Engineered 4-1BBL variants and methods of use thereof

Abstract

The present disclosure relates to engineered 4-1BBL variants and methods of use thereof.

Description

[0001] Cross - Reference to Related Applications

[0002] This disclosure claims the priority and benefit of U.S. Provisional Patent Application Serial No. 63 / 420,400, filed Oct. 28, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been submitted electronically as an XML file named 52246-0010WO1_ST26_SL.XML. The XML file was created on Oct. 24, 2023, and is 112,677 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0005] This disclosure relates to engineered 4-1BBL variants and methods of using the same. BACKGROUND ART

[0006] The immune system can distinguish normal cells in the body from those that are considered “foreign,” which allows the immune system to attack foreign cells while leaving normal cells alone. This mechanism sometimes involves proteins called immune checkpoints. Immune checkpoints are molecules that enhance signals (co-stimulatory molecules) or attenuate signals in the immune system.

[0007] The interaction between 4-1BB and 4-1BBL provides co-stimulatory signals for a variety of T cells, and these co-stimulatory signals can be used to discover cancer immunotherapies. The 4-1BB / 4-1BBL complex, together with the signal provided by the T cell receptor, can provide co-stimulatory signals to CD4+ and CD8+ T cells in mice, resulting in the activation of CD4+ and CD8+ T cells. The activation of CD8+ T cells is crucial in anti-tumor immunity. Thus, targeting 4-1BB / 4-1BBL contributes to cancer immunotherapy. However, some anti-4-1BBL antibodies (e.g., urelumab) can induce hepatotoxicity. Therefore, it is necessary to develop cancer therapies targeting the 4-1BB / 4-1BBL pathway with limited toxicity. SUMMARY OF THE INVENTION

[0008] The present disclosure relates to engineered 4-1BBL variants, protein complexes, and methods of using the same. The variants or protein complexes can be used to target the 4-1BB / 4-1BBL pathway. Results show that some variants and protein complexes can effectively bind to 4-1BB-expressing cells (e.g., T cells) and induce the activity of 4-1BB-mediated downstream signal transduction pathways (e.g., NFκB activity). Additionally, the protein complexes can stimulate the activation, proliferation, and cytokine release of 4-1BB-expressing immune cells (e.g., T cells). In particular, the protein complexes do not overinduce cytokine release that may lead to hepatotoxicity, which has been observed with the anti-4-1BB antibody urelumab.

[0009] Accordingly, the protein complexes described herein can be used for cancer treatment, and these protein complexes have a 4-1BB binding affinity that is similar to or higher than, and an agonistic ability to activate the 4-1BB / 4-1BBL signal transduction pathway that is similar to or lower than. In some embodiments, the protein complexes have an enhanced T cell binding ability compared to wild-type 4-1BBL.

[0010] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:2 or SEQ ID NO:59. In some embodiments, the engineered 4-1BBL polypeptide comprises one or more amino acid mutations at the AA' loop, CD loop, and / or GH loop. In some embodiments, the amino acid corresponding to S62 of SEQ ID NO:2 is E, T, P, A, N, T, or H. In some embodiments, the engineered 4-1BBL polypeptide described herein comprises one or more of the following: (a) the amino acid corresponding to G106 of SEQ ID NO:2 is Q, K, H, R, F, or S; and (b) the amino acid corresponding to E107 of SEQ ID NO:2 is T, Q, A, R, L, M, S, or I. In some embodiments, the engineered 4-1BBL polypeptide described herein comprises one or more of the following: (a) the amino acid corresponding to A176 of SEQ ID NO:2 is S or Q; (b) the amino acid corresponding to W177 of SEQ ID NO:2 is L, M, or F; (c) the amino acid corresponding to L179 of SEQ ID NO:2 is F, A, or M; (d) the amino acid corresponding to T180 of SEQ ID NO:2 is R, S, A, or E; and (e) the amino acid corresponding to A183 of SEQ ID NO:2 is Q, R, or K. In some embodiments, the engineered 4-1BBL polypeptide described herein comprises one or more of the following: (a) the amino acid corresponding to W60 of SEQ ID NO:2 is F; and (b) the amino acid corresponding to P64 of SEQ ID NO:2 is N. In some embodiments, the amino acid corresponding to L100 of SEQ ID NO:2 is V. In some embodiments, the engineered 4-1BBL polypeptide described herein further comprises one or more of the following: (a) the amino acid corresponding to S18 of SEQ ID NO:2 is I; and (b) the amino acid corresponding to L98 of SEQ ID NO:2 is V.In some embodiments, the engineered 4-1BBL polypeptides described herein comprise one or more of the following: (a) the amino acid corresponding to position 61 of SEQ ID NO:2 is Y; (b) the amino acid corresponding to position 63 of SEQ ID NO:2 is D; (c) the amino acid corresponding to position 65 of SEQ ID NO:2 is G; (d) the amino acid corresponding to position 66 of SEQ ID NO:2 is L; (e) the amino acid corresponding to position 101 of SEQ ID NO:2 is R; (f) the amino acid corresponding to position 102 of SEQ ID NO:2 is R; (g) the amino acid corresponding to position 103 of SEQ ID NO:2 is V; (h) the amino acid corresponding to position 104 of SEQ ID NO:2 is V; (i) the amino acid corresponding to position 105 of SEQ ID NO:2 is A; (j) the amino acid corresponding to position 178 of SEQ ID NO:2 is Q; and (k) the amino acid corresponding to position 181 of SEQ ID NO:2 is Q. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 85%, 90%, 95% or 100% identical to SEQ ID NO:2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 85%, 90%, 95% or 100% identical to SEQ ID NO:59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93.

[0011] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide that comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 6, 59 or 60. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO:2 is E; (b) the amino acid corresponding to E107 of SEQ ID NO:2 is T; and (c) the amino acid corresponding to W177 of SEQ ID NO:2 is L. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO:6 or 60.

[0012] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 7, 59, or 61, and in some embodiments, the amino acid corresponding to T180 of SEQ ID NO: 2 is R. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 7 or 61.

[0013] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 8, 59, or 62, and in some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is T; and (b) the amino acid corresponding to T180 of SEQ ID NO: 2 is S. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 8 or 62.

[0014] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 9, 59, or 63, and in some embodiments, the amino acid corresponding to G106 of SEQ ID NO: 2 is Q. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 or 63.

[0015] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 10, 59, or 64, and in some embodiments, the amino acid corresponding to T180 of SEQ ID NO: 2 is A. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 10 or 64.

[0016] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 11, 59, or 65, and in some embodiments, the amino acid corresponding to T180 of SEQ ID NO: 2 is E. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 11 or 65.

[0017] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 12, 59, or 66. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to G106 of SEQ ID NO: 2 is K; and (b) the amino acid corresponding to A183 of SEQ ID NO: 2 is Q. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 or 66.

[0018] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 13, 59, or 67. In some embodiments, the amino acid corresponding to S62 of SEQ ID NO: 2 is P. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 13 or 67.

[0019] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 14, 59, or 68. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to E107 of SEQ ID NO: 2 is Q; and (b) the amino acid corresponding to A183 of SEQ ID NO: 2 is R. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 14 or 68.

[0020] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 15, 59, or 69. In some embodiments, the amino acid corresponding to L98 of SEQ ID NO: 2 is V. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 15 or 69.

[0021] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 16, 59, or 70. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to G106 of SEQ ID NO: 2 is H; and (b) the amino acid corresponding to T180 of SEQ ID NO: 2 is A. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 16 or 70.

[0022] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 17, 59, or 71. In some embodiments, the amino acid corresponding to E107 of SEQ ID NO: 2 is A. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 17 or 71.

[0023] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 18, 59, or 72. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is A; and (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is Q. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or 72.

[0024] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 19, 59, or 73. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is N; and (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is Q. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or 73.

[0025] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 20, 59, or 74. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is T; and (b) the amino acid corresponding to A183 of SEQ ID NO: 2 is R. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 20 or 74.

[0026] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 21, 59, or 75. In some embodiments, the amino acid corresponding to S62 of SEQ ID NO: 2 is H. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 21 or 75.

[0027] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 22, 59, or 76. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is P; (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is R; and (c) the amino acid corresponding to L179 of SEQ ID NO: 2 is F. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 22 or 76.

[0028] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 23, 59, or 77. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to E107 of SEQ ID NO: 2 is L; and (b) the amino acid corresponding to W177 of SEQ ID NO: 2 is M. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 23 or 77.

[0029] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide that comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 24, 59, or 78. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is P; (b) the amino acid corresponding to G106 of SEQ ID NO: 2 is R; and (c) the amino acid corresponding to W177 of SEQ ID NO: 2 is M. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 24 or 78.

[0030] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide that comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 25, 59, or 79. In some embodiments, the amino acid corresponding to S18 of SEQ ID NO: 2 is I. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 or 79.

[0031] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide that comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 26, 59, or 80. In some embodiments, the amino acid corresponding to L179 of SEQ ID NO: 2 is A. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 26 or 80.

[0032] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide that comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 27, 59, or 81. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is A; and (b) the amino acid corresponding to L100 of SEQ ID NO: 2 is V. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 27 or 81.

[0033] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 28, 59, or 82. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to W60 of SEQ ID NO: 2 is F; (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is T; and (c) the amino acid corresponding to L179 of SEQ ID NO: 2 is M. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 28 or 82.

[0034] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 29, 59, or 83. In some embodiments, the amino acid corresponding to E107 of SEQ ID NO: 2 is Q. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 29 or 83.

[0035] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 30, 59, or 84. In some embodiments, the amino acid corresponding to A183 of SEQ ID NO: 2 is K. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 30 or 84.

[0036] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 31, 59, or 85. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to W60 of SEQ ID NO: 2 is F; and (b) the amino acid corresponding to G106 of SEQ ID NO: 2 is R. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or 85.

[0037] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 32, 59, or 86. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is T; (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is M; and (c) the amino acid corresponding to A176 of SEQ ID NO: 2 is S. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 32 or 86.

[0038] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 33, 59, or 87. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is A; and (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is S. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 33 or 87.

[0039] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 34, 59, or 88. In some embodiments, the amino acid corresponding to A176 of SEQ ID NO: 2 is Q. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 34 or 88.

[0040] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 35, 59, or 89. In some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is P; (b) the amino acid corresponding to G106 of SEQ ID NO: 2 is F; and (c) the amino acid corresponding to A176 of SEQ ID NO: 2 is S. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 35 or 89.

[0041] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 36, 59, or 90, and in some embodiments, the amino acid corresponding to E107 of SEQ ID NO: 2 is S. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 36 or 90.

[0042] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 37, 59, or 91, and in some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to G106 of SEQ ID NO: 2 is S; and (b) the amino acid corresponding to W177 of SEQ ID NO: 2 is F. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 37 or 91.

[0043] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 38, 59, or 92, and in some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to P64 of SEQ ID NO: 2 is N; and (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is I. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 38 or 92.

[0044] In one aspect, the present disclosure relates to an engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 39, 59, or 93, and in some embodiments, the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is P; and (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is A. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 39 or 93.

[0045] In some embodiments, the engineered 4-1BBL polypeptide further comprises a CH2 domain and a CH3 domain. In some embodiments, the engineered 4-1BBL polypeptide further comprises a hinge region. In some embodiments, the CH2 domain is an IgG CH2 domain, and the CH3 domain is an IgG CH3 domain. In some embodiments, the engineered 4-1BBL polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 41 to 58.

