be a powerful strategy for cancer immunotherapy. 【0007】 For the purpose of using the recombinant LIGHT protein as a therapeutic agent, human LIGHT was engineered to selectively interact with its receptor. 【Summary of the Invention】 【Means for Solving the Problems】 【0008】 For the above purpose, novel LIGHT mutants are provided herein. In some embodiments, the LIGHT mutant has the sequence set forth in SEQ ID NO: 87, or SEQ ID NO: 88, or SEQ ID NOs: 1-85, or SEQ ID NOs: 89-93. 【0009】 In some embodiments, LIGHT mutants having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence set forth in SEQ ID NO: 86 are provided herein. In some embodiments, LIGHT mutants having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence set forth in SEQ ID NO: 87 are provided herein. In some embodiments, LIGHT mutants having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence set forth in SEQ ID NO: 88 are provided herein. In some embodiments, LIGHT mutants having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequences set forth in SEQ ID NOs: 1-85 or SEQ ID NOs: 89-93 are provided herein. 【0010】 Provided herein are novel LIGHT mutants selected from the group consisting of proteins having a homology greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to the sequence set forth in SEQ ID NO: 87. In some embodiments, a novel LIGHT mutant comprising one or more amino acid mutations as compared to the amino acid sequence set forth in SEQ ID NO: 86. An amino acid mutation selected from one or more positions selected from the group consisting of 95, 103, 117, 125, 150, 152, 155, 157, 158, 160, 161, 175, 184, 189, 190, 198, 202, 208, 214, 220, 221, 227, 228, and any combination thereof, wherein the positions are defined with reference to SEQ ID NO: 86. 【0011】 In some embodiments, the LIGHT mutant comprises one or more amino acid mutations as compared to the amino acid sequence set forth in SEQ ID NO: 87. An amino acid mutation selected from one or more positions selected from the group consisting of 95, 103, 117, 125, 150, 152, 155, 157, 158, 160, 161, 175, 184, 189, 190, 198, 202, 208, 214, 220, 221, 227, 228, and any combination thereof, wherein the positions are defined with reference to SEQ ID NO: 87, and the position of the first amino acid of SEQ ID NO: 87 is defined as position 74. 【0012】 In some embodiments, the LIGHT mutant comprises one or more amino acid mutations as compared to the amino acid sequence set forth in SEQ ID NO: 88. An amino acid mutation selected from one or more positions selected from the group consisting of 95, 103, 117, 125, 150, 152, 155, 157, 158, 160, 161, 175, 184, 189, 190, 198, 202, 208, 214, 220, 221, 227, 228, and any combination thereof, wherein the positions are defined with reference to SEQ ID NO: 88, and the position of the first amino acid of SEQ ID NO: 88 is defined as position 87. 【0013】 In some embodiments, provided herein are LIGHT mutants having the sequence set forth in SEQ ID NO: 86, 87, or 88, and having one or more amino acid mutations selected from the group consisting of S103N, Q117E / Q117N / Q117H / Q117R, L126M, G150A / G150S / G150R, V152M, L158Q / L158P / L158M, S160G / S160N / S160T / S160A, T161G / T161P / T161S / T161N, L166M, E175K, Q184R, R189S, A190T / A190V, W198Q, F202Y, H208Y / H208R, K214E, L220S / L220N / L220T / L220R / L220M, D221G, E222K / E222S, L227T / L227M, R228L, R232H, and any combination thereof. 【0014】 In some embodiments, provided herein are LIGHT mutants having the sequence set forth in SEQ ID NO: 100, and having one or more amino acid mutations selected from the group consisting of A95T, A101D, N102R, S103N, Q117E / Q117N / Q117H / Q117R, L126M, V135I, T136S, G150A / G150S / G150R, V152M, P155R, G157S, L158Q / L158P / L158M, S160G / S160N / S160T / S160A, T161G / T161P / T161S / T161N, L166M, P174L, E175K, Q184R, R189S, A190T / A190V, W198Q, F202Y, H208Y / H208R, E213D, K214E, L220S / L220N / L220T / L220R / L220M, D221G, E222K / E222S / E222Q / E222D / E222N, L227T / L227M, R228L, R232H, and any combination thereof, wherein the positions are defined with reference to SEQ ID NO: 87, and the position of the first amino acid of SEQ ID NO: 87 is defined as position 74. 【0015】 In some embodiments, provided herein is a LIGHT mutant having the sequence set forth in SEQ ID NO: 88 and having one or more amino acid mutations selected from the group consisting of A95T, A101D, N102R, S103N, S104P, L105P, T116S, Q117E / Q117N / Q117H / Q117R, L126M, V135I, T136S, G150A / G150S / G150R, V152M, P155R, L156P, G157S, L158Q / L158P / L158M, S160G / S160N / S160T / S160A, T161G / T161P / T161S / T161N, L166M, P174L, E175K, Q184R, R189S, A190T / A190V, W198Q, F202Y, H208Y / H208R, E213D, K214E, L220S / L220N / L220T / L220R / L220M, D221G, E222K / E222S / E222Q / E222D / E222N, L227T / L227M, R228L, R232H, and any combination thereof, wherein the positions are defined with reference to SEQ ID NO: 88, and the position of the first amino acid of SEQ ID NO: 87 is defined as position 87. 【0016】 In some embodiments, provided herein is an LTβR-binding LIGHT mutant selected from the group consisting of LIGHT mutants having the sequences set forth in SEQ ID NOs: 1-75, 76-85, and SEQ ID NOs: 89-93. Alternatively, the mutant is selected from proteins having the sequence set forth in SEQ ID NO: 87 or SEQ ID NO: 88 and having at least one amino acid difference as compared to SEQ ID NO: 87 or SEQ ID NO: 88. 【0017】 In some embodiments, the LTβR-binding LIGHT mutant is non-binding to HVEM (e.g., human HVEM), and the LIGHT mutant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-11. 【0018】 In some embodiments, the LTβR-binding LIGHT mutant is HVEM-binding, and the LIGHT mutant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12-75, SEQ ID NOs: 77-85, and SEQ ID NOs: 89-93. 【0019】 In some embodiments, the LTβR-binding LIGHT mutant binds to DcR3 with a reduced affinity compared to wild-type LIGHT, and the LIGHT mutant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 52, 58, 61, and SEQ ID NOs: 89-93. 【0020】 In some embodiments, provided herein is an LTβR-binding LIGHT mutant that is an mHVEM (mouse HVEM)-binding protein and an hHVEM (human HVEM)-nonbinding protein. For example, the sequence set forth in SEQ ID NO: 1, 2, 9, or 11. 【0021】 In some embodiments, provided herein is a human LTβR-binding LIGHT mutant selected from the group consisting of LIGHT mutants having the sequences set forth in SEQ ID NOs: 1, 2, 9, 11, 12, 22, 37, 52, 53-85. 【0022】 In some embodiments, provided herein is a mouse LTβR-binding LIGHT mutant selected from the group consisting of LIGHT mutants having the sequences set forth in SEQ ID NOs: 1, 9, 11, 12, 22, 38, and 52. In some embodiments, provided herein is an LTβR-binding and hHVEM-binding LIGHT mutant selected from the group consisting of LIGHT mutants having the sequences set forth in SEQ ID NOs: 21, 22, 37, and 51. In some embodiments, provided herein is an LTβR-binding and hHVEM-nonbinding LIGHT mutant selected from the group consisting of LIGHT mutants having the sequences set forth in SEQ ID NOs: 1, 2, 9, and 11. 