4-1BB-binding proteins and their uses

Abstract

To provide a 4-1BB-binding protein, and to provide uses thereof.SOLUTION: A 4-1BB binding protein comprises a heavy chain variable region that contains HCDR1, HCDR2 and HCDR3, the HCDR1 and HCDR2 comprising a specific amino acid sequence or a variant thereof, the HCDR3 comprising a specific amino acid sequence or a variant thereof. The 4-1BB binding protein of the present invention is a fully human derived antibody, has activity with human 4-1BB and cynomolgus monkey 4-1BB binding. Some 4-1BB-binding proteins are only half the size of conventional IgG antibodies and can be well used for bispecific antibodies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application requests priority from Chinese patent application 202010619500.9, filed on 2020 / 6 / 30. This application cites the entire text of the aforementioned Chinese patent application.

[0002] This application relates to the biopharmaceutical field, and more specifically to 4-1BB-binding proteins and their applications. [Background technology]

[0003] 4-1BB, also known as CD137 or tumor necrosis factor receptor superfamily member 9 (TNFRSF 9), is an activation-inducible costimulatory receptor molecule. 4-1BB is primarily expressed in lymphocytes such as activated T cells, activated spontaneously disrupted (NK) cells, activated thymocytes, and intradermal lymphocytes. It is also expressed in dendritic cells, mononuclear cells, neutrophil cells, and eosinophilic cells. T cell activation requires not only the specific binding of antigen-recognition receptors (TCRs) on the T cell surface to MHC molecular antigen peptides, but also a costimulatory signal provided by the binding of costimulatory molecules on the antigen-presenting cell (APC) surface to the corresponding receptors on the T cell surface. Binding of 4-1BB and its ligand, 4-1BBL, can provide a costimulatory signal to activate T cells, enhancing cytokine and immune function. 4-1BB has also been shown to promote central memory T cell responses, which may support the therapeutic persistence and resistance to desaturation of tumor-specific T cells in patients receiving 4-1BB agonist therapy. [1,2] Overexpression of anti-4-1BB single-chain antibody fragment (scFv) on tumors leads to CD4+ T cell and NK cell-dependent tumor removal. [3、4、5] This leads to... Systemic injection of anti-4-1BB antibodies in a mouse model has also been shown to cause a delay in tumor growth. [6] .

[0004] Activated 4-1BB antibodies have been shown to activate the downstream 4-1BB pathway in place of its ligand. Currently, activated 4-1BB antibodies being studied clinically include Urelumab (BMS-663513) and Utomilumab (PF-05082566). Utomilumab is a ligand-blocking IgG2 antibody, while Urelumab is a non-ligand-blocking IgG4 antibody. Both Utomilumab and Urelumab can enhance T cell function and promote antitumor effects both in vivo and in vitro. However, clinical trials have shown that some patients developed pneumonia toxicity when Urelumab doses were greater than 1 mg / kg (see NCT00309023, NCT00612664, NCT01471210). Compared to Urelumab, Utomilumab is safer but has lower activity in activating 4-1BB. Therefore, there is an urgent need to develop new, safer, and more effective therapies targeting 4-1BB.

[0005] To address the lack of antitumor therapeutic efficacy in conventional technologies and to resolve technical issues such as safety, the present invention provides a whole-human antibody targeting 4-1BB, a bispecific antibody based on this antibody and a tumor-specific target, and its applications. This application provides one or more anti-4-1BB antigen-binding proteins having the following properties: 1) capable of binding to human and monkey-derived 4-1BB proteins; 2) capable of stimulating immune cells to secrete IFN-γ, IL2, and / or TNFα; 3) capable of inhibiting tumor growth and / or tumor cell proliferation; and 4) capable of activating the 4-1BB signaling pathway. This application also provides the use of antigen-binding proteins in the prevention and treatment of tumors. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The technical problem to be solved by the present invention is to provide a 4-1BB-targeting binding protein and use thereof, in particular a fully human-derived antibody targeting 4-1BB, a bispecific antibody based on said antibody and a tumor-specific target, and use thereof, so as to overcome the defects such as insufficient anti-tumor therapeutic effect and poor safety of antibody drugs in the prior art. [Means for Solving the Problems]

[0007] In a first aspect of the present invention, there is provided a 4-1BB binding protein, wherein said 4-1BB binding protein comprises a heavy chain variable region; said heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprises the sequence set forth in SEQ ID NO: 15 or variant 1 thereof, or SEQ ID NO: 16, said HCDR2 comprises the sequence set forth in SEQ ID NO: 60 or variant 2 thereof, or SEQ ID NO: 50 or variant 4 thereof, and said HCDR3 comprises the sequence set forth in SEQ ID NO: 103 or variant 3 thereof, SEQ ID NO: 333 or variant 5 thereof, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 116, SEQ ID NO: 326, SEQ ID NO: 331, SEQ ID NO: 334 or SEQ ID NO: 96; wherein: the mutation of said variant 1 comprises one or more of T3I, S6N / G / R and Y7F; preferably, the sequence of said variant 1 is preferably as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 300 or SEQ ID NO: 24 in the sequence listing; the mutation of said variant 2 comprises one or more of S1G / N / D, G2S / A, S3D, G5D / N / F / S / V and S6T / N / D; the sequence of said variant 2 is preferably as shown in any one of SEQ ID NO: 57-59, SEQ ID NO: 61, SEQ ID NO: 49, SEQ ID NO: 308-317 and SEQ ID NO: 63-71 in the sequence listing; The mutations in mutant 3 include one or more of the following: G2R / D / A / K, S3A / T, S4G / N / A / T / H, E5T / V / M / G, T6A, D7G / S, H9Y / S / N, Y10H, Y11F, N12G / D, and V13I / M / T; the amino acid sequence of mutant 3 is preferably one of the sequences shown in SEQ ID NOs. 101, 102, 104-106, 108, 109, and 112-115 in the sequence listing; The mutation of mutant 4 comprises N1I or N1Q; the amino acid sequence of mutant 4 is preferably shown in sequence number 53 or 54 in the sequence listing; The mutation of mutant 5 comprises E4A / P and / or N13Y; the amino acid sequence of mutant 5 is preferably shown in sequence numbers 327-330 in the sequence listing; The aforementioned mutants 1, 2, 3, 5, and 4 all possess at least the function of the sequence before the mutation.

[0008] Preferably, HCDR1, HCDR2, and HCDR3 are sequences shown in sequence numbers 16, 50, and 96, respectively, or sequences shown in sequence numbers 16, 53, and 96, respectively, or sequences shown in sequence numbers 16, 54, and 96, respectively, or sequences shown in sequence numbers 19, 57, and 101, respectively, or sequences shown in sequence numbers 15, 58, and 102, respectively, or sequences shown in sequence numbers 20, 59, and 103, respectively, or sequences shown in sequence numbers 20, 60, and 104, respectively, or sequences shown in sequence numbers 15, 61, and 105, respectively, or sequences shown in sequence numbers 15, 60, and 106, respectively, or sequences shown in sequence numbers 20, 63, and 108, respectively, or sequences shown in sequence numbers 20, 60, and 108, respectively, or sequences shown in sequence numbers 22, 64, and 109, respectively, or sequences shown in sequence numbers 15, 60, and 1 The sequence shown in 10, or the sequence shown in sequence number 15, sequence number 65 and sequence number 111 respectively, or the sequence shown in sequence number 22, sequence number 66 and sequence number 112 respectively, or the sequence shown in sequence number 15, sequence number 49 and sequence number 113 respectively, or the sequence shown in sequence number 20, sequence number 60 and sequence number 103 respectively, or the sequence shown in sequence number 15, sequence number 63 and sequence number 104 respectively, or the sequence shown in sequence number 15, sequence number 60 and sequence number 114 respectively, or the sequence shown in sequence number 23, sequence number 67 and sequence number 105 respectively, or the sequence shown in sequence number 24, sequence number 68 and sequence number 103 respectively, or the sequence shown in sequence number 15, sequence number 60 and sequence number 105 respectively, or the sequence shown in sequence number 20, sequence number 69 and sequence number 115 respectively, or the sequence shown in sequence number 15, sequence number 70 and sequence number 116 respectively, or the sequence shown in sequence number 20, sequence number 71 and sequence number 115 respectively, or the sequence shown in sequence number 300, sequence number 308 and sequence number 326 respectively, or sequence number 300,The sequence includes the sequence shown in SEQ ID NO: 309 and SEQ ID NO: 327, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 310 and SEQ ID NO: 328, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 308 and SEQ ID NO: 329, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 311 and SEQ ID NO: 330, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 312 and SEQ ID NO: 331, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 308 and SEQ ID NO: 332, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 313 and SEQ ID NO: 330, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 314 and SEQ ID NO: 329, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 315 and SEQ ID NO: 331, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 314 and SEQ ID NO: 327, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 316 and SEQ ID NO: 331, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 308 and SEQ ID NO: 333, or the sequence shown in SEQ ID NO: 300, SEQ ID NO: 317 and SEQ ID NO: 334. Refer to Table a below.

[0009] [Table A]

[0010] The gene encoding the FR of the heavy chain variable region is preferably derived from embryonic line V gene IGHV4-34, IGHV3-23, IGHV3-11, or IGHV3-74; among these: HFWR1 preferably comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5-8, SEQ ID NO: 294-299, and SEQ ID NO: 10-13; HFWR2 preferably comprises the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 32-36, SEQ ID NO: 301-307, and SEQ ID NO: 38-46; HFWR3 preferably comprises the amino acid sequence shown in SEQ ID NO: 74, SEQ ID NO: 79-83, SEQ ID NO: 318-325, and SEQ ID NO: 85-92; and HFWR4 preferably comprises the amino acid sequence shown in SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 335, SEQ ID NO: 336, or SEQ ID NO: 123. Preferably, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 3, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 27, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 74, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 118; the amino acid sequence of HFWR1 is shown in SEQ ID NO: 5, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 32, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 79, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119; the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 33, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 80, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119; or the HFWR1 The amino acid sequence of is shown in SEQ ID NO: 7, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 32, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 81, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 8, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 82, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 35, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 83, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, and the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6,The amino acid sequence of HFWR2 is shown in SEQ ID NO: 36, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 80, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 38, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 80, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 39, and the amino acid sequence of HFWR3 is shown in SEQ ID NO: 8 The amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 1, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 40, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 85, the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 10, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 41, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 86, the amino acid sequence of HFWR4 is shown in SEQ ID NO: 118, and the amino acid sequence of HFWR1 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 10, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 41, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 86, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 118, and the amino acid sequence of HFWR1 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 86, and the amino acid sequence of HFWR4 is shown in The sequence is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 35, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 80, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 11, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 42, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 87, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 5, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 32, and the amino acid sequence of HFWR3 is The amino acid sequence is shown in SEQ ID NO: 80, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 8, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 88, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 12, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 89, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 123,The amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 88, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 43, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 90, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, and the amino acid sequence of HFWR2 is shown in sequence As shown in number 44, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 91, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 13, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 90, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 45, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 80, and the HFWR The amino acid sequence of 4 is the sequence shown in SEQ ID NO: 119, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 1, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 46, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 92, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 301, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 318, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 335, or the The amino acid sequence of HFWR1 is shown in SEQ ID NO: 295, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 302, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 319, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 302, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 320, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294,The amino acid sequence of HFWR2 is shown in SEQ ID NO: 302, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 321, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 335, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 296, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 302, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 322, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 335, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 297, and the amino acid sequence of HFWR2 is shown in SEQ ID NO: 303, The amino acid sequence of HFWR3 is shown in SEQ ID NO: 318, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 335, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 301, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 322, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 302, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 323, and the The amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 298, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 302, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 322, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 301, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 318, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 120, Alternatively, the amino acid sequence of HFWR1 is shown in SEQ ID NO: 298, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 302, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 322, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 335, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 301, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 318, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294,The amino acid sequence of HFWR2 is shown in SEQ ID NO: 304, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 324, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 118; or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 294, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 305, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 325, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 336; or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 299, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 306, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 318, and the amino acid sequence of HFWR4 is the sequence shown in SEQ ID NO: 335; or the amino acid sequence of HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of HFWR2 is shown in SEQ ID NO: 307, the amino acid sequence of HFWR3 is shown in SEQ ID NO: 80, and the amino acid sequence of HFWR4 is shown in SEQ ID NO: 119. See Table b below for details. ,

[0011] [Table B]

[0012] In the present invention, the heavy chain variable region preferably includes any of the amino acid sequences shown in SEQ ID NO: 168, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 175-180, SEQ ID NO: 182-196, SEQ ID NO: 337-352, and SEQ ID NO: 198 in the sequence listing.

[0013] As described above, the 4-1BB binding protein described in the present invention further contains a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 133, LCDR2 comprises the sequence shown in SEQ ID NO: 145, and LCDR3 comprises the sequence shown in SEQ ID NO: 158. Preferably, the gene encoding FR in the light chain variable region is derived from embryonic line V gene IGKV3-15; in which: LFWR1 preferably comprises the amino acid sequence shown in SEQ ID NO: 126 or SEQ ID NO: 128, LFWR2 preferably comprises the amino acid sequence shown in SEQ ID NO: 140, LFWR3 preferably comprises the amino acid sequence shown in SEQ ID NO: 151, and LFWR4 preferably comprises the amino acid sequence shown in SEQ ID NO: 164; preferably, the light chain variable region comprises the sequence shown in SEQ ID NO: 201 in the sequence listing or a variant thereof, the variant being based on a mutation in one or more amino acid residues in the sequence shown in SEQ ID NO: 201; the sequence of the light chain variable region obtained after the mutation is preferably shown in SEQ ID NO: 204.

[0014] In one embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 168 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 201; or the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 171 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 204; or the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 172 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 204. See Table c below for details.

[0015] [Table C]

[0016] Furthermore, the 4-1BB binding protein described in the present invention may include a heavy chain constant region and / or a light chain constant region, preferably the heavy chain constant region being selected from hIgG1, hIgG2, hIgG3, or hIgG4 or a variant thereof, and the light chain constant region being selected from a κ chain or a λ chain or a variant thereof.

[0017] Among these, the mutation in the hIgG1 variant is preferably one or more of L234A, L235A, E345R, and P329G, more preferably E345R, or a combination of L234A, L235A, and E345R, or a combination of L234A, L235A, and P329G; the mutation in the hIgG4 variant is preferably S228P.

[0018] In a preferred embodiment of the present invention, the heavy chain of the 4-1BB binding protein includes any of the sequences shown in SEQ ID NOs: 209, 212, 213, 216-222, 224-238, 353-368, and 240 in the sequence listing, and / or its light chain includes the sequence shown in SEQ ID NOs: 246 or 243 in the sequence listing. For example: the amino acid sequence of the heavy chain is shown in SEQ ID NOs: 209 and the amino acid sequence of the light chain is shown in SEQ ID NOs: 243; or the amino acid sequence of the heavy chain is shown in SEQ ID NOs: 212 and the amino acid sequence of the light chain is shown in SEQ ID NOs: 246; or the amino acid sequence of the heavy chain is shown in SEQ ID NOs: 213 and the amino acid sequence of the light chain is shown in SEQ ID NOs: 246; or the amino acid sequence of the heavy chain is shown in SEQ ID NOs: 216 and the amino acid sequence of the light chain is shown in SEQ ID NOs: 246. See Table d below.

[0019] [Table D]

[0020] The 4-1BB-binding protein in the present invention may be in the form of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, heavy chain antibody, single-domain antibody, or single-region antibody, or it may be a monoclonal antibody or multiclonal antibody obtained from the said antibody.

[0021] If the 4-1BB-binding protein is a full-length antibody, its heavy chain contains the sequence shown in SEQ ID NO: 209 and its light chain contains the sequence shown in SEQ ID NO: 243; or it contains the sequence shown in SEQ ID NO: 212 and its light chain contains the sequence shown in SEQ ID NO: 246; or it contains the sequence shown in SEQ ID NO: 213 and its light chain contains the sequence shown in SEQ ID NO: 246; or it contains the sequence shown in SEQ ID NO: 216 and its light chain contains the sequence shown in SEQ ID NO: 246.

[0022] A second aspect of the present invention provides a bispecific antibody comprising a first protein functional domain and a second protein functional domain, wherein the first protein functional domain is a 4-1BB binding protein as described above, and the second protein functional domain targets a tumor antigen; preferably a HER2 antibody or a PD-L1 antibody; where the HER2 antibody is preferably trastuzumab or pertuzumab, and the PD-L1 antibody is preferably atezolizumab or PR000265, where the heavy chain of PR000265 is shown in SEQ ID NO: 211 and the light chain is shown in SEQ ID NO: 245. Therein, for further information on PR000265, refer to application CN201910944996.4.