[0046] In one aspect, the present disclosure relates to a protein construct comprising the engineered 4-1BBL polypeptide described herein. In some embodiments, the protein construct described herein comprises two or more engineered 4-1BBL polypeptides. In some embodiments, at least two of the engineered 4-1BBL polypeptides are the same. In some embodiments, at least two of the engineered 4-1BBL polypeptides are different. In some embodiments, the protein construct described herein further comprises an Fc region. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region (e.g., having an LALA mutation or an LALA-PG mutation). In some embodiments, the engineered 4-1BBL polypeptide is optionally linked to the C-terminus of the Fc region via a linker peptide. In some embodiments, the engineered 4-1BBL polypeptide is optionally linked to the N-terminus of the Fc region via a linker peptide.

[0047] In one aspect, the present disclosure relates to a protein construct comprising a first fusion polypeptide and a second fusion polypeptide, the first fusion polypeptide comprising the engineered 4-1BBL polypeptide, a first CH2 domain, and a first CH3 domain described herein, the second fusion polypeptide comprising a second CH2 domain and a second CH3 domain. In some embodiments, the first fusion polypeptide and the second fusion polypeptide associate with each other to form a dimer. In some embodiments, the second fusion polypeptide further comprises a second engineered 4-1BBL polypeptide.

[0048] In one aspect, the present disclosure relates to a pharmaceutical composition comprising the engineered 4-1BBL polypeptide or protein construct described herein; and a pharmaceutically acceptable carrier.

[0049] In one aspect, the present disclosure relates to a nucleic acid encoding the engineered 4-1BBL polypeptide or protein construct described herein.

[0050] In one aspect, the present disclosure relates to a vector comprising the nucleic acid described herein. In one aspect, the present disclosure relates to a cell comprising the nucleic acid described herein. In some embodiments, the cell is a CHO cell.

[0051] In one aspect, the present disclosure relates to a method for producing an engineered 4-1BBL polypeptide or a protein construct comprising the engineered 4-1BBL polypeptide, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the engineered 4-1BBL polypeptide or protein construct; and (b) collecting the engineered 4-1BBL polypeptide or protein construct produced by the cell.

[0052] In one aspect, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the engineered 4-1BBL polypeptide or protein construct described herein. In some embodiments, the subject has a solid tumor or a hematological cancer. In some embodiments, the cancer is breast cancer, oropharyngeal cancer, ovarian cancer, B-cell lymphoma or non-Hodgkin lymphoma, non-small cell lung cancer (NSCLC), melanoma, B-cell non-Hodgkin lymphoma, colorectal cancer or multiple myeloma.

[0053] In one aspect, the present disclosure relates to a method for reducing the tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising the engineered 4-1BBL polypeptide or protein construct described herein.

[0054] In one aspect, the present disclosure relates to a method for killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising the engineered 4-1BBL polypeptide or protein construct described herein.

[0055] As used herein, the term "engineered 4-1BBL polypeptide" refers to a polypeptide derived from a wild-type 4-1BBL polypeptide or a portion thereof (e.g., the extracellular domain of 4-1BBL), optionally having one or more mutations (e.g., insertions, deletions or substitutions). In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of the extracellular domain of 4-1BBL or a variant thereof. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of the TNF homology domain (THD) of 4-1BBL or a variant thereof. In some embodiments, the engineered 4-1BBL polypeptide is a modified THD domain. In some embodiments, the 4-1BBL extracellular domain has one or more mutations. In some embodiments, the THD domain has one or more mutations.

[0056] As used herein, the term "protein construct" refers to a complex having one or more polypeptides. In some embodiments, the protein construct has two or more polypeptides, wherein the polypeptides can associate with each other to form dimers or multimers (e.g., trimers).

[0057] As used herein, the term "cancer" refers to cells having the ability of autonomous growth without control. Examples of such cells include cells having an abnormal state or condition characterized by rapid proliferative cell growth. The term is meant to include cancerous growths, e.g., tumors; carcinogenic processes, metastatic tissues, and cells, tissues, or organs undergoing malignant transformation, regardless of the pathological type or stage of invasion. Also included are malignancies of different organ systems, such as the respiratory system, cardiovascular system, renal system, reproductive system, hematological system, nervous system, hepatic system, gastrointestinal system, and endocrine system; and adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, and small intestine cancers. "Spontaneously occurring" cancers include any cancers not experimentally induced by implanting cancer cells into a subject, and include, for example, cancers occurring spontaneously, cancers caused by a subject's exposure to carcinogens, cancers resulting from the insertion of transgenic oncogenes or the knockout of tumor suppressor genes, and cancers caused by infections, e.g., viral infections. The term "carcinoma" is recognized in the art and refers to malignancies of epithelial or endocrine tissues. The term also encompasses carcinosarcomas, which include malignancies composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is known in the art and refers to malignancies of mesenchymal origin. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic disorders can originate from myeloid, lymphoid, or erythroid lineages, or their precursor cells. Blood cancers are cancers that begin in hematopoietic tissues (such as the bone marrow) or cells of the immune system. Examples of blood cancers include, for example, leukemia, lymphoma, and multiple myeloma, etc.

[0058] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human, or non-human to which treatment is provided according to the methods of the present invention. Both veterinary and non-veterinary applications are contemplated in the present disclosure. A human patient can be an adult or an adolescent (e.g., a person under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic animals, farm animals, and zoo animals.

[0059] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acids of any length of at least two amino acids.

[0060] As used herein, the terms "polynucleotide", "nucleic acid molecule" and "nucleic acid sequence" are used interchangeably herein to refer to a polymer of nucleotides of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids and their modifications.

[0061] 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 invention belongs. Methods and materials for this invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0062] Other features and advantages of the invention will become apparent from the following detailed description, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Amino acid residues of the extracellular domain of human 4-1BBL are shown. Residues at the binding interface between 4-1BBL and its receptor 4-1BB are shaded. Amino acid positions in SEQ ID NO:1 are labeled on top of the selected residues.

[0064] Figure 2A Results of screening the first-round selected 4-1BBL variant clones are shown.

[0065] Figure 2B Results of screening the second-round selected 4-1BBL variant clones are shown.

[0066] Figure 2C Results of 4-1BBL phagemid screening are summarized.

[0067] Figure 3 A table showing the residues after mutation of the selected 4-1BBL variants is shown.

[0068] Figure 4A Results of screening 34 unique 4-1BBL variants are shown.

[0069] Figure 4B Characteristics of 34 unique 4-1BBL variants on 4-1BB binding activity and 4-1BB-mediated NF-κB reporter gene activity are summarized.

[0070] Figure 5 The HPLC-SEC analysis results of the Fc-fused 4-1BBL variants are shown.

[0071] Figures 6A - 6B The binding activity of the G4Fc-4-1BBL variants to activated T cells is shown.

[0072] Figures 6C - 6D The activity of the G4Fc-4-1BBL variants in inducing 4-1BB-mediated NF-κB reporter gene activity is shown.

[0073] Figure 6E The characteristics of the selected G4Fc-4-1BBL variants in terms of 4-1BB binding activity and 4-1BB-mediated NF-κB reporter gene activity are summarized.

[0074] Figure 7A The effect of the selected G4Fc-4-1BBL variants on T cell proliferation is shown. SIRPα-G4Fc-wt (Trillium; SEQ ID NO:4) was used as a negative control. PF05082566 and BMS663513 were used as positive controls.

[0075] Figure 7B The effect of the selected G4Fc-4-1BBL variants on IFN-γ production is shown. SIRPα-G4Fc-wt (Trillium) was used as a negative control. PF05082566 and BMS663513 were used as positive controls.

[0076] Figure 8A The effect of the selected G4Fc-4-1BBL variants on T cell proliferation is shown. SIRPα-G4Fc-wt (Trillium) was used as a negative control. PF05082566 and BMS663513 were used as positive controls.

[0077] Figure 8B The effect of the selected G4Fc-4-1BBL variants on IFN-γ production is shown. SIRPα-G4Fc-wt (Trillium) was used as a negative control. PF05082566 and BMS663513 were used as positive controls.

[0078] Figure 8C The effect of the selected G4Fc-4-1BBL variants on IL-2 production is shown. SIRPα-G4Fc-wt (Trillium) was used as a negative control. PF05082566 and BMS663513 were used as positive controls.

[0079] Figure 9AShows the binding activity of the selected G4Fc-4-1BBL variants to activated T cells. The anti-Her2 antibody (HLX22, developed by Henlix) was used as a negative control.

[0080] Figure 9B Shows the activity of the selected G4Fc-4-1BBL variants in inducing 4-1BB-mediated NF-κB reporter gene activity. SIRPα-G4Fc-wt (Trillium) was used as a negative control.

[0081] Figure 10A Shows the effect of the selected G4Fc-4-1BBL variants on T cell proliferation. SIRPα-G4Fc-wt (Trillium) was used as a negative control.

[0082] Figure 10B Shows the effect of the selected G4Fc-4-1BBL variants on IL2 production. SIRPα-G4Fc-wt (Trillium) was used as a negative control.

[0083] Figure 10C Shows the effect of the selected G4Fc-4-1BBL variants on IFN-γ production. SIRPα-G4Fc-wt (Trillium) was used as a negative control.

[0084] Figure 11A Shows the effect of the selected G4Fc-4-1BBL variants on IL-27 production. Urelumab (BMS663513) was used as a positive control. Utomliumab (PF05082566) and SIRPα-G4Fc-wt (Trillium) were used as negative controls.

[0085] Figure 11B Shows the effect of the selected G4Fc-4-1BBL variants on TNF-α production. Urelumab (BMS663513) and Utomliumab (PF05082566) were used as positive controls. SIRPα-G4Fc-wt (Trillium) was used as a negative control.

[0086] Figure 12 Lists the amino acid sequences of the wild-type 4-1BBL extracellular domain and its variants. The sequences correspond to amino acids 50 to 254 of human 4-1BBL (SEQ ID NO:1).

[0087] Figure 13 Lists the amino acid sequences of the wild-type 4-1BBL TNF homology domain and its variants. The sequences correspond to amino acids 90 to 241 of human 4-1BBL (SEQ ID NO:1).

[0088] Figure 14 Lists the protein sequences discussed in the present disclosure. Detailed implementation mode

[0089] TNFSF9 (also known as 4-1BB ligand, 4-1BBL, CD137L, tumor necrosis factor ligand superfamily member 9 (TNFSF9)) is a type II transmembrane protein of the TNF superfamily, mainly on antigen-presenting cells such as IFN-γ-activated macrophages, CD40 ligand-activated B cells, monocytes, T cells, dendritic cells (DCs), and B cells. TNFSF9 on the cell membrane can transmit reverse signals, thereby inhibiting the proliferation of activated T cells and inducing their apoptosis. The reverse signal can also induce monocyte activation, promote the secretion of IL-6, IL-8, and TNF-Ade, and prolong cell survival. In addition, the reverse signal can stimulate the maturation of DCs derived from CD34+ hematopoietic stem cells. Northern blot analysis shows that multiple TNFSF9 transcripts are present in the brain, placenta, lung, skeletal muscle, and kidney, as well as in activated T cells, transformed B cells, and monocyte lines.

[0090] The membrane form of 4-1BBL exists as a trimer, and when it binds to the receptor on T cells, it transmits a strong co-stimulatory signal. 4-1BBL is found to be expressed in professional APCs (including DCs and macrophages) of humans and mice, as well as in activated B cells after stimulation. Human 4-1BBL messages were detected as early as 30 minutes after stimulation with immobilized CD3 monoclonal antibody (mAb) and reached a peak at 1 hour. 4-1BBL is also present at high levels in the sera of some patients with hematological diseases 35 and on some cancer cell lines.