【0023】 In some embodiments, provided herein are truncated LTβR-binding LIGHT and muteins thereof that comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1-85. In some embodiments, provided herein are truncated LTβR-binding and mHVEM-binding LIGHT and muteins thereof that comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1-85. Some embodiments provide truncated LTβR-binding and mHVEM-binding LIGHT and muteins thereof that do not bind hHVEM and that comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1-11. 【0024】 In some embodiments, provided herein are LTβR-binding and HVEM-binding LIGHT muteins that comprise an amino acid sequence set forth in any one of SEQ ID NOs: 12-75, SEQ ID NOs: 77-85, and SEQ ID NOs: 89-93. 【0025】 In another aspect, provided herein is an isolated polynucleotide encoding a LIGHT mutein provided herein. 【0026】 In another aspect, provided herein is an isolated vector comprising a polynucleotide encoding a LIGHT mutein. 【0027】 In another aspect, provided herein is a host cell comprising an isolated polynucleotide or an isolated vector encoding a LIGHT mutein. 【0028】 In another aspect, provided herein is a pharmaceutical composition comprising a LIGHT mutein, an isolated polynucleotide, an isolated vector, or a host cell. 【0029】 In another aspect, provided herein is the use of a LIGHT mutein, an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition in the manufacture of a therapeutic agent for diagnosing, preventing, or treating a disease disorder, or condition. 【0030】 The present disclosure provides a method of performing the same in a subject in need of diagnosis, prevention, or treatment of a disease, disorder, or condition, the method comprising administering to the subject a therapeutically effective amount of a LIGHT mutein, or an isolated polynucleotide, or an isolated vector, or a host cell, or a pharmaceutical composition. The following is a brief description of the drawings, which are presented for the purpose of illustrating exemplary embodiments disclosed herein and are not intended to limit the same. 【Brief Description of the Drawings】 【0031】 【Figure 1】 Shows an ELISA analysis of LIGHT muteins that bind to the extracellular domain of human LTβR. LIGHT-9, LIGHT-11, LIGHT-21, LIGHT-22, LIGHT-52, LIGHT-3, LIGHT-29, and LIGHT-20 exhibit stronger binding affinity than wild-type human LIGHT. The X-axis is the concentration of LIGHT protein (ng / ml), and the Y-axis is the absorbance at 450 nm. 【Figure 2】 Shows an ELISA analysis of LIGHT muteins that bind to the extracellular domain of mouse LTβR. LIGHT-3, LIGHT-9, LIGHT-11, LIGHT-29, LIGHT-22, and LIGHT-52 exhibit stronger binding affinity than wild-type human LIGHT. The X-axis is the concentration of LIGHT protein (ng / ml), and the Y-axis is the absorbance at 450 nm. 【Figure 3】 Shows an ELISA analysis of LIGHT muteins that bind to the extracellular domain of human HVEM. LIGHT-20, LIGHT-21, LIGHT-22, LIGHT-29, and LIGHT-52 exhibit stronger binding affinity than wild-type human LIGHT, while LIGHT-3, LIGHT-9, and LIGHT-11 do not bind to human HVEM. The X-axis is the concentration of LIGHT protein (ng / ml), and the Y-axis is the absorbance at 450 nm. 【Figure 4A】 Shows the alignment results of amino acids 1-60 and 61-120 of LIGHT-1 to LIGHT-90 with full-length human LIGHT, respectively. 【Figure 4B】 The alignment results of amino acids 1 - 60 and 61 - 120 of LIGHT - 1 to LIGHT - 90 with full - length human LIGHT are shown respectively. 【Figure 5A】 The alignment results of amino acids 121 - 180 and 181 - 240 of LIGHT - 1 to LIGHT - 90 with full - length human LIGHT are shown respectively. 【Figure 5B】 The alignment results of amino acids 121 - 180 and 181 - 240 of LIGHT - 1 to LIGHT - 90 with full - length human LIGHT are shown respectively. 【Figure 6】 The ELISA analysis of LIGHT mutants binding to the extracellular domain of human LTβR is shown. LIGHT - 1, LIGHT - 2, LIGHT - 58, LIGHT - 52, LIGHT - 86, and LIGHT - 88 show binding affinities similar to wild - type human LIGHT. The X - axis is the concentration (nM) of the trimeric LIGHT protein, and the Y - axis is the absorbance at 450 nm. 【Figure 7】 The ELISA analysis of LIGHT mutants binding to the extracellular domain of mouse LTβR is shown. LIGHT - 58, LIGHT - 60, LIGHT - 61, LIGHT - 86, LIGHT - 87, LIGHT - 88, and LIGHT - 89 show stronger binding affinities than wild - type human LIGHT. The X - axis is the concentration (nM) of the trimeric LIGHT protein, and the Y - axis is the absorbance at 450 nm. 【Figure 8】 The ELISA analysis of LIGHT mutants binding to the extracellular domain of human HVEM is shown. LIGHT - 58, LIGHT - 60, LIGHT - 61, and LIGHT - 52 show stronger binding affinities than wild - type human LIGHT, while LIGHT - 1, LIGHT - 2, LIGHT - 88, and LIGHT - 89 do not bind to human HVEM. The X - axis is the concentration (nM) of the trimeric LIGHT protein, and the Y - axis is the absorbance at 450 nm. 【Figure 9】Shows the ELISA analysis of LIGHT mutein binding to human DcR3 (Uniprot O95407). LIGHT-1, LIGHT-2, LIGHT-86, LIGHT-87, LIGHT-88, LIGHT-89, and LIGHT-90 show reduced binding affinity compared to wild-type human LIGHT. The X-axis is the concentration of the LIGHT protein (nM), and the Y-axis is the absorbance at 450 nm. 【Figure 10】 Shows the ELISA analysis of LIGHT-60, LIGHT-61, and LIGHT-90 binding to the extracellular domain of human LTβR. LIGHT-60, LIGHT-61, and LIGHT-90 show similar affinity for the extracellular domain of human LTβR compared to LIGHT(74~240) (LIGHTwt, LIGHT wild-type ECD). The X-axis is the concentration of the LIGHT protein (nM), and the Y-axis is the absorbance at 450 nm. 【Figure 11】 Shows the functional activation of human LTβR by LIGHT90, LIGHT60, and LIGHT63 in Hela-NK-kB reporter cells. The EC50 of each variant is shown in the table. 【Mode for Carrying Out the Invention】 【0032】 The present disclosure is described in more detail below. This description is not intended to be an exhaustive list of all the different ways in which the invention can be implemented or all the features that can be added to the invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the present disclosure and do not depart from the invention. Accordingly, the following description is intended to illustrate some particular embodiments of the invention and is not intended to comprehensively specify all permutations, combinations, and variations thereof. 【0033】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any methods and materials similar or equivalent to those described herein can be used in the practice for testing of this disclosure, but the preferred methods and materials are described herein. In the description and claims of this disclosure, the following terms are used. 