[0023] Specifically, the first or second protein functional region may be in the form of scFv, VHH, immunoglobulin, Fab, Fab', F(ab')2, or a heavy chain variable region. For example, the first protein functional region may be Fab and the second protein functional region may be VHH; or the first protein functional region may be Fab and the second protein functional region may be immunoglobulin; or the first protein functional region may be immunoglobulin and the second protein functional region may be a heavy chain variable region.

[0024] Preferably, the first protein functional region and the second protein functional region and / or the heavy chain variable region and the light chain variable region of the scFv are linked by a conjugate, the amino acid sequence of the conjugate is shown in either GS or sequence numbers 273-293 in the sequence listing.

[0025] In certain embodiments of the present invention, the bispecific antibody comprises polypeptide chain 1 and polypeptide chain 2, and optionally further comprises polypeptide chain 3; preferably: The amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 244, and the amino acid sequence of polypeptide chain 2 is shown in any of SEQ ID NOs: 251-265; or the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 245, and the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO: 271; or the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 249, and the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO: 272; or the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 245, and the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO: 270; or the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 245, and the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO: 269; Alternatively, the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 245, and the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO: 268; or the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 245, and the amino acid sequence of polypeptide chain 2 is shown in any of SEQ ID NOs: 369-382; or the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 267, the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO: 266, and the amino acid sequence of polypeptide chain 3 is shown in SEQ ID NO: 246; or the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 250, the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO: 249, and the amino acid sequence of polypeptide chain 3 is shown in SEQ ID NO: 246.

[0026] A third aspect of the present invention provides an isolated nucleic acid encoding the 4-1BB binding protein described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0027] A fourth aspect of the present invention provides an expression vector comprising the isolated nucleic acid described in the third aspect.

[0028] A fifth aspect of the present invention provides a host cell comprising the expression vector described in the fourth aspect of the present invention; preferably, the host cell includes a prokaryotic cell or a eukaryotic cell, wherein the eukaryotic cell is preferably a mammalian cell.

[0029] A sixth aspect of the present invention provides a method for producing a 4-1BB-binding protein, comprising culturing the host cells of the fifth aspect and obtaining the 4-1BB-binding protein from the culture.

[0030] A seventh aspect of the present invention provides a chimeric antigen receptor comprising the 4-1BB binding protein described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0031] The eighth aspect of the present invention provides an antibody-drug conjugate comprising a cytotoxic agent and a 4-1BB-binding protein as described in the first aspect of the present invention or a bispecific antibody as described in the second aspect of the present invention.

[0032] The cytotoxic agent is preferably a cytoxin, a chemotherapeutic agent, a radioisotope, a therapeutic nucleic acid, an immunomodulator, an antitubulin-forming agent, an antiproliferative apoptotic agent, or a cell-lysinus enzyme. More preferably, the cytotoxic agent is a tubulin synthase inhibitor, such as methyl orestatin F (MMAF) or methyl orestatin E (MMAE).

[0033] The method for producing the antibody-drug conjugate described above is a common method in this art, and is preferably the method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124. Preferably, the method produces an antibody-drug conjugate with a minimum low complex fraction (LCF) of less than 10%.

[0034] The antibody-drug complex can exist in any physical form known in the art, preferably as a clear solution.

[0035] The ninth aspect of the present invention provides a drug composition comprising a 4-1BB-binding protein as described in the first aspect of the present invention, a bispecific antibody as described in the second aspect of the present invention and / or an antibody-drug conjugate as described in the eighth aspect of the present invention, and a pharmaceutically acceptable vector.

[0036] The drug composition preferably comprises other antitumor antibodies as an active ingredient, and / or further comprises one or more from the group consisting of hormone preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, lysogenic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0037] The pharmaceutically acceptable vector may be a conventional vector in the art, and this vector may be any suitable physiologically or pharmaceutically acceptable drug excipient. The drug excipient is a conventional drug excipient in the art, and preferably includes pharmaceutically acceptable excipients, fillers, stabilizers, or diluents. More preferably, the drug composition comprises 0.01 to 99.99% of the protein and / or the antibody-drug complex and 0.01 to 99.99% of the drug vector, where the percentages are by mass percentages of the drug composition.

[0038] Preferably, the drug composition is an antitumor agent. More preferably, it is a drug for gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and / or leukemia.

[0039] The administration routes of the drug composition according to the present invention are preferably extra-enterectogastric, intravenous, or oral. Intravenous administration preferably includes routes such as intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The drug composition is in various conventional dosage forms in the art, preferably in solid, semi-solid, or liquid form, i.e., aqueous, non-aqueous, or suspension, and more preferably in the form of tablets, capsules, pellets, injections, or infusions. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the drug composition can be administered as an aerosol or coarse spray, i.e., nasally, or intracavitally, intramedullarily, or intravenously. More preferably, the drug composition can be administered transdermally, percutaneously, topically, intraintestinally, intravaginally, sublingually, or rectally.

[0040] The dosage level of the drug composition according to the present invention can be adjusted according to the amount of composition that achieves the desired diagnostic or therapeutic outcome. The administration scheme may be a single injection or multiple injections, or may be modified. The selected dose level and scheme are reasonably adjusted depending on various factors, including the activity and stability (i.e., half-life) of the drug composition, formulation, route of administration, combination with other drugs or treatments, the disease or disorder being detected and / or treated, and the health status and prior medical history of the subject being treated.

[0041] The therapeutically effective dose of the drug composition of the present invention can first be estimated in cell culture experiments or animal models, such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine appropriate dose ranges and routes. These can then be used to determine useful doses and routes for administration to humans. Generally, the determination and adjustment of effective doses or dosages, and the evaluation of the timing and methods of such adjustments, are known to those skilled in the art.

[0042] In combination therapy, the 4-1BB binding protein, the antibody-drug conjugate, and / or additional therapeutic or diagnostic agents can each be used as a single agent within any time range suitable for performing the desired treatment or diagnosis. Therefore, these single agents can be administered substantially simultaneously (i.e., as a single formulation, or within minutes or hours) or sequentially. For example, these single agents can be administered within one year, or within 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or within 5, 4, 3, 2, or 1 day.

[0043] For additional guidance on formulations, dosages, administration methods, and measurable therapeutic outcomes, please refer to works such as Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey, and Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0044] The tenth aspect of the present invention relates to the use of the 4-1BB binding protein described in the first aspect, the bispecific antibody described in the second aspect of the present invention, and the drug composition described in the seventh aspect in the preparation of drugs for treating and / or preventing cancer; the cancer is preferably a cancer associated with one or more of HER2, PD-L1, and 4-1BB, such as breast cancer, melanoma, lung cancer, gastric cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumor, or colorectal cancer.

[0045] In some embodiments, the present disclosure provides a method for treating a disease associated with one or more of HER2, PD-L1, and 4-1BB, the method comprising administering a pharmaceutically effective amount of the 4-1BB-binding protein to a subject, or using a drug composition or a nucleic acid molecule containing the HER2, PD-L1, and 4-1BB, the disease being preferably a tumor or cancer.

[0046] Here, the tumor or cancer is a tumor or cancer with abnormal expression of one or more of the common HER2, PD-L1, and 4-1BB in the art, for example, scaly cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and cervical scaly cell carcinoma (HNSCC), glioma, He Jiejin lymphoma, non-He Jiejin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelocytic leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), Small lymphocytic lymphoma (SLL), large B-cell lymphoma rich in T-cells / tissue cells, multiple myeloma, medullary cell leukemia-1 protein (Mcl-1), myeloproliferative syndrome (MDS), gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroprogenitor cell tumor, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), pellucid cell renal cell carcinoma (RCC), head and neck cancer, pharyngeal cancer, hepatobiliary cancer Cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloid degeneration, lymphocytic lymphomaLymphoma, myeloproliferative syndrome, myeloproliferative neoplasm, neuroendocrine neoplasm, Mekell cell carcinoma, testicular cancer and skin cancer, PD-L1-positive scaly cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), scaly cell carcinoma of the head and neck (HNSCC), glioma, He Jiejin lymphoma, non-He Jiejin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic Lymphoma (SLL), large B-cell lymphoma rich in T-cells / tissue cells, multiple myeloma, medullary cell leukemia-1 protein (Mcl-1), myeloproliferative syndrome (MDS), gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuronocyte tumor, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, eugenics sarcoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), pellucid cell renal cell carcinoma (RCC), head and neck cancer, pharyngeal cancer, hepatobiliary cancer Cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloid degeneration, lymphocytic lymphoma, myeloproliferative syndrome, myeloproliferative neoplasm, neuroendocrine neoplasm, Merck cell carcinoma, testicular cancer, skin cancer.

[0047] An eleventh aspect of the present invention provides a reagent kit comprising a 4-1BB binding protein as described in the first aspect of the present invention, a bispecific antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the seventh aspect of the present invention, an antibody-drug complex as described in the eighth aspect of the present invention, and / or a drug composition as described in the ninth aspect of the present invention.

[0048] Preferably, the reagent kit further includes (i) a device for administering an antibody or its antigen-binding fragment or an antibody-drug complex or drug composition; and / or (ii) instructions for use.

[0049] A twelfth aspect of the present invention provides a drug kit comprising drug cassette A and drug cassette B, wherein: The drug cassette A comprises a 4-1BB binding protein according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a chimeric antigen receptor according to the seventh aspect of the present invention, an antibody-drug complex according to the eighth aspect of the present invention, and / or a drug composition according to the ninth aspect of the present invention; The drug cassette B includes other antitumor antibodies or drug compositions containing the other antitumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, lytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0050] To solve the aforementioned technical problems, the 4-1BB binding protein described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the chimeric antigen receptor described in the seventh aspect of the present invention, the antibody-drug complex described in the eighth aspect of the present invention, and / or the drug composition described in the ninth aspect of the present invention may be used in combination with other drugs, such as hormone preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, lysotherapy agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, and / or other antitumor antibodies (or drug compositions containing the other antitumor antibodies) and administered in combination.

[0051] A thirteenth aspect of the present invention provides a method for diagnosing, treating and / or preventing a disease or disorder mediated by 4-1BB, the method comprising administering to a patient in need a therapeutically effective amount of the 4-1BB-binding protein described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the chimeric antigen receptor described in the seventh aspect of the present invention, the antibody-drug complex described in the eighth aspect of the present invention, or the drug composition described in the ninth aspect of the present invention, or treating the patient in need using the drug kit described in the twelfth aspect of the present invention.

[0052] Preferably, the disease or disorder is a tumor, preferably a 4-1BB positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and / or leukemia.

[0053] A fourteenth aspect of the present invention provides a method for immunodetection or measurement of 4-1BB, the method comprising using a 4-1BB-binding protein as described in the first aspect of the present invention, a bispecific antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the seventh aspect of the present invention, an antibody-drug complex as described in the eighth aspect of the present invention, or a drug composition as described in the ninth aspect of the present invention; preferably, the detection is not for diagnostic and / or therapeutic purposes.

[0054] Fifteenth aspect of the present invention provides a combination therapy comprising administering to a patient in need a 4-1BB binding protein described in the first aspect of the present invention, a bispecific antibody described in the second aspect of the present invention, a chimeric antigen receptor described in the seventh aspect of the present invention, an antibody-drug complex described in the eighth aspect of the present invention, or a drug composition described in the ninth aspect of the present invention, and a second therapeutic agent; the second therapeutic agent preferably comprises one or more from the group consisting of hormonal preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, tumor drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0055] The aforementioned preferred conditions may be combined in any way that is consistent with common sense in the art, that is, any preferred example of the present invention can be obtained.

[0056] All reagents and raw materials used in this invention are commercially available. [Effects of the Invention]

[0057] The positive progressive effects of this invention are as follows: 1. This application provides a whole-human antibody of anti-4-1BB binding protein having at least one of the following properties: 1) The whole-human antibody of anti-4-1BB binding protein of the present invention comprises a full-length antibody and a completely novel whole-human antibody containing only the "heavy chain," and has the activity to bind to human 4-1BB and cynomolgus monkey 4-1BB; among these, the heavy chain antibody of the 4-1BB binding protein is only half the size of a conventional IgG antibody and does not contain a light chain, so this antibody can be used as a bispecific antibody and solves the problems of light chain mismatch and heterodimerization; 2) The binding epitope is different from that of Urelumab; 3) It can activate the 4-1BB signaling pathway, stimulating the secretion of IFNγ, IL2 and / or TNFα in immune cells and suppressing tumor growth and / or tumor cell proliferation; the activity was significantly stronger than that of Utomilumab.

[0058] 2. The PD-L1×4-1BB bispecific antibody provided by the present invention enhances antitumor efficacy and safety through one or more mechanisms of action. Firstly, the PD-L1×4-1BB bispecific antibody can activate T cells by blocking the PD-1 / PD-L1 signaling pathway. Secondly, the PD-L1 molecule, which is highly expressed on the surface of tumor cells, utilizes the bispecific antibody molecule to promote cross-linking and trimerization of 4-1BB molecules on the surface of T cells, activating downstream signaling pathways and further promoting T cell activation and proliferation. Its ability to activate T cells is superior to that of urelumab. Thirdly, the activating effect of the PD-L1×4-1BB bispecific antibody on T cells is specifically PD-L1-dependent, and the T cell activation mediated by the bispecific antibody molecule is limited to the tumor microenvironment. In contrast to conventional HCAb monoclonal antibodies that rely on cross-linked anti-4-1BB and cannot directly activate T cells, the present invention demonstrates that a PD-L1×4-1BB biantibody constructed using an HCAb monoclonal antibody can specifically activate T cells in the presence of cells that highly express PD-L1, thereby avoiding the toxic side effects caused by the excessive activation of T cells in normal tissues by monoclonal antibodies such as urelumab. Fourthly, this embodiment constructed bispecific anti-PD-L1×4-1BB antibody molecules with various structures by utilizing the antigen-binding domain Fab of an anti-PD-L1 IgG antibody and the antigen-binding domain VH of an anti-4-1BB HCAb antibody. It demonstrates flexibility in constructing bispecific antibody molecular structures based on HCAb, and modulates the functional activity that activates T cells by different structural types, relative positions, and binding valencies. HCAb-based biantibody structures, particularly IgG-VH tetravalent symmetric biantibody molecules and Fab-HCAb biantibody molecules, retained PD-L1 terminal activity and exhibited stronger T cell activation ability than corresponding anti-PD-L1 parental monoclonal antibodies in MLR experiments; on the other hand, PD-L1 molecules highly expressed in target cells can transmit T cell activation signals by mediating 4-1BB crosslinking and trimerization, and their T cell activation ability is superior to that of urelumab.Furthermore, the biantibody molecules with IgG-VH tetravalent symmetric structure and Fab-HCAb symmetric structure exhibit stronger T cell activation ability than the biantibody molecule with FIT-Ig structure.

