LIGHT mutant protein and application thereof

Abstract

The invention provides an LIGHT mutant protein and an LT [beta] R binding LIGHT mutant protein, and also provides a related polynucleotide, an isolated vector, a host cell and a pharmaceutical composition. Furthermore, the use of the LIGHT mutant protein or the isolated polynucleotide, the isolated vector, the host cell or the pharmaceutical composition in the preparation of a medicament for preventing or treating a disease, and a method of preventing or treating a disease in a subject in need thereof are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to PCT application number PCT / CN2022 / 097735 filed on June 8, 2022. The contents of the prior PCT application are considered part of this disclosure and are incorporated herein in their entirety.

[0003] Indexing of sequence listings

[0004] The sequence listing named DCF230337WO-Sequencelisting.xml, created on June 6, 2023 and 95,646 bytes in size, is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to a LIGHT mutant protein (or "LIGHT mutant"), an isolated vector comprising a polynucleotide encoding the LIGHT mutant protein, a host cell comprising the isolated polynucleotide encoding the LIGHT mutant protein or the isolated vector, a pharmaceutical composition comprising the LIGHT mutant protein, use of the LIGHT mutant protein, the isolated polynucleotide, the isolated vector, the host cell or the pharmaceutical composition in the preparation of a medicament for preventing or treating a disease, and a method for preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the LIGHT mutant protein or the isolated polynucleotide, the isolated vector, the host cell or the pharmaceutical composition. Background Art

[0006] The documents in this section are only for the purpose of providing background information related to the present disclosure and the information presented in this section does not necessarily constitute prior art.

[0007] LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediators, receptor expressed by T cells) is known as tumor necrosis factor superfamily member 14 (TNFSF14), also known 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, especially immature dendritic cells (DCs) and activated T cells. The membrane-anchored form of LIGHT can be cleaved by proteases to generate a soluble functional structure (Yu et al., 2004).

[0008] LIGHT has three receptors: herpes virus invasion 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, stromal, immature DCs, and myeloid cells, but not on lymphocytes. The LIGHT-LTβR interaction leads to 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 triggered by LIGHT (Liu et al., 2021). Introducing LIGHT into tumors or the tumor microenvironment may be an effective strategy for cancer immunotherapy.

[0009] We have engineered human LIGHT to selectively interact with its receptor with the goal of utilizing the recombinant LIGHT protein as a therapeutic agent. Summary of the invention

[0010] For the above purposes, a novel LIGHT mutant protein is provided herein. In some embodiments, the LIGHT mutant protein has a sequence shown in SEQ ID NO: 87 or SEQ ID NO: 88 or SEQ ID NO: 1-85 or SEQ ID NO: 89-93.

[0011] In some embodiments, provided herein is a LIGHT mutant protein having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 86. In some embodiments, provided herein is a LIGHT mutant protein having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 87. In some embodiments, provided herein is a LIGHT mutant protein having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 88. In some embodiments, provided herein is a LIGHT mutant protein having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 1-85 or SEQ ID NO: 89-93.

[0012] Provided herein is a novel LIGHT mutant protein, the novel LIGHT mutant protein is selected from the group consisting of proteins having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 87. In some embodiments, the novel LIGHT mutant protein comprises one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 86. The amino acid mutation is selected from one or more positions, the one or more positions are 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 a combination of any one of them, wherein the position is defined with reference to SEQ ID NO: 86.

[0013] In some embodiments, the LIGHT mutant protein comprises one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 87. The amino acid mutation is selected from one or more positions, and the one or more positions are 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 a combination of any one of them, wherein 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.

[0014] In some embodiments, the LIGHT mutant protein comprises one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 88. The amino acid mutation is selected from one or more positions, and the one or more positions are 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 a combination of any one of them, wherein the position is 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.

[0015] In some embodiments, provided herein is a LIGHT mutant protein having a sequence as shown in SEQ ID No. 86, SEQ ID No. 87 or SEQ ID No. 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 / T 161N, 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.

[0016] In some embodiments, provided herein is a LIGHT mutant protein having a sequence as shown 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 a combination of any one of them, wherein 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.

[0017] In some embodiments, provided herein is a LIGHT mutant protein having a sequence shown 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 / T 161P / 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 a combination of any one of them, wherein the position is 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.

[0018] In some embodiments, provided herein is a LTβR binding LIGHT mutant protein, the LTβR binding LIGHT mutant protein is selected from the group consisting of LIGHT mutant proteins having sequences shown in SEQ ID NO: 1-75, SEQ ID NO: 76-85 and SEQ ID NO: 89-93. Or the mutant protein is selected from a protein having a sequence shown in SEQ ID NO: 87 or SEQ ID NO: 88 and having at least one amino acid difference compared to SEQ ID NO: 87 or SEQ ID NO: 88.

[0019] In some embodiments, the LTβR-binding LIGHT mutein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-11 is HVEM (eg, human HVEM) non-binding.

[0020] In some embodiments, the LTβR-binding LIGHT mutant protein is HVEM-binding, and the LIGHT mutant protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-75, SEQ ID NOs: 77-85, and SEQ ID NOs: 89-93.

[0021] In some embodiments, the LTβR binding LIGHT mutant protein binds to DcR3 with reduced affinity compared to wild-type LIGHT, and the LIGHT mutant protein comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 61 and SEQ ID NO: 89-93.