【0034】 Definition As used herein, the term "naturally occurring" refers to sequences of natural origin, which means that all or a part thereof is not synthetic and is present or produced in nature. More preferably, as used herein, the term "naturally occurring" refers to sequences of natural origin, which means that the entire sequence is not synthetic and is present or produced in nature. 【0035】 The terms "mutated", "mutation", "mutant", and "variant" are used interchangeably herein. Typically and preferably, a mutation is a substitution, insertion, deletion, or combination thereof of one amino acid by one or more amino acids. More preferably, a mutation is a substitution of a single amino acid by a different single amino acid. 【0036】 The term "LIGHT" has the meaning generally understood in the art and refers to a protein expressed on activated CD4 / CD8 T cells, dendritic cells (DCs), monocytes, and natural killer (NK) cells. Binding of LIGHT to herpesvirus entry mediator (HVEM) expressed on resting T cells, DCs, and monocytes, or to lymphotoxin beta receptor (LTβR) expressed on DCs and stromal cells promotes T cell activation, proliferation, and cytokine production. The full amino acid sequence of LIGHT is shown in SEQ ID NO: 86. 【0037】 As used herein, "LIGHT mutein" means a mutein derived from the sequence set forth in SEQ ID NO: 86 that has a mutation in one or more amino acids, where the mutation is a substitution, insertion, deletion, or combination thereof of one amino acid by one or more amino acids. More preferably, the mutation is a substitution of a single amino acid by a different single amino acid. The LIGHT mutein includes at least the sequence set forth in SEQ ID NO: 87 and has one or more amino acids substituted by different single amino acids. 【0038】 As used herein, "LIGHT mutein" also includes "cleaved LIGHT mutein", which refers to a shorter LIGHT that covers the major functional regions of LIGHT without the transmembrane domain of LIGHT, compared to the naturally occurring LIGHT set forth in SEQ ID NO: 86, and includes, as examples used herein, the sequences of LIGHT 74-240 (SEQ ID NO: 87), LIGHT(87-240) (SEQ ID NO: 88), and LIGHT mutein (SEQ ID NOs: 1-85), or a cleaved LIGHT mutein that is a combination thereof. 【0039】 "LTβR" means lymphotoxin beta receptor. 【0040】 "mLTβR" means a mouse-derived lymphotoxin beta receptor, such as Uniport P50284. 【0041】 "hLTβR" means a lymphotoxin beta receptor supplied from a human, such as Uniport P36941. 【0042】 "LTβR-binding LIGHT mutein" means a LIGHT mutein protein that can bind to LTβR. 【0043】 "HVEM" means herpesvirus entry mediator. 【0044】 "mHVEM" refers to a herpesvirus entry mediator derived from a mouse, for example, Uniport Q80WM9. 【0045】 "hHVEM" refers to a herpesvirus entry mediator supplied from a human, for example, Uniport Q92956. 【0046】 "LTβR-binding and mHVEM-binding LIGHT mutant" refers to a LIGHT mutant protein that can bind to both LTβR and mouse HVEM. 【0047】 LIGHT mutant The present disclosure provides a LIGHT mutant that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 86, 87, or 88. 【0048】 In some embodiments, the LIGHT mutant provided herein is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 88. 【0049】 Homology to a sequence is well known to those skilled in the art. Methods for measuring homology to a sequence include, but are not limited to, BLAST on the NCBI website. 【0050】 The LIGHT mutant provided herein contains at least one amino acid mutation as compared to the amino acid sequence set forth in SEQ ID NO: 86, 87, or 88. 【0051】 In some embodiments, the LIGHT mutein comprises an amino acid mutation at a position selected from the group consisting of 95, 101, 102, 103, 104, 116, 117, 120, 126, 135, 136, 150, 152, 155, 156, 157, 158, 160, 161, 166, 174, 175, 184, 189, 190, 198, 202, 208, 213, 214, 220, 221, 222, 223, 227, 228, 232, and combinations thereof, the position being defined with reference to SEQ ID NO: 87. 【0052】 The position of the first amino acid of SEQ ID NO: 87 is defined as position 74. 【0053】 In some embodiments, the LIGHT mutein comprises one or more amino acid mutations selected from the group consisting of A95T, A101D, N102R, S103N, S104P, L105P, T116S, Q117E / Q117N / Q117H / Q117R, L120P / L120Q, L126M, V135I, T136S, G150A / G150S / G150R, V152M, P155R, L156P, G157S, L158Q / L158P / L158M, S160G / S160N / S160T / S160A / S160H / S160R, T161G / T161P / T161S / T161N, L166M, P174L, E175K, Q184R, R189S, A190T / A190V, W198Q, F202Y, H208Y / H208R, E213D, K214E, L220S / L220N / L220T / L220R / L220M / L220Q, D221G, E222K / E222S / E222Q / E222D / E222N, R223H, L227T / L227M, R228L, R232H, and combinations thereof, as compared to the amino acid sequence set forth in SEQ ID NO: 87. 【0054】 In the present disclosure, the mutation "A95T" means that the amino acid A at position 95 has mutated to the amino acid T. The mutation "L120P / L120Q" means the mutation L120P or L120Q. Other mutations described in the present disclosure have similar meanings. 【0055】 In the present disclosure, amino acids are represented as standard one-letter codes according to the standard IUPAC (International Union of Pure and Applied Chemistry) amino acid abbreviations. 【0056】 In some embodiments, the LIGHT mutant contains one or more amino acid mutations selected from Q117E / Q117N / Q117H, G150A / G150S, S160G / S160N / S160T / S160H / S160A, T161G / T161P / T161S / T161N, W198Q, K214E, L220S / L220N / L220T / L220M / L220R / L220Q, E222K / E222S / E222Q / E222D / E222N, R228L, and R232H. 【0057】 In some embodiments, the LIGHT mutant contains one or more amino acid mutations selected from A95T, A101D, N102R, S103N, S104P, L105P, T116S, Q117E / Q117N / Q117H / Q117R, L126M, V135I, T136S, G150A / G150S / G150R, V152M, P155R, L156P, G157S, L158Q / L158P / L158M, S160G / S160N / S160T / S160A, T161G / T161P / T161S / T161N, L166M, P174L, E175K, Q184R, R189S, A190T / A190V, W198Q, F202Y, H208Y / H208R, E213D, K214E, L220S / L220N / L220T / L220R / L220M, D221G, E222K / E222S / E222Q / E222D / E222N, L227T / L227M, R228L, and R232H. 