[0059] 3. The HER2×4-1BB bispecific antibody provided by the present invention enhances antitumor efficacy and safety through one or more mechanisms of action. Firstly, the HER2×4-1BB bispecific antibody retains the mechanism of action of conventional HER2 inhibitors (inhibiting HER2 dimerization, promoting HER2 internalization and degradation, and suppressing downstream phosphorylation signals). Secondly, HER2 molecules highly expressed on the tumor cell surface promote cross-linking and trimerization of 4-1BB molecules on the T cell surface, activating downstream signaling pathways, and further promoting T cell activation and proliferation. Its ability to activate T cells is superior to that of urelumab. Thirdly, the activating effect of the HER2×4-1BB bispecific antibody on T cells is HER2-dependent expression, and T cell activation mediated by the bispecific antibody molecule is limited to the tumor microenvironment. In contrast to conventional HCAb monoclonal antibodies that rely on cross-linked anti-4-1BB and cannot directly activate T cells, the present invention provides a HER2×4-1BB biantibody constructed using an HCAb monoclonal antibody that can specifically activate T cells in the presence of cells that highly express HER2, thereby avoiding the toxic side effects caused by the overactivation of T cells in normal tissues by monoclonal antibodies such as urelumab. Fourthly, this embodiment constructs two bispecific anti-HER2×4-1BB antibody molecules with the structures IgG-VH and IgG-scFv, and modulates the functional activity that activates T cells through different structural types, relative positions, and valencies. At the same time, it demonstrates the flexibility of constructing bispecific antibody molecular structures based on HCAb. [Brief explanation of the drawing]

[0060] [Figure 1A] Figures 1A-1B: FACS detection of binding of 4-1BB H2L2 antibody to human or cynomolgus monkey 4-1BB overexpressing CHO-K1 cells. [Figure 1B] Same as above. [Figure 2] Figure 2: FACS detection of the blockade of binding between 4-1BB ligand and CHO-K1 / human 4-1BB cells by the 4-1BB H2L2 antibody. [Figure 3A] Figures 3A-3B: Activation of the 4-1BB H2L2 antibody against the 4-1BB signaling pathway is detected using HEK293 / 4-1BB / NF-κb reporter gene cells. [Figure 3B] Same as above. [Figure 4] Figure 4: The 4-1BB H2L2 antibody activates the 4-1BB pathway in vitro, inducing and activating IFN-γ secretion in T cells. [Figure 5A] Figures 5A-5B: The 4-1BB H2L2 antibody activates the 4-1BB pathway in vitro, inducing and activating IFN-γ secretion in CD8+ T cells and CD4+ T cells. [Figure 5B] Same as above. [Figure 6A] Figures 6A-6C: Tumor growth inhibitory activity of the 4-1BB H2L2 antibody PR000448 in the B6-h4-1BB transgenic mouse MC38 subcutaneous colon cancer model. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7] Figure 7: Pharmacokinetics of PR000448 in C57BL / 6J mice. [Figure 8A] Figures 8A-8C: The PR000980 antibody activates the 4-1BB pathway in vitro, inducing and activating the secretion of IFN-γ, TNF-α, and IL-2 by T cells. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] Figures 9A-9D: BIACORE detection of affinity between 4-1BB H2L2 antibody and human 4-1BB protein or monkey 4-1BB protein. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A] Figures 10A-10B: Tumor growth inhibitory activity of the 4-1BB H2L2 antibody PR000980 in the B6-h4-1BB transgenic mouse MC38 subcutaneous colon cancer model. [Figure 10B] Same as above. [Figure 11A] Figures 11A-11M: FACS detection of binding of 4-1BB HCAb antibody to CHO-K1 cells overexpressing human 4-1BB. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 11G] Same as above. [Figure 11H] Same as above. [Figure 11I] Same as above. [Figure 11J] Same as above. [Figure 11K] Same as above. [Figure 11L] Same as above. [Figure 11M] Same as above. [Figure 12A] Figures 12A-12L: FACS detection of binding of 4-1BB HCAb antibody to CHO-K1 cells overexpressing cynomolgus monkey 4-1BB. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 12F] Same as above. [Figure 12G] Same as above. [Figure 12H] Same as above. [Figure 12I] Same as above. [Figure 12J] Same as above. [Figure 12K] Same as above. [Figure 12L] Same as above. [Figure 13A]Figures 13A-13J: Activation of the 4-1BB HCAb antibody against the 4-1BB signaling pathway is detected using HEK293 / 4-1BB / NF-κb reporter gene cells. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 13H] Same as above. [Figure 13I] Same as above. [Figure 13J] Same as above. [Figure 14A] Figures 14A-14K: FACS detection of the blocking of binding between 4-1BB HCAb antibodies and CHO-K1 / human 4-1BB cells. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 14E] Same as above. [Figure 14F] Same as above. [Figure 14G] Same as above. [Figure 14H] Same as above. [Figure 14I] Same as above. [Figure 14J] Same as above. [Figure 14K] Same as above. [Figure 15A] Figures 15A-15D: 4-1BB HCAb antibodies activate the 4-1BB pathway in vitro, inducing and activating IFN-γ secretion in T cells. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 16A]Figures 16A-16B: When CHO-K1 / CD32b crosslinking is present or absent, 4-1BB HCAb antibodies activate the 4-1BB pathway in vitro, inducing and activating IFN-γ secretion in T cells. [Figure 16B] Same as above. [Figure 17A] Figures 17A-17D: FACS detection of specific binding of 4-1BB HCAb antibody to CHO-K1 / human 4-1BB cells. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 18A] Figures 18A-18C: Schematic diagrams of the structure of the HER2×4-1BB bispecific antibody. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19A] Figures 19A-19C: FACS detection of binding of HER2×4-1BB bispecific antibody to SK-BR-3 cells. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 20A] Figures 20A-20E: FACS detection of binding of HER2×4-1BB bispecific antibody to human 4-1BB overexpressing CHO-K1 cells. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 20E] Same as above. [Figure 21A] Figures 21A-21B: FACS detection of binding of HER2×4-1BB bispecific antibody to CHO-K1 cells overexpressing cynomolgus monkey 4-1BB. [Figure 21B] Same as above. [Figure 22A]Figures 22A-22D: The HER2 × 4-1BB bispecific antibody cross-links SK-BR-3 cells, activating the 4-1BB pathway in vitro and inducing and activating IFN-γ secretion in T cells. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above. [Figure 23A] Figures 23A-23E: Schematic diagrams of the structure of PD-L1×4-1BB bispecific antibodies. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 23E] Same as above. [Figure 24A] Figures 24A-24E: FACS detection of binding of PD-L1×4-1BB bispecific antibody to CHO-K1 / hPD-L1 cells. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 24E] Same as above. [Figure 25A] Figures 25A-25E: FACS detection of binding of PD-L1×4-1BB bispecific antibody to human 4-1BB overexpressing CHO-K1 cells. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above. [Figure 26A] Figures 26A-26B: FACS detection of binding of PD-L1×4-1BB bispecific antibody to CHO-K1 cells overexpressing cynomolgus monkey 4-1BB. [Figure 26B] Same as above. [Figure 27A]Figures 27A-27I: PD-L1 × 4-1BB bispecific antibodies, when cross-linked with CHO-K1 / hPD-L1 or MDA-MB-231 cells, activate the 4-1BB pathway in vitro, inducing and activating IFN-γ secretion in T cells. [Figure 27B] Same as above. [Figure 27C] Same as above. [Figure 27D] Same as above. [Figure 27E] Same as above. [Figure 27F] Same as above. [Figure 27G] Same as above. [Figure 27H] Same as above. [Figure 27I] Same as above. [Figure 28A] Figures 28A-28K: The activating effect of the PD-L1×4-1BB biantibody molecule on T cells is studied using the mixed lymphocyte reaction (MLR). [Figure 28B] Same as above. [Figure 28C] Same as above. [Figure 28D] Same as above. [Figure 28E] Same as above. [Figure 28F] Same as above. [Figure 28G] Same as above. [Figure 28H] Same as above. [Figure 28I] Same as above. [Figure 28J] Same as above. [Figure 28K] Same as above. [Figure 29] Figure 29: Pharmacokinetics of 4-1BB bispecific antibody in PD-L1×C57BL / 6J mice. [Modes for carrying out the invention]

[0061] The following provides specific definitions of certain technical and scientific terms. Unless otherwise clearly stated, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this invention pertains.

[0062] The three-letter and one-letter codes for the amino acids used are described in J. Biol. Chem, 243, p3558 (1968).

[0063] The term "antibody" refers to immunoglobulins, which are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Because the amino acid composition and sequence order of the constant region of the heavy chains of immunoglobulins differ, their antigenicity also differs. This allows immunoglobulins to be classified into five types, or into homogeneous immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subtypes depending on the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are divided into κ or λ chains depending on the differences in the constant region. Of the five types of Ig, each type of Ig may have either a κ or λ chain.

[0064] The antibody light chain antibody may further include a light chain constant region, which may include human-derived κ, λ chains or their variants.

[0065] The antibody heavy chain antibody may further include a heavy chain constant region, which may include human-derived IgG1, 2, 3, 4 or its variants.

[0066] The amino acid sequences near the N-terminus of the antibody heavy and light chains undergo significant changes and are known as the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are known as the constant region (C region). The variable regions of the light and heavy chains each consist of approximately 110 amino acids, and the changes in amino acid residues in some of these regions are greater than in other parts of the variable region, for example, positions 24-34, 50-56, and 89-97 of the light chain and positions 31-35, 50-65, and 95-102 of the heavy chain. These regions are called hypervariable regions (HVRs), and because they are the sites where the antibody directly contacts the antigen epitope, they are also called complementarity-determining regions (CDRs). Non-hypervariable regions within the variable region undergo relatively little change in amino acid composition and sequence, and these amino acid residues constitute the stable three-dimensional structure of the variable region, i.e., the framework region or stent structure (FR). The variable regions include three highly variable regions (HVRs) and four relatively conserved frame regions (FRs). Each light-chain variable region (VL) and heavy-chain variable region (VH) consists of three CDR regions and four FR regions, arranged sequentially from the amino end to the carboxyl end as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3. The three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. In this application, the classification is made using the Chothia definition rules. However, those skilled in the art can define the CDR of an antibody in various ways in the art, such as the Kabat definition rule based on sequence variability (see Kabat et al., Immunological Protein Sequences, 5th edition, National Institutes of Health, Bethesda, Maryland (1991)) and the Chothia definition rule based on the position of the structural ring region (see Jmol Biol 273:927-48, 1997). In aspects of the present invention, a Combined definition rule, which includes both the Kabat and Chothia definitions, can also be used to determine the amino acid residues in the variable domain sequence. The Combined definition rule combines the ranges of the Kabat and Chothia definitions.Those skilled in the art will understand, unless otherwise specified, that the terms “CDR” and “complementary determination region” of a particular antibody or region (e.g., variable region) encompass the complementary determination region as defined by any known embodiment described herein. The scope of protection claimed by this invention is the sequence shown under the Chothia definition rules, but the corresponding amino acid sequence according to other CDR definition rules should also be included in the scope of protection of this invention.

[0067] In this application, the term "whole-human antibody" generally refers to an antibody expressed in an animal that has been genetically modified to contain all of the genes encoding human antibodies. All parts of the antibody (including the variable and constant regions) are encoded by human-derived genes. Whole-human antibodies can significantly reduce adverse immune reactions in the human body caused by heterologous antibodies. Methods for obtaining whole-human antibodies in this field include phage display technology and transgenic mouse technology.

[0068] The term "single-chain antibody" refers to a single-chain recombinant protein in which the heavy chain variable region (VH) and light chain variable region (VL) of an antibody are linked by a connecting peptide, and it is the smallest antibody fragment that has a complete antigen-binding site.

[0069] The term "epitope" refers to a site in an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged in parallel by tertiary folding of a protein. Epitopes formed from adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. Epitopes typically contain at least 3 to 15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to specific antibodies are well known in the art and include immunoimaging and immunoprecipitation detection analysis. Methods for determining the spatial conformation of epitopes include techniques in the art and techniques described in the text, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0070] The term "nucleic acid" refers to DNA and RNA molecules. They may be single-stranded or double-stranded, but preferably double-stranded DNA. When nucleic acids are functionally related to another nucleic acid sequence, they are "effectively bound." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively bound to the coding sequence.

[0071] In this application, the term "specifically binds" generally means that an antibody binds to an epitope via its antigen-binding region, and that such binding requires a certain degree of complementarity between the antigen-binding region and the epitope. According to this definition, if an antibody is more likely to bind to an epitope via its antigen-binding region than to bind to an unrelated epitope randomly, it is said to "specifically bind" to that antigen.

[0072] In this application, the term "Fab" generally refers to the antigen-binding portion in a typical antibody (e.g., IgG), which includes the heavy chain variable region VH, the light chain variable region VL, the heavy chain constant region domain CH1, and the light chain constant region CL. In a typical antibody, the C-end of VH binds to the N-end of CH1 to form a heavy chain Fd fragment, the C-end of VL binds to the N-end of CL to form a light chain, and the C-end of CH1 further binds to the hinge region and other constant region domains of the heavy chain to form a heavy chain. In some embodiments, "Fab" also refers to a variant structure of Fab. For example, in some embodiments, the C-end of VH and the N-end of CL bind to form one polypeptide chain, and the C-end of VL and the N-end of CH1 bind to form another polypeptide chain, forming the structure Fab(cross VH / VL); in some embodiments, CH1 of Fab is not bound to the hinge region, and the C-end of CL is bound to the hinge region of the heavy chain, forming the structure Fab(cross Fd / LC).

[0073] In this application, the term "VH" usually refers to the VH domain of the heavy chain variable region of an antibody, which may be the VH of a normal antibody (H2L2 structure) of a human or other animal, the VHH of a heavy chain antibody (HCAb structure) of an animal such as a camelid, or the VH of a whole-human heavy chain antibody (HCAb structure) produced using Harbour HCAb transgenic mice.

[0074] Example 1. Acquisition of whole-human derived 4-1BB H2L2 antibody. Example 1.1. Preparation of monoclonal antibodies Harbour H2L2 mice (Harbour Antibodies BV) are transgenic mice possessing a human immunoglobulin immune library, and their produced antibodies have a complete human antibody variable domain and a rat constant domain. Harbour H2L2 mice were immunized in multiple rounds using a soluble recombinant human 4-1BB-Fc fusion protein. After detecting that the 4-1BB-specific antibody titer in mouse serum reached a specific level, mouse spleen cells were isolated and fused with myeloma cell lines to obtain hybridoma cells. After multiple rounds of screening and cloning of the hybridoma cells, two hybridoma cell lines expressing the anti-4-1BB monoclonal antibody molecule, 65D4G5G11 and 79B10G8D4, were isolated. The nucleotide sequences encoding the antibody molecule variable domain and the corresponding amino acid sequences were obtained using conventional hybridoma sequencing methods. In this example, the sequences of the monoclonal antibody molecule variable domains obtained from immunized Harbour H2L2 mice are human-derived antibody sequences. The CDR sequences of the antibody variable domain can be analyzed using Kabat, Chothia, or a combined definition rule combining Kabat and Chothia definitions. In embodiments of the present invention, the CDR sequences are segmented according to the Chothia definition rule.

[0075] Example 1.2. Preparation of recombinant whole-human antibodies After obtaining the light and heavy chain variable domain sequences encoding the antibody molecule, the corresponding human antibody light and heavy chain constant domain sequences can be fused and expressed using conventional recombinant DNA technology to obtain recombinant antibody molecules.

[0076] The antibody number for the 65D4G5G11 clone is PR000196, and the antibody number for the 79B10G8D4 clone is PR000197.

[0077] Simultaneously, this application produces a positive control antibody against 4-1BB, Urelumab, and an analogue of Utomilumab. The antibody number corresponding to Urelumab is PR000628, and the antibody number corresponding to Utomilumab is PR000483. Their corresponding amino acid sequences are derived from the IMGT database.

[0078] In this example, the antibody light chain variable domain sequence (VL) is cloned by gene synthesis into a mammalian cell expression plasmid vector encoding the constant domain sequence of the human antibody κ light chain, thereby encoding and producing the full-length light chain of the antibody. In this example, the antibody heavy chain variable domain sequence (VH) is cloned by gene synthesis into a mammalian cell expression plasmid vector encoding the constant domain sequence of the human IgG4 antibody heavy chain, thereby encoding and producing the full-length heavy chain of the IgG4 antibody. Furthermore, the stability of the IgG4 antibody is enhanced by introducing an S228P mutation (substitution of serine at position 228 according to EU number with proline) into the constant region of the IgG4 heavy chain. In this example, the antibody heavy chain variable domain sequence (VH) is cloned by gene synthesis into a mammalian cell expression plasmid vector encoding the constant domain sequence of the human IgG1 or human IgG2 antibody heavy chain, thereby encoding and producing the full-length heavy chain of the IgG1 or IgG2 antibody. In this example, since the sequence of the monoclonal antibody molecule variable domain obtained from immunized Harbour H2L2 mice is a human-derived antibody sequence, a whole-human-derived anti-4-1BB recombinant antibody was also obtained in this example.