[0022] In some embodiments, the present invention provides a LTβR binding LIGHT mutant protein, which is a mHVEM (mouse HVEM) binding protein and a hHVEM (human HVEM) non-binding protein. For example, the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or SEQ ID NO: 11.

[0023] In some embodiments, provided herein is a human LTβR binding LIGHT mutant protein, which is selected from the group consisting of LIGHT mutant proteins having sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 37, SEQ ID NO: 52, and SEQ ID NO: 53-85.

[0024] In some embodiments, provided herein is a mouse LTβR binding LIGHT mutant protein, the mouse LTβR binding LIGHT mutant protein is selected from the LIGHT mutant protein having a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 38 and SEQ ID NO: 52. In some embodiments, provided herein is a LTβR binding and hHVEM binding LIGHT mutant protein, the LTβR binding and hHVEM binding LIGHT mutant protein is selected from the LIGHT mutant protein having a sequence as shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 37 and SEQ ID NO: 51. In some embodiments, provided herein is a LTβR-binding and hHVEM-non-binding LIGHT mutant protein selected from the group consisting of LIGHT mutant proteins having sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 11.

[0025] In some embodiments, provided herein is a truncated form of LTβR binding to LIGHT and a mutant protein thereof, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 85. In some embodiments, provided herein is a truncated form of LTβR binding to and mHVEM binding to LIGHT and a mutant protein thereof, which comprises an amino acid sequence as shown in any one of SEQ ID NOs 1 to 85. In some embodiments, provided herein is a truncated form of LTβR binding to and mHVEM binding to LIGHT and a mutant protein thereof, which is hHVEM non-binding, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-11.

[0026] In some embodiments, provided herein is a LTβR-binding and HVEM-binding LIGHT mutant protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 12-75, SEQ ID NOs: 77-85, and SEQ ID NOs: 89-93.

[0027] In another aspect, provided herein is an isolated polynucleotide encoding a LIGHT mutant protein provided herein.

[0028] In another aspect, provided herein is an isolated vector comprising a polynucleotide encoding a LIGHT mutant protein.

[0029] In another aspect, provided herein is a host cell comprising an isolated polynucleotide or an isolated vector encoding a LIGHT mutant protein.

[0030] In another aspect, provided herein is a pharmaceutical composition comprising a LIGHT mutant protein, an isolated polynucleotide, an isolated vector or a host cell.

[0031] In another aspect, provided herein is a use of a LIGHT mutant protein or an isolated polynucleotide or an isolated vector or a host cell or a pharmaceutical composition in the preparation of a therapeutic agent for diagnosing, preventing or treating a disease disorder or condition.

[0032] The present disclosure provides a method of diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a LIGHT mutant protein, an isolated polynucleotide, an isolated vector, a host cell or a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following is a brief description of the drawings, which are presented for the purpose of illustrating the exemplary embodiments disclosed herein and not for the purpose of limiting these exemplary embodiments.

[0034] Figure 1 ELISA analysis of LIGHT mutant proteins binding to the extracellular domain of human LTβR is shown. LIGHT-9, LIGHT-11, LIGHT-21, LIGHT-22, LIGHT-52, LIGHT-3, LIGHT-29, and LIGHT-20 showed 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.

[0035] Figure 2 ELISA analysis of LIGHT mutant proteins binding to the mouse LTβR extracellular domain is shown. LIGHT-3, LIGHT-9, LIGHT-11, LIGHT-29, LIGHT-22, and LIGHT-52 showed 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.

[0036] Figure 3ELISA analysis of LIGHT mutant proteins binding to the extracellular domain of human HVEM is shown. LIGHT-20, LIGHT-21, LIGHT-22, LIGHT-29 and LIGHT-52 showed stronger binding affinity than wild-type human LIGHT, while LIGHT-3, LIGHT-9 and LIGHT-11 did 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.

[0037] Figure 4A and 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.

[0038] Figure 5A and 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.

[0039] Figure 6 ELISA analysis of LIGHT mutant proteins binding to the extracellular domain of human LTβR is shown. LIGHT-1, LIGHT-2, LIGHT-58, LIGHT-52, LIGHT-86 and LIGHT-88 showed similar binding affinity to wild-type human LIGHT. The X-axis is the concentration of trimeric LIGHT protein (nM), and the Y-axis is the absorbance at 450 nm.

[0040] Figure 7 ELISA analysis of LIGHT mutant proteins binding to the mouse LTβR extracellular domain is shown. LIGHT-58, LIGHT-60, LIGHT-61, LIGHT-86, LIGHT-87, LIGHT-88, and LIGHT-89 showed stronger binding affinity than wild-type human LIGHT. The X-axis is the concentration of trimeric LIGHT protein (nM), and the Y-axis is the absorbance at 450 nm.

[0041] Figure 8 ELISA analysis of LIGHT mutant proteins binding to the extracellular domain of human HVEM is shown. LIGHT-58, LIGHT-60, LIGHT-61 and LIGHT-52 showed stronger binding affinity than wild-type human LIGHT, while LIGHT-1, LIGHT-2, LIGHT-88 and LIGHT-89 did 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.

[0042] Fig. 9 ELISA analysis of LIGHT mutant proteins binding to human DcR3 (Uniprot O95407) is shown. LIGHT-1, LIGHT-2, LIGHT-86, LIGHT-87, LIGHT-88, LIGHT-89, and LIGHT-90 showed reduced binding affinity compared to wild-type human LIGHT. The X-axis is the concentration of LIGHT protein (nM), and the Y-axis is the absorbance at 450 nm.