【0058】 In some embodiments, the LIGHT mutein is a combination of mutations: V152M, W198Q, and R228L; R189S, W198Q, and R228L; G150A and L220S; V152M, T161P, and R228L; T161P, R189S, W198Q, and R228L; W198Q, L220N, and D221G; P155R, L220Q, and R232H; G150S, S160G, and L220S; G150S, T161G, and L220S; G150S, T161P, and L220S; G150S and L220S; L158Q and K214E; S104P, G157S, H208Y, and L220R; L158Q and L166M; Q117E, E175K, K214E, and L227T; Q117H, L158M, and E213D; H208Y and Q117N; L158Q, K214E, and E222K; L156P, S160G, and L220M; S160A, L220S, and E222D; S160G, T161P, and L220T; S160G, T161S, and L220S; S160G and L220S; S160G, A190T, and L220S; S160H and L220S; S160H, L220S, and E222Q; S160N, T161S, L220S, and E222D; S160N, K214E, and L227M; S160N and L220S; S160R, T161N, and L220S; S160R, T161S, and L220S; S160R and L220S; T161P and L220S; T161S and L220S; T161S, L220S, and E222N; H208Y; A190T, F202Y, and K214E; Q184R and W198Q; L158P, L166M, and L220M; L156P and H208Y; G150R, S160G, and L220S; G150R and L220S; G150R, S160T, and L220S; G150R, L158P, and L220S; G150S, S160P, T161S, L220S, and R223H; L126M and K214E; L126M, A190V, and H208Y; L120P and L220R; L105P, Q117R, and L220Q; N102R, L120Q, H208R, and K214E; A101D, S160N, and L220S; L220S; Q117N, L220S, and L227T;Q117E, L220S, and L227T; Q117N, L220T, and L227T; Q117E, L220T, and L227T; L220S and L227T; S160G, T161G, and L220S; S160G, T161P, and L220S; S160G, T161G, L220S, and E222S; S160G, T161P, L220S, and E222S; S160G, T161G, and L220T; T161G, L220S, and L227T; T161P, L220S, and L227T; S160G, L220S, and L227T; S160G, L220T, and L227T; T161S, L220S, and L227T; S160G, T161G, L220S, and L227T; S160G, T161P, L220S, and L227T; Q117N, T161G, L220S, and L227T; Q117N, T161P, L220S, and L227T; Q117N, S160G, L220S, and L227T; Q117E, T161G, L220S, and L227T; Q117N, S160G, T161G, L220S, and L227T; Q117N, S160G, T161P, L220S, and L227T; Q117E, S160G, T161P, L220S, and L227T; Q117N, S160G, T161S, L220S, and L227T; Q117E, S160G, T161S, L220S, and L227T; Q117N, S160G, T161G, L220S, E222S, and L227T; Q117N, S160G, T161P, L220S, E222S, and L227T; Q117E, S160G, T161G, L220S, E222S, and L227T; Q117E, S160G, T161P, L220S, E222S, and L227T; S160G, T161S, L220S, E222S, and L227T; Q117E, S160G, T161S, L220S, E222S, and L227T; G150R, S160R, and L220S; G150R, S160R, W198Q, L220S, and R228L; A95T, G150R, S160R, P174L, W198Q, L220S, and R228L; G150R, S160R, P174L, W198Q, and R228L; or any one of S160G, T161G, L220T, and E222S.; 【0059】 In some embodiments, the LIGHT mutein has an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence set forth in any one of SEQ ID NOs: 1-75, 77-85, and 89-93. In some embodiments, the LIGHT mutein has the amino acid sequence set forth in any one of SEQ ID NOs: 1-75, 77-85, and 89-93. 【0060】 In some embodiments, the LIGHT mutein has an amino acid sequence that differs from any one of the sequences of SEQ ID NOs: 1-75, 77-85, and 89-93 by 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acids. 【0061】 The LIGHT mutein selectively binds to the LIGHT receptor, LTβR, HVEM, or DcR3. In some embodiments, the LIGHT mutein is LTβR binding. In some embodiments, the LIGHT mutein is HVEM binding. In some embodiments, the LIGHT mutein is non-HVEM binding. 【0062】 In some embodiments, compared to wild-type LIGHT, the LIGHT mutein binds to LTβR with improved affinity. In some embodiments, compared to wild-type LIGHT, the LIGHT mutein binds to HVEM with improved affinity. In some embodiments, compared to wild-type LIGHT, the LIGHT mutein binds to DcR3 with reduced affinity. In some embodiments, compared to wild-type LIGHT, the LIGHT mutein binds to LTβR or HVEM with improved affinity and binds to DcR3 with reduced affinity. 【0063】 In some embodiments, the LIGHT mutein maintains cross-reactivity across different species. 【0064】 In some embodiments, the LTβR (e.g., human LTβR)-binding LIGHT mutein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-75, 77-85, and 89-93. 【0065】 In some embodiments, the LIGHT mutein binds to human LTβR with an EC50 value of 1000 ng / ml, 800 ng / ml, 600 ng / ml, 400 ng / ml, 200 ng / ml, 100 ng / ml, 80 ng / ml, 70 ng / ml, 60 ng / ml, or 50 ng / ml or less. In some embodiments, the LIGHT mutein binds to human LTβR with an EC50 value of 2 nM, 1.8 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM or less. 【0066】 In some embodiments, the LTβR (e.g., mouse LTβR)-binding LIGHT mutein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-75, 77-85, and 89-93. 【0067】 In some embodiments, the LIGHT mutein binds to mouse LTβR with an EC50 value of 300 ng / ml, 200 ng / ml, 100 ng / ml, 80 ng / ml, 60 ng / ml, 50 ng / ml, 40 ng / ml, or 30 ng / ml or less. In some embodiments, the LIGHT mutein binds to mouse LTβR with an EC50 value of 3 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM or less. 【0068】 In some embodiments, the HVEM (e.g., human HVEM)-binding LIGHT mutein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12-75, 77-85, and 89-93. 【0069】 In some embodiments, the LIGHT mutein binds to human HVEM with an EC50 value of 200 ng / ml, 150 ng / ml, 100 ng / ml, 80 ng / ml, 70 ng / ml, 60 ng / ml, 50 ng / ml, or 40 ng / ml or less. In some embodiments, the LIGHT mutein binds to human HVEM with an EC50 value of 10 nM, 8 nM, 7 nM, 6 nM, 3 nM, 2 nM, 1 nM, 0.8 nM, 0.7 nM, 0.6 nM, or 0.5 nM or less. 【0070】 In some embodiments, the LIGHT mutein does not bind or substantially does not bind to human HVEM. In some embodiments, the HVEM (e.g., human HVEM) non-binding LIGHT mutein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-11. 【0071】 In some embodiments, the HVEM (e.g., mouse HVEM) binding LIGHT mutein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-52. 【0072】 In some embodiments, the HVEM (e.g., mouse HVEM) non-binding LIGHT mutein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-2. 【0073】 In some embodiments, the LIGHT mutein having a reduced affinity for DcR3 (e.g., human DcR3) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 52, 58, 61, and 89-93. 【0074】 In some embodiments, the LIGHT mutein does not bind or substantially does not bind to human DcR3. In some embodiments, the LIGHT mutein binds to human DcR3 with an EC50 value exceeding 0.1 nM, 0.2 nM, 0.3 nM, or 0.5 nM. 【0075】 Improved affinity for LTβR or HVEM is beneficial for enhancing the efficacy of LIGHT mutants when preventing, treating, or diagnosing LIGHT-related diseases, disorders, or conditions. Reduced affinity for DcR3 helps minimize the toxicity caused by LIGHT-DcR3 interactions. 【0076】 The LIGHT mutants provided herein optimize efficacy while minimizing potential toxicity. 【0077】 Isolated polynucleotide Isolated polynucleotides encoding the above LIGHT mutants are encompassed within the present disclosure. Aspects of the present disclosure include polynucleotide variants (e.g., by degeneracy) encoding the amino acid sequences described herein. 【0078】 The nucleotide sequences corresponding to the amino acid sequences described herein can be obtained by "reverse translation" from the amino acid sequences. The DNA sequences encoding the LIGHT mutants can be isolated and amplified using well-known polymerase chain reaction (PCR) procedures. 【0079】 The isolated polynucleotide includes both single-stranded and double-stranded DNA and RNA, as well as corresponding complementary sequences. An "isolated nucleic acid" is, in the case of a nucleic acid isolated from a naturally occurring source, the nucleic acid separated from the adjacent gene sequences present in the genome of the organism from which the nucleic acid was isolated. In the case of a nucleic acid enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a process is understood to be an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of separate fragments or as a component of a larger nucleic acid construct. The nucleic acid molecule is preferably in a substantially pure form and in an amount or concentration that enables the identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)) and is derived from at least one isolated DNA or RNA. 