[0079] By simultaneously transfecting mammalian host cells such as human embryonic kidney cells (HEK293) with plasmids encoding antibody heavy chains and antibody light chains, and utilizing conventional recombinant protein expression and purification techniques, purified recombinant antibodies with correctly combined light and heavy chains can be obtained. Specifically, HEK293 cells can be transfected using FreeStyle (商標) Cells were cultured in F17 Expression Medium (Thermo, A1383504). Before initiating instantaneous transfection, the cell concentration was increased to 6-8 × 10⁶. 5 The cells were adjusted to a concentration of 1.2 × 10⁶ cells / ml and cultured for 24 hours in an 8% CO₂ rocker at 37°C. 6 The cell density is cells / ml. 30 ml of cultured cells were prepared. A total of 30 μg of plasmids encoding the antibody heavy chain and antibody light chain were mixed in a 2:3 ratio and dissolved in 1.5 ml of Opti-MEM serum-reduced medium (Thermo, 31985088). The mixture was filtered and sterilized using a 0.22 μm filter membrane. Furthermore, 1.5 ml of Opti-MEM was dissolved in 120 μl of 1 mg / ml PEI (Polysciences Inc, 23966-2) and allowed to stand for 5 minutes. The PEI was slowly added to the plasmid, incubated at room temperature for 10 minutes, and the plasmid-PEI mixture was slowly added dropwise while shaking the culture bottle. The culture was incubated at 37°C in an 8% CO2 rocking bed for 5 days. Cell viability was observed after 5 days. The culture was collected and centrifuged at 3300 G for 10 minutes, after which the supernatant was collected. The supernatant was then centrifuged at high speed to remove impurities. MabSelect was used with PBS (pH 7.4). (商標)A gravity column (Bio-Rad, 7311550) equilibrated with (GE Healthcare Life Science, 71-5020-91 AE) was washed with 2-5 column volumes. Load the supernatant sample onto the column. Wash the column with 5 to 10 column volumes of PBS. After eluting the target protein with 0.1M glycine at pH 3.5, adjust the pH to neutral with Tris-HCl at pH 8.0, and finally concentrate and exchange into PBS buffer using an ultrafiltration tube (Millipore, UFC 901024) to obtain a purified antibody solution. Then measure with NanoDrop (Thermo Scientific) (商標) NanoDrop (商標) One) to measure the concentration, then aliquot and store as a reserve.

[0080] Example 1.3. Antibody Sequence Analysis and Optimization The heavy chain variable domain sequence of an antibody is derived from events such as genetic recombination of germline V, D, and J gene fragments of the heavy chain gene cluster on the chromosome and somatic high-frequency mutations; the light chain variable domain sequence is derived from events such as genetic recombination of germline V and J gene fragments of the κ or λ light chain gene cluster and somatic high-frequency mutations. Genetic recombination and somatic high-frequency mutations are the main factors that increase the diversity of antibodies. Antibodies derived from the same germline V gene fragment may also produce different sequences, but overall have high similarity. Computational methods such as IMGT / DomainGapAlign (http: / / imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) or NCBI / IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) can be used to infer possible germline gene fragments when genetic recombination occurs from the variable domain sequence of an antibody. The antibody sequences obtained in Example 1.1 were analyzed, and the germline V gene fragments of the heavy chain variable domain (VH) and light chain variable domain (VL) are shown in Table 1.

[0081] When protein or polypeptide amino acid chains are translated and synthesized in cells, they may undergo chemical modifications called post-translational modifications (PTMs). For antibodies, some PTM sites are highly conservative; for example, asparagine (Asn), the conservative amino acid at position 297 (EU number) of the constant domain of human IgG1 antibodies, usually undergoes glycosylation to form a sugar chain, and this sugar chain structure is crucial for the antibody structure and associated effector function. However, when PTMs are present in the variable domains of antibodies, particularly antigen-binding regions such as CDRs, the presence of these PTMs can significantly affect antigen binding and potentially alter the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, and oxidation all increase the instability or heterogeneity of the antibody molecule, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding some potential PTMs is crucial for the development of therapeutic antibodies. With the accumulation of experience, it has been discovered that some PTMs are highly correlated with the composition of the amino acid sequence, particularly the "patterns" of adjacent amino acid compositions, allowing for the prediction of potential PTMs from the primary amino acid sequence of a protein. For example, the N-linked glycosylation site is predicted from the sequence pattern of NxS / T (position 1 is asparagine, position 2 is any amino acid other than proline, and position 3 is serine or threonine). The amino acid sequence pattern that causes PTM may originate from embryonic gene sequences; for example, the human embryonic gene fragment IGHV3-33 naturally has the glycosylation pattern NST in the FR3 region; it may also originate from somatic high-frequency mutations. Table 1 shows the predicted PTM of the variable domains VH and VL of the antibody of Example 1.1. Specifically, NHS may be the glycosylation site and NG may be the deamide site.

[0082] [Table 1]

[0083] Amino acid mutations can disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of specific PTMs. Different mutation design methods exist depending on the antibody sequence and PTM sequence pattern. One method involves substituting a "hotspot" amino acid (e.g., N or S in NS mode) with a physicochemically similar amino acid (e.g., mutating N to Q). If the PTM sequence pattern originates from somatic high-frequency mutations and does not exist in the embryonic gene sequence, another method is to substitute the sequence pattern with the corresponding embryonic gene sequence. In practice, multiple mutation design methods can be employed for the same PTM sequence pattern.

[0084] The effector functions mediated by the antibody Fc domain, such as ADCC and CDC, also have very important biological functions. Different IgG subtypes have different ADCC or CDC functions; for example, IgG1 and IgG3 have strong ADCC and CDC activity, while IgG2 and IgG4 have relatively weak activity. Furthermore, changes in the binding ability of Fc to the Fc receptor due to amino acid mutations or modifications can also regulate the intrinsic effector function of Fc. For example, the "LALA" double mutant (L234A / L235A) in IgG1 can reduce its affinity for FcγRIIIA (CD16 A) and further reduce its ADCC activity. In this embodiment, the variable region derived from the same antibody can be recombined with different IgG subtypes or mutants to regulate effector function, such as human IgG4 (S228P), human IgG2, and human IgG1 (LALA). D. Zhang et al. discovered that introducing the E345R mutation into the E345 site of the Fc molecule in human IgG effectively increases the activating effect of agonist-type OX40 antibodies (The Journal of Biological Chemistry, 291:27134-27146, 2016).

[0085] This application also found that the E345R mutation can effectively increase the activating effect of agonist-type 4-1BB antibodies. In this example, the anti-4-1BB hybridoma monoclonal antibodies shown in Table 1 were sequence-optimized by IgG subtype conversion, variable region mutation, and Fc mutation to obtain a series of recombinant antibodies or their variants (see Table 2). Tables 3.1, 3.2, and a show the light chain and heavy chain variable domain amino acid sequences, the full-length light chain amino acid sequence, the full-length heavy chain amino acid sequence, and the amino acid sequence of the CDR defined according to the Chothia definition rules of the recombinant antibodies obtained in this example.

[0086] [Table 2]

[0087] [Table 3-1]

[0088] [Table 3-2]

[0089] Example 1.4.4-1BB H2L2 antibody and 4-1BB binding to the cell surface Human 4-1BB-expressing CHO-K1 cells and cynomolgus monkey 4-1BB-expressing CHO-K1 cells were amplified and cultured, then washed three times with PBS, resulting in 3 × 10⁶ cells. 5The antigen-binding protein was added to a 96-well plate (Corning, #3799) at a concentration of / well. Next, 100 μl of the target antigen-binding protein or the positive control antibody Utomilumab or Urelumab was added, and the mixture was incubated at a maximum concentration of 200 nM, 5-fold dilution, and incubated at 4°C for 1 hour. The mixture was washed twice with FACS buffer, and AF488 conjugation antibody of sheep anti-human IgG (Life Technologies, #A11013), diluted 1:1000, was added, and the mixture was incubated at 4°C for 1 hour. The mixture was washed twice with FACS buffer, resuspended in 100 μl of PBS, and a FACS (BD Biosciences, Canto II) test was performed, and the fluorescence signal was read. From the measurement results, the maximum concordance (EC50) was calculated as the basis for evaluating relative binding activity.

[0090] The results are shown in Figure 1 and Table 4 below. Figure 1A shows the binding activity of the antigen-binding protein to human 4-1BB, and Figure 1B shows the binding activity of the antigen-binding protein to monkey 4-1BB. Both PR000447 and PR000448 can bind to human and cynomolgus monkey 4-1BB, and their binding activity is superior to that of the positive control Urelumab. Urelumab cannot cross-bind to cynomolgus monkey 4-1BB.

[0091] [Table 4]

[0092] Example 1.5.4-1BB H2L2 antibody blocks the binding of 4-1BB ligand to 4-1BB. To study the activity of human 4-1BB-binding protein in vitro in blocking the binding of human 4-1BB to human 4-1BBL, we conducted cellular-level human 4-1BB / human 4-1BBL binding blocking experiments using the CHO-K1 cell line (CHO-K1 / hu 4-1BB) overexpressing human 4-1BB. In short, CHO-K1 / hu 4-1BB cells were digested, resuspended in F-12K complete medium, and the cell density was increased to 1 × 10⁶. 6The cells were adjusted to the required concentration per mL. 100 μL of cells / well were inoculated into a 96-well V plate (Corning, #3894), and then the target antigen-binding protein, diluted with a 3x concentration gradient (2x the final concentration) in 100 μL / wells, was added and mixed uniformly. The highest final concentration of the antigen-binding protein was 100 nM, with a total of 8 concentrations, hIgG1 used as the control. The cells were placed at 4°C, protected from light, and incubated for 1 hour. Subsequently, the cells were centrifuged at 4°C for 5 minutes, the supernatant discarded, and then 1 μg / mL of biotin-labeled human 4-1BBL protein (Acro, #41L-H82F9) was added in 50 μL / wells and incubated at 4°C, protected from light, for 30 minutes. The cells were rinsed twice with 100 μL / well of pre-cooled PBS, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. 100 μL / well was added to the cell with fluorescent secondary antibody (PE Streptavidin, BD, #554061, 1:200), and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with 200 μL / well of pre-cooled PBS, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-cooled PBS, fluorescence emission signal values ​​were read using BD FACS CANTO II, IC50 was calculated, and the inhibition rate % = 1 - MFI(4 - 1BB Ab) / MFI(iso).

[0093] The results are shown in Figure 2. PR000448 blocks the binding of the 4-1BB ligand to 4-1BB, and its blocking effect is similar to that of Utomilumab.

[0094] Example 1.6. Detection of stimulating effects on the 4-1BB signaling pathway using a reporter gene cell system. CHO-K1 cells expressing CD32b, namely CHO-K1 / CD32b (Genscript, #M00587) or CHO-K1 (ATCC, #CCL-61), were placed in a 96-well plate (Perkin Elmer, #6005225), with a cell volume of 1.5 × 10⁶. 4The sample was divided into 100 μL / well and incubated overnight at 37°C in a 5% CO2 environment. The supernatant was removed, and 2x dilution of the antigen-binding protein (40 μL / well) was added. The initial concentration was 200 nM, and at a 5-fold dilution, hlgG1 was the control group. 4.5 × 10 4 HEK293 reporter cells (HEK293 / 4-1BB / NF-kb reporter cells, BPS Biosciences, #79289), which can continuously express the 4-1BB and NF-Kb reaction elements, were added at 40 μL / well. The cells were cultured at 37°C in a 5% CO2 environment for 6 hours. ONE-Glo TM Luciferase reagent (Promega, #E6110) was added, incubated at room temperature for 5 minutes, and the luminescence value was measured using a microplate reader analyzer.

[0095] As shown in Figures 3A, 3B, and Table 5 below, the 4-1BB antigen-binding protein PR000448 of this application is CHO-K1 / CD32b crosslinking-dependent. As shown in Figure 3A, under CHO-K1 / CD32b crosslinking, the activating effect of PR000448 described in this application on the 4-1BB-mediated NF-Kb signaling pathway increases in a positive correlation with its concentration. Compared to the reference antibody (Utomilumab), the EC50 of this antibody is lower and the maximum luminescence value is higher than that of the reference antibody, indicating that this antibody can promote NF-Kb activation at lower concentrations. As shown in Figure 3B, the reference antibody Urelumab does not depend on crosslinking for 4-1BB signaling pathway activation and can similarly activate the 4-1BB-mediated NF-Kb signaling pathway in the absence of CHO-K1 / CD32b crosslinking. On the other hand, PR000448 cannot activate the 4-1BB-mediated NF-Kb signaling pathway in the absence of the CHO-K1 / CD32b bridge.

[0096] [Table 5]

[0097] Example 1.7.4-1BB In vitro functional detection of H2L2 antibody Example 1.7.1.4-1BB H2L2 antibody activates the 4-1BB pathway in vitro. CHO-K1-CD32b cells (CHO-K1 cells overexpressing human CD32b) were treated with 10 μg / ml mitomycin (Ruitaibio, 10107409001) and left at 37°C for 30 minutes. The cells were then washed four times with 10% FBS F-12K culture medium. The treated cells were placed in a 96-well plate, with 1.5 × 10⁶ cells per well. 4 The cells were cultured overnight in an insulated box at 37°C. The following day, human CD3-positive T cells were isolated from human PBMCs using a MACS kit (Miltenyi Biotec, #130-096-535). First, the cell count was confirmed, and then MACS buffer and Pan-T cell biotin antibody were added according to the cell count, mixed, and allowed to stand at 4°C for 5 minutes. Then, a corresponding amount of microbeads was added and allowed to stand at 4°C for 10 minutes. Only CD3-positive T cells passed through the LS column. The culture medium from the previous day's 96-well plate was washed, and purified T cells were added, with 1 × 10⁶ cells per well. 5 The sample size was [number]. Then, the corresponding concentration of 4-1BB antigen-binding protein or control antibody Utomilumab, PR000196 was added, followed by the addition of OKT3 (eBiosciences, #16-0037-85) to a final concentration of 0.3 μg / ml. The samples were incubated in a 37°C incubator for 72 hours. After 72 hours, the supernatant was collected and the IFN-γ content was detected using an ELISA kit (Invitrogen, #88-7316-88). Coated antibody was added to a 96 flat-bottom plate and left overnight at 4°C. The next day, ELISA buffer was added and left at room temperature for 1 hour. The received supernatant was added and incubated at room temperature for 2 hours. The plate was washed twice, the detection antibody was added, and left at room temperature for 1 hour. The plate was washed twice, HRP-streptavidin was added, and incubated at room temperature for 1 hour. Next, TMB substrate was added, followed by ELISA stop solution (BBI, #E661006-0200). Absorbance values ​​at 450 nm and 570 nm were read using a microplate reader (Perkin ElemerEnspire), and the IFN-γ concentration was calculated using OD450-OD570.

[0098] As shown in Figure 4, the results indicate that the PR000197, PR000447, and PR000448 antibodies all activate the 4-1BB pathway, activating T cells and inducing IFN-γ secretion. Furthermore, their activating effects are stronger than those of Utomilumab and PR000196.

[0099] Example 1.7.2.4-1BB H2L2 antibody induces the activity of CD4+ and CD8+ T cells. CHO-K1 or CHO-K1 / CD32b cells were processed according to the method of Example 1.7.1, plated, and incubated overnight in a 37°C incubator. The following day, sorting reagents (Miltenyi Biotec, #130-096-495, #130-096-533) were used to sort CD8+ and CD4+ T cells. Then, 4-1BB antibody or control antibody at the corresponding concentration was added, followed by OKT3 (eBiosciences, #16-0037-85) to a final concentration of 0.3 μg / ml. The cells were incubated in a 37°C incubator for 72 hours. After 72 hours, the supernatant was collected, and the IFN-γ content was detected using an ELISA kit (Invitrogen, #88-7316-88).

[0100] CD8+ T cell induction is shown in Figure 5A, and CD4+ T cell induction is shown in Figure 5B.

[0101] As a result, PR000447 and PR000448 were able to significantly activate IFN-γ secretion from CD8+ T cells, but their activation function on CD4+ T cells was not significant. This may be because 4-1BB expression is higher in CD8+ T cells than in CD4+ T cells.

[0102] Example 1.8.4-1BB H2L2 antibody exhibits activity in suppressing tumor growth in the body. A subcutaneous colon cancer model was constructed using B6-h4-1BB recombinant mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd, #11004) containing MC38 cells. First, mouse colon cancer cells MC38 (Kerafast, ENH204-FP) were resuscitated. Culture conditions: RPMI-1640 medium (Gibco, 22400089) + 100 U / mL penicillin (AMRESCO). (登録商標) ,#0242-100MU)+100μg / mL streptomycin (AMRESCO (登録商標) ,#0382)+10%FBS(GIBCO (登録商標) Invitrogen (商標) ,#10099); 37°C, saturated humidity, 5% CO2. Logarithmic growth phase MC38 cells (passage count recorded) were collected, the culture medium was removed, and the cells were inoculated after being washed twice with PBS (pretumor cell viability: 98.9%, posttumor cell viability: 92%), inoculation volume: 5 × 10 5 / 100μl / mice (without matrix adhesive), inoculation site: right subcutaneous tissue. Mice were divided into 5 groups according to grouping criteria, with 6 mice in each group. The average size of each group was similar, with an average range of 80-120mm. 3 The criteria for inclusion in a group are: the volume of the tumor in a single mouse within each group should be as close to 150 mm as possible. 3 The SEM values ​​within each group were not to exceed 1 / 10 of the average value. The day of group assignment was defined as day 0, and administration began from day 0.