[0043] Fig.10 ELISA analysis of LIGHT-60, LIGHT-61 and LIGHT-90 binding to the human LTβR extracellular domain is shown. LIGHT-60, LIGHT-61 and LIGHT-90 showed similar affinity to the human LTβR extracellular domain compared to LIGHT (74-240) (LIGHTwt, LIGHT wild-type ECD). The X-axis is the concentration of LIGHT protein (nM), and the Y-axis is the absorbance at 450 nm.

[0044] Fig.11 Functional activation of human LTßR by LIGHT90, LIGHT60 and LIGHT63 in Hela-NK-kB reporter cells is shown. The EC50 for each variant is shown in the table. DETAILED DESCRIPTION

[0045] The present disclosure is explained in more detail below. This description is not intended to be an exhaustive list of all the different ways in which the present invention may be implemented or all the features that may be added to the present invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a specific embodiment may be deleted from that embodiment. In addition, multiple variations and additions to the various embodiments proposed herein will be apparent to those skilled in the art, in accordance with the present disclosure without departing from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can also be used in the practice of the testing of the present disclosure, preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terms will be used.

[0047] definition

[0048] As used herein, the term "naturally occurring" refers to a sequence of natural origin, which means that all or part of it is not synthetic and exists or occurs in nature. More preferably, as used herein, the term "naturally occurring" refers to a sequence of natural origin, which means that the entire sequence is not synthetic and exists or occurs in nature.

[0049] The terms "mutated", "mutation", "mutant" and "mutant" are used interchangeably herein. Typically and preferably, a mutation is a substitution, insertion, deletion or combination thereof of one amino acid with one or more amino acids. More preferably, a mutation is a substitution of a single amino acid with a different single amino acid.

[0050] The term "LIGHT" has a 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 cells (NKs). Binding of LIGHT to herpes virus invasion 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 complete amino acid sequence of LIGHT is shown in SEQ ID NO: 86.

[0051] As used herein, "LIGHT mutant protein" means a mutant protein derived from the sequence shown in SEQ ID NO: 86, which has a mutation in one or more amino acids, wherein the mutation is one amino acid replaced, inserted, deleted or a combination thereof by one or more amino acids. More preferably, the mutation is a single amino acid replaced by a different single amino acid. The LIGHT mutant protein comprises at least the sequence shown in SEQ ID NO: 87, and has one or more amino acids replaced by different single amino acids.

[0052] As used herein, "LIGHT mutant protein" also includes "truncated form of LIGHT mutant protein", and "truncated form of LIGHT mutant protein" refers to LIGHT that is shorter than the naturally occurring LIGHT shown in SEQ ID NO: 86, and the shorter LIGHT covers the main functional region of LIGHT but does not have the transmembrane domain of LIGHT. As the example used herein, the truncated form of LIGHT mutant protein comprises the sequence of LIGHT 74-240 (SEQ ID NO: 87), LIGHT (87-240) (SEQ ID NO: 88) and LIGHT mutant protein (SEQ ID NO: 1-85) or a combination thereof.

[0053] "LT[beta]R" means lymphotoxin beta receptor.

[0054] "mLTβR" means mouse derived lymphotoxin beta receptor, eg, Uniport P50284.

[0055] "hLTβR" refers to lymphotoxin β receptor derived from human, such as Uniport P36941.

[0056] "LTβR-binding LIGHT mutant protein" means a LIGHT mutant protein that can bind to LTβR.

[0057] "HVEM" means herpes virus entry mediator.

[0058] "mHVEM" means mouse-derived herpesvirus entry mediator, eg, Uniport Q80WM9.

[0059] "hHVEM" means herpes simplex virus entry mediator of human origin, for example Uniport Q92956.

[0060] "LTβR-binding and mHVEM-binding LIGHT mutant protein" means a LIGHT mutant protein that can bind to both LTβR and mouse HVEM.

[0061] LIGHT mutant protein

[0062] The present disclosure provides a LIGHT mutant protein, which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88.

[0063] In some embodiments, the LIGHT mutant proteins provided herein are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 88.

[0064] Homology to a sequence is well known to those skilled in the art. Methods for determining homology to a sequence include, but are not limited to, BLAST on the NCBI website.

[0065] Compared to the amino acid sequence shown in SEQ ID NO: 86, SEQ ID NO: 87 or SEQ ID NO: 88, the LIGHT mutant protein provided herein includes at least one amino acid mutation.

[0066] In some embodiments, the LIGHT mutant protein includes 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, wherein the position is defined with reference to SEQ ID NO: 87.

[0067] The position of the first amino acid of SEQ ID NO: 87 is defined as position 74.

[0068] In some embodiments, the LIGHT mutant protein 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, compared to the amino acid sequence shown in SEQ ID NO: 87. / 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.

[0069] In the present disclosure, the mutation "A95T" means that the amino acid A at position 95 is 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.

[0070] In this disclosure, amino acids are referred to by the standard single-letter codes according to the standard IUPAC (International Union of Pure and Applied Chemistry) amino acid abbreviations.

[0071] In some embodiments, the LIGHT mutant protein includes 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.

[0072] In some embodiments, the LIGHT mutant protein includes 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 / T one or more amino acid mutations of 161P / 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.