【0080】 As will be understood by those skilled in the art, due to the degeneracy of the genetic code, each LIGHT mutant is encoded by a very large number of nucleic acids, each of which is within the scope of the present disclosure and can be made using standard techniques. Thus, given an identified amino acid sequence, those skilled in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the encoded protein. 【0081】 Isolated vector The present disclosure also provides an isolated vector comprising the above polynucleotide, and the isolated vector acts as an expression system in the form of a transcription or expression cassette comprising at least one of the above polynucleotides, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome. 【0082】 In some embodiments, the isolated vector also contains sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences, such as promoters, one or more enhancer sequences, origins of replication, transcription termination sequences, complete intron sequences containing donor and acceptor splice sites, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, polylinker regions for inserting nucleic acids encoding the polypeptide to be expressed, and selectable marker elements). Sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences can be obtained by any of several methods well known in the art. 【0083】 The constructed isolated vector can be inserted into a host cell suitable for amplification and / or polypeptide expression. Transformation of the expression vector into the selected host cell can be accomplished by well-known methods including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected is, in part, a function of the type of host cell used. These and other suitable methods are well known to those skilled in the art (e.g., Sambrook et al., 2001 (supra)). 【0084】 Host cell The host cells containing the isolated vectors provided herein can be eukaryotic or prokaryotic. 【0085】 In some embodiments, the host cell is a mammalian cell line. Examples of mammalian cell lines include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), and any cell line used in expression systems known in the art can be used to generate the LIGHT mutants of the present disclosure. 【0086】 Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), Chinese hamster ovary (CHO) cells, or derivatives thereof, such as Veggie CHO, and related cell lines that grow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, human fetal kidney cells, such as 293, 293 EBNA, or MSR 293. 【0087】 Pharmaceutical composition The present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of LIGHT mutein, together with a pharmaceutically acceptable carrier such as a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Pharmaceutical compositions of the present disclosure include, but are not limited to, liquid, frozen, and lyophilized compositions. 【0088】 Preferably, the pharmaceutically acceptable carrier that acts as a formulation material is non-toxic to the recipient at the dosages and concentrations used. Optimal pharmaceutical compositions are determined by those skilled in the art, for example, according to the intended route of administration, delivery format, and desired dosage. 【0089】 In some embodiments, the pharmaceutical composition can be selected for parenteral delivery. Preparation of such pharmaceutically acceptable compositions is within the scope of the art. 【0090】 In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine), antibacterial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), salt-forming counterions (such as sodium), and preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide). Pharmaceutically acceptable carriers improve the effectiveness of pharmaceutical compositions and maximize their shelf life. 【0091】 Use of LIGHT Mutant The present disclosure provides the use of LIGHT Mutant, an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition in the manufacture of a therapeutic agent (e.g., a drug) for diagnosing, preventing, or treating a disease, disorder, or condition. 【0092】 As used herein, the term "treatment" of any disease refers to alleviating or improving the disease, disorder, or condition (i.e., slowing or arresting the progression of the disease or at least one of its clinical symptoms), or alleviating or improving at least one physical parameter or biomarker associated with the disease, which may not be recognized by the patient. For cancer, "treating" can refer to weakening or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, delaying the occurrence of the tumor, or some combination thereof. 【0093】 As used herein, the term "prevention" of any disease refers to prophylactic treatment of the disease, or to delaying the onset or progression of a disease, disorder, or condition. 【0094】 In another aspect, the disclosure provides a method of doing so in a subject in need of diagnosing, preventing, or treating a disease, disorder, or condition, the method comprising administering to the subject a therapeutically effective amount of the above LIGHT mutein, or the isolated polynucleotide, or the isolated vector, or the host cell, or the pharmaceutical composition. 【0095】 The therapeutically effective amount of the LIGHT mutein or LIGHT mutein-containing pharmaceutical composition used depends, for example, on the context and purpose of the treatment. One of ordinary skill in the art will understand that the appropriate dosage level for treatment will vary in part depending on the molecule being delivered, the indication for which the LIGHT mutein is being used, the route of administration, and the size (body weight, body surface area, or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dosage and modify the route of administration to obtain an optimal therapeutic effect. 【0096】 Subject refers to mammals, primates (e.g., male or female humans), dogs, rabbits, guinea pigs, pigs, rats, and mice. In certain embodiments, the subject is a primate. In still other embodiments, the subject is a human. 【0097】 In some embodiments, the disease, disorder, or condition can be LIGHT-related, such as cancer. In some embodiments, at least one cell in the tumor or tumor microenvironment expresses a LIGHT receptor, such as LTβR, HVEM. 【0098】 Other suitable modifications and adaptations of the present application described herein will be apparent, and it will be readily apparent to those skilled in the art that they can be made using suitable equivalents without departing from the scope of the present disclosure or the embodiments disclosed herein. 