[0091] A detailed description of TNFSF9 and its functions can be found, for example, in Cheuk, Adam TC, et al., "4-1BB: Role of 4-1BB ligand in cancer immunotherapy," Cancer Gene Therapy 11.3 (2004): 215-226; and Li, Yan, et al., "Limited cross-linking of 4-1BB by 4-1BB ligand and the agonist monoclonal antibody Utomilumab," Cell Reports 25.4 (2018): 909-920, each of which is incorporated herein by reference in its entirety.

[0092] The present disclosure provides engineered 4-1BBL variants. These engineered 4-1BBL variants can be used to target the 4-1BB / 4-1BBL pathway, and the interaction between the engineered 4-1BBL variants and 4-1BB is carefully regulated.

[0093] Engineered 4-1BBL variants

[0094] 4-1BBL belongs to the TNF superfamily and is expressed on the surface of antigen-presenting cells, including, for example, dendritic cells, B cells, and macrophages. The co-stimulatory signal generated by the interaction between 4-1BB and its receptor 4-1BBL induces the activation and proliferation of T cells and NK cells, as well as the production of cytokines. Human 4-1BBL includes a cytoplasmic region, a transmembrane region, and an extracellular region from the N-terminus to the C-terminus. According to the UniProt database (UniProt ID: P41273), the cytoplasmic region of human 4-1BBL corresponds to amino acids 1 to 28 of SEQ ID NO: 1, the transmembrane region of human 4-1BBL corresponds to amino acids 29 to 49 of SEQ ID NO: 1, and the extracellular region of human 4-1BBL corresponds to amino acids 50 to 254 of SEQ ID NO: 1. The extracellular region of human 4-1BB includes a TNF homology domain (or THD domain), which corresponds to amino acids 90 to 241 of SEQ ID NO: 1. In some embodiments, the sequence of the THD domain (also referred to as the 4-1BBLv2 sequence) is shown in SEQ ID NO: 59. In some embodiments, the sequence corresponding to amino acids 64 to 254 of SEQ ID NO: 1 is also referred to as the 4-1BBLv1 sequence (SEQ ID NO: 94).

[0095] The 4-1BB / 4-1BBL complex consists of three monomers of 4-1BB bound to trimeric 4-1BBL. Each 4-1BB monomer binds to two 4-1BBL via a cysteine-rich domain (CRD). The interaction between 4-1BB and the second 4-1BBL is necessary to stabilize their interaction. Based on a detailed study of the binding between the 4-1BB and 4-1BBL interface, the linkage to 4-1BBL is mainly composed of amino acids from the dynamic loop of CRD2 and the β-sheet of CRD3 of 4-1BB. The CRD2 amino acids (T61, Q67, and K69) interact with the AA' loop (e.g., Y110 and G114) and the GH loop (e.g., Q227 and Q230) of 4-1BBL, forming various hydrogen bond interactions. Details can be found, for example, in Li, Y. et al., "Limited Crosslinking of 4-1BB by the 4-1BB Ligand and the Agonist Monoclonal Antibody Urelumab", Cell Reports 25.4 (2018): 909-920, which is incorporated herein by reference in its entirety.

[0096] Based on the structure of human 4-1BB complexed with human 4-1BBL, the binding interface between 4-1BBL and 4-1BB can be determined. Residues located around the binding interface are selected for mutagenesis analysis, for example, by constructing a phagemid library to generate random mutations. As Figure 1 shown, multiple interacting residues are located within the AA' loop (corresponding to amino acids 109 to 117 of SEQ ID NO:1 or amino acids 60 to 68 of SEQ ID NO:2), the CD loop (corresponding to amino acids 149 to 157 of SEQ ID NO:1 or amino acids 100 to 108 of SEQ ID NO:2), and the GH loop (corresponding to amino acids 225 to 232 of SEQ ID NO:1 or amino acids 176 to 183 of SEQ ID NO:2). These regions are targets for mutagenesis. Thus, in some embodiments, engineered 4-1BBL variants (e.g., any of the engineered 4-1BBL polypeptides described herein) comprise or consist of one or more amino acid mutations at the AA' loop, CD loop, and / or GH loop.

[0097] In addition, the present disclosure indicates that Val100, Tyr110, Asp112, Gly114, Leu115, Val140, Tyr142, Leu147, Arg150, Arg151, Val152, Val153, Ala154, Asn194, Phe199, Gln227, and Gln230 in SEQ ID NO:1 may be important for maintaining the homotrimeric contact interface of human 4-1BBL. Thus, in some embodiments, these amino acid residues are retained.

[0098] In summary, Ser67, Trp109, Ser111, Pro113, Leu147, Leu149, Gly155, Glu156, Ala225, Trp226, Leu228, Thr229, and Ala232 in SEQ ID NO:1 were identified as candidate amino acids for human 4-1BBL mutation screening. These residues correspond to Ser18, Trp60, Ser62, Pro64, Leu98, Leu100, Gly106, Glu107, Ala176, Trp177, Leu179, Thr180, and Ala183 in SEQ ID NO:2, respectively.

[0099] Thus, in one aspect, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93. In some embodiments, the engineered 4-1BBL polypeptides described herein comprise or consist of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1 or SEQ ID NO:94, wherein the amino acid sequence comprises one or more of the mutations described herein.

[0100] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7 or 61. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9 or 63. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16 or 70. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18 or 72. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:23 or 77. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26 or 80.In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27 or 81.

[0101] In some embodiments, compared to any one of SEQ ID NO:2, 6 to 39, and 59 to 93, the engineered 4-1BBL variant may have at least or about 1 (e.g., at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40) amino acid insertions, deletions or substitutions.

[0102] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0103] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is E, T, P, A, N, T or H;

[0104] (b) The amino acid corresponding to G106 of SEQ ID NO:2 is Q, K, H, R, F or S; and

[0105] (c) The amino acid corresponding to E107 of SEQ ID NO:2 is T, Q, A, R, L, M, S or I.

[0106] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0107] (a) The amino acid corresponding to A176 of SEQ ID NO:2 is S or Q;

[0108] (b) The amino acid corresponding to W177 of SEQ ID NO:2 is L, M or F;

[0109] (c) The amino acid corresponding to L179 of SEQ ID NO:2 is F, A or M;

[0110] (d) The amino acid corresponding to T180 of SEQ ID NO:2 is R, S, A or E; and

[0111] (e) The amino acid corresponding to A183 of SEQ ID NO:2 is Q, R or K.

[0112] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0113] (a) The amino acid corresponding to W60 of SEQ ID NO:2 is F; and

[0114] (b) The amino acid corresponding to P64 of SEQ ID NO:2 is N.

[0115] In some embodiments, the amino acid corresponding to L100 of SEQ ID NO:2 is V.

[0116] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0117] (a) The amino acid corresponding to S18 of SEQ ID NO:2 is I; and

[0118] (b) The amino acid corresponding to L98 of SEQ ID NO:2 is V.

[0119] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following residues:

[0120] (a) The amino acid corresponding to position 61 of SEQ ID NO:2 is Y;

[0121] (b) The amino acid corresponding to position 63 of SEQ ID NO:2 is D;

[0122] (c) The amino acid corresponding to position 65 of SEQ ID NO:2 is G;

[0123] (d) The amino acid corresponding to position 66 of SEQ ID NO:2 is L;

[0124] (e) The amino acid corresponding to position 101 of SEQ ID NO:2 is R;

[0125] (f) The amino acid corresponding to position 102 of SEQ ID NO:2 is R;

[0126] (g) The amino acid corresponding to position 103 of SEQ ID NO:2 is V;

[0127] (h) The amino acid corresponding to position 104 of SEQ ID NO:2 is V;

[0128] (i) The amino acid corresponding to position 105 of SEQ ID NO:2 is A;

[0129] (j) The amino acid corresponding to position 178 of SEQ ID NO:2 is Q; and

[0130] (k) The amino acid corresponding to position 181 of SEQ ID NO:2 is Q.

[0131] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0132] (a) The amino acid corresponding to S18 of SEQ ID NO:2 is I;

[0133] (b) The amino acid corresponding to W60 of SEQ ID NO:2 is F;

[0134] (c) The amino acid corresponding to S62 of SEQ ID NO:2 is E, T, P, A, N, T or H;

[0135] (d) The amino acid corresponding to P64 of SEQ ID NO:2 is N;

[0136] (e) The amino acid corresponding to L98 of SEQ ID NO:2 is V;

[0137] (f) The amino acid corresponding to L100 of SEQ ID NO:2 is V;

[0138] (g) The amino acid corresponding to G106 of SEQ ID NO:2 is Q, K, H, R, F or S;

[0139] (h) The amino acid corresponding to E107 of SEQ ID NO:2 is T, Q, A, R, L, M, S or I;

[0140] (i) The amino acid corresponding to A176 of SEQ ID NO:2 is S or Q;

[0141] (j) The amino acid corresponding to W177 of SEQ ID NO:2 is L, M or F;

[0142] (k) The amino acid corresponding to L179 of SEQ ID NO:2 is F, A or M;

[0143] (l) The amino acid corresponding to T180 of SEQ ID NO:2 is R, S, A or E; and

[0144] (m) The amino acid corresponding to A183 of SEQ ID NO:2 is Q, R or K.

[0145] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0146] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is E;

[0147] (b) The amino acid corresponding to E107 of SEQ ID NO:2 is T; and

[0148] (c) The amino acid corresponding to W177 of SEQ ID NO:2 is L.

[0149] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 6, 59 or 60).

[0150] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to T180 of SEQ ID NO:2 is R. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2 and 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 7, 59 or 61).

[0151] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following:

[0152] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is T; and

[0153] (b) The amino acid corresponding to T180 of SEQ ID NO:2 is S.

[0154] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 8, 59 or 62).

[0155] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to G106 of SEQ ID NO:2 is Q. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2 and 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 9, 59 or 63).

[0156] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to T180 of SEQ ID NO:2 is A. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 10, 59 or 64).

[0157] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to T180 of SEQ ID NO:2 is E. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 11, 59 or 65).

[0158] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following:

[0159] (a) the amino acid corresponding to G106 of SEQ ID NO:2 is K; and

[0160] (b) the amino acid corresponding to A183 of SEQ ID NO:2 is Q.

[0161] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 12, 59 or 66).

[0162] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to S62 of SEQ ID NO:2 is P. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 13, 59 or 67).

[0163] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following:

[0164] (a) the amino acid corresponding to E107 of SEQ ID NO:2 is Q; and

[0165] (b) The amino acid of A183 corresponding to SEQ ID NO:2 is R.

[0166] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO:2, 14, 59 or 68).

[0167] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to L98 of SEQ ID NO:2 is V. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO:2, 15, 59 or 69).

[0168] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following:

[0169] (a) The amino acid corresponding to G106 of SEQ ID NO:2 is H; and

[0170] (b) The amino acid corresponding to T180 of SEQ ID NO:2 is A.

[0171] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO:2, 16, 59 or 70).

[0172] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to E107 of SEQ ID NO:2 is A. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO:2, 17, 59 or 71).

[0173] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0174] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is A; and

[0175] (b) The amino acid corresponding to E107 of SEQ ID NO:2 is Q.

[0176] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 18, 59 or 72).

[0177] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0178] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is N; and

[0179] (b) The amino acid corresponding to E107 of SEQ ID NO:2 is Q.

[0180] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 19, 59 or 73).

[0181] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following:

[0182] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is T; and

[0183] (b) The amino acid corresponding to A183 of SEQ ID NO:2 is R.

[0184] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 20, 59 or 74).

[0185] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to S62 of SEQ ID NO:2 is H. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NOs: 2, 21, 59 or 75).