[0073] In some embodiments, the LIGHT mutant protein includes any one of the following mutation combinations: 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, L220 S 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 E2 22D; 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, L158 P 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; A1 01D, 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, T 161P, 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; Q11 7N, 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; S160 G, 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 S160G, T161G, L220T, and E222S. ;

[0074] In some embodiments, the LIGHT mutant protein has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 75, SEQ ID NOs: 77 to 85, and SEQ ID NOs: 89 to 93. In some embodiments, the LIGHT mutant protein has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 75, SEQ ID NOs: 77 to 85, and SEQ ID NOs: 89 to 93.

[0075] In some embodiments, the LIGHT mutant protein has an amino acid sequence that differs from any one of SEQ ID NOs: 1 to 75, SEQ ID NOs: 77 to 85, and SEQ ID NOs: 89 to 93 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0076] The LIGHT mutant protein selectively binds to the LIGHT receptor, LTβR, HVEM or DcR3. In some embodiments, the LIGHT mutant protein is LTβR-bound. In some embodiments, the LIGHT mutant protein is HVEM-bound. In some embodiments, the LIGHT mutant protein is HVEM-unbound.

[0077] In some embodiments, the LIGHT mutant protein binds to LTβR with improved affinity compared to wild-type LIGHT. In some embodiments, the LIGHT mutant protein binds to HVEM with improved affinity compared to wild-type LIGHT. In some embodiments, the LIGHT mutant protein binds to DcR3 with reduced affinity compared to wild-type LIGHT. In some embodiments, the LIGHT mutant protein binds to LTβR or HVEM with improved affinity and binds to DcR3 with reduced affinity compared to wild-type LIGHT.

[0078] In some embodiments, the LIGHT muteins retain cross-reactivity across different species.

[0079] In some embodiments, the LTβR (eg, human LTβR) binding LIGHT mutein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-75, SEQ ID NOs: 77-85, and SEQ ID NOs: 89-93.

[0080] In some embodiments, the EC50 value of LIGHT mutant protein binding to human LTßR is no more than 1000ng / ml, 800ng / ml, 600ng / ml, 400ng / ml, 200ng / ml, 100ng / ml, 80ng / ml, 70ng / ml, 60ng / ml or 50ng / ml. In some embodiments, the EC50 value of LIGHT mutant protein binding to human LTßR is no more than 2nM, 1.8nM, 1.5nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM or 0.4nM.

[0081] In some embodiments, the LTβR (eg, mouse LTβR) binding LIGHT mutant protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-75, SEQ ID NOs: 77-85, and SEQ ID NOs: 89-93.

[0082] In some embodiments, the EC50 value of LIGHT mutant protein binding to mouse LTßR is no more than 300ng / ml, 200ng / ml, 100ng / ml, 80ng / ml, 60ng / ml, 50ng / ml, 40ng / ml or 30ng / ml. In some embodiments, the EC50 value of LIGHT mutant protein binding to mouse LTßR is no more than 3nM, 2nM, 1.5nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM or 0.4nM.

[0083] In some embodiments, the HVEM (eg, human HVEM) binding LIGHT mutant protein comprises the 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.

[0084] In some embodiments, the EC50 value of LIGHT mutant protein binding to human HVEM is no more than 200ng / ml, 150ng / ml, 100ng / ml, 80ng / ml, 70ng / ml, 60ng / ml, 50ng / ml or 40ng / ml. In some embodiments, the EC50 value of LIGHT mutant protein binding to human HVEM is no more than 10nM, 8nM, 7nM, 6nM, 3nM, 2nM, 1nM, 0.8nM, 0.7nM, 0.6nM or 0.5nM.

[0085] In some embodiments, the LIGHT mutant protein does not bind to human HVEM or does not substantially bind to human HVEM.In some embodiments, the HVEM (eg, human HVEM) non-binding LIGHT mutant protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-11.

[0086] In some embodiments, the HVEM (eg, mouse HVEM) binding LIGHT mutant protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-52.

[0087] In some embodiments, the HVEM (eg, mouse HVEM) non-binding LIGHT mutant protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-2.

[0088] In some embodiments, a LIGHT mutant protein with reduced affinity for DcR3 (eg, human DcR3) comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-2, SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 61, and SEQ ID NOs: 89-93.

[0089] In some embodiments, the LIGHT mutant protein does not bind to human DcR3 or does not substantially bind to human DcR3. In some embodiments, the LIGHT mutant protein binds to human DcR3 with an EC50 value of no more than 0.1 nM, 0.2 nM, 0.3 nM or 0.5 nM.

[0090] Improved affinity for LTβR or HVEM is beneficial in enhancing the efficacy of LIGHT mutant proteins when preventing, treating or diagnosing LIGHT-related diseases, disorders or conditions. Reduced affinity for DcR3 helps minimize toxicity caused by LIGHT-DcR3 interactions.

[0091] The LIGHT muteins provided herein optimize efficacy while minimizing potential toxicity.

[0092] Isolated polynucleotides

[0093] Contemplated within the present disclosure are isolated polynucleotides encoding the above-described LIGHT mutant proteins.Aspects of the present disclosure include polynucleotide variants (eg, due to degeneracy) encoding the amino acid sequences described herein.

[0094] The nucleotide sequence corresponding to the amino acid sequence described herein can be obtained by "reverse translation" from the amino acid sequence.The well-known polymerase chain reaction (PCR) method can be used to isolate and amplify the DNA sequence encoding the LIGHT mutant protein.