【Examples】 【0099】 The following examples, both of actual and predictive embodiments, are provided for the purpose of illustrating certain embodiments or features of the present invention and are not intended to limit its scope. 【0100】 Example 1: Design and construction of phage library The phagemid pComb3XSS (#VPT4013, Creative Biogene) was engineered to display the human LIGHT (87 - 240) protein on the surface of M13 phage particles. The original sequence encoding TrxA in the pComb3XSS vector was replaced with an open reading frame encoding human LIGHT (87 - 240), a (G4S)3 linker, and the GCN4 peptide. The assembly of LIGHT as a homotrimer was stabilized by the GCN4 peptide on the phage surface. The modified construct serves as a template for library construction. 【0101】 Three libraries designated Library - 1, Library - 2, and Library - 3 were constructed (Table - 1). Library - 1 was generated using the GeneMorph II Random Mutagenesis Kit (#200550, Agilent) according to the manufacturer's protocol. The DNA fragment encoding human LIGHT (87 - 240) was subjected to 3 rounds of error - prone PCR using Mutazyme II DNA polymerase. The resulting PCR products were gel - extracted, purified, and cloned into the phage vector. Library - 2 and Library - 3 were constructed separately using mutagenic oligonucleotides designed to introduce diversity at specific residues (Q117, G150, S160, T161, P171, E175, L220, D221, E222, L227) (Table - 1). Degenerate primers with a mixture of bases (70 - 10 - 10 - 10) that favor the wild - type sequence were synthesized (Genewiz, China). The resulting PCR products were inserted into the pComb3XSS phage display vector and the reaction was transformed into XL1 - Blue cells (#DL1030, Weidi Bio, China). 【0102】 E. coli XL1 - blue cells carrying phagemid were further infected with M13KO7 helper phage (New England Biolabs, N0315SVIAL) and incubated overnight at 30 °C in 2YT medium supplemented with 50 μg / ml of ampicillin and 50 μg / ml of kanamycin. Phage was precipitated from the culture medium using 20% PEG and 2.5 M NaCl and then resuspended in 0.5 mL of PBS supplemented with 20% glycerol. The phage concentration was determined using the following formula: Phage concentration = (OD 269 - OD 320 ) × 5 × 10 12 cfu / ml (cfu: colony forming unit). 【0103】 【Table 1】 * The position is defined with reference to SEQ ID NO: 87, and the position of the first amino acid of SEQ ID NO: 87 is defined as position 74. 【0104】 Example 2: Phage panning and ELISA analysis Two panning strategies were implemented using a phage library. A) The aim was to obtain mutants that could bind to HVEM and LTβR. hHVEM, mHVEM, hLTβR, and mLTβR were used as targets for performing consecutive phage selections. B) The aim was to identify mutants that maintained LTβR binding while reducing HVEM binding. For the first 3 rounds of panning, hLTβR and mLTβR were used as targets. In the 4th round, phage that did not bind to plates coated with human and mouse - HVEM were used for subsequent positive selection using hLTβR and mLTβR. 【0105】 To reduce DcR3 binding, phage particles from Strategies A and B were subjected to an additional round of negative selection using plates coated with human DcR3 protein. 【0106】 Phage panning HVEM and LTβR proteins containing extracellular domains fused to an Fc fragment at the C-terminus were obtained from SinoBiological (#10567-M03S), Novoprotein (#CX78), and Acrobiosystems (#HVM-H5258 and #LTR-H5251). Recombinant DcR3 protein was generated by linking human DcR3 residues 33 - 300 to the N-terminus of rabbit Fc. The recombinant protein was expressed in expi293F cells (A14527, ThermoFisher) and affinity purified using protein A resin as previously described. 【0107】 To screen for the desired variants, 96-well immunoplates were coated overnight at 4°C with the antigens listed in Tables 2-1 and 2-2 (5 μg / ml). The next morning, the plates were blocked with 2% BSA (bovine serum albumin, Sangon Biotech China) for 2 hours at room temperature. Phage solution (1×10 11 phage) was added to the coated immunoplates and incubated for 1 hour at room temperature. The plates were washed 5 times with PBST (PBS, 0.5% Tween® 20) and 5 times with PBS. Bound phage were eluted by adding 50 μl / well of 100 mM glycine (pH 2.2) for 5 minutes. The eluate was transferred to a new tube and neutralized by adding 1 / 10 volume of 1 M Tris buffer (pH 8.5). The eluted phage were amplified in E. coli XL1-Blue and used for further rounds of selection. 【0108】 【Table 2-1】 【0109】 【Table 2-2】 【0110】 Phage ELISA Individual clones from the fourth selection round were grown for 5 hours at 37 °C and 220 rpm in 96-well deep well blocks in 100 μl of 2YT broth supplemented with 50 μg / ml ampicillin. Then, 5 μl of helper phage was added to each well and incubated at 37 °C for 30 minutes. After centrifugation at 3000 rpm for 10 minutes, the cells were resuspended and grown overnight in 150 μl / well of 2YT broth supplemented with 50 μg / ml ampicillin and 50 μg / ml kanamycin. The next day, the supernatant was collected by centrifugation at 3000 rpm for 20 minutes at 4 °C and used in an ELISA assay to screen for phage-displayed LIGHT mutants that bind to the antigen but not to BSA. 【0111】 For ELISA screening, 96-well ELISA plates were coated overnight at 4 °C with the target protein (1 μg / ml in PBS), and then blocked with 2% BSA for 2 hours at room temperature. The supernatant containing phage particles was added to the plates and incubated for 1 hour at room temperature. After incubation, the plates were washed 5 times with PBST. Anti-M13 antibody conjugated to HRP (horseradish peroxidase) (Sino Biological, China) was added to the wells and incubated for 1 hour at room temperature. After a further round of washing 5 times with PBST, 50 μl of TMB (3,3’,5,5”-tetramethylbenzidine, #34029, ThermoFisher) was added to each well. After 5 minutes, the reaction was stopped by adding 50 μl of stop solution (#C1058, Solarbio, China). Absorbance at 450 nM (OD 450) was measured. Wells coated with BSA were used as negative controls. Phage clones having an ELISA score ratio (target / BSA) exceeding 3 were regarded as positive clones. 【0112】 Example 3: Sequence analysis of positive clones. Phagemids were extracted from XL1-Blue cells that produced positive phage clones (BioSune, China) and sequenced. A total of 52 unique sequences with 1 to 5 amino acid mutations were identified when compared with the wild-type human LIGHT sequence (SEQ ID NO: 87) (SEQ ID NOs: 1 to 52, Table 3). Of these sequences, 11 mutants were derived from the HVEM depletion group and were found not to bind to the HVEM protein. The remaining mutants showed cross-reactivity to human and mouse HVEM and LTβR receptors (Table 3). 