[0103] The sample to be measured was prepared in PBS, administered by intraperitoneal injection, with a dose volume of 10 μl / g and a dose of 5 mg / kg, administered twice a week for a total of 6 times over a period of 3 weeks. The body weight and tumor volume of the mice were measured on the day of administration, and the average tumor volume was 700-800 mm³. 3 Measurements were taken twice a week until the average tumor volume reached 700-800 mm³. 3 If it exceeds this value, the tumor volume was changed to be measured three times a week. The method for calculating tumor volume is: Tumor volume (mm 3 ) = 0.5 × Tumor length × Tumor width 2 .

[0104] Mouse weight monitoring is shown in Figure 6A, and tumor volume monitoring is shown in Figure 6B. After 27 days, the mice were euthanized, the tumors were removed and photographed, and the tumor sizes are shown in Figure 6C.

[0105] The results showed that PR000448 had a clear tumor-suppressing effect, and during the experiment, the body weight of all groups increased, indicating good tolerance to the test substance in the animals. There were no obvious toxic effects in the animals, and the safety was relatively good.

[0106] Example 1.9. Pharmacokinetic study The pharmacokinetics of PR000448 were evaluated in female C57BL / 6J mice. PR000448 and Utomilumab were administered intravenously as a single dose at 5 mg / kg (n=5), and blood was collected before injection and on days 1, 2, 4, 7, 10, and 14 after injection. The collected blood was immediately centrifuged at 15000 rpm at 4°C for 15 minutes to obtain plasma, which was stored in a refrigerator at -20°C or below. The concentrations of the target antibodies in the plasma were measured using the ELISA method. Figure 7 shows the changes in plasma concentrations of PR000448 and Utomilumab. The obtained plasma concentration change data were analyzed using the drug dynamics analysis software WinNolin, and the drug half-life (t) was determined. 1 / 2 ) was calculated. l / 2 This was calculated using the final 3-point or the concentration in the final-phase plasma, which is automatically set by the software. 1 / 2 This is shown in Figure 7 and Table 6 below.

[0107] As a result, it was revealed that PR000448 has a longer half-life in the blood compared to Utomilumab.

[0108] [Table 6]

[0109] Example 1.10. In vitro activation function of Fc point mutation 4-1BB H2L2 antibody To evaluate the activating effect of the Fc mutant antibody PR000980 on the 4-1BB pathway, the content of IFN-γ, TNF-α, and IL-2 was detected using ELISA kits (Invitrogen#88-7316-88, Invitrogen#88-7346-88, Invitrogen#88-7025-77) according to the method of Example 1.6.1. Utomilumab, IgG1, IgG2, and IgG4 were used as controls.

[0110] As shown in Figures 8A, 8B, and 8C, the measured antigen-binding proteins were all able to stimulate cytokine secretion from activated T cells, and more strongly than Utomilumab. Furthermore, PR000980 after Fc mutation possesses the ability to stimulate cytokine (IFN-γ, TNF-α, IL-2) secretion from T cells of the parent antibody PR000448.

[0111] Example 1.11. Epitope identification of antigen-binding proteins Using the ForteBioOctet platform, the 4-1BB H2L2 antibodies (PR000448 and PR000980) obtained in Example 1, as well as Utomilumab and Urelumab, were epitope-identified. Here, Urelumab is the Bristol-MyersSquibb anti-4-1BB antibody BMS-663513 (CAS:934823-49-1), obtained by expression purification according to the method of Example 1.2. In the first step, the histidine-labeled 4-1BB antigen was captured with a sensor, and the sensor was immersed in the antibody to obtain a 100% signal of the antibody. In the second step, the first antibody was loaded onto multiple AHC tips, and a baseline of 60 seconds was obtained by measuring the pH of buffer at 7.5. Then, to enable antigen binding, the tips were exposed to histidine-labeled 4-1BB for 180 seconds. The tip was transferred to a well containing the second antibody in the measurement buffer and continued for 90 seconds. The final signal was recorded as the signal for the second antibody. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (AB) / A * 100, where A: 100% signal of a certain antibody (obtained from the first step), and B: signal indicating this antibody as the second antibody (obtained from the second step).

[0112] If the second antibody shows clear binding (i.e., inhibition rate is less than 40%), it is considered a non-competitive agent (i.e., located in a different epitope region than the first antibody). If the second antibody does not show clear binding (i.e., inhibition rate is 40% or higher), it is considered a competitive agent (i.e., located in the same epitope region as the first antibody). Binding was measured by comparing the binding of the second antibody to 4-1BB in the presence of the first antibody with the blockade of the first antibody itself.

[0113] As a result, as shown in Table 7 below, there is high overlap between the 4-1BB binding sites of PR000448 and PR000980 and Utomilumab, but low overlap between the binding sites of Urelumab.

[0114] [Table 7]

[0115] Example 1.12.4-1BB H2L2 antibody affinity test with human or monkey 4-1BB protein The 4-1BB H2L2 antibodies (PR000448 and PR000980) obtained in Example 1, along with Utomilumab and Urelumab, were subjected to affinity identification using the Biacore platform. HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P2O, pH 7.4, GE Healthcare, BR-1006-69) was used as the operating buffer for the tests, and Series S CM5 (GE Healthcare, BR-1005-30) was used as the experimental chip. The flow rate was set to 10 μl / min, and Protein A was conjugated into the four channels of CM5 using the following procedure: 1) The injection time was set to 800 s, and 50 mM NHS and 200 mM EDC were freshly mixed in a 1:1 volume ratio and injected into the four channels; 2) Protein A was diluted to 20 μg / ml with pH 4.5 sodium acetate (GE Healthcare, BR-1003-50) and injected into each channel for 800 s; 3) 1 M pH 8.5 ethanolamine was injected for 800 s to seal the remaining active carboxyl groups on the tip surface. After sealing, the instrument was equilibrated with 1 × HBS-EP+ buffer for 2 hours, and the final conjugation volume of Protein A was approximately 2000 RU. Furthermore, a multi-cycle dynamics mode was set, and antibody-protein affinity measurements were performed, with each cycle including antibody capture, analyte binding, and tip regeneration. All antibodies were diluted to 1 μg / ml and injected into channels 2, 3, and 4 at a flow rate of 10 μl / min for 30 s. Each antibody was captured by pre-conjugated Protein A, with a capture volume of approximately 120 RU. Human 4-1BB or cynomolgus monkey 4-1BB were injected into the four channels sequentially with concentration gradients of 0 nM, 0.391 nM, 0.781 nM, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, and 25 nM at a flow rate of 30 μl / min. The dissociation time was set to 600 s for PR000448 and PR000980, and to 80 s for Uremulab and Utomilumab, with an injection time of 120 s for all.Finally, to remove the test antibody from the surface, 10 mM glycine hydrochloride pH 1.5 (GE Life Sciences, BR-1003-54) was injected for 30 seconds at a similar flow rate, and the tip was regenerated. The experimental results were analyzed using Biacore T200 analysis software 2.0, with one channel deducted as the reference channel, and a 1:1 dynamics fitting model was selected for the analysis model.

[0116] The results, as shown in Table 8 and Figures 9A-9D, are the affinity results for the 4-1BB H2L2 antibody against histidine-labeled human 4-1BB protein and histidine-labeled monkey 4-1BB protein. The results showed that, regardless of the binding affinity to human or monkey 4-1BB protein, the binding affinity of antibodies PR000448 and PR000980 was higher than that of Uremulab and Utomilumab, respectively, and it was revealed that Urelumab does not bind to monkey 4-1BB protein.

[0117] [Table 8]

[0118] Example 1.13. Activity of Fc point mutation antibody in suppressing in vivo tumor growth. A B6-h4-1BB transgenic mouse MC38 subcutaneous colon cancer model was constructed according to the method of Example 5. The average tumor volume was 99.14 mm². 3 When the target was reached, the mice were randomly grouped based on tumor volume, with 6 mice per group. The day of grouping was defined as day 0, and administration began on day 0. The sample to be measured was prepared in PBS, administered by intraperitoneal injection, and the dose was 2 mg / kg. After the start of administration, body weight was measured twice a week. Tumor volume was measured twice a week, and the tumor volume (mm²) was measured. 3 ) = 0.5 × Tumor length × Tumor width 2 .

[0119] As shown in Figures 10A and 10B, when measured 29 days after administration, the Fc mutant PR000980 of PR000448 showed stronger tumor growth inhibitory activity.

[0120] summary This application provides an antigen-binding protein having one or more of the following properties: 1) capable of binding to human and monkey-derived 4-1BB proteins; 2) capable of stimulating the secretion of IFN-γ, IL2, and / or TNFα from immune cells; 3) capable of inhibiting tumor growth and / or proliferation of tumor cells; and 4) capable of activating the 4-1BB signaling pathway. This application also provides the use of said antigen-binding protein in the prevention and treatment of tumors.

[0121] Example 2. Acquisition of whole-human derived 4-1BB HCAB antibody. Example 2.1. Immunization of whole-human HCAb mice and acquisition of 4-1BB antibody. The Harbour HCAb mouse (HarbourAntibodiesBV, WO2002 / 085945A3) is a transgenic mouse with a human immunoglobulin immune bank. It produces antibodies that are half the size of conventional IgG antibodies and consist solely of a novel "heavy chain." These antibodies possess only the variable domain of the human antibody "heavy chain" and the mouse Fc constant domain. Due to the absence of a light chain, these antibodies largely overcome the problems of light chain mismatch and heterodimerization, enabling the development of products that are difficult to achieve with conventional antibody platforms.

[0122] Example 2.1.1. Immune HCAb mice Harbour human antibody transgenic mice aged 6-8 weeks were immunized in multiple rounds against Harbour HCAb mice using two immunization schemes. Immune scheme 1 involved immunization with recombinant human 4-1BB-ECD-Fc (ChemPartner, Shanghai) antigen protein. Each mouse received a total injection volume of 100 microliters via subcutaneous, inguinal, or intraperitoneal injection. In the first immunization, each mouse was immunized with an immunogen reagent prepared by mixing 50 micrograms of antigen protein with complete Freund adjuvant (Sigma, #F5881) in a 1:1 volume ratio. In subsequent rounds of immunization enhancement, each mouse was immunized with an immunogen reagent prepared by mixing 25 micrograms of antigen protein with Ribi adjuvant (SigmaAdjuvantSystem, Sigma, #S6322). Immunization scheme 2 stabilizes the cell line using NIH3T3-h4-1BB (ChemPartner, Shanghai), which overexpresses human 4-1BB, and then immunizes the mouse. Each mouse is immunized 2 × 10⁶ times. 6 The cell suspension was administered by intraperitoneal injection. The interval between immunization rounds was at least two weeks, and usually did not exceed five rounds. Immunization times were 0, 14, 28, 42, 56, and 70 days, and mouse serum antibody titers were measured on days 49 and 77. The final immunization was performed with a dose of 25 micrograms of antigen protein per mouse, five days before isolation of HCAb mouse spleen B cells.

[0123] Example 2.1.2. Acquisition of the HCAb antibody sequence for anti-4-1BB. Mouse blood was collected and diluted 10-fold to obtain six concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000). ELISA detection was performed on ELISA plates coated with human 4-1BB-ECD-Fc (Chempartner, Shanghai) to determine the antibody titer against human 4-1BB in the mouse blood. Then, flow cytometry was used to measure the specific reactivity of two concentrations of mouse blood (1:100, 1:1000) to 4-1BB-highly expressing CHO-K1 / h4-1BB cells (Chempartner, Shanghai) and CHO-K1 progenitor cells. The blank control group (PB) consisted of serum from pre-immunized mice. After detecting that the 4-1BB-specific antibody titer in mouse serum had reached a specific level, mouse spleen cells were isolated, and B cells were separated. CD138-positive serological cells and human 4-1BB antigen-positive B cell populations were sorted using a BD FACS AriaIICellSorter. RNA from the B cells was extracted, and cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, 18091200) was reverse transcribed. Subsequently, the human VH gene was amplified using specific primer PCR. PCR primers used were 5'-GGTCCCAGTGTSAGAGAGTGTG-3' and 5'-AATCCCTGGCACTGAGACTGACC-3'. The amplified VH gene fragment was constructed in a mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc domain sequence.

[0124] Mammalian host cells (e.g., human embryonic kidney cells HEK293) were transfected with the constructed plasmid and expressed to obtain antibodies against HCAb. Binding of the supernatant expressing HCAb to a CHO-K1 / hu 4-1BB (ChemPartner, Shanghai) stable cell line overexpressing human 4-1BB was detected, and Acumen screening was performed simultaneously using CHO-K1 cells as a negative control. The obtained positive monoclonal antibodies were further tested for their binding and cross-binding activity with a CHO-K1 / cyno4-1BB (ChemPartner, Shanghai) stable cell line overexpressing cynomolgus monkey 4-1BB. Expression supernatants of 683 monoclonals binding to CHO-K1 / hu 4-1BB and CHO-K1 / cyno4-1BB were obtained, and NF-κB function tests were performed. Simultaneously, nucleotide sequences encoding the antibody molecule's variable domain and corresponding amino acid sequences were obtained using conventional sequence recognition methods. After removing repeating sequences, 323 functional, unique whole-human 4-1BB monoclonal antibodies were obtained that simultaneously bind CHO-K1 / hu 4-1BB and CHO-K1 / cyno4-1BB. Based on the results of human monkey cell binding ability and NF-Kb function tests, the top 38 antibodies were selected and subjected to recombinant expression.

[0125] [Table 9]

[0126] A novel antibody molecule (called a PTM mutant) was obtained by amino acid mutation from a single antibody having a potential PTM site derived from Example 2.1.2.

[0127] [Table 10]

[0128] [Table 11]

[0129] Example 2.1.3. Preparation of whole-human recombinant antibody against 4-1BB By transfecting mammalian host cells (e.g., human embryonic kidney cells HEK293) with a plasmid encoding an HCAb antibody, purified anti-4-1BB recombinant heavy chain antibody can be obtained using conventional recombinant protein expression and purification techniques. Specifically, HEK293 cells can be processed using FreeStyle. (商標) Cells were cultured in F17 Expression Medium (Thermo, A1383504). The cell concentration was increased to 6 × 10⁶ before the start of instantaneous transfection. 5 The cells were adjusted to a concentration of 1.2 × 10⁶ cells / ml and cultured for 24 hours in a 37°C, 8% CO₂ rocker until the cell concentration reached 1.2 × 10⁶ cells. 6 The cell count was 4 / ml. 30 ml of cultured cells were prepared. 30 μg of the plasmid encoding the HCAb heavy chain was dissolved in 1.5 ml of Opti-MEM serum-reduced medium (Thermo, #31985088), and then 1.5 ml of Opti-MEM was dissolved in 120 μl of 1 mg / ml PEI (Polysciences, Inc, #23966-2). The mixture was allowed to stand for 5 minutes. The PEI was slowly added to the plasmid, incubated at room temperature for 10 minutes, and the plasmid-PEI mixture was slowly added dropwise while shaking the culture bottle. The cells were cultured at 37°C in an 8% CO2 rocking bed for 5 days. Cell viability was observed after 5 days. The culture was collected and centrifuged at 3300 G for 10 minutes, after which the supernatant was collected. The supernatant was then centrifuged at high speed to remove impurities. MabSelect was used with PBS (pH 7.4). (商標)A gravity column (Bio-Rad, #7311550) containing (GE Healthcare Life Science, #71-5020-91AE) was equilibrated and washed with 2-5 times the column volume. The supernatant sample was passed through the column. The column was washed with 5-10 times the column volume of PBS. The target protein was eluted with 0.1 M glycine at pH 3.5, neutralized with Tris-HCl at pH 8.0, and finally concentrated in PBS buffer using an ultrafiltration tube (Millipore, UFC901024) to obtain a purified anti-4-1BB heavy chain antibody solution. The antibody concentration was obtained by measuring the absorbance at 280 nm with NanoDrop, and the antibody purity was obtained by SEC-HPLC and SDS-PAGE.