[0095] The polynucleotide of separation includes DNA and RNA in both single-stranded form and double-stranded form, and corresponding complementary sequence.In the case of nucleic acid separation from naturally occurring sources, "isolated nucleic acid" is a nucleic acid separated from the adjacent genetic sequence present in the genome of the organism from which nucleic acid is separated.For example, in the case of enzymatic synthesis or chemical synthesis of nucleic acid from a template, such as PCR product, cDNA molecule or oligonucleotide, it should be understood that the nucleic acid produced from such process is an isolated nucleic acid.Isolated nucleic acid molecules refer to nucleic acid molecules in the form of isolated fragments or nucleic acid molecules as components of larger nucleic acid constructs.Nucleic acid molecules are preferably derived from DNA or RNA, which is separated at least once in a substantially pure form and in an amount or concentration that enables identification, manipulation and recovery of its constituent nucleotide sequences by standard biochemical methods (such as those summarized in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

[0096] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, each LIGHT mutant protein is encoded by a very large number of nucleic acids, each of which is within the scope of the present disclosure and can be prepared using standard techniques. Thus, having identified a specific amino acid sequence, one skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein.

[0097] Separated vector

[0098] The present disclosure also provides an isolated vector comprising the above polynucleotide, which serves as an expression system in the form of a plasmid, phagemid, phage, baculovirus, cosmid or artificial chromosome, transcription or expression cassette comprising at least one of the above polynucleotides.

[0099] In some embodiments, the isolated vector also contains sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences, such as a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a marker element capable of selection. The 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.

[0100] The constructed isolated vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. The transformation of the expression vector to the selected host cell can be accomplished by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, liposome infection, DEAE-dextran-mediated transfection or other known techniques. The selected method will depend in part on the type of host cell to be used. These methods and other suitable methods are well known to the technician, for example in Sambrook et al., 2001, supra.

[0101] Host cells

[0102] Host cells containing the isolated vectors provided herein can be eukaryotic or prokaryotic.

[0103] In some embodiments, the host cell is a mammalian cell line. Mammalian cell lines include, but are not limited to, immortalized cell lines purchased from the American Type Culture Collection (ATCC), and any cell line used in expression systems known in the art can be used to prepare the LIGHT mutant proteins of the present disclosure.

[0104] 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 their derivatives (such as VeggieCHO ​​and related cell lines grown in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31)), HeLa cells, human embryonic kidney cell lines (such as 293, 293 EBNA or MSR 293).

[0105] Pharmaceutical composition

[0106] The present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a LIGHT mutant protein and a pharmaceutically acceptable carrier, such as a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. The pharmaceutical composition of the present disclosure includes but is not limited to a liquid composition, a frozen composition and a lyophilized composition.

[0107] Preferably, pharmaceutically acceptable carriers used as formulation materials are non-toxic to recipients at the dosages and concentrations employed. The optimal pharmaceutical composition will be determined by one skilled in the art based on, for example, the intended route of administration, delivery form, and desired dosage form.

[0108] In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0109] In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide). Pharmaceutically acceptable carriers improve the effect of the pharmaceutical composition and maximize the shelf life of the pharmaceutical composition.

[0110] Uses of LIGHT mutant proteins

[0111] The present disclosure provides uses of LIGHT mutant proteins, isolated polynucleotides, isolated vectors, host cells or pharmaceutical compositions in the preparation of therapeutic agents (eg, drugs) for diagnosing, preventing or treating diseases, disorders or conditions.

[0112] As used herein, the term "treating" any disease refers to alleviating or ameliorating the disease, disorder or condition (i.e., slowing or arresting the development of at least one of the disease or its clinical symptoms); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease, including those that may not be discernible by the patient. For cancer, "treating" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation or metastasis, or some combination thereof. For tumors, "treating" includes resection of all or part of a tumor, inhibiting or slowing tumor growth and metastasis, delaying the development of a tumor, or some combination thereof.

[0113] As used herein, the term "preventing" any disease refers to prophylactic treatment of a disease; or delaying the onset or progression of a disease, disorder, or condition.

[0114] In another aspect, the present disclosure provides a method for diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-mentioned LIGHT mutant protein, isolated polynucleotide, isolated vector, host cell or pharmaceutical composition.

[0115] The therapeutically effective amount of the LIGHT mutant protein or pharmaceutical composition containing the LIGHT mutant protein to be used will depend on, for example, the treatment context and purpose. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the delivered molecule, the indication for which the LIGHT mutant protein is being used, the route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and general health). In certain embodiments, the clinician can titrate the dose and modify the route of administration to obtain the best therapeutic effect.

[0116] A subject refers to a mammal, a primate (e.g., a human, male or female), a dog, a rabbit, a guinea pig, a pig, a rat, and a mouse. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

[0117] In some embodiments, the disease, disorder or condition may be LIGHT related, such as cancer. In some embodiments, at least one cell in a tumor or tumor microenvironment expresses a LIGHT receptor, eg, LTβR, HVEM.

[0118] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the present application described herein will be apparent, and suitable equivalents may be used, without departing from the scope of the disclosure or embodiments disclosed herein.

[0119] Example

[0120] The following examples, both actual and hypothetical, are provided to illustrate specific embodiments or features of the invention and are not intended to limit the scope of the invention.