[0186] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0187] (a) the amino acid corresponding to S62 of SEQ ID NO:2 is P;

[0188] (b) the amino acid corresponding to E107 of SEQ ID NO:2 is R; and

[0189] (c) the amino acid corresponding to L179 of SEQ ID NO:2 is F.

[0190] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NOs: 2, 22, 59 or 76).

[0191] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0192] (a) the amino acid corresponding to E107 of SEQ ID NO:2 is L; and

[0193] (b) the amino acid corresponding to W177 of SEQ ID NO:2 is M.

[0194] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NOs: 2, 23, 59 or 77).

[0195] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0196] (a) the amino acid corresponding to S62 of SEQ ID NO:2 is P;

[0197] (b) The amino acid at position G106 corresponding to SEQ ID NO:2 is R; and

[0198] (c) The amino acid at position W177 corresponding to SEQ ID NO:2 is M.

[0199] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NOs: 2, 24, 59, or 78).

[0200] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to S18 of SEQ ID NO:2 is I. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NOs: 2, 25, 59, or 79).

[0201] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to L179 of SEQ ID NO:2 is A. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NOs: 2, 26, 59, or 80).

[0202] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0203] (a) The amino acid at position S62 corresponding to SEQ ID NO:2 is A; and

[0204] (b) The amino acid at position L100 corresponding to SEQ ID NO:2 is V.

[0205] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NOs: 2, 27, 59, or 81).

[0206] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0207] (a) The amino acid corresponding to W60 of SEQ ID NO:2 is F;

[0208] (b) The amino acid corresponding to E107 of SEQ ID NO:2 is T; and

[0209] (c) The amino acid corresponding to L179 of SEQ ID NO:2 is M.

[0210] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NOs: 2, 28, 59 or 82).

[0211] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to E107 of SEQ ID NO:2 is Q. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NOs: 2, 29, 59 or 83).

[0212] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to A183 of SEQ ID NO:2 is K. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NOs: 2, 30, 59 or 84).

[0213] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0214] (a) The amino acid corresponding to W60 of SEQ ID NO:2 is F; and

[0215] (b) The amino acid corresponding to G106 of SEQ ID NO:2 is R.

[0216] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 2, 6 to 39 and 59 to 93 (e.g., SEQ ID NOs: 2, 31, 59 or 85).

[0217] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0218] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is T;

[0219] (b) The amino acid corresponding to E107 of SEQ ID NO:2 is M; and

[0220] (c) The amino acid corresponding to A176 of SEQ ID NO:2 is S.

[0221] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 32, 59 or 86).

[0222] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0223] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is A; and

[0224] (b) The amino acid corresponding to E107 of SEQ ID NO:2 is S.

[0225] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 33, 59 or 87).

[0226] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to A176 of SEQ ID NO:2 is Q. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39 and 59 to 93 (e.g., SEQ ID NO:2, 34, 59 or 88).

[0227] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0228] (a) The amino acid corresponding to S62 of SEQ ID NO:2 is P;

[0229] (b) The amino acid corresponding to G106 of SEQ ID NO:2 is F; and

[0230] (c) The amino acid corresponding to A176 of SEQ ID NO:2 is S.

[0231] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO:2, 35, 59 or 89).

[0232] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence in which the amino acid corresponding to E107 of SEQ ID NO:2 is S. In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO:2, 36, 59 or 90).

[0233] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0234] (a) The amino acid corresponding to G106 of SEQ ID NO:2 is S; and

[0235] (b) The amino acid corresponding to W177 of SEQ ID NO:2 is F.

[0236] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO:2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO:2, 37, 59 or 91).

[0237] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0238] (a) The amino acid corresponding to P64 of SEQ ID NO:2 is N; and

[0239] (b) The amino acid corresponding to E107 of SEQ ID NO:2 is I.

[0240] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO: 2, 38, 59 or 92).

[0241] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of one or more of the following mutations:

[0242] (a) The amino acid corresponding to S62 of SEQ ID NO: 2 is P; and

[0243] (b) The amino acid corresponding to E107 of SEQ ID NO: 2 is A.

[0244] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO: 2, 39, 59 or 93).

[0245] In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 61 or SEQ ID NO: 70) having any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mutations as shown Figure 3 In some embodiments, the engineered 4-1BBL polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NO: 2, 6 to 39, and 59 to 93 (e.g., SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 61 or SEQ ID NO: 70) having any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mutations as shown

[0246] Engineered 4-1BBL polypeptides can have additional modifications. In some embodiments, engineered 4-1BBL polypeptides can have the CH2 domain and / or CH3 domain of Fc. In some embodiments, engineered 4-1BBL polypeptides can be linked to the N-terminus of the CH2 domain (e.g., via an optional hinge region or GS linker). In some embodiments, engineered 4-1BBL polypeptides can be linked to the C-terminus of the CH3 domain (e.g., via an optional GS linker). In some embodiments, the hinge region is an IgG hinge region (e.g., IgG4 hinge region). In some embodiments, the CH2 domain is an IgG CH2 domain (e.g., IgG4 CH2 domain). In some embodiments, the CH3 domain is an IgG CH3 domain (e.g., IgG4 CH3 domain). In some embodiments, the hinge region, CH2 domain, and CH3 domain have a sequence that is at least 80%, 85%, 90%, 95%, 100% identical to SEQ ID NO: 40.

[0247] In some embodiments, the engineered 4-1BBL polypeptides described herein can also include a tag (e.g., His tag) to facilitate screening and / or detection. In some embodiments, the tag has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 95 to 98. In some embodiments, the tag is linked to the N-terminus or C-terminus of any of the engineered 4-1BBL polypeptides described herein.

[0248] 4-1BBL protein construct

[0249] The present disclosure provides engineered 4-1BBL protein constructs that can specifically bind to 4-1BB. In some embodiments, these protein constructs have a similar affinity for 4-1BB but reduced agonistic ability. In some embodiments, these protein constructs have a higher affinity for 4-1BB but a similar agonistic ability. In some embodiments, these protein constructs have better thermal stability than wild-type 4-1BBL.

[0250] In some embodiments, an engineered 4-1BBL protein construct can comprise any of the engineered 4-1BBL variants described herein. In some embodiments, an engineered 4-1BBL protein construct can have a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 59-93. In some embodiments, an engineered 4-1BBL protein construct can comprise or consist of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 41-58.

[0251] The present disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 2, 6-39, 59-93 or any of SEQ ID NOs: 41-58.

[0252] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment and, for comparison purposes, non-homologous sequences can be disregarded). Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of positions shared by the sequences taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of the percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4 and a frameshift gap penalty of 5.

[0253] Engineered 4-1BBL protein constructs may further comprise the Fc region of an antibody. These antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class, or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2). In some embodiments, the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is an IgG4 Fc region (e.g., human IgG4 Fc region).

[0254] In some embodiments, the engineered 4-1BBL variant is linked to the Fc region via an antibody hinge region (e.g., IgG, IgE hinge region). Additionally, the Fc region can be modified to provide the desired effector function or serum half-life.

[0255] The engineered 4-1BBL variants and protein constructs described herein can block the binding between endogenous 4-1BB and endogenous 4-1BBL expressed on immune cells. In some embodiments, by binding to 4-1BB, the engineered 4-1BBL variants and protein constructs can inhibit 4-1BB (e.g., 4-1BB expressed on T cells) from binding to endogenous 4-1BBL expressed on antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells). Due to the retained 4-1BB binding affinity and reduced agonistic ability, the 4-1BBL variants and protein complexes described herein can maintain the potency to induce T cell activation and proliferation while having minimal hepatotoxicity.

[0256] In some embodiments, the engineered 4-1BBL variants and protein constructs described herein can increase the immune response, activity, or number of immune cells (e.g., T cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0257] In some embodiments, the engineered 4-1BBL variants and protein constructs can bind to 4-1BB (e.g., human 4-1BB, monkey 4-1BB (e.g., cynomolgus macaque (Macaca fascicularis)), mouse 4-1BB), and the dissociation rate (k off ) is less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 or less than 0.00001 s -1 . In some embodiments, the dissociation rate (koff ) greater than 0.01 s -1 、 greater than 0.001 s -1 、 less than 0.0001 s -1 、 greater than 0.00001 s -1 or greater than 0.000001 s -1 。

[0258] In some embodiments, the kinetic association rate (k on ) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, less than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1x 10 6 / Ms. In some embodiments, the kinetic association rate (k on ) is less than 1×10 5 / Ms, less than 1×10 6 / Ms, or less than 1x 10 7 / Ms.

[0259] The affinity can be derived from the quotient of the kinetic rate constants (KD = k off / k on ). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 M. In some embodiments, KD is less than 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM or 10 pM. In some embodiments, KD is greater than 1×10-7 M, greater than 1×10-8 M, greater than 1×10-9 M, greater than 1×10-10 M, greater than 1×10-11 M or greater than 1×10-12 M.

[0260] General techniques for measuring affinity include, for example, ELISA, radioimmunoassay (RIA), and surface plasmon resonance technology (SPR). In some embodiments, the engineered 4-1BBL variants and protein constructs can bind to monkey 4-1BB and / or mouse 4-1BB. In some embodiments, the engineered 4-1BBL variants and protein constructs cannot bind to monkey 4-1BB and / or mouse 4-1BB.

[0261] In some embodiments, thermal stability is determined. The Tm of the engineered 4-1BBL variants and protein constructs as described herein can be greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0262] In some embodiments, the tumor growth inhibition percentage (TGI%) of the engineered 4-1BBL variants and / or protein constructs as described herein is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the tumor growth inhibition percentage of the engineered 4-1BBL variants and / or protein constructs as described herein is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0263] TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100

[0264] Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

[0265] In some embodiments, the tumor suppressive effects of the engineered 4-1BBL variants and / or protein constructs as described herein are comparable to those of an anti-4-1BB reference antibody. In some embodiments, compared to the anti-4-1BB reference antibody, the tumor suppressive effects of the engineered 4-1BBL variants and / or protein constructs as described herein are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold.

[0266] In some embodiments, the protein constructs as described herein have a functional Fc region. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the protein constructs as described herein have an Fc region that does not have effector function. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (L234A mutation and L235A mutation under EU numbering) or an LALA-PG mutation (L234A mutation, L235A mutation, P329G mutation under EU numbering).

[0267] In some embodiments, the engineered 4-1BBL variant (e.g., any of the engineered 4-1BBL variants described herein) is linked to the N-terminus or C-terminus of the Fc region. In some embodiments, the engineered 4-1BBL variant is linked to the Fc region via a linker peptide. In some embodiments, the linker peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to either SEQ ID NO:99 or 100. In some embodiments, the linker peptide comprises a sequence that comprises 1, 2, 3, 4, 5, 6, 7, or 8 repeat sequences of GGGGS (SEQ ID NO:5).

[0268] In some embodiments, provided herein are protein constructs that include, from the N-terminus to the C-terminus, a 4-1BBL variant (e.g., any of the 4-1BBL described herein), a linker peptide (e.g., any of the linker peptides described herein), and a human IgG4 hinge region and Fc region (e.g., SEQ ID NO: 40). In some embodiments, the 4-1BBL variant includes a sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 2, 6 to 39, and 59 to 93. In some embodiments, the 4-1BBL variant includes one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the mutations described herein.

[0269] Some other modifications to the Fc region can be made. For example, cysteine residues can be introduced into the Fc region, allowing for the formation of interchain disulfide bonds in this region. The resulting homodimeric fusion protein may have any increase in its half-life in vitro and / or in vivo.