[0130] Example 2.1.4. Analysis of protein purity and polymers using HPLC-SEC Analytical molecular size exclusion chromatography (SEC) was used to analyze the purity and polymer morphology of protein samples. An analytical column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 μm, 7.8 mm x 30 cm) was connected to a high-pressure liquid chromatography (HPLC) system (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated in PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg, adjusted to a sample concentration of 1 mg / ml) was filtered through a 0.22 μm filtration membrane and injected into the system. The HPLC program was set: the sample was flowed through the column at a flow rate of 1.0 ml / min with PBS (pH 7.4) buffer for a maximum time of 20 minutes; the detection wavelength was 280 nm. After collection, the chromatogram was integrated using ChemStation software, relevant data were calculated, and an analysis report was generated, reporting the residence times of different molecular size components in the sample.

[0131] Example 2.1.5. Analysis of protein purity and hydrophobicity using HPLC-HIC The purity and hydrophobicity of protein samples were analyzed using analytical hydrophobic interaction chromatography (HIC). An analytical column, TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6 mm × 3.5 cm), was connected to a high-pressure liquid chromatography (HPLC) system (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. The setup involved a linear gradient from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0) within 16 minutes, with a flow rate of 0.7 ml / min, a protein sample concentration of 1 mg / ml, an injection volume of 20 μl, and a measurement wavelength of 280 nm. After sample collection, chromatograms were integrated using ChemStation software, relevant data were calculated, and an analysis report was generated, reporting the residence times of different molecular size components within the sample.

[0132] Example 2.1.6. Measurement of the thermal stability of antibody molecules using DSF Differential scanning fluorescence (DSF) is a common high-flux method for measuring the thermal stability of proteins. Using a real-time fluorescence quantitative PCR instrument, it reflects the process of protein denaturation and the thermal stability of protein molecules by monitoring the change in fluorescence intensity of the dye bound to the unfolded protein molecule. In this example, the thermal denaturation temperature (Tm) of a protein molecule was measured using the DSF method. 10 μg of protein was added to a 96-well PCR plate (Thermo, AB-0700 / W), followed by the addition of 2 μl of 100X diluted dye SYPRO™ (Invitrogen, 2008138), and then buffer was added to a final volume of 40 μl / well. The PCR plates were sealed and placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad CFX96 PCR System). After incubation at 25°C for 5 minutes, the temperature was gradually increased from 25°C to 95°C with a gradient of 0.2°C / 0.2 mins, and then lowered to 25°C at the end of the test. Using FRET scan mode, data analysis was performed using Bio-Rad CFX Maestro software to calculate the Tm of the samples.

[0133] [Table 12] Table 12 shows that most 4-1BB HCABs have good physicochemical properties.

[0134] Example 2.2.4-1BB: Binding of HCAb antibody to cell surface 4-1BB This example aims to study the in vitro binding activity of an anti-human 4-1BB HCAb monoclonal antibody to human and cynomolgus monkey 4-1BB cells. Antibody binding experiments were performed at the cell level using CHO-K1 cell lines overexpressing human 4-1BB (CHO-K1 / hu 4-1BB, Genescript) and CHO-K1 cell lines overexpressing cynomolgus monkey 4-1BB (CHO-K1 / cyno4-1BB, Genescript). In short, CHO-K1 / hu 4-1BB cells and CHO-K1 / cyno4-1BB cells were digested, resuspended in F12K complete medium, and the cell density was set to 1 × 10⁶ cells. 6 The cells were adjusted to the desired concentration per ml. 100 μL of cells were inoculated into a 96-well V plate (Corning, #3894), followed by the addition of 100 μL of the target antibody, diluted with a 3x concentration gradient (2x the final concentration). The cells were incubated at 4°C in the dark for 1 hour. Then, 100 μL of pre-cooled PBS was added to rinse the cells twice, and the cells were centrifuged at 500 g at 4°C for 5 minutes, discarding the supernatant. Finally, 100 μL of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-06, 1:500 dilution) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with pre-cooled PBS in 100 μl / well, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in pre-cooled PBS in 200 μl / well, and the fluorescence emission signal values ​​were read using a BD FACS CANTO II.

[0135] As shown in Figures 11A to 11M, all of the anti-4-1BB HCAb antibodies of the present invention can bind to human 4-1BB, and the detected antibody binding ability increases in a positive correlation with the antibody concentration. These antibodies can bind to human 4-1BB more sensitively at lower concentrations compared to the reference antibodies (Urelumab and Utomilumab). Here, PR001758-PR001760, PR001764, PR001830, PR001831, PR001833, PR001836, and PR001838 are optimal, and their EC50s are all less than 0.3 nM, which is equivalent to the EC50s of the reference antibodies Utomilumab and Urelumab. The maximum fluorescence values ​​of PR007287, PR007292, PR007293, PR007294, and PR007298 when binding to CHO-K1 / hu 4-1BB cells are higher compared to the reference antibodies Utomilumab and Urelumab.

[0136] As shown in Figures 12A to 12L, all of the anti-4-1BB HCAb antibodies of the present invention can bind to monkey 4-1BB cells, and the detected antibody binding ability increases in a positive correlation with antibody concentration. These antibodies can bind sensitively to monkey 4-1BB cells at lower concentrations compared to the reference antibody Tab (Utomilumab), and their EC50 is equivalent to or better than that of the reference antibody Utomilumab. The maximum fluorescence values ​​of PR007287, PR007292, PR007293, PR007294, and PR007298 when binding to CHO-K1 / hu 4-1BB cells are higher compared to the reference antibodies Utomilumab and Urelumab. On the other hand, the reference antibody Urelumab does not have the activity to cross-bind with monkey 4-1BB cells.

[0137] Example 2.3. Detection of stimulating effects on the 4-1BB signaling pathway using a reporter gene cell system. CHO-K1 cells expressing CD32b (CHO-K1 / CD32b) were placed in a 96-well plate (Perkin Elmer, #6005225), with a cell volume of 1.5 × 10⁶. 4The solution was 100 μL / well. The solution was incubated overnight at 37°C in a 5% CO2 environment. The supernatant was removed, and 40 μL / well of a 2x dilution of the antigen-binding protein was added. The initial concentration was 200 nM, followed by a 3-fold dilution, or the initial concentration was 30 nM, followed by a 5-fold dilution. hlgG1 served as the control group. 4.5 × 10 4 HEK293 reporter cells (HEK293 / 4-1BB / NF-kb reporter cells, BPS Biosciences, #79289), a luciferase reporter gene capable of continuous expression of 4-1BB and NF-Kb reaction elements per well, were added at 40 μL / well. The cells were cultured at 37°C under 5% CO2 conditions for 6 hours. ONE-Glo TM Luciferase reagent (Promega, #E6110) was added, incubated at room temperature for 5 minutes, and the luminescence value was measured using a microplate reader analyzer.

[0138] The results, as shown in Figures 13A to 13I, showed that under CHO-K1 / CD32b crosslinking, the promotion of the 4-1BB-mediated NF-Kb signaling pathway by most of the 4-1BB antigen-binding proteins of this application increased in a positive correlation with their concentration. Compared to the reference antibody Tab (Utomilumab), the EC50 was equivalent to or lower than that of the reference antibody, and the maximum fluorescence value was also equivalent to or higher than that of the reference antibody, indicating that these antibodies can promote NF-Kb activation at lower concentrations. Here, PR00758, PR001759 and PR001760, PR007286, PR007291, PR007295, PR007296, PR007297, PR007299, and PR007300 were optimal, with their EC50 all being less than 0.8 nM, equivalent to the EC50 of the reference antibody.

[0139] As shown in Figure 13J, without CHO-K1 / CD32b crosslinking, none of the 4-1BB antigen-binding proteins described in this application activated the 4-1BB-mediated NF-KB signaling pathway. The reference antibody Tab (Urelumab) still exhibited strong activating activity.

[0140] Example 2.4. The antigen-binding protein blocks the binding of the 4-1BB ligand to 4-1BB. To study the activity of human 4-1BB-binding protein in vitro in blocking the binding of human 4-1BB to human 4-1BBL, we conducted cellular-level human 4-1BB / human 4-1BBL binding blocking experiments using the CHO-K1 cell line (CHO-K1 / hu 4-1BB) overexpressing human 4-1BB. In short, CHO-K1 / hu 4-1BB cells were digested, resuspended in F-12K complete medium, and the cell density was increased to 1 × 10⁶. 6 The cells were adjusted to the required concentration per mL. 100 μL of cells / well were inoculated into a 96-well V plate (Corning, #3894), followed by 100 μL / well of the antigen-binding protein diluted with a 3x concentration gradient (2x to 3x) to the final concentration. The mixture was homogeneous, with eight concentrations of antigen-binding protein, the highest final concentration being 100 nM, and hIgG1 was used as a control. The cells were incubated at 4°C for 1 hour in the dark. Then, they were centrifuged at 4°C for 5 minutes, the supernatant discarded, and then 50 μL / well of biotin-labeled human 4-1BBL protein (ACRO, 41L-H82F9) at a concentration of 1 μg / mL was added. The cells were incubated at 4°C for 30 minutes in the dark. 100 μL / well of pre-cooled PBS was added to rinse the cells twice, and the cells were centrifuged at 500 g at 4°C for 5 minutes. The supernatant was discarded. 100 μL / well was added to the cell with fluorescent secondary antibody (PEStreptavidin, BD, #554061, 1:200), and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with 200 μL / well of pre-cooled PBS, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-cooled PBS, the fluorescence emission signal values ​​were read using BD FACS CANTO II, the IC50 was calculated, and the inhibition rate % = 1 - MFI(4 - 1BBAb) / MFI(iso).

[0141] The results are shown in Figures 14A to 14K. The 4-1BB antigen-binding proteins of this application can be classified into two types. Class I was able to block the binding of human 4-1BBL to human 4-1BB on the cell surface, showing a blocking effect equivalent to the reference antibody Tab (Utomilumab) (PR001758, PR001759, PR001760, PR001764, PR001766, PR001776, PR001776, PR001830, PR001831, PR001833, PR001836, PR001837, PR001838, PR007287, PR007289, PR007290, PR007291, PR007292, PR007293, PR007294, PR007295, PR007296, PR007298, PR007299, PR007300). Another group of antibodies are similar to the reference antibody (Urelumab) and have weak or no blocking effect (PR001763, PR001767, PR001768, PR001771, PR001774, PR001780, PR001781, PR001840, PR001842, PR007286, PR007297).

[0142] Example 2.5. Antigen-binding protein can activate the 4-1BB pathway in vitro. CHO-K1 (ATCC, #CCL-61) or CHO-K1 / CD32b (CHO-K1 cells overexpressing human CD32b) cells were treated with 10 μg / ml mitomycin (Beijing Zhongsheng Ruitai Technology, #10107409001) at 37°C for 30 minutes. The cells were then washed four times with 10% FBS F-12K culture medium. The treated cells were placed in a 96-well plate, with 1.5 × 10⁶ cells per well. 4The cells were cultured overnight in an insulated box at 37°C. The following day, human CD3-positive T cells were isolated from human PBMCs using the MACS kit (Miltenyi Biotec, #130-096-535). First, the cell count was confirmed, and then MACS buffer and Pan-T cell biotin antibody were added according to the cell count, mixed, and allowed to stand at 4°C for 5 minutes. Subsequently, a corresponding amount of microbeads was added, and the mixture was allowed to stand at 4°C for 10 minutes. Only CD3-positive T cells passed through the LS column. The culture medium from the previous day's 96-well plate was washed, and purified T cells were placed in 1 × 10⁶ wells. 5 The antibody was added individually. Then, the corresponding concentration of 4-1BB antibody or control antibody was added, and OKT3 (eBiosciences, #16-0037-85) was added to bring the final concentration to 0.3 μg / ml. The mixture was incubated in a 37°C incubator for 72 hours. After 72 hours, the supernatant was collected and the IFN-γ content was detected using an ELISA kit (Invitrogen, #88-7316-88). Coated antibody was added to a 96 flat-bottom plate and left overnight at 4°C. The next day, ELISA buffer was added and left at room temperature for 1 hour. The received supernatant was added and incubated at room temperature for 2 hours. The plate was washed twice, the detection antibody was added and left at room temperature for 1 hour. The plate was washed twice, HRP-streptavidin was added and incubated at room temperature for 1 hour. Next, TMB substrate was added, followed by ELISA stop solution (BBI, E661006-0200). The microplate reader (Perkin ElemerEnspire) read the absorbance value at 450 nm (OD450) and calculated the IFN-γ concentration.

[0143] As a result, as shown in Figures 15A to 15D, most of the 4-1BB HCABs in this example have a stronger activating effect than Utomilumab. For example, PR001758, PR001759, PR001760, PR001764, PR001830, PR001833, PR001834, PR001836, PR001837, and PR001838 have a stronger T cell activating ability than the reference antibody Utomilumab at a low concentration of 1 nM.

[0144] As shown in Figures 16A and 16B, the PR0001758, PR001759, and PR001760 antibodies all activate the CD32b crosslinking-dependent 4-1BB pathway and induce the function of activated T cells. In the case of CHO-K1 / CD32b cell crosslinking, their activation effect was stronger than Utomilumab and slightly weaker than Urelumab. In the absence of CHO-K1 / CD32b cell crosslinking, the HCAb antibodies in this example could not activate T cell function, while Urelumab could, which is also considered to be the cause of Urelumab toxicity. The activation effect increased in a positive correlation with antibody concentration, and the EC50 was lower for all antibodies than for the reference antibody Utomilumab, indicating that the 4-1BB pathway can be activated at low concentrations. The maximum cytokine interferon gamma release was also higher than that of Utomilumab.

[0145] Example 2.6. Measurement of binding affinity of 4-1BB antibody to recombinant 4-1BB protein Affinity was measured using an OctetRED96 instrument (Fortiebio) and an anti-human IgGFc avidin sensor (AHC sensor, PallForteBio, #18-5060) according to the detailed operating instructions provided by the manufacturer. Specifically, human 4-1BB protein with His tag (Acrobiosystem, #41B-H5227) or cynomolgus monkey 4-1BB protein with his tag (Acrobiosystem, #41B-C52H4) was diluted to 400 nM with PBS buffer (pH 7.4) containing 0.1% (w / w) BSA and 0.02% (v / v) TWEEN20, and incubated with the AHC sensor. 40 nM of 4-1BB antibody was incubated with the AHC sensor containing human 4-1BB protein or monkey 4-1BB protein at 30°C for 3 minutes. The reaction mixture was incubated at 30°C for 5 minutes in PBS buffer (pH 7.4) containing 0.1% (v / w) BSA and 0.02% (v / v) TWEEN20. OctetRed96 recorded the binding and separation signals of the 4-1BB antibody and 4-1BB protein in real time. Affinity, correlation, and dissociation constants were confirmed using Octet software, and the results are shown in Tables 13 and 14.

[0146] As a result, among the detected antibodies, the KD values of PR001836 and PR001838 antibodies were relatively low regardless of the binding to human 4-1BB or cynomolgus monkey 4-1BB, indicating a stronger 4-1BB binding affinity.

[0147]

Table 13

[0148]

Table 14

[0149] Example 2.7. Epitope Identification of Antigen-binding Protein (Epitope Binning) Using the ForteBio Octet platform, epitope identification was performed on the antigen-binding protein obtained in Example 1, utomilab and urelumab. Briefly, the first antibody was loaded onto multiple AHC tips, then a 60-second baseline was obtained in measurement at buffer pH 7.5. Next, to allow antigen binding, the tips were exposed to histidine-tagged 4-1BB for 180 seconds. The tips were transferred to a well containing the second antibody in measurement buffer for 90 seconds. If the second antibody shows obvious binding, it is considered a non-competing agent (i.e., a different epitope bin from the first antibody). If the second antibody does not show obvious binding, it is considered a competing agent (i.e., the same epitope bin as the first antibody). The binding assay was performed by comparing the binding of the second antibody to 4-1BB in the presence of the first antibody with the blocking by the first antibody itself. The inhibition rate was calculated by the formula: inhibition rate (%)=(A-B) / A*100 (Note: A: 100% signal of a certain antibody, B: signal of this antibody when used as the second antibody).

[0150] A suppression rate exceeding 80% means that the two antibodies have very close epitopes; a suppression rate between 40% and 80% means that the two antibodies have relatively close but not completely overlapping epitopes; and a suppression rate of less than 40% means that the two antibodies have non-overlapping epitopes.

[0151] As shown in Table 15 below, PR001760, PR001779, PR001838, and PR001840 have high overlap with Utomiluab at the 4-1BB binding site, but low overlap with Urelumab. On the other hand, the binding site of PR001767 has a specificity different from that of Utomiluab and Urelumab.