[0121] Example 1: Phage library design and construction

[0122] Phagemid pComb3XSS (#VPT4013, Creative Biogene) was engineered to display human LIGHT (87-240) protein on the surface of M13 phage particles. The original sequence encoding TrxA of the pComb3XSS vector was replaced by an open reading frame encoding human LIGHT (87-240), a (G4S)3 linker, and a GCN4 peptide. The assembly of LIGHT as a homotrimer is stabilized by the GCN4 peptide on the phage surface. The modified construct was used as a template for library construction.

[0123] Three libraries were constructed, referred to as library-1, library-2, and library-3 (Table-1). Library-1 was generated using the GeneMorph II random mutagenesis kit (#200550, Agilent) according to the manufacturer's protocol. Three rounds of error-prone PCR were performed on the DNA fragment encoding human LIGHT (87-240) using Mutazyme II DNA polymerase. The resulting PCR products were gel extracted, purified, and cloned into a phage vector. Library-2 and library-3 (Table-1) were constructed using mutagenic oligonucleotides designed to introduce diversity at specific residues (Q117, G150, S160, T161, P171, E175, L220, D221, E222, L227), respectively. Degenerate primers with a base mixture (70-10-10-10) that favored the wild-type sequence were synthesized (Genewiz, China). The resulting PCR products were inserted into the pComb3XSS phage display vector, and the reactions were transformed into XL1-Blue cells (#DL1030, Weidi Bio, China).

[0124] E. coli XL1-blue cells carrying the phagemid were further infected with M13KO7 helper phage (New England Biolabs, N0315SVIAL) and incubated overnight at 30°C in 2 YT medium supplemented with 50µg / ml ampicillin and 50µg / ml kanamycin. Phages were precipitated from the culture medium using 20% ​​PEG and 2.5M NaCl and then resuspended in 0.5ml PBS supplemented with 20% glycerol. The concentration of phage was determined using the following formula: Phage concentration = (OD 269 -OD 320 )×5×10 12 cfu / ml (cfu: colony forming units).

[0125] Table-1. Phage library design .

[0126]

[0127] *wherein 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.

[0128] Example 2: Phage panning and ELISA analysis

[0129] Two panning strategies were implemented using phage libraries. A) The goal was to obtain mutants that could bind to HVEM and LTβR. Sequential phage selections were performed using hHVEM, mHVEM, hLTβR, and mLTβR as targets. B) The goal was to identify mutant proteins that maintained LTβR binding while reducing HVEM binding. For the first three rounds of panning, hLTβR and mLTβR were used as targets. In the fourth round, phage that did not bind to plates coated with human HVEM and mouse HVEM were used in subsequent positive selections using hLTβR and mLTβR.

[0130] To reduce DcR3 binding, phage particles from strategy A and strategy B were subjected to an additional round of negative selection using plates coated with human DcR3 protein.

[0131] Phage panning

[0132] HVEM and LTβR proteins include an extracellular domain fused to an Fc fragment at the C-terminus and are available from SinoBiological (#10567-M03S), Novoprotein (#CX78), and Acrobiosystems (#HVM-H5258 and #LTR-H5251). Recombinant DcR3 protein was produced by linking human DcR3 residues 33-300 to the N-terminus of rabbit Fc. Recombinant proteins were expressed in expi293F cells (A14527, ThermoFisher) and affinity purified using protein A resin as described previously.

[0133] To screen the desired mutants, 96-well immunoplates were coated with the antigens (5 µg / ml) listed in Tables 2-1 and 2-2 at 4°C overnight. 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 immunoplate and incubated for 1 hour at room temperature. The plate was washed five times with PBST (PBS, 0.5% Tween 20) and five times with PBS. Bound phage were eluted by adding 50µl / well of 100mM glycine (pH 2.2) for 5 minutes. The eluate was transferred to a new tube and neutralized by adding 1 / 10 volume of 1M Tris buffer (pH 8.5). The eluted phage was amplified in E. coli XL1-Blue and used for further selection rounds.

[0134] Table 2-1. Phage panning strategy A and target proteins in different rounds .

[0135]

[0136] Table 2-2. Phage panning strategy B and target proteins in different rounds .

[0137]

[0138] Phage ELISA

[0139] Individual clones from the fourth round of selection were grown in 96-well deep well blocks in 100µl 2YT broth supplemented with 50µg / ml ampicillin at 37°C and 220rpm for 5 hours. Then, 5µl of helper phage was added to each well and incubated at 37°C for 30 minutes. After centrifugation at 3,000rpm for 10 minutes, the cells were resuspended and grown overnight in 150µl / well 2YT broth supplemented with 50µg / ml ampicillin and 50µg / ml kanamycin. The next day, the supernatant was collected by centrifugation at 3,000rpm for 20 minutes at 4°C and used for ELISA assay to screen phage-displayed LIGHT mutant proteins that bound to the antigen but not to BSA.

[0140] For ELISA screening, 96-well ELISA plates were coated with target protein (1 µg / ml in PBS) overnight at 4°C and subsequently blocked with 2% BSA for 2 h at room temperature. The supernatant containing phage particles was added to the plate and incubated for 1 h at room temperature. After incubation, the plate was washed five times with PBST. HRP (horseradish peroxidase)-conjugated anti-M13 antibody (SinoBiological, China) was added to the wells and incubated for 1 h at room temperature. After another round of five washes with PBST, 50 µl of TMB (3,3',5,5''-tetramethylbenzidine, #34029, ThemoFisher) was added to each well. After 5 min, the reaction was stopped by adding 50 µl of stop solution (#C1058, Solarbio, China). The absorbance at 450 nM (OD 450 Wells coated with BSA were used as negative controls. Phage clones with an ELISA score ratio (target / BSA) > 3 were considered positive clones.