[0270] In some embodiments, IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0271] In some embodiments, an Fc region is provided having a carbohydrate structure that lacks (directly or indirectly) fucose linked to the Fc region. For example, the amount of fucose in such an Fc region composition can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex mannose structures, hybrid mannose structures, and high mannose structures) linked to Asn297 as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in the Fc region sequence, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region can be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0272] In some embodiments, the binding affinity between 4-1BB (e.g., human 4-1BB, monkey 4-1BB, mouse 4-1BB, or its extracellular domain) and an engineered 4-1BBL variant and / or protein construct as described herein is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold compared to the binding affinity between 4-1BB and wild-type 4-1BBL or its protein construct.

[0273] In some embodiments, the B / E ratio (4-1BB binding OD 450 to expression OD 450 ) of an engineered 4-1BBL variant and / or protein construct as described herein is between 0.1 and 0.5, between 0.5 and 1, between 1 and 1.25, between 1.25 and 1.5, between 1.5 and 1.75, between 1.75 and 2, or greater than 2. In certain cases, the B / E ratio is greater than 0.4. In some embodiments, the B / E ratio is determined at 25 °C or 45 °C.

[0274] In some embodiments, the R / E ratio (reporter gene assay (RLU) to expression OD 450 ) of an engineered 4-1BBL variant and / or protein construct as described herein is between 0.5 and 1, between 1 and 1.5, between 1.5 and 2, between 2 and 2.5, between 2.5 and 3, between 3 and 3.5, between 3.5 and 4, or higher than 4. In certain cases, the R / E ratio is greater than 0.5.

[0275] In some embodiments, the main peak of HPLC-SEC accounts for at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of an engineered 4-1BBL variant and / or protein construct as described herein.

[0276] In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can bind to human 4-1BB with an affinity that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold higher than the affinity of wild-type 4-1BBL or its protein constructs (e.g., 4-1BBL (SEQ ID NO:1); 4-1BBL extracellular domain (SEQ ID NO:2); 4-1BBLv2 (SEQ ID NO:59); or 4-1BBLv1 (SEQ ID NO:94)). In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can bind to 4-1BB with an affinity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% higher than the affinity of wild-type 4-1BBL or its protein constructs (e.g., 4-1BBL (SEQ ID NO:1); 4-1BBL extracellular domain (SEQ ID NO:2); 4-1BBLv2 (SEQ ID NO:59); or 4-1BBLv1 (SEQ ID NO:94)).

[0277] In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can bind to human 4-1BB-expressing T cells (e.g., T cells isolated from human PBMCs) with an affinity that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold higher than the affinity of wild-type 4-1BBL or its protein constructs (e.g., G4Fc-4-1BBLv2). In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can bind to 4-1BB-expressing T cells (e.g., T cells isolated from human PBMCs) with an affinity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% higher than the affinity of wild-type 4-1BBL or its protein constructs (e.g., G4Fc-4-1BBLv2).

[0278] In some embodiments, compared to wild-type 4-1BBL or its protein constructs (e.g., G4Fc-4-1BBLv2), the engineered 4-1BBL variants and / or their protein constructs as described herein can induce 4-1BB-mediated NFκB activity in a comparable manner.

[0279] In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can induce the proliferation of T cells (e.g., pre-activated T cells isolated from human PBMCs) at a proliferation rate that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 400% or at least 500% compared to wild-type 4-1BBL or its protein constructs (e.g., G4Fc-4-1BBLv2) or protein constructs targeting different antigens (e.g., SIRPα-G4Fc-wt).

[0280] In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can induce the release of cytokines (e.g., IFN-γ or IL2) at a proliferation rate that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold or at least 10000-fold compared to wild-type 4-1BBL or its protein constructs (e.g., G4Fc-4-1BBLv2) or protein constructs targeting different antigens (e.g., SIRPα-G4Fc-wt).

[0281] In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can induce less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of hepatotoxicity-related cytokines (e.g., IL-27 or TNF-α) compared to urelumab or utomilumab.

[0282] In some embodiments, the engineered 4-1BBL variants and / or their protein constructs as described herein can inhibit tumor growth.

[0283] Methods for preparing engineered 4-1BBL variants and protein constructs

[0284] Variants of 4-1BBL as described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the 4-1BBL peptide or a portion thereof or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. In some embodiments, random mutations can be introduced into residues within the AA' loop, CD loop, and / or GH loop of human 4-1BBL. In some embodiments, random mutations are introduced by constructing a phagemid library.

[0285] Screening can be performed. Among a population of such variants, some engineered 4-1BBL variants will have an increased affinity for 4-1BB. Any combination of deletions, insertions, and / or combinations can be made to obtain variants with increased binding affinity for the target. The amino acid changes introduced into the variants can also alter or introduce new post-translational modifications into the polypeptide, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that different sugars are linked by enzymes present in the cell), or introducing new glycosylation sites.

[0286] Engineered 4-1BBL variants can be derived from any animal species, including mammals. Non-limiting examples of 4-1BBL variants include 4-1BBL variants derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0287] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which a combination vector has been introduced (i.e., such that the host cell contains the polynucleotide and / or the vector comprising the polynucleotide), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

[0288] As used herein, a "vector" is any construct that is capable of delivering one or more polynucleotides of interest into a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked to regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in a host cell into which the expression vector has been introduced.

[0289] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0290] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of a non-pathogenic (defective), replication-competent virus, or a replication-defective virus may be used. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be "naked". Uptake of naked DNA can be enhanced by coating the DNA onto biodegradable beads, which are efficiently transported into cells.

[0291] For expression, a DNA insert containing a polynucleotide encoding a polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage λPL promoter, the Escherichia coli (E. coli) lac, trp, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs, among others. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is the cytomegalovirus (CMV) promoter. The expression construct can further contain sites for transcription initiation, termination, and a ribosome binding site for translation within the transcribed region. The coding portion of the mature transcript expressed by the construct can include translation starting at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0292] As indicated, the expression vector can contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera frugiperda Sf9 cells; animal cells such as CHO, COS, human melanoma, and HK 293 cells; and plant cells. Suitable media and conditions for the host cells described herein are known in the art.

[0293] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9 available from Qiagen; pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0294] Non-limiting bacterial promoters suitable for use include the E. coli lacI promoter and lacZ promoter, T3 promoter and T7 promoter, gpt promoter, λPR promoter and PL promoter, and trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs (such as the promoter of Rous Sarcoma Virus (RSV)), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0295] In the yeast Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters (such as alpha factor, alcohol oxidase, and PGH) can be used.

[0296] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0297] By inserting enhancer sequences into the vector, transcription of DNA encoding the polypeptides of the present disclosure in higher eukaryotes can be increased. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp in length, that serve to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located downstream of the origin of replication at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located downstream of the origin of replication, and the adenovirus enhancer.

[0298] To enable the secreted protein to be secreted into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be endogenous to the polypeptide, or it can also be a heterologous signal.

[0299] Polypeptides (e.g., 4-1BBL variants) can be expressed in a modified form, such as a fusion protein (e.g., GST fusion) or expressed with a histidine tag, and can include not only a secretion signal but also additional heterologous functional regions. For example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent processing and storage. Similarly, a peptide moiety can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding peptide moieties to polypeptides to effect secretion or excretion, improve stability and facilitate purification (among other things) is well-known and conventional in the art.

[0300] Therapeutic methods

[0301] The engineered 4-1BBL variants and protein constructs of the present disclosure can be used for a variety of therapeutic purposes.

[0302] In one aspect, the present disclosure provides methods for treating cancer in a subject, methods for reducing the rate of increase in the volume of a tumor in a subject over time, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment can halt, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment can reduce the number, severity, and / or duration of one or more cancer symptoms in the subject.

[0303] In one aspect, the present disclosure features methods that include administering a therapeutically effective amount of an engineered 4-1BBL variant and protein construct disclosed herein to a subject in need thereof (e.g., a subject having cancer or identified or diagnosed with cancer such as, for example, breast cancer (e.g., triple-negative breast cancer), adenocarcinoma, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies). In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial cancer, Merkel cell carcinoma, or head and neck cancer.

[0304] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Patients having cancer can be identified by a variety of methods known in the art.

[0305] As used herein, "effective amount" means an amount or dose sufficient to produce a beneficial or desired result, the beneficial or desired result including halting, slowing, delaying, or inhibiting a disease, e.g., the progression of cancer. The effective amount will depend, for example, on the age and weight of the subject to whom the engineered 4-1BBL variant and protein construct, the vector comprising the polynucleotide encoding the engineered 4-1BBL variant and protein construct, and / or their composition are to be administered, the severity of the symptoms, and the route of administration, and thus dosing can be determined on an individual basis.

[0306] An effective amount can be administered in one or more administrations. For example, an effective amount of an engineered 4-1BBL variant and / or protein construct is an amount sufficient to improve, halt, stabilize, reverse, inhibit, slow down, and / or delay the progression of cancer in a patient or an amount sufficient to improve, halt, stabilize, reverse, slow down, and / or delay the proliferation of any cancer cell or cell line in vitro (e.g., biopsy cells, cancer cells described herein, or cell lines (e.g., cancer cell lines)). As understood in the art, the effective amount can vary, particularly depending on the patient's medical history and other factors such as the type (and / or dose) of the engineered 4-1BBL variant and protein construct used.

[0307] The effective amount and schedule of administration of an engineered 4-1BBL variant and protein construct, a polynucleotide encoding an engineered 4-1BBL variant and protein construct, and / or a composition disclosed herein can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will understand that the dose to be administered will vary depending on, for example, the mammal to which the engineered 4-1BBL variant and protein construct, polynucleotide, and / or composition disclosed herein is to be administered, the route of administration, the particular type of polynucleotide, and / or the composition disclosed herein being used, and other drugs being administered to the mammal.

[0308] The typical daily dose of an effective amount of an engineered 4-1BBL variant and / or protein construct is from 0.1 mg / kg to 100 mg / kg (mg / kg of patient body weight). In some embodiments, the dose can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg. In some embodiments, the dose is about 1 to 10 mg / kg, about 1 to 5 mg / kg, or about 2 to 5 mg / kg.

[0309] In any of the methods described herein, the engineered 4-1BBL variants and protein constructs can be administered to a subject at least once per week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0310] In some embodiments, one or more additional therapeutic agents can be administered to the subject before or after administering the engineered 4-1BBL variants and protein constructs. In some embodiments, the one or more additional therapeutic agents are administered to the subject such that there is an overlap in the bioactive periods of the one or more additional therapeutic agents and the engineered 4-1BBL variants and protein constructs in the subject's body.

[0311] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent(s) can comprise one or more inhibitors selected from the group consisting of: B-Raf inhibitor, EGFR inhibitor, MEK inhibitor, ERK inhibitor, K-Ras inhibitor, c-Met inhibitor, anaplastic lymphoma kinase (ALK) inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, Akt inhibitor, mTOR inhibitor, dual PI3K / mTOR inhibitor, Bruton's tyrosine kinase (BTK) inhibitor, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0312] In some embodiments, the additional therapeutic agent(s) can comprise one or more inhibitors selected from the group consisting of: HER3 inhibitor, LSD1 inhibitor, MDM2 inhibitor, BCL2 inhibitor, CHK1 inhibitor, an inhibitor of the activated hedgehog signaling pathway, and a reagent for selectively degrading the estrogen receptor.

[0313] In some embodiments, the additional therapeutic agent can include one or more therapeutic agents selected from the group consisting of: Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Pazopanib (Votrient), Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0314] In some embodiments, the additional therapeutic agent can include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapeutics targeting CX3CL1, therapeutics targeting CXCL9, therapeutics targeting CXCL10, therapeutics targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0315] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to a subject.