【0113】 Example 4: NGS sequencing and LIGHT variant design by machine learning For further affinity improvement through deep sequencing, phagemids were isolated from phages that could bind to the target protein and phages that showed non-specific binding to BSA. Subsequently, segments of the LIGHT protein were PCR amplified and purified (DC301, Vazyme, China). Amplicons were prepared using the VATHS Universal DNA Library Prep Kit (Vazyme #ND607-01) according to the standard library preparation protocol. The library ligated to the adapter was subjected to 1 cycle of PCR and then sequenced on an Illumina Miseq system using paired-end 300 bp reads to cover the full length of the amplicon. Using the sequencing results, high-affinity clones for the receptor were predicted by comparing the frequency of a given mutation at specific positions in the enriched sample against the negative control. A total of 33 LIGHT mutants (Table 3) (SEQ ID NOs: 53-85) containing 3 to 7 mutation sites were expressed and further characterized. The amino acid mutations and positions of all LIGHT mutants (SEQ ID NOs: 1-85 and 89-93) are summarized in Table 4 (see Figures 4 and 5). The LIGHT mutants, LIGHT86-89 (SEQ ID NOs: 89-93), were identified through DcR3-depleted phage panning. 【0114】 【Table 3-1】 【0115】 【Table 3-2】 【0116】 【Table 3-3】 【0117】 【Table 3-4】 【0118】 【Table 3-5】 【0119】 【Table 3-6】 【0120】 【Table 3-7】 【0121】 【Table 3-8】 +: Strong binding, -: No binding, + / -: Weak binding, N.A: Unavailable / data not available 【0122】 【Table 4】 【0123】 Example 5: Expression and purification of LIGHT mutein A DNA fragment of the human LIGHT variant was synthesized by Genewiz and cloned into a pCI vector (E1731, Promega) with an N-terminal His tag. The construct was transfected into Expi293F cells (A14527, ThermoFisher) and cultured in suspension for about 5 days. The supernatant was collected by centrifugation at 7000 RPM for 20 minutes at 4°C and filtered through a 0.22 μm filter. Subsequently, the filtered supernatant was incubated with magnetic nickel-NTA beads (Ni Smart Beads 6FF, Smart-Lifesciences, China) for 1 hour. After washing with 10 CV (column volume) of PBS, the LIGHT mutein was eluted using PBS containing 300 mM imidazole and 0.3 M NaCl. Finally, the eluted protein was dialyzed against PBS (pH 6.5) and 5% glycerol. 【0124】 Example 6: Affinity of LIGHT Mutants with HVEM and LTβR A Nunc MaxiSorp 96-well plate (Thermofisher) was coated with the target protein (1 μg / ml) and then blocked with 2% BSA-PBS buffer for 1 hour. Serial dilutions of the LIGHT mutants were added at a maximum concentration of 200 ng / ml. The plate was incubated for 1 hour, washed 4 times with PBST, and then incubated with a mouse anti-His tag antibody (#105327, Sino Biological) for 1 hour. The plate was further incubated with a goat anti-mouse secondary antibody conjugated to HRP (#SSA006, Sino Biological), washed 3 times with PBST, and treated with a TMB substrate (#34029, ThermoFisher). The plate was read at 450 nM on a SpectraMax M5 microplate reader (Molecule Devices). The ELISA results were analyzed using GraphPad Prism 9.0 software, and the EC50 (half maximal effective concentration) was summarized in Table 5-1 and Table 5-2. 【0125】 【Table 5-1】 【0126】 Most of the LIGHT mutants showed higher affinity for LTβR (both human and mouse) than wild-type human LIGHT (74-240). Some LIGHT mutants, such as LIGHT-1, LIGHT-3, LIGHT-9, and LIGHT-11, had reduced affinity for human HVEM compared to the wild type (Figures 1-3, Figures 6-8, and Figure 10). 【0127】 In addition to HVEM and LTβR, LIGHT also interacts with the decoy receptor, DcR3, which lacks transmembrane and cytoplasmic segments. This interaction has the potential to disrupt signaling pathways by sequestering LIGHT from HVEM and LTβR. DcR3 expression is typically low in healthy human tissues but is often significantly upregulated in cancer patients (Wu et al., 2003; Yoo et al., 2022). 【0128】 To test whether the mutein could bind to DcR3, a recombinant DcR3 protein was generated by linking human DcR3 residues 33 - 300 to the N-terminus of rabbit Fc. The recombinant protein was expressed in Expi293F and affinity purified using protein A beads as previously described. The human DcR3 protein was immobilized on a MaxiSorp 96-well ELISA plate (Thermo Fisher) at a concentration of 0.5 μg / mL and then blocked with 2% BSA-PBS buffer for 1 hour. LIGHT variants were added to the plate at various dilutions with a maximum concentration of 10 nM. The plate was incubated for 1 hour, washed 4 times with PBST, and then incubated with a mouse anti-His tag antibody (#105327, Sino Biological) for 1 hour. The plate was further incubated with a goat anti-mouse secondary antibody conjugated to HRP (#SSA006, Sino Biological), washed 3 times with PBST, and treated with TMB substrate (#34029, ThermoFisher). The plate was read at 450 nM on a SpectraMax M5 microplate reader (Molecule Devices). ELISA results were analyzed using GraphPad Prism 9.0 software, and the EC50 (half maximal effective concentration) was summarized in Table 5-2. 【0129】 【Table 5-2】 【0130】 As shown in Table 5-2 and FIG. 9, the affinities of LIGHT-1, LIGHT-2, LIGHT-86, LIGHT-87, LIGHT-88, and LIGHT-89 for DcR3 were observed to be reduced. 【0131】 Example 7: Functional Assay The HeLa-NF-κB reporter cell line was generated by transfecting cells with the pNL3.2.NF-κB-RE[NlucP / NF-κB-RE / Hygro] vector (Promega #N1111) using Lipofectamine 3000. After 3 days, the cells were treated with hygromycin B (Sigma) and cultured at 37 °C and 5% CO2 for 14 days. The obtained HeLa-NF-κB reporter cells were used to evaluate the downstream signaling of LTβR activation by treatment with the LIGHT mutants. The HeLa-NF-κB reporter cells were exposed to serial dilutions of the LIGHT mutants. After 24 hours, the cells were lysed (Promega #E397A), and luciferase activity was measured using Promega #E4500 with a SpectraMax M5 microplate reader (Molecule Devices). 【0132】 As shown in FIG. 11, the hLTβR-stimulating activities of LIGHT90, LIGHT60, and LIGHT63 were consistent with the binding affinities. 【0133】 Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are not presently anticipated or might not be anticipated may occur to the applicant or other persons skilled in the art. Accordingly, the appended claims, as filed and as they may be amended, are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

[Claim 1] LIGHT mutain selected from the group consisting of proteins that have greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to the sequence described in SEQ ID NO: 87, 86, or 88, and that have at least one amino acid difference compared to the amino acid sequence described in SEQ ID NO: 86, 87, or 88. [Claim 2] LIGHT mutain according to claim 1, selected from the group consisting of proteins having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology to the sequence from the position of SEQ ID NO: 87, and having at least one amino acid difference compared to the amino acid sequence described in SEQ ID NO:

87. [Claim 3] It contains one or more amino acid mutations compared to the amino acid sequence described in Sequence ID No.