[0141] Example 3: Sequence analysis of positive clones

[0142] Phagemids were extracted from XL1-Blue cells that produced positive phage clones (BioSune, China) and sequenced. A total of 52 unique sequences (SEQ ID NO: 1-52, Table 3) with 1 to 5 amino acid mutations compared to the wild-type human LIGHT sequence (SEQ ID NO: 87) were identified. Among these sequences, 11 mutants were derived from the HVEM deletion group and were found to not bind to the HVEM protein. The remaining mutants showed cross-reactivity to human and mouse HVEM and LTβR receptors (Table 3).

[0143] Example 4: NGS sequencing and LIGHT mutant design by machine learning

[0144] In order to further improve affinity by deep sequencing, phagemids were isolated from phages that could bind to the target protein and phages that showed non-specific binding to BSA. The fragments of the LIGHT protein were then PCR amplified and purified (DC301, Vazyme, China). The VATHS Universal DNA Library Preparation Kit (Vazyme #ND607-01) was used to prepare amplicons according to the standard library preparation protocol. The adapter-connected library was subjected to a single cycle of PCR and then sequenced using paired-end 300bp reads on the Illumina Miseq system to cover the entire length of the amplicon. The sequencing results were used to predict high-affinity clones for the receptor by comparing the frequency of a given mutation at a specific position in the enriched sample with a negative control. A total of 33 LIGHT mutants containing 3 to 7 mutation sites (Table 3) (SEQ ID NO: 53-85) were expressed and further characterized. The amino acid mutations and positions of all LIGHT mutant proteins (SEQ IN NO: 1-85 and SEQ IN NO: 89-93) are summarized in Table 4 (see Figures 4 and 5). The LIGHT mutant proteins, LIGHT86-89 (SEQ IN NO: 89-93), were identified by DcR3 deletion phage panning.

[0145] Table 3. LIGHT mutant proteins from phage panning and machine learning

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] +: strong binding; -: no binding; + / -: weak binding; NA: not available / no data

[0158] Table 4. Amino acid mutations and mutated positions

[0159]

[0160] Example 5: Expression and purification of LIGHT mutant protein

[0161] DNA fragments of human LIGHT mutants were synthesized by Genewiz and cloned into pCI vector (E1731, Promega) with an N-terminal His tag. The constructs were transfected into Expi293F cells (A14527, ThermoFisher) and cultured in suspension culture for about five days. The supernatant was harvested by centrifugation at 7,000RPM for 20 minutes at 4°C and filtered through a 0.22µm 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 10CV (column volume) of PBS, the LIGHT mutant protein was eluted using PBS containing 300mM imidazole and 0.3M NaCl. Finally, the eluted protein was dialyzed with PBS (pH 6.5) and 5% glycerol.

[0162] Example 6: Affinity of LIGHT mutant protein to HVEM and LTβR

[0163] Nunc MaxiSorp 96-well plates (Thermofisher) were coated with target protein (1 µg / ml) and then blocked with 2% BSA-PBS buffer for one hour. Serial dilutions of LIGHT mutant proteins were added with a maximum concentration of 200 ng / ml. The plates were incubated for one hour, washed four times with PBST, and then incubated with mouse anti-His tag antibody (#105327, SinoBiological) for 1 hour. The plates were further incubated with HRP-conjugated goat anti-mouse secondary antibody (#SSA006, SinoBiological), then washed three times with PBST and treated with TMB substrate (#34029, ThermoFisher). The plates were read at 450 nM on a SpectraMax M5 microplate reader (Molecule Devices). ELISA results were analyzed using GraphPadPrism 9.0 software, and EC50 (half maximal effective concentration) is summarized in Table 5-1 and Table 5-2.

[0164] Table 5-1. Calculated EC50 results of LIGHT mutant proteins .

[0165]

[0166]

[0167] Most LIGHT mutant proteins showed higher affinity for LTβR (both human and mouse) than wild-type human LIGHT (74-240). Some LIGHT mutant proteins, such as LIGHT-1, LIGHT-3, LIGHT-9, and LIGHT-11, had reduced affinity for human HVEM compared to wild-type ( Figure 1-3 , Figure 6-8 and Fig.10 ).

[0168] In addition to HVEM and LTβR, LIGHT also interacts with the decoy receptor DcR3, which lacks a transmembrane segment and a cytoplasmic segment. This interaction has the potential to disrupt signaling pathways by sequestering LIGHT from HVEM and LTßR. Although DcR3 expression is usually low in healthy human tissues, it is often significantly upregulated in cancer patients (Wu et al., 2003; Yoo et al., 2022).