[0316] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, an anti-CD47 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody or an anti-GITR antibody. In some embodiments, the additional therapeutic agent is an anti-CD20 antibody (e.g., rituximab) or an anti-EGF receptor antibody (e.g., cetuximab).

[0317] Drug Compositions and Routes of Administration

[0318] Also provided herein are drug compositions comprising the engineered 4-1BBL variants and protein constructs described herein. The drug compositions can be formulated in any manner known in the art.

[0319] The drug compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The compositions can include sterile diluents (e.g., sterile water or saline), fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial or antifungal agents such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate, citrate or phosphate and isotonic agents such as sugars (e.g., dextrose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride) or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and encapsulated in ampoules, disposable syringes or multi-dose vials. Where necessary (e.g., in injectable formulations), appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin or surfactants. The absorption of the agent can be extended by including a drug that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implant and microcapsule delivery systems, which can include biodegradable biocompatible polymers (e.g., ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid).

[0320] Compositions comprising the engineered 4-1BBL variants and protein constructs described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined quantity of the active compound, which are convenient for administration and dosage uniformity).

[0321] The pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic, and is produced under good manufacturing practice (GMP) conditions. The pharmaceutical composition can be provided in unit dosage forms (i.e., the dosage for a single administration). One or more physiologically acceptable carriers, diluents, excipients or adjuvants can be used to formulate the pharmaceutical composition. The formulation depends on the selected route of administration. For injection, the engineered 4-1BBL variants and protein constructs can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution can contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents. Alternatively, the engineered 4-1BBL variants and protein constructs can be in a lyophilized form before use, so as to be constituted with a suitable vehicle such as sterile pyrogen-free water.

[0322] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents with a high therapeutic index are preferably exhibited. When an agent exhibits adverse side effects, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). The toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0323] Exemplary doses include amounts of milligrams or micrograms per kilogram of subject body weight of any one of the engineered 4-1BBL variants and protein constructs described herein (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; about 1 μg / kg to about 50 μg / kg; about 1 mg / kg to about 10 mg / kg; or about 1 mg / kg to about 5 mg / kg). Although these doses cover a wide range, those of ordinary skill in the art will understand that the potency of the therapeutic agent can vary and the effective amount can be determined by methods known in the art. Generally, a relatively low dose is administered first, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still in the development stage) can subsequently gradually increase the dose until an appropriate response is obtained. Additionally, it can be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health status, gender and diet of the subject, the time of administration, the route of administration, the excretion rate, and the half-life in the body of the engineered 4-1BBL variants and protein constructs.

[0324] The pharmaceutical composition may be included in a container, package, or dispenser together with the administration instructions. The present disclosure also provides methods for preparing engineered 4-1BBL variants and protein constructs for various uses as described herein.

[0325] Examples

[0326] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0327] Example 1. Design of the Engineered Extracellular Domain of Human 4-1BBL

[0328] The extracellular domain of 4-1BBL is from residues 50 to 254 of the wild-type human 4-1BBL protein (SEQ ID NO: 1), and this extracellular domain belongs to the TNF-α homology domain. The Fold-A, AA' loop, CD loop, and GH loop are the main regions involved in 4-1BB interaction. As Figure 1 shown, the residues marked with star symbols are those that directly interact with 4-1BB, and the residues marked in light gray are the residues designed for random mutagenesis.

[0329] To obtain different 4-1BBL variants with random mutations at residues within the binding interface around 4-1BBL and 4-1BB (based on PDB identifiers 6A3V and 2X29), a phagemid library with a library size of 2.8×10 7 was constructed. 4-1BBL variants with different properties (e.g., binding activity and agonist function) were selected from this phagemid library by screening. The expression of 4-1BBL variants was induced by 1 mM IPTG. The supernatant was collected to determine the expression and binding activity of 4-1BBL variants. The supernatant was further incubated at 45 °C for 30 minutes to evaluate the thermal stability of 4-1BBL variants.

[0330] Specifically, 20 μl of ECOM competent cells (TG1) were transformed with the phagemid library expressing 4-1BBL variants. The transformed cells were spread onto LB plates (containing 150 μg / ml ampicillin), and then the plates were incubated overnight at 37 °C. Single colonies from the plates were used to inoculate 900 μl of 2YT medium (containing 150 μg / ml ampicillin and 10% phosphate buffer), and the plates were incubated at 37 °C for 3 to 4 hours until the OD600 value of the bacterial culture reached 0.9 to 1. The expression of 4-1BBL variants was induced by adding 1 mM IPTG, and then the culture was incubated overnight at 30 °C.

[0331] After centrifugation, the culture supernatant was collected to determine the expression and binding activity of the 4-1BBL variants. To determine the expression of the 4-1BBL variants, a 96-well plate (flat bottom) was coated with 2 μg / ml anti-His tag antibody overnight. Then the pre-coated plate was blocked with PBS containing 5% milk at room temperature (RT) for 1 hour. After blocking, 30 μl of the bacterial supernatant was added to each well of the plate, and the plate was incubated at room temperature for 1 hour. After incubation, anti-c-myc-HRP was added, and the expression level of the 4-1BBL variants could be determined by measuring OD450 in a plate reader. To determine the binding activity of the 4-1BBL variants, a 96-well plate (flat bottom) was coated with 0.5 μg / ml of 4-1BB overnight. Then the pre-coated plate was blocked with PBS containing 5% milk at room temperature for 1 hour. After blocking, 30 μl of the bacterial supernatant was added to each well of the plate, and the plate was incubated at room temperature for 1 hour. After incubation, anti-c-myc-HRP was added, and the 4-1BB binding activity of the 4-1BBL variants could be determined by measuring OD450 in a plate reader.

[0332] For the thermal stability test, the supernatant was incubated at 45 °C for 30 minutes, and then the expression and 4-1BB binding activity could be determined using the method described above.

[0333] After two rounds of screening, 56 potential 4-1BBL variants with different characteristics were obtained ( Figures 2A - 2B ). As Figure 2C shown, 2 clones showed a 5-fold enhancement in 4-1BB binding, while 6 clones showed a 2- to 4-fold enhancement in 4-1BB binding. Compared with wild-type 4-1BBL (peG4Fc-4-1BBLv2; SEQ ID NO:3), 35 clones showed similar binding activity, while 13 clones showed weaker binding activity. Compared with wild-type 4-1BBL, 3 clones showed better thermal stability, while 16 clones showed similar thermal stability.

[0334] Example 2. Verification of 4-1BB binding activity and reporter gene activity

[0335] After sequencing, 34 unique sequences were obtained from the 56 potential clones. The mutated residues are shown in Figure 3 .

[0336] The 4-1BB binding activity and 4-1BB-mediated reporter gene activity of the 34 unique 4-1BBL variants were verified as follows. Briefly, the expression of the 34 unique 4-1BBL variants was induced by 1 mM IPTG overnight at 30 °C. After centrifugation, the culture supernatant was further collected to determine the 4-1BB binding activity and 4-1BB reporter gene activity.

[0337] 4-1BB binding activity was measured using the same method as described above. Regarding 4-1BB-mediated NF-κB reporter gene activity, 96-well plates (flat bottom) were coated overnight at 4 °C with 5 μg / ml anti-c-myc antibody. The induced 4-1BBL variant supernatant was added to the pre-coated plates, and the plates were incubated at 37 °C for 2 hours. The above steps were repeated six times. Thereafter, 1×10 5 / well of transfected NF-κB Jurkat cells expressing 4-1BB were added to the plates, and then the plates were incubated at 37 °C for 5 hours. After incubation, the luminescence signal was detected to determine 4-1BB reporter gene activity.

[0338] The results are listed in Figure 4A and the characteristics of 34 unique 4-1BBL variants are listed in Figure 4B Six clones showed higher 4-1BB binding than wild-type 4-1BBL and exhibited different potencies in inducing 4-1BB-mediated NF-κB reporter gene activity. Ten clones showed similar 4-1BB binding activity compared to wild-type 4-1BBL and exhibited different potencies in inducing 4-1BB-mediated NF-κB reporter gene activity. The remaining 18 clones showed relatively weak 4-1BB binding activity and exhibited lower potencies in inducing 4-1BB-mediated NF-κB reporter gene activity.

[0339] As a result, 16 potential candidates with strong or moderate 4-1BB binding activity were selected to construct their respective Fc fusion proteins. To increase the diversity of 4-1BBL variants, two candidates with weaker 4-1BB binding activity (8G3 and 8G7) were also selected to construct their respective Fc fusion proteins.

[0340] Example 3. Characterization of 4-1BBL Variants Fused to IgG4 Fc

[0341] The results of HPLC-SEC (high performance liquid chromatography - size exclusion chromatography) analysis of IgG4 Fc (G4Fc)-fused 4-1BBL variants are summarized in Figure 5 Peak-1 represents trimeric 4-1BBL, while peak-2 represents dimeric 4-1BBL.

[0342] The activated T cell binding activity of G4Fc-4-1BBL variants was determined by flow cytometry ( Figures 6A - 6B ) and the 4-1BB-mediated reporter gene activity was determined by reporter gene assay ( Figures 6C - 6D ).

[0343] The activated T cell (whole cell) binding activity was determined as follows. By Ficoll- T cells were isolated from human PBMC donors by density gradient centrifugation. The isolated T cells were activated with CD3 / CD28 at a cell-bead ratio of 1:2 for 4 days to induce 4-1BB expression. 5×10 4 activated T cells per well were incubated with G4Fc-4-1BBL variants at the indicated concentrations at 4 °C for 30 minutes, and then incubated with a PE-conjugated anti-human Fc secondary antibody at 4 °C for 30 minutes. Binding activity was analyzed by CytoFlex TM flow cytometry.

[0344] For 4-1BB-mediated NF-κB reporter gene assays, 5×10 4 transfected NF-κB Jurkat cells expressing 4-1BB per well were incubated with 5×10 4 FcγRIIb-expressing 293F cells per well and G4Fc-4-1BBL variants at the indicated concentrations at 37 °C for 5 hours. Luminescence signals were detected to determine 4-1BB reporter gene activity.

[0345] As Figure 6E shown, G4Fc-4-1BBL variants 4C5 and 5B10 exhibited higher T cell binding activity than wild-type 4-1BBL (peG4Fc_4-1BBLv2 or G4Fc-4-1BBL-wt; SEQ ID NO:3), and showed moderate to low potency in inducing 4-1BB-mediated NF-κB reporter gene activity. Compared with wild-type 4-1BBL, G4Fc-4-1BBL variants 2B2, 2E9, 3D9, 3F4, and 5D10 exhibited similar T cell binding activity and showed low potency in inducing 4-1BB-mediated NF-κB reporter gene activity. 8G3 and 8G7 were excluded from the subsequence experiments due to poor purification and production titers.

[0346] Example 4. Determination of the potency of selected G4Fc-4-1BBL variant-1 in T cell responses

[0347] The potency of G4Fc-4-1BBL variants in T cell responses was determined in plate-bound form. Briefly, 96-well plates (U-bottom) were coated with G4Fc-4-1BBL variants at the indicated concentrations (4 to 100 nM), and T cell proliferation ( ) was measured by CellTiter- Figure 7A Cell viability assay (Promega). Secretion of IFN-γ ( Figure 7B ) was measured by ELISAMAX TM Deluxe Set human IFN-γ kit (BioLegend) according to the manufacturer's protocol.