87. The amino acid mutation is selected from one or more positions selected from the group consisting of 152, 198, 228, 95, 101, 102, 103, 104, 116, 117, 120, 126, 135, 136, 150, 155, 156, 157, 158, 160, 161, 166, 174, 175, 184, 189, 190, 202, 208, 213, 214, 220, 221, 222, 223, 227, 232, and combinations thereof. The LIGHT mutain according to claim 1, wherein the aforementioned position is defined with reference to Sequence ID No. 87, and the position of the first amino acid in Sequence ID No. 87 is defined as position 74. [Claim 4] The one or more amino acid mutations mentioned above are selected from the group consisting of W198Q, R228L, Q117E / Q117N / Q117H, G150A / G150S, S160G / S160N / S160T / S160H / S160A, T161G / T161P / T161S / T161N, K214E, L220S / L220N / L220T / L220M / L220R / L220Q, E222K / E222S / E222Q / E222D / E222N, R232H, and combinations thereof. The LIGHT mutain according to claim 3, wherein the aforementioned position is defined with reference to Sequence ID No. 87, and the position of the first amino acid in Sequence ID No. 87 is defined as position 74. [Claim 5] The one or more amino acid mutations mentioned above are V152M, W198Q, R228L, A95T, A101D, N102R, S103N, S104P, L105P, T116S, Q117E / Q117N / Q117H / Q117R, L126M, V135I, T136S, G150A / G150S / G150R, P155R, L156P, G157S, L158Q / L158P / L158M, S160G / S160N / S160T / S160A, T161 Selected from the group consisting of G / T161P / T161S / T161N, L166M, P174L, E175K, Q184R, R189S, A190T / A190V, F202Y, H208Y / H208R, E213D, K214E, L220S / L220N / L220T / L220R / L220M, D221G, E222K / E222S / E222Q / E222D / E222N, L227T / L227M, R232H, and combinations thereof, The LIGHT mutain according to claim 3, wherein the aforementioned position is defined with reference to Sequence ID No. 87, and the position of the first amino acid in Sequence ID No. 87 is defined as position 74. [Claim 6] The LIGHT mutein is a combination of mutations: V152M, W198Q, and R228L; R189S, W198Q, and R228L; G150A and L220S; V152M, T161P, and R228L; T161P, R189S, W198Q, and R228L; W198Q, L220N, and D221G; P155R, L220Q, and R232H; G150S, S160G, and L220S; G150S, T161G, and L220S; G150S, T161P, and L220S; G150S and L220S; L158Q and K214E ;S104P, G157S, H208Y, and L220R;L158Q and L166M;Q117E, E175K, K214E, and L227T;Q117H, L158M, and E213D;H208Y and Q117N;L158Q, K214E, and E222K;L156P, S160G, and L220M;S160A, L220S, and E222D;S160G, T161P, and L220T;S160G, T161S, and L220S;S160G and L220S;S160G, A190T, and L220S;S160H and L220S;S 160H, L220S, and E222Q; S160N, T161S, L220S, and E222D; S160N, K214E, and L227M; S160N and L220S; S160R, T161N, and L220S; S160R, T161S, and L220S; S160R and L220S; T161P and L220S; T161S and L220S; T161S, L220S, and E222N; H208Y; A190T, F202Y, and K214E; Q184R and W198Q; L158P, L166M, and L220M; L156P and H20 8Y; G150R, S160G, and L220S; G150R and L220S; G150R, S160T, and L220S; G150R, L158P, and L220S; G150S, S160P, T161S, L220S, and R223H; L126M and K214E; L126M, A190V, and H208Y; L120P and L220R; L105P, Q117R, and L220Q; N102R, L120Q, H208R, and K214E; A101D, S160N, and L220S; L220S; Q117N, L220S, and L227T;Q117E, L220S, and L227T; Q117N, L220T, and L227T; Q117E, L220T, and L227T; L220S and L227T; S160G, T161G, and L220S; S160G, T161P, and L220S; S160G, T161G, L220S, and E222S; S160G, T161P, L220S, and E222S; S160G, T161G, and L220T; T161G, L220S, and L227T; T161P, L220S, and L227T; S160G, L220S, and L227T; S160G, L220T, and L227T; T161S, L220S, and L227T; S160G, T161G, L220S, and L227T; S160G, T161P, L220S, and L227T; Q117N, T161G, L220S, and L227T; Q117N, T161P, L220S, and L227T; Q117N, S160G, L220S, and L227T; Q117E, T161G, L220S, and L227T; Q117N, S160G, T161G, L220S, and L227T; Q1 17N, S160G, T161P, L220S, and L227T; Q117E, S160G, T161P, L220S, and L227T; Q117N, S160G, T161S, L220S, and L227T; Q117E, S160G, T161S, L220S, and L227T; Q117N, S160G, T161G, L220S, E222S, and L227T; Q117N, S160G, T161P, L220S, E222S, and L227T; Q117E, S160G, T161G, L220S, E222S, and L22 7T; Q117E, S160G, T161P, L220S, E222S, and L227T; S160G, T161S, L220S, E222S, and L227T; Q117E, S160G, T161S, L220S, E222S, and L227T; G150R, S160R, and L220S; G150R, S160R, W198Q, L220S, and R228L; A95T, G150R, S160R, P174L, W198Q, L220S, and R228L; G150R, S160R, P174L, W198Q, and R228L;Or, the LIGHT Mutein according to claim 3, comprising any one of S160G, T161G, L220T, and E222S. [Claim 7] LIGHT mutain according to claim 1, comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% homology to any one of the amino acid sequences described in SEQ ID NOs: 1-75, 77-85, and 89-93. [Claim 8] LTβR-binding LIGHT mutain containing the amino acid sequence described in any one of SEQ ID NOs: 1-75, 77-85, and 89-93. [Claim 9] The LTβR-bound LIGHT mutain according to claim 8, wherein the LIGHT mutain is HVEM-unbound and comprises an amino acid sequence described in any one of SEQ ID NOs: 1 to 11, or the LIGHT mutain is HVEM-bound and comprises an amino acid sequence described in any one of SEQ ID NOs: 12 to 75, SEQ ID NOs: 77 to 85, and SEQ ID NOs: 89 to 93. [Claim 10] The LTβR-binding LIGHT mutain according to claim 8, wherein the LIGHT mutain binds to DcR3 with reduced affinity compared to wild-type LIGHT, and the LIGHT mutain comprises an amino acid sequence described in any one of SEQ ID NOs: 1, 2, 52, 58, 61, and 89-93. [Claim 11] An isolated polynucleotide encoding the LIGHT mutaine described in claim 1. [Claim 12] An isolated vector comprising the polynucleotide described in claim 11. [Claim 13] A host cell comprising the isolated polynucleotide described in claim 11. [Claim 14] A pharmaceutical composition comprising LIGHT mutein as described in claim 1 and a pharmaceutically acceptable carrier. [Claim 15] Use of LIGHT mutaine according to claim 1 in the manufacture of a therapeutic agent for diagnosing, preventing, or treating a disease, disorder, or condition. [Claim 16] A composition comprising the LIGHT mutain described in Claim 1, for use in a subject requiring diagnosis, prevention, or treatment of a disease, disorder, or condition.

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