[0169] To test whether the mutant proteins can bind to DcR3, recombinant DcR3 proteins were generated by linking human DcR3 residues 33-300 to the N-terminus of rabbit Fc. Recombinant proteins were expressed in Expi293F and affinity purified using protein A beads as described previously. Human DcR3 proteins were immobilized on maxiSorp 96-well ELISA plates (Thermo Fisher) at a concentration of 0.5 µg / mL and subsequently blocked with 2% BSA-PBS buffer for one hour. LIGHT variants were added to the plates at various dilutions with a maximum concentration of 10 nM. The plates were incubated for one hour, washed four times with PBST, and then incubated with mouse anti-His tag antibody (#105327, Sino Biological) for 1 hour. The plates were further incubated with HRP-conjugated goat anti-mouse secondary antibody (#SSA006, SinoBiological), then washed three times with PBST and treated with TMB substrate (#34029, ThermoFisher). The plates were read at 450 nM on a SpectraMax M5 microplate reader (Molecule Devices). ELISA results were analyzed using GraphPadPrism 9.0 software and the EC50 (half maximal effective concentration) is summarized in Table 5-2.

[0170] Table 5-2. EC50 (nM) for LIGHT binding to LTßR, HVEM and human DcR3 .

[0171]

[0172] As shown in Table 5-2 and Fig. 9 As shown, it has been observed that LIGHT-1, LIGHT-2, LIGHT-86, LIGHT-87, LIGHT-88, and LIGHT-89 have reduced affinity for DcR3.

[0173] Example 7: Functional assay

[0174] 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. Three days later, the cells were treated with hygromycin B (Sigma) and cultured for 14 days at 37°C, 5% CO2. The resulting HeLa-NF-κB reporter cells were used to evaluate downstream signaling of LTβR activation by LIGHT mutant protein treatment. HeLa-NF-κB reporter cells were exposed to serial dilutions of LIGHT mutant proteins. After 24 hours, cells were lysed (Promega #E397A) and luciferase activity was measured using Promega #E4500 by a SpectraMax M5 microplate reader (Molecule Devices).

[0175] like Fig.11 As shown, LIGHT90, LIGHT60, and LIGHT63 stimulated hLTßR activity consistent with the binding affinity.

[0176] Although specific embodiments have been described, the applicant or other persons skilled in the art may think of alternatives, modifications, variations, improvements, and substantial equivalents that are not currently foreseeable or may not be foreseeable. Therefore, the appended claims submitted and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. A LIGHT mutant protein, wherein the LIGHT mutant protein is selected from the group consisting of proteins having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 86, SEQ ID NO: 87 or SEQ ID NO: 88, and has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO: 86, SEQ ID NO: 87 or SEQ ID NO:

88.

2. The LIGHT mutant protein according to claim 1, wherein the LIGHT mutant protein is selected from the group consisting of proteins having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology with the sequence from the position of SEQ ID NO: 87, and has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:

87.

3. The LIGHT mutant protein according to claim 1 or 2, comprising one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 87, wherein the amino acid mutation is selected from one or more positions 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, 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.

4. The LIGHT mutant protein according to any one of claims 1 to 3, wherein the one or more amino acid mutations are selected from the group consisting of 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, R232H and combinations 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.

5. The LIGHT mutant protein according to any one of claims 1 to 4, wherein the one or more amino acid mutations are 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 / S 160A, 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 a 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.

6. The LIGHT mutant protein according to any one of claims 1 to 5, wherein the LIGHT mutant protein comprises any one of the following mutation combinations: 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; 61P 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; T 161S 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, S16 0T 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, H208 R 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 L22 7T; 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; 1S, 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 S160G, T161G, L220T and E222S.

7. The LIGHT mutant protein according to any one of claims 1 to 6, comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence shown in any one of SEQ ID NOs: 1-75, SEQ ID NOs: 77-85 and SEQ ID NOs: 89-93.

8. A LTβR-binding LIGHT mutant protein, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-75, SEQ ID NOs: 77-85 and SEQ ID NOs: 89-93. 9 . The LTβR-binding LIGHT mutant protein according to claim 8 , wherein the LIGHT mutant protein is HVEM non-binding, and the LIGHT mutant protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-11.

10. The LTβR-binding LIGHT mutant protein according to claim 8 or 9, wherein the LIGHT mutant protein is HVEM-binding, and the LIGHT mutant protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-75, SEQ ID NOs: 77-85 and SEQ ID NOs: 89-93.

11. The LTβR-binding LIGHT mutant protein according to any one of claims 8 to 10, wherein the LIGHT mutant protein binds to DcR3 with reduced affinity compared to wild-type LIGHT, and the LIGHT mutant protein comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 61 and SEQ ID NO: 89-93.

12. An isolated polynucleotide encoding the LIGHT mutant protein according to any one of claims 1 to 11.

13. An isolated vector comprising the polynucleotide according to claim 12.

14. A host cell comprising the isolated polynucleotide according to claim 12 or the isolated vector according to claim 13.

15. A pharmaceutical composition comprising the LIGHT mutant protein according to any one of claims 1 to 11, the isolated polynucleotide according to claim 12, the isolated vector according to claim 13, or the host cell according to claim 14, and a pharmaceutically acceptable carrier.

16. Use of the LIGHT mutant protein according to any one of claims 1 to 11, or the isolated polynucleotide according to claim 12, or the isolated vector according to claim 13, or the host cell according to claim 14, or the pharmaceutical composition according to claim 15 in the preparation of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

17. A method for diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a LIGHT mutant protein according to any one of claims 1 to 11, or an isolated polynucleotide according to claim 12, or an isolated vector according to claim 13, or a host cell according to claim 14, or a pharmaceutical composition according to claim 15.

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