[0348] Specifically, T cells were isolated from human PBMC donors by Ficoll- density gradient centrifugation. The isolated T cells were activated with CD3 / CD28 at a cell-bead ratio of 1:10 for 3 days to induce 4-1BB expression. A 96-well plate was pre-coated with 1 μg / ml anti-CD3 antibody (OKT-3) at 37 °C for 3 hours, and then blocked with PBS containing 1% FBS at room temperature for 1 hour. The G4Fc-4-1BBL variants were incubated overnight in the plate at the indicated concentrations at 4 °C. After incubation, 5×10 4 / well of pre-activated T cells were added, and the plate was cultured for another 4 days. The supernatant was collected for cytokine detection, and T cell proliferation was determined by CellTiter- Cell Viability Assay (Promega).

[0349] As Figures 7A - 7B shown, the G4Fc-4-1BBL variants 2B2, 3D9, 4C5, and 5B10 exhibited higher potency in inducing T cell proliferation and cytokine production than wild-type 4-1BBL (peG4Fc_4-1BBLv2; SEQ ID NO:3) and SIRPα-G4Fc-wt (Trillium; SEQ ID NO:4).

[0350] Example 5. Determination of the T cell response potency of selected G4Fc-4-1BBL variants-2

[0351] The potency of the G4Fc-4-1BBL variants on T cell response was also determined by cross-linking with anti-human Fc antibody. Briefly, a 96-well plate (U-bottom) was coated with anti-human Fc antibody, and the G4Fc-4-1BBL variants were cross-linked at the indicated concentrations (6 to 150 μM G4Fc-4-1BBL variants) by interaction with the pre-coated anti-human Fc. T cell proliferation ( ) was determined by CellTiter- Figure 8A ) Cell Viability Assay (Promega). The secretion of cytokines ( Figures 8B - 8C ) was determined by ELISAMAX TM Deluxe Set Human IFN-γ and IL-2 Kit (BioLegend) according to the manufacturer's protocol.

[0352] Specifically, T cells were isolated from human PBMC donors by Ficoll- density gradient centrifugation. The isolated T cells were activated with CD3 / CD28 Activate for 3 days at a cell-bead ratio of 1:10 to induce 4-1BB expression. Pre-coat a 96-well plate with 1 μg / ml anti-CD3 antibody (OKT-3) and 3 μg / ml anti-human Fc at 37 °C for 3 hours, and then block with PBS containing 1% FBS at room temperature for 1 hour. Incubate the G4Fc-4-1BBL variant overnight at the indicated concentration in the plate at 4 °C. After incubation, add 5×10 4 / well of pre-activated T cells, and culture the plate for another 4 days. Collect the supernatant for cytokine detection, and determine T cell proliferation by CellTiter- Cell Viability Assay (Promega).

[0353] As Figures 8A - 8C shown, the G4Fc-4-1BBL variants 2B2, 3D9, 4C5, and 5B10 showed higher potency in inducing T cell proliferation and IFN-γ production than wild-type 4-1BBL (peG4Fc_4-1BBLv2; SEQ ID NO: 3). Further, the G4Fc-4-1BBL variants 4C5 and 5B10 showed higher potency in inducing IL-2 production than other tested G4Fc-4-1BBL variants.

[0354] Example 6. Verification of T cell binding activity and reporter gene activity

[0355] Further verify the activated T cell binding activity and 4-1BB-mediated reporter gene activity of the G4Fc-4-1BBL variants 2B2, 3D9, 4C5, and 5B10 by flow cytometry ( Figure 9A ) and reporter gene analysis ( Figure 9B ), respectively. Perform the same methods as the activated T cell (whole cell) binding assay and 4-1BB-mediated NF-κB reporter gene assay described in Example 3.

[0356] Compared with wild-type 4-1BBL ((peG4Fc_4-1BBLv2; SEQ ID NO: 3)), the G4Fc-4-1BBL variants 2B2, 3D9, and 4C5 showed higher T cell binding activity and similar agonist function. Since 4C5 showed poor purification by HPLC-SEC ( Figure 5 ), the G4Fc-4-1BBL variants 2B2 and 3D9 were selected for subsequent experiments.

[0357] Example 7. Determination of the T cell response potency of G4Fc-4-1BBL

[0358] The potency of the G4Fc-4-1BBL variant on T cell responses was further verified by crosslinking with anti-human Fc antibody. Briefly, the G4Fc-4-1BBL variant was crosslinked at the indicated concentrations (0.003858 to 30 nM) by interaction with FcγRIIb 293F cells. T cell proliferation ( TM ) was determined by measuring the percentage of Violet 低 CD3+ T cells by CytoFlex Figure 10A . Secretion of cytokines ( Figures 10B - 10C ) was determined by ELISA MAX TM Deluxe Set Human IFN-γ and IL-2 kits (BioLegend) according to the manufacturer's protocol.

[0359] Specifically, T cells were isolated from human PBMC donors by Ficoll- density gradient centrifugation. The isolated T cells were activated for 1 day with CD3 / CD28 at a cell-bead ratio of 1:1 to induce 4-1BB expression. A 96-well plate (U-bottom) was pre-coated with 1 μg / ml anti-CD3 antibody (OKT-3) at 37 °C for 3 hours and then blocked with PBS containing 1% FBS at room temperature for 1 hour. The G4Fc-4-1BBL variant at the indicated concentrations was incubated with mitomycin C-treated FcγRIIb293F and CellTrace TM Violet-labeled CD3+ T cells for 3 days. The supernatant was collected for cytokine detection, and T cell proliferation was determined by CytoFlex TM flow cytometry.

[0360] Compared with wild-type G4Fc-4-1BBL (peG4Fc_4-1BBLv2; SEQ ID NO:3), 2B2 and 3D9 showed higher T cell binding activity and reduced ability to induce T cell proliferation and cytokine release.

[0361] Example 8. Determination of the hepatotoxicity risk of the G4Fc-4-1BBL variant

[0362] The potential hepatotoxicity risk was determined by inducing the production of IL-27 and TNF-α. Specifically, MDM (monocyte-derived macrophages) cells at 2.5×10 4 / well were stimulated with 1.5 μg / ml LPS and incubated with the G4Fc-4-1BBL variant at the indicated concentrations for 48 hours. The production of IL-27 and TNF-α was determined by Human IL-27 DuoSet ELISA kit (R&D) and ELISA MAXTM The Deluxe Set human TNF-α kit (BioLegend) was assayed according to the manufacturer's protocol.

[0363] As Figures 11A - 11B shown, the G4Fc-4-1BBL variants 2B2 and 3D9 did not enhance the production of IL-27 and TNF-α relative to wild-type G4Fc-4-1BBL (peG4Fc_4-1BBLv2; SEQ ID NO:3). The results indicate that the 4-1BBL variants 2B2 and 3D9 do not show a risk of hepatotoxicity.

[0364] Other embodiments

[0365] It should be understood that although the present invention has been described in connection with its foregoing detailed description, the foregoing description is intended to be illustrative and not limiting of the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:2 or SEQ ID NO:59, wherein the engineered 4-1BBL polypeptide comprises one or more amino acid mutations at the AA' loop, CD loop, and / or GH loop.

2. The engineered 4-1BBL polypeptide according to claim 1, wherein the amino acid corresponding to S62 of SEQ ID NO:2 is E, T, P, A, N, T, or H.

3. The engineered 4-1BBL polypeptide according to claim 1 or 2, comprising one or more of the following: (a) The amino acid corresponding to G106 of SEQ ID NO:2 is Q, K, H, R, F, or S; and (b) The amino acid corresponding to E107 of SEQ ID NO:2 is T, Q, A, R, L, M, S, or I.

4. The engineered 4-1BBL polypeptide according to any one of claims 1 to 3, comprising one or more of the following: (a) The amino acid corresponding to A176 of SEQ ID NO:2 is S or Q; (b) The amino acid corresponding to W177 of SEQ ID NO:2 is L, M, or F; (c) The amino acid corresponding to L179 of SEQ ID NO:2 is F, A, or M; (d) The amino acid corresponding to T180 of SEQ ID NO:2 is R, S, A, or E; and (e) The amino acid corresponding to A183 of SEQ ID NO:2 is Q, R, or K.

5. The engineered 4-1BBL polypeptide according to any one of claims 1 to 4, comprising one or more of the following: (a) The amino acid corresponding to W60 of SEQ ID NO:2 is F; and (b) The amino acid corresponding to P64 of SEQ ID NO:2 is N.

6. The engineered 4-1BBL polypeptide according to any one of claims 1 to 5, wherein the amino acid corresponding to L100 of SEQ ID NO:2 is V.

7. The engineered 4-1BBL polypeptide according to any one of claims 1 to 6, further comprising one or more of the following: (a) The amino acid corresponding to S18 of SEQ ID NO:2 is I; and (b) The amino acid corresponding to L98 of SEQ ID NO:2 is V.

8. The engineered 4-1BBL polypeptide according to any one of claims 1 to 7, comprising one or more of the following: (a) The amino acid at position 61 corresponding to SEQ ID NO:2 is Y; (b) The amino acid at position 63 corresponding to SEQ ID NO:2 is D; (c) The amino acid at position 65 corresponding to SEQ ID NO:2 is G; (d) The amino acid at position 66 corresponding to SEQ ID NO:2 is L; (e) The amino acid at position 101 corresponding to SEQ ID NO:2 is R; (f) The amino acid corresponding to position 102 of SEQ ID NO:2 is R; (g) The amino acid corresponding to position 103 of SEQ ID NO:2 is V; (h) The amino acid corresponding to position 104 of SEQ ID NO:2 is V; (i) The amino acid corresponding to position 105 of SEQ ID NO:2 is A; (j) The amino acid corresponding to position 178 of SEQ ID NO:2 is Q; and (k) The amino acid corresponding to position 181 of SEQ ID NO:2 is Q.

9. The engineered 4-1BBL polypeptide according to any one of claims 1 to 8, which comprises an amino acid sequence that is at least 85%, 90%, 95% or 100% identical to SEQ ID NO:2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

10. The engineered 4-1BBL polypeptide according to any one of claims 1 to 8, which comprises an amino acid sequence that is at least 85%, 90%, 95% or 100% identical to SEQ ID NO:59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93.

11. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 6, 59 or 60, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to S62 of SEQ ID NO:2 is E; (b) The amino acid corresponding to E107 of SEQ ID NO:2 is T; and (c) The amino acid corresponding to W177 of SEQ ID NO:2 is L.

12. The engineered 4-1BBL polypeptide according to claim 11, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:6 or 60.

13. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 7, 59 or 61, wherein the amino acid corresponding to T180 of SEQ ID NO:2 is R.

14. The engineered 4-1BBL polypeptide according to claim 13, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:7 or 61.

15. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 8, 59 or 62, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to S62 of SEQ ID NO:2 is T; and (b) The amino acid corresponding to T180 of SEQ ID NO:2 is S.

16. The engineered 4-1BBL polypeptide according to claim 15, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:8 or 62.

17. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 9, 59 or 63, wherein the amino acid corresponding to G106 of SEQ ID NO:2 is Q.

18. The engineered 4-1BBL polypeptide according to claim 17, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:9 or 63.

19. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 10, 59 or 64, wherein the amino acid corresponding to T180 of SEQ ID NO:2 is A.

20. The engineered 4-1BBL polypeptide according to claim 19, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:10 or 64.

21. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 11, 59 or 65, wherein the amino acid corresponding to T180 of SEQ ID NO:2 is E.

22. The engineered 4-1BBL polypeptide according to claim 21, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:11 or 65.

23. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 12, 59 or 66, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to G106 of SEQ ID NO:2 is K; and (b) The amino acid corresponding to A183 of SEQ ID NO:2 is Q.