[0152] [Table 15]

[0153] Example 2.8. Specific binding of antigen-binding protein to 4-1BB 4-1BB belongs to the TNF tumor necrosis factor receptor superfamily, which consists of a large class of multifunctional receptors that mediate immune and non-immune cell functions. Six receptors, including CD40, OX40, 41BB, CD27, GITR, and CD30, have been identified as common immune stimulants that play important roles. Similarly, inducible T cell coefficientstimuli (ICOS) are another receptor that plays a crucial role in the function and survival of activated T cells or memory T cells.

[0154] This example investigates the specificity of in vitro binding of an anti-human 4-1BB HCAb monoclonal antibody to three receptors of the TNF tumor necrosis factor receptor superfamily and ICOS by flow cytometry detection. Antibody binding experiments were performed at the cell level using CHO-K1 cell lines overexpressing human 4-1BB (CHO-K1 / hu 4-1BB, Genescript), CHO-K1 cell lines overexpressing human CD40 (CHO-K1 / hu CD40, Beijing Kangyuan Bochuang, #KC-1286), CHO-K1 cell lines overexpressing human OX40 (CHO-K1 / hu OX40, Genescript, #M00561), and HEK293 cell lines overexpressing human ICOS (HEK293T / ICOS, Genescript, #KC-0210). Simply put, digest these cells, resuspend them in F12K or DMEM complete medium, and reduce the cell density to 1 × 10⁶ each. 6 The cells were adjusted to the desired concentration per ml. 100 μL of cells were inoculated into a 96-well V plate (Corning, #3894), followed by the addition of 100 μL of the target antibody, diluted with a 3x concentration gradient (2x the final concentration). The cells were incubated at 4°C in the dark for 1 hour. Then, 100 μL of pre-cooled PBS was added to rinse the cells twice, and the cells were centrifuged at 500 g at 4°C for 5 minutes, discarding the supernatant. Finally, 100 μL of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific, Jackson, #109-545-06, 1:1000 dilution) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with pre-cooled PBS in 100 μl / well, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in pre-cooled PBS in 200 μl / well, and the fluorescence emission signal values ​​were read using a BD FACS CANTO II.

[0155] As shown in Figures 17A to 17D, the results indicate that PR001758, PR001760, PR001836, and PR001838 described in this application specifically bind to CHO-K1 / hu 4-1BB cells without binding to other members of the TNF tumor necrosis factor receptor superfamily.

[0156] summary The positive progressive effects of this invention are as follows: The 4-1BB antibody of the present invention is a completely novel whole-human antibody containing only the "heavy chain," and has the activity to specifically bind to human 4-1BB and cynomolgus monkey 4-1BB. The size of this 4-1BB heavy chain antibody is only half that of conventional IgG antibodies, and since it does not contain a light chain, this antibody can be used as a bispecific antibody, solving the problems of light chain mismatch and heterodimerization.

[0157] Example 3. HER2×4-1BB bispecific antibody Human epidermal growth factor receptor 2 (HER2), also known as ERBB2, HER-2, HER-2 / neu, NEU, NGL, TKR1, and c-erbB2, and in rats as ErbB2 or neu, is a member of the ErbB protein family and is commonly referred to as the epidermal growth factor receptor family. It is a highly invasive protein in breast cancer. HER2 is a tyrosine kinase bound to the cell membrane surface and is normally involved in signaling pathways that lead to cell growth and differentiation. HER2 is considered an orphan receptor and cannot be activated by the EGF ligand family. Approximately 30% of breast cancers have HER2 gene amplification or overexpression of its protein product, and overexpression of this receptor in breast cancer is associated with disease recurrence and poor prognosis. HER2 plays a role in development, cancer, neuromuscular junction communication, and regulation of cell growth and differentiation.

[0158] The HER2×4-1BB biantibody aims to promote 4-1BB aggregation by conjugating T cells and HER2-positive tumor cells, providing effective costimulatory signals to tumor antigen-specific T cells, further enhancing T cell receptor (TCR)-mediated activity, and inducing tumor disintegration. Therefore, HER2×4-1BB-mediated 4-1BB activation is biased towards co-localization of T cells and tumor cells in vivo, for example, in primary tumors and lymph nodes containing tumor-infiltrating lymphocytes (TILs) or tumor metastases.

[0159] In this example, the aim is to construct a bispecific antibody that simultaneously targets HER2 and 4-1BB, thereby enhancing antitumor efficacy and safety through one or more mechanisms of action. Firstly, the HER2×4-1BB biantibody enriches tumor tissue with high HER2 expression, and T cells conjugate with HER2-positive tumor cells to promote 4-1BB aggregation, providing effective costimulatory signals to tumor antigen-specific T cells. Furthermore, it enhances T cell activity mediated by the T cell receptor (TCR), thereby increasing antitumor activity. Therefore, HER2×4-1BB-mediated 4-1BB activation is biased towards the co-localization of T cells and tumor cells in vivo, for example, in primary tumors and lymph nodes containing tumor-infiltrating lymphocytes (TILs) or tumor metastases. Secondly, the function of the anti-4-1BB agonist antibody used in this example is dependent on molecular cross-linking, which means it can only mediate T cell activation by utilizing target cells in the tumor microenvironment, thus avoiding toxic side effects caused by the excessive activation of T cells in normal tissues, as can occur with monoclonal antibodies such as urelumab.

[0160] Example 3.1. Structure and design of a HER2×4-1BB bispecific antibody This example used trastuzumab, an anti-HER2 IgG antibody whose corresponding amino acid sequence was derived from the IMGT database. The manufactured antibody number was PR000210.

[0161] [Table 16]

[0162] The fully human-derived anti-4-1BB H2L2 antibody PR000448 and the derived scFv used in this example are derived from Harbour H2L2 mice, as described in Example 1.

[0163] The fully human-derived anti-4-1BB HCAb antibody used in this example is derived from Harbour HCAb mice, as described in Example 2.

[0164] In this example and the following examples, the positive control molecule is the anti-HER2 IgG monoclonal antibody PR000210 (a trastuzumab analog), which is the parent monoclonal antibody at the HER2 end of the HER2×4-1BB bispecific antibody molecule.

[0165] In this example and the following examples, the positive control molecules are the anti-4-1BB IgG monoclonal antibodies Uremulab and Utomilumab

[0166] Example 3.1.1. Construction of bispecific antibodies with IgG-VH tetravalent symmetric structure using anti-HER2 IgG antibody and anti-4-1BB HCAb antibody The binding protein with IgG-VH tetravalent symmetric structure (shown in Figure 18A) comprises two polypeptide chains: polypeptide chain 1, also called the light chain, which comprises VL_A-CL from the amino terminus to the carboxy terminus; and polypeptide chain 2, also called the heavy chain, which comprises VH_A-CH1-h-CH2-CH3-L-VH_B from the amino terminus to the carboxy terminus.

[0167] In one embodiment, CH3 of polypeptide chain 2 is directly fused and bound to VH_B, that is, the length of L is 0. In another embodiment, CH3 of polypeptide chain 2 is bound to VH_B via a connecting peptide L; L may be any of the sequences listed in Table 17.

[0168]

Table 17

[0169] Using an anti-HER2 IgG antibody and an anti-4-1BB HCAb heavy chain antibody, we designed an IgG-VH tetravalent symmetric HER2×4-1BB biantibody molecule, which is summarized in Table 18; the physicochemical properties of the prepared biantibody molecule samples are summarized in Table 19.

[0170] [Table 18]

[0171] [Table 19]

[0172] Example 3.1.2. Constructing a bispecific antibody with an IgG-scFv tetravalent symmetric structure using an anti-HER2 IgG antibody and an anti-4-1BB H2L2 antibody. The binding protein of the IgG-scFv tetravalent symmetric structure (shown in Figure 18B) contains two polypeptide chains: polypeptide chain 1, also called the short chain, containing VL_A-CL from the amino terminus to the carboxyl terminus; and polypeptide chain 2, also called the long chain, containing VH_A-CH1-h-CH2-CH3-L1-VH_B L2-VL_B from the amino terminus to the carboxyl terminus. or

[0173] The binding protein of the IgG-scFv tetravalent symmetric structure (shown in Figure 18C) contains two polypeptide chains: polypeptide chain 1, also called the short chain, containing VL_A-CL from the amino terminus to the carboxyl terminus; and polypeptide chain 2, also called the long chain, containing VH_A-CH1-h-CH2-CH3-L1-VL_B L2-VH_B from the amino terminus to the carboxyl terminus.

[0174] The linking peptides L1 and L2 of polypeptide chain 2 may be the sequences listed in Table 17.

[0175] Using an anti-HER2 IgG antibody and an anti-4-1BB H2L2 antibody, we designed HER2×4-1BB biantibody molecules with an IgG-scFv tetravalent symmetric structure, which are summarized in Table 20; the physicochemical properties of the prepared biantibody molecule samples are summarized in Table 21.

[0176] [Table 20]

[0177] [Table 21]

[0178] [Table 22]

[0179] Example 3.2. FACS detection of the binding ability of HER2×4-1BB biantibody to SK-BR-3 cells. This example aims to study the in vitro binding activity of the 4-1BB bispecific antibody to SK-BR-3 cells. SK-BR-3 cells are HER2-highly expressing breast cancer cells, and antibody binding experiments were performed at the cell level using SK-BR-3 cells. In short, SK-BR-3 cells were digested, resuspended in complete medium, and the cell density was set to 1 × 10⁶. 6 Prepare the cells / ml. Inoculate 100 μL of cells / well into a 96-well V plate (Corning, #3894), then add 100 μL / well of the target antibody diluted with a 3x concentration gradient (2x the final concentration). Incubate the cells at 4°C in the dark for 1 hour. Then, rinse the cells twice with 100 μL / well of pre-cooled PBS, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Further inoculation with 100 μL / well of fluorescent secondary antibody (Alexa Fluor (登録商標)647 Affini Pure Goat Anti-Human IgG, F(ab')2 fragment specific, Jackson Immunoresearch, #109-605-006 (1:1000 dilution) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with pre-cooled PBS in 100 μl / well, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in pre-cooled PBS in 200 μl / well, and the fluorescence emission signal values ​​were read using a BD FACS CANTO II.

[0180] As shown in Figure 19, all of the HER2×4-1BB bispecific antibodies of the present invention can bind to human SK-BR-3, and the detected antibody binding capacity increases in a positive correlation with the antibody concentration. Compared to the reference antibody (Trastuzumab), PR002813 shows a lower EC50 than the reference antibody at the same concentration, while the other antibodies correspond to the reference antibody.

[0181] Example 3.3. FACS detection of the binding ability of HER2×4-1BB biantibody to CHO-K1 / hu 4-1BB cells. This example aims to study the in vitro binding activity of a bispecific antibody against 4-1BB to 4-1BB. Antibody binding experiments were performed at the cell level using a CHO-K1 cell line expressing human 4-1BB (CHO-K1 / hu 4-1BB, Genescript). In short, CHO-K1 / hu 4-1BB cells were digested, resuspended in F12K complete medium, and the cell density was set to 1 × 10⁶. 6 The cells were adjusted to the desired concentration per ml. 100 μL of cells / well were inoculated into a 96-well V plate (Corning, #3894), followed by the addition of 100 μL / well of the target antibody, diluted with a 3x concentration gradient (2x the final concentration). The cells were incubated at 4°C in the dark for 1 hour. Then, 100 μL / well of pre-cooled PBS was added to rinse the cells twice, and the cells were centrifuged at 500 g at 4°C for 5 minutes. The supernatant was discarded. Finally, 100 μL / well of the fluorescent secondary antibody (Alexa Fluor) was added. (登録商標)647 Affini Pure Goat Anti-Human IgG, F(ab')2 fragment specific, Jackson Immunoresearch, #109-605-006 (1:1000 dilution) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with pre-cooled PBS in 100 μl / well, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in pre-cooled PBS in 200 μl / well, and the fluorescence emission signal values ​​were read using a NovoCyte flow cytometer (ACEABiosciences).

[0182] As shown in Figure 20, all of the HER2×4-1BB bispecific antibodies of the present invention bind well to human 4-1BB, and the detected antibody binding capacity increases in a positive correlation with antibody concentration. These antibodies can bind sensitively to human 4-1BB at low concentrations, with PR002811, PR001212, PR002813, and PR002824 being the most preferred, and their EC50 was superior to that of the reference antibody Utomilumab. The other bispecific antibodies were equivalent to or slightly weaker than Utomilumab. However, their maximum MFI was higher than that of the reference antibody Utomilumab.

[0183] Example 3.4. FACS detection of the binding ability of HER2×4-1BB biantibody to CHO-K1 / cyno4-1BB cells. This example aims to study the in vitro binding activity of a bispecific antibody against 4-1BB to 4-1BB. Antibody binding experiments were performed at the cell level using a CHO-K1 cell line expressing cynomolgus monkey 4-1BB (CHO-K1 / cyno4-1BB, Genescript). In short, CHO-K1 / cyno4-1BB cells were digested, resuspended in F12K complete medium, and the cell density was set to 1 × 10⁶. 6The cells were adjusted to the desired concentration per ml. 100 μL of cells / well were inoculated into a 96-well V plate (Corning, #3894), followed by the addition of 100 μL / well of the target antibody, diluted with a 3x concentration gradient (2x the final concentration). The cells were incubated at 4°C in the dark for 1 hour. Then, 100 μL / well of pre-cooled PBS was added to rinse the cells twice, and the cells were centrifuged at 500 g at 4°C for 5 minutes. The supernatant was discarded. Finally, 100 μL / well of the fluorescent secondary antibody (Alexa Fluor) was added. (登録商標) 647 Affini Pure Goat Anti-Human IgG, F(ab')2 fragment specific, Jackson Immunoresearch, #109-605-006 (1:1000 dilution) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with pre-cooled PBS in 100 μl / well, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in pre-cooled PBS in 200 μl / well, and the fluorescence emission signal values ​​were read using a NovoCyte flow cytometer (ACEABiosciences).

[0184] As shown in Figure 21, all of the HER2×4-1BB biantibodies of the present invention bind well to cynomolgus monkey 4-1BB, and the detected antibody binding capacity increases in a positive correlation with the antibody concentration. Compared with the reference antibody (Utomilumab), the binding of PR001212, PR002824, and PR002826-PR002829 to cynomolgus monkey 4-1BB is equivalent.

[0185] Example 3.5. HER2 / 4-1BB biantibody can activate the T cell pathway in vitro. 1 mg / ml of OKT3 (eBiosciences, #16-0037-85) was diluted in PBS, and 100 μL of 0.08 μg / ml of OKT3 was placed per well and wrapped around a 96-well plate (Corning, #3599) to a final concentration of 10 μg / ml. The plate was covered overnight at 4°C. The following day, SK-BR-3 cells were digested, resuspended in complete medium, and the cell density was set to 4 × 10⁶ each. 5Prepare the volume to cells / ml and set aside as a reserve. Human CD3-positive T cells were isolated from human PBMCs using the MACS kit (Miltenyi Biotec, #130-096-535). First, the cell count was confirmed, and then MACS buffer and Pan-T cell biotin antibody were added according to the cell count, mixed, and allowed to stand at 4°C for 5 minutes. Then, the corresponding amount of microbeads was added and allowed to stand at 4°C for 10 minutes. Only CD3-positive T cells passed through the LS column. The OKT3 coating on the 96-well plate from the previous day was washed off, and 50 μL of purified T cells were added, resulting in 1 × 10⁶ cells per well. 5 The cells were then divided into individual cells. Furthermore, 50 μL of SK-BR-3 cells were placed in a 96-well plate, with 2 × 10⁶ cells per well. 4 The cells were then divided into individual cells. Next, a bispecific antibody or control monoclonal antibody of HER2 / 4-1BB at the corresponding concentration was added, and the cells were cultured in a 5% CO2 culture box at 37°C. After 72 hours of incubation, the supernatant was collected, and the IFN-γ content was detected using an ELISA kit (Invitrogen, #88-7316). The ELISA test was performed according to the manufacturer's instructions. In short, the coated antibody was added to a 96 flat-bottom plate and left overnight at 4°C. The next day, ELISA buffer was added and left at room temperature for 1 hour. The received supernatant was added and cultured at room temperature for 2 hours. The plate was washed twice, the detection antibody was added, and left at room temperature for 1 hour. The plate was washed twice, HRP-streptavidin was added, and incubated at room temperature for 1 hour. Next, TMB substrate was added, and ELISA stop solution (BBI life sciences, #E661006-0200) was added after 10-30 minutes. OD450-570 values ​​were read using a board reader (Molecular Devices, #SpectraMaxPlus). The values ​​were analyzed and graphs were created using Graphad 8.0.