24. The engineered 4-1BBL polypeptide according to claim 23, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:12 or 66.

25. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 13, 59 or 67, wherein the amino acid corresponding to S62 of SEQ ID NO:2 is P.

26. The engineered 4-1BBL polypeptide according to claim 25, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:13 or 67.

27. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 14, 59 or 68, wherein the polypeptide comprises one or more of the following: (a) The amino acid of E107 corresponding to SEQ ID NO:2 is Q; and (b) The amino acid of A183 corresponding to SEQ ID NO:2 is R.

28. The engineered 4-1BBL polypeptide according to claim 27, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:14 or 68.

29. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 15, 59 or 69, wherein the amino acid of L98 corresponding to SEQ ID NO:2 is V.

30. The engineered 4-1BBL polypeptide according to claim 29, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:15 or 69.

31. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 16, 59 or 70, wherein the polypeptide comprises one or more of the following: (a) The amino acid of G106 corresponding to SEQ ID NO:2 is H; and (b) The amino acid of T180 corresponding to SEQ ID NO:2 is A.

32. The engineered 4-1BBL polypeptide according to claim 31, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:16 or 70.

33. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 17, 59 or 71, wherein the amino acid of E107 corresponding to SEQ ID NO:2 is A.

34. The engineered 4-1BBL polypeptide according to claim 33, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:17 or 71.

35. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 18, 59 or 72, wherein the polypeptide comprises one or more of the following: (a) The amino acid of S62 corresponding to SEQ ID NO:2 is A; and (b) The amino acid of E107 corresponding to SEQ ID NO:2 is Q.

36. The engineered 4-1BBL polypeptide according to claim 35, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:18 or 72.

37. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 19, 59 or 73, wherein the polypeptide comprises one or more of the following: (a) The amino acid of S62 corresponding to SEQ ID NO:2 is N; and (b) The amino acid of E107 corresponding to SEQ ID NO:2 is Q.

38. The engineered 4-1BBL polypeptide according to claim 37, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or 73.

39. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 20, 59 or 74, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to S62 of SEQ ID NO: 2 is T; and (b) The amino acid corresponding to A183 of SEQ ID NO: 2 is R.

40. The engineered 4-1BBL polypeptide according to claim 39, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 20 or 74.

41. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 21, 59 or 75, wherein the amino acid corresponding to S62 of SEQ ID NO: 2 is H.

42. The engineered 4-1BBL polypeptide according to claim 41, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21 or 75.

43. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 22, 59 or 76, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to S62 of SEQ ID NO: 2 is P; (b) The amino acid corresponding to E107 of SEQ ID NO: 2 is R; and (c) The amino acid corresponding to L179 of SEQ ID NO: 2 is F.

44. The engineered 4-1BBL polypeptide according to claim 43, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 22 or 76.

45. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 23, 59 or 77, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to E107 of SEQ ID NO: 2 is L; and (b) The amino acid corresponding to W177 of SEQ ID NO: 2 is M.

46. The engineered 4-1BBL polypeptide according to claim 45, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 23 or 77.

47. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 24, 59 or 78, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to S62 of SEQ ID NO: 2 is P; (b) The amino acid corresponding to G106 of SEQ ID NO: 2 is R; and (c) The amino acid corresponding to W177 of SEQ ID NO: 2 is M.

48. The engineered 4-1BBL polypeptide according to claim 47, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24 or 78.

49. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 25, 59 or 79, wherein the amino acid corresponding to S18 of SEQ ID NO: 2 is I.

50. The engineered 4-1BBL polypeptide according to claim 49, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 or 79.

51. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 26, 59 or 80, wherein the amino acid corresponding to L179 of SEQ ID NO: 2 is A.

52. The engineered 4-1BBL polypeptide according to claim 51, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26 or 80.

53. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 27, 59 or 81, wherein the polypeptide comprises one or more of the following: (a) the amino acid corresponding to S62 of SEQ ID NO: 2 is A; and (b) the amino acid corresponding to L100 of SEQ ID NO: 2 is V.

54. The engineered 4-1BBL polypeptide according to claim 53, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27 or 81.

55. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 28, 59 or 82, wherein the polypeptide comprises one or more of the following: (a) the amino acid corresponding to W60 of SEQ ID NO: 2 is F; (b) the amino acid corresponding to E107 of SEQ ID NO: 2 is T; and (c) the amino acid corresponding to L179 of SEQ ID NO: 2 is M.

56. The engineered 4-1BBL polypeptide according to claim 55, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28 or 82.

57. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 29, 59 or 83, wherein the amino acid corresponding to E107 of SEQ ID NO: 2 is Q.

58. The engineered 4-1BBL polypeptide according to claim 57, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29 or 83.

59. An engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 30, 59, or 84, wherein the amino acid corresponding to A183 of SEQ ID NO: 2 is K.

60. The engineered 4-1BBL polypeptide according to claim 59, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 30 or 84.

61. An engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 31, 59, or 85, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to W60 of SEQ ID NO: 2 is F; and (b) The amino acid corresponding to G106 of SEQ ID NO: 2 is R.

62. The engineered 4-1BBL polypeptide according to claim 61, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or 85.

63. An engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 32, 59, or 86, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to S62 of SEQ ID NO: 2 is T; (b) The amino acid corresponding to E107 of SEQ ID NO: 2 is M; and (c) The amino acid corresponding to A176 of SEQ ID NO: 2 is S.

64. The engineered 4-1BBL polypeptide according to claim 63, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 32 or 86.

65. An engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 33, 59, or 87, wherein the polypeptide comprises one or more of the following: (a) The amino acid corresponding to S62 of SEQ ID NO: 2 is A; and (b) The amino acid corresponding to E107 of SEQ ID NO: 2 is S.

66. The engineered 4-1BBL polypeptide according to claim 65, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33 or 87.

67. An engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 34, 59, or 88, wherein the amino acid corresponding to A176 of SEQ ID NO: 2 is Q.

68. The engineered 4-1BBL polypeptide according to claim 67, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 34 or 88.

69. An engineered 4-1BBL polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, 35, 59, or 89, wherein the polypeptide comprises one or more of the following: (a) The amino acid at position S62 corresponding to SEQ ID NO:2 is P; (b) The amino acid at position G106 corresponding to SEQ ID NO:2 is F; and (c) The amino acid at position A176 corresponding to SEQ ID NO:2 is S.

70. The engineered 4-1BBL polypeptide according to claim 69, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:35 or 89.

71. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 36, 59 or 90, wherein the amino acid at position E107 corresponding to SEQ ID NO:2 is S.

72. The engineered 4-1BBL polypeptide according to claim 71, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:36 or 90.

73. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 37, 59 or 91, wherein the polypeptide comprises one or more of the following: (a) The amino acid at position G106 corresponding to SEQ ID NO:2 is S; and (b) The amino acid at position W177 corresponding to SEQ ID NO:2 is F.

74. The engineered 4-1BBL polypeptide according to claim 73, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:37 or 91.

75. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 38, 59 or 92, wherein the polypeptide comprises one or more of the following: (a) The amino acid at position P64 corresponding to SEQ ID NO:2 is N; and (b) The amino acid at position E107 corresponding to SEQ ID NO:2 is I.

76. The engineered 4-1BBL polypeptide according to claim 75, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:38 or 92.

77. An engineered 4-1BBL polypeptide, which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 39, 59 or 93, wherein the polypeptide comprises one or more of the following: (a) The amino acid at position S62 corresponding to SEQ ID NO:2 is P; and (b) The amino acid at position E107 corresponding to SEQ ID NO:2 is A.

78. The engineered 4-1BBL polypeptide according to claim 77, which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:39 or 93.

79. The engineered 4-1BBL polypeptide according to any one of claims 1 to 78, wherein the engineered 4-1BBL polypeptide further comprises a CH2 domain and a CH3 domain.

80. The engineered 4-1BBL polypeptide according to claim 79, wherein the engineered 4-1BBL polypeptide further comprises a hinge region.

81. The engineered 4-1BBL polypeptide according to claim 79 or 80, wherein the CH2 domain is an IgG CH2 domain and the CH3 domain is an IgG CH3 domain.

82. The engineered 4-1BBL polypeptide according to any one of claims 79 to 81, wherein the engineered 4-1BBL polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 41 to 58.

83. A protein construct comprising the engineered 4-1BBL polypeptide according to any one of claims 1 to 82.

84. The protein construct according to claim 83, which comprises two or more engineered 4-1BBL polypeptides.

85. The protein construct according to claim 84, wherein at least two of the engineered 4-1BBL polypeptides are identical.

86. The protein construct according to claim 84, wherein at least two of the engineered 4-1BBL polypeptides are different.

87. The protein construct according to claim 83, which further comprises an Fc region.

88. The protein construct according to claim 87, wherein the Fc region is an IgG4 Fc region.

89. The protein construct according to claim 87, wherein the Fc region is an IgG1 Fc region (e.g., having a LALA mutation or a LALA-PG mutation).

90. The protein construct according to any one of claims 87 to 89, wherein the engineered 4-1BBL polypeptide is optionally linked to the C-terminus of the Fc region via a linker peptide.

91. The protein construct according to any one of claims 87 to 89, wherein the engineered 4-1BBL polypeptide is optionally linked to the N-terminus of the Fc region via a linker peptide.

92. A protein construct comprising: a first fusion polypeptide comprising the engineered 4-1BBL polypeptide according to any one of claims 1 to 82, a first CH2 domain and a first CH3 domain; and a second fusion polypeptide comprising a second CH2 domain and a second CH3 domain; wherein the first fusion polypeptide and the second fusion polypeptide associate with each other to form a dimer.

93. The protein construct according to claim 92, wherein the second fusion polypeptide further comprises a second engineered 4-1BBL polypeptide.

94. A pharmaceutical composition comprising the engineered 4-1BBL polypeptide according to any one of claims 1 to 82 or the protein construct according to any one of claims 83 to 93; and a pharmaceutically acceptable carrier.

95. A nucleic acid encoding an engineered 4-1BBL polypeptide according to any one of claims 1 to 82 or a protein construct according to any one of claims 83 to 93.

96. A vector comprising the nucleic acid according to claim 95.

97. A cell comprising the nucleic acid according to claim 95.

98. The cell according to claim 97, wherein the cell is a CHO cell.

99. A method for producing an engineered 4-1BBL polypeptide or a protein construct comprising the engineered 4-1BBL polypeptide, the method comprising: (a) culturing the cell according to claim 97 or 98 under conditions sufficient to cause the cell to produce the engineered 4-1BBL polypeptide or the protein construct; and (b) collecting the engineered 4-1BBL polypeptide or the protein construct produced by the cell.

100. A method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an engineered 4-1BBL polypeptide according to any one of claims 1 to 82 or a protein construct according to any one of claims 83 to 93.

101. The method according to claim 100, wherein the subject has a solid tumor or a hematological cancer.

102. The method according to claim 100, wherein the cancer is breast cancer, oropharyngeal cancer, ovarian cancer, B-cell lymphoma or non-Hodgkin lymphoma, non-small cell lung cancer (NSCLC), melanoma, B-cell non-Hodgkin lymphoma, colorectal cancer or multiple myeloma.

103. A method for reducing the tumor growth rate, the method comprising: contacting tumor cells with an effective amount of a composition comprising an engineered 4-1BBL polypeptide according to any one of claims 1 to 82 or a protein construct according to any one of claims 83 to 93.

104. A method for killing tumor cells, the method comprising: contacting tumor cells with an effective amount of a composition comprising an engineered 4-1BBL polypeptide according to any one of claims 1 to 82 or a protein construct according to any one of claims 83 to 93.

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