[0186] As shown in Figures 22A to 22D, the HER2×4-1BB biantibodies of the present invention all have the ability to activate the 4-1BB-mediated T cell pathway under SK-BR-3 crosslinking in tumor cells. Here, PR002813 and PR002827 exhibit low EC50 and high IFN gamma release values.

[0187] summary In this example, a HER2×4-1BB biantibody molecule with an IgG-VH tetravalent symmetric structure was constructed using the antigen-binding domain Fab of an anti-HER2 IgG antibody and the antigen-binding domain VH of an anti-4-1BB HCAb antibody. Simultaneously, a bispecific antibody with an IgG-scFv tetravalent symmetric structure was constructed using the antigen-binding domain Fab of an anti-HER2 IgG antibody and an anti-4-1BB H2L2 antibody.

[0188] The bispecific anti-HER2×4-1BB antibody molecule with two different structures modulates its T cell activation activity through parameters such as different structural types, relative positions, and binding valencies. Furthermore, it demonstrates the flexibility in constructing bispecific antibody molecular structures based on HCAb.

[0189] The lack of target specificity in T cell activation by urelumab is one of the causes of clinical toxic side effects. However, the effect of HER2×4-1BB biantibody on T cell activation is specifically dependent on HER2 expression. When cells that highly express HER2 are present, HER2×4-1BB biantibody can specifically activate T cells.

[0190] Based on the above, this example constructed a HER2×4-1BB bispecific antibody molecule with outstanding functional activity and excellent molecular stability.

[0191] Example 4. PD-L1×4-1BB bispecific antibody Programmed death receptor 1 (PD-1) is primarily expressed on immune cells such as T cells and has two ligands: programmed death ligand 1 (PD-L1) and PD-L2. PD-L1 is mainly expressed on antigen-presenting cells and several tumor cells. The interaction between PD-L1 and PD-1 reduces T cell activity, weakens cytokine secretion, and exerts an immunosuppressive effect. PD-L1 protein expression has been detected in many human tumor tissues, and the microenvironment of the tumor site can induce PD-L1 expression on tumor cells. Expressed PD-L1 is advantageous for tumor development and growth, induces apoptosis of anti-tumor T cells, and further protects tumor cells from immune attack.

[0192] 4-1BB (TNFRSF 9, CD137) is a transmembrane protein belonging to the TNF receptor superfamily. 4-1BB is a costimulatory molecule expressed on multiple immune cells and is a multifunctional modulator of immune activity. It is induced and expressed on activated immune cells such as T cells and NK cells. 4-1BB activates T cells through its ligand, 4-1BBL-mediated trimerization, promoting cell proliferation and cytokine release. Anti-4-1BB agonist antibodies have tumor-suppressing properties, and the first 4-1BB whole-human monocolonial antibody to enter clinical trials was Urelumab (BMS-663513) from Bristol-Myers Squibb (BMS). The initial clinical results for Urelumab were published in 2008, showing encouraging therapeutic effects in some patients, but the data indicated that Urelumab caused hepatotoxicity, which was related to the target and dose. Furthermore, the clinical trial was discontinued after two patients died from hepatotoxicity.

[0193] In this example, a bispecific antibody targeting both PD-L1 and 4-1BB was constructed, improving antitumor efficacy and safety through one or more mechanisms of action. Firstly, the PD-L1 × 4-1BB biantibody can activate T cells by blocking the PD-1 / PD-L1 signaling pathway. Secondly, the PD-L1 molecule, which is highly expressed on the surface of tumor cells, can utilize the biantibody molecule to promote cross-linking and trimerization of 4-1BB molecules on the surface of T cells, activating the downstream signaling pathway and further promoting T cell activation and proliferation. Thirdly, biantibody molecule-mediated T cell activation is limited to the tumor microcycle, thereby avoiding toxic side effects caused by excessive T cell activation in normal tissues by monoclonal antibodies such as urelumab.

[0194] Example 4.1. Structure and design of a PD-L1×4-1BB bispecific antibody This example uses PR000151 (an atesolizumab analog), an anti-PD-L1 IgG antibody whose corresponding amino acid sequence is derived from the IMGT database.

[0195] The whole-human anti-PD-L1 IgG antibody PR000265 (Table 4.2) used in this example is derived from Harbour H2L2 mice, as described below.

[0196] Harbour H2L2 mice (HarbourAntibodiesBV) are transgenic mice possessing a human immunoglobulin immune library, and their produced antibodies have a complete human antibody variable domain and a rat constant domain. Harbour H2L2 mice were subjected to multi-round immunization with soluble recombinant human PD-L1 protein (NovoProtein, #C764). After detecting that PD-L1-specific antibody titers in mouse serum reached a specific level, mouse spleen cells were isolated and fused with myeloma cell lines to obtain hybridoma cells. After multi-round screening and cloning of the hybridoma cells, several monoclonal antibody molecules capable of specifically identifying PD-L1 were identified. These monoclonal antibodies were further identified, and several preferred candidate antibody molecules were selected based on parameters such as binding ability to human PD-L1, binding ability to cynomolgus monkey PD-L1, and inhibitory ability to suppress the binding of PD-L1 and PD-1. Subsequently, sequence analysis and optimization of the candidate antibody molecules were performed, yielding several mutant sequences. Recombinant whole-human antibody molecules were obtained by fusion expression of the VL and VH sequences of antibodies with the corresponding human κ light chain constant region and IgG1 heavy chain constant region sequences. Recombinant whole-human IgG antibodies against PD-L1 are shown in Table 23.

[0197] [Table 23]

[0198] The anti-4-1BB whole-human IgG antibodies PR000197 and PR000448 used in this example are derived from Harbour H2L2 mice, as described in Example 1.

[0199] The whole-human anti-4-1BB HCAb antibodies PR001758, PR001760, and PR001836 used in this example are derived from Harbour HCAB mice, as described in Example 2.

[0200] In this example and subsequent examples, the positive control molecule was the anti-PD-L1 IgG monoclonal antibody PR000265.

[0201] In this example and subsequent examples, the positive control molecules are the anti-4-1BB IgG monoclonal antibodies Uremulab and Utomilumab.

[0202] Example 4.1.1. Construct a bispecific antibody molecule having a FIT-Ig structure using an anti-PD-L1 IgG antibody and an anti-4-1BB IgG antibody. This embodiment constructs a bispecific anti-PD-L1 × 4-1BB antibody molecule having a FIT-Ig structure using the antigen-binding domain Fab of an anti-PD-L1 IgG antibody PR000265 or PR000151 (atezolizumab analog) and the antigen-binding domain Fab of an anti-4-1BB IgG antibody PR000197 or PR000448. The design of the FIT-Ig structure can be found in patent WO2015 / 103072A1, and the structure is shown in Figure 23A.

[0203] The constructed molecules are summarized in Table 24. Then, antibody molecule samples were prepared and analyzed according to the methods described in Examples 1 and 2, and the results are summarized in Table 25. Table 26 shows the sequence numbers corresponding to the polypeptide chain sequences of the biantibody molecules with the FIT-Ig structure.

[0204] [Table 24]

[0205] [Table 25]

[0206] [Table 26]

[0207] Example 4.1.2. Constructing a bispecific antibody molecule with a Fab-HCAb structure using an anti-PD-L1 IgG antibody and an anti-4-1BB HCAb antibody. This example constructs bispecific anti-PD-L1 × 4-1BB antibody molecules with various structures using the antigen-binding domain Fab of the anti-PD-L1 IgG antibody PR000265 and the antigen-binding domain VH of the anti-4-1BB HCAb antibody PR001758, PR001760, or PR001836.

[0208] In this example and subsequent examples, the positive control molecule is the anti-PD-L1 IgG monoclonal antibody PR000265, which is the parent monoclonal antibody at the PD-L1 end of the PD-L1×4-1BB biantibody molecule.

[0209] In this example and subsequent examples, the positive control molecule is the anti-4-1BB IgG monoclonal antibody urelumab (IgG4) or utomilumab (IgG2).

[0210] As shown in Figures 23B and 23C, the Fab end is derived from the anti-PD-L1 IgG antibody used in this embodiment. The VH end is derived from the antigen-binding domain VH of the anti-4-1BB HCAb antibody PR001758, PR001760, or PR001836. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. L1 and L2 are the first and second linkage peptides, respectively.

[0211] Fab(CL)-VH-Fc The binding protein in the Figure 23B structure contains both polypeptide chains: polypeptide chain 1, also called the short chain, contains VH_A-CH1 from the amino terminus to the carboxyl terminus; polypeptide chain 2, also called the long chain, contains VL_A-CL-L1-VH_B-L2-CH2-CH3 from the amino terminus to the carboxyl terminus. In the structure Fab(CL)-VH-Fc, the VL_A of antibody A and the VH_B of heavy chain antibody B are fused to the same single polypeptide chain, thereby avoiding mismatch byproducts that occur during the association of VL_A and VH_B.

[0212] VH_B of polypeptide chain 2 is bound to CH2 via the linking peptide L2; L2 may be the hinge region of IgG or a linking peptide sequence derived from the hinge region, preferably the human IgG1 hinge or the human IgG1 hinge (C220S) or G5-LH sequence.

[0213] In one embodiment, CL and VH_B of polypeptide chain 2 are directly fused and bound, i.e., the length of L1 is 0. In another embodiment, CL of polypeptide chain 2 is bound to VH_B via a connecting peptide L1; L1 is the sequence listed in Table 17.

[0214] Fab(CH1)-VH-Fc The binding protein in the Figure 23C structure contains two polypeptide chains: polypeptide chain 1, also called the short chain, contains VL_A-CL from the amino terminus to the carboxyl terminus; polypeptide chain 2, also called the long chain, contains VH_A-CH1-L1-VH_B-L2-CH2-CH3 from the amino terminus to the carboxyl terminus.

[0215] VH_B of polypeptide chain 2 is bound to CH2 via the linking peptide L2; L2 may be the hinge region of IgG or a linking peptide sequence derived from the hinge region, preferably the human IgG1 hinge or the human IgG1 hinge (C220S) or G5-LH sequence.

[0216] In one embodiment, CH1 of polypeptide chain 2 and VH_B are directly fused and bound, i.e., the length of L1 is 0. In another embodiment, CH1 of polypeptide chain 2 is bound to VH_B via a connecting peptide L1; L1 is the sequence listed in Table 17.

[0217] Using an anti-PD-L1 IgG antibody and an anti-4-1BB heavy chain antibody, we designed a PD-L1×4-1BB biantibody molecule with a Fab-HCAb symmetric structure, which is summarized in Table 27; the physicochemical properties of the fabricated biantibody molecules are summarized in Table 28.

[0218] [Table 27]

[0219] [Table 28]

[0220] Example 4.1.3. Construction of an IgG-VH tetravalent symmetric structure molecule Using an anti-PD-L1 IgG antibody and an anti-4-1BB heavy chain antibody, a PD-L1×4-1BB biantibody molecule with an IgG-VH tetravalent symmetric structure was designed according to the structure described in Example 3.1.1 (Figure 23D).

[0221] Alternatively, the VH of the anti-4-1BB HCAb antibody is linked to the N-terminus of the heavy chain of the PD-L1 IgG antibody via a connecting peptide (shown in Figure 23E). The binding protein in the Figure 23E structure contains two different polypeptide chains: polypeptide chain 1, also called the short chain, contains VL_A-CL from the amino terminus to the carboxyl terminus; polypeptide chain 2, also called the long chain, contains VH_B-L-VH_A-CH1-h-CH2-CH3 from the amino terminus to the carboxyl terminus. In one embodiment, VH_B and VH_A of polypeptide chain 2 are directly fused and linked, i.e., the length of L is 0. In another embodiment, VH_B of polypeptide chain 2 is linked to VH_A via a connecting peptide L; L may be one of the sequences listed in Table 17.

[0222] Using an anti-PD-L1 IgG antibody and an anti-4-1BB heavy chain antibody, we designed PD-L1×4-1BB biantibody molecules with an IgG-VH symmetric structure, which are summarized in Table 29; the physicochemical properties of the fabricated biantibody molecules are summarized in Table 30.

[0223] [Table 29]

[0224] [Table 30]

[0225] Example 4.1.4. Sequence list of PD-L1×4-1BB biantibody molecule and control molecule Table 31 shows the sequence numbers corresponding to the sequences of the PD-L1×4-1BB biantibody molecules constructed in this embodiment.

[0226] [Table 31]

[0227] Example 4.2. Binding to cells that highly express human PD-L1. This example is intended to study the binding activity of the PD-L1×4-1BB biantibody molecule to PD-L1.

[0228] The binding ability of antibody molecules to CHO-K1 / hPDL1 (Nanjing Jinsrui, M00543), a CHO-K1 cell line highly expressing human PD-L1, or to MDA-MB-231 (ATCC, HTB-26), a MDA-MB-231 cell line highly expressing human PD-L1, was tested using flow cytometry (FACS). Specifically, CHO-K1 / hPDL1 cells or MDA-MB-231 cells were digested and resuspended in complete medium; the cell density was set to 1 × 10⁶. 6The cells were adjusted to the required concentration per mL. Next, 100 μL / well of cells were seeded into a 96-well V plate (Corning, #3894), centrifuged at 4°C for 5 minutes, and the supernatant was discarded. Subsequently, gradient-diluted antibody molecules were added to the 96-well plate at 100 μL / well and mixed uniformly. The antibody molecules were diluted to a total of 12 concentrations, starting from a maximum final concentration of 200 nM and progressing through a 3-fold concentration gradient. hIgG1iso (CrownBio, #C0001) was used as an isotyped control. The cells were incubated at 4°C in the dark for 1 hour. Then, 100 μL / well of pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) was added to rinse the cells twice, and 500 g was centrifuged at 4°C for 5 minutes, after which the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goathuman IgG(H+L)Alexa Fluor488 Conjunction, Thermo, #A11013, 1:1000 dilution) was added, and the cells were incubated at 4°C for 1 hour in the dark. Then, the cells were rinsed twice with 200 μL / well of pre-cooled FACS buffer, followed by centrifugation of 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended with 200 μL / well of pre-cooled FACS buffer. The fluorescence emission signal values ​​were read using a BD FACS CANTO II flow cytometer.

[0229] Using the GraphPadPrism8 software, data processing and plotting analysis were performed, and nonlinear fitting of four parameters was used to obtain the antibody binding curve to target cells and parameters such as the EC50 value.

[0230] In this embodiment, the positive control molecule is the anti-PD-L1 monoclonal antibody PR000265, which is also the parent monoclonal antibody of the PD-L1 end of the PD-L1x4-1BB biantibody molecule.

[0231] As shown in Figure 24A, the ability of the IgG-VH (C-terminal) tetravalent symmetric biantibody molecules (PR003549, PR003550, PR003551) to bind PD-L1 is similar to that of the parent monoclonal antibody PR000265, and the EC50 value and MFI maximum value for binding PD-L1 are slightly better than those of the FIT-Ig structure biantibody molecules (PR000701, PR003052).

[0232] As shown in Figure 24B, the ability of the Fab-HCAb symmetric bivalent antibody molecule PR004270 and the IgG-VH (N-terminal) tetravalent symmetric bivalent antibody molecule PR004268 to bind PD-L1 is similar to that of the parent monoclonal antibody. Their EC50 values ​​for binding to PD-L1 are slightly weaker than those of the parent monoclonal antibody, but their maximum binding capacity to MFI is higher than that of the parent monoclonal antibody.

[0233] As shown in Figures 24C-24E, the ability of the biantibody molecules with a quadrivalent symmetric structure at the IgG-VH (C-terminus) (PR007130, PR007132, PR007133, PR007135, PR007136, PR007137, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) to bind PD-L1 is similar to that of the parent monocolonial antibody PR000265.

[0234] Example 4.3.4-1 Binding with CHO-K1 cells overexpressing BB This example is intended to study the binding activity of the PD-L1×4-1BB biantibody molecule to 4-1BB.

[0235] The binding ability of antibody molecules to CHO-K1 cell lines that highly express human 4-1BB, such as CHO-K1 / hu 4-1BB (Nanjing Jinsirui, M00538), and CHO-K1 cell lines that highly express cynomolgus monkey 4-1BB, such as CHO-K1 / cyno4-1BB (Nanjing Jinsirui, M00569), was tested using flow cytometry (FACS). Specifically, cells were digested and resuspended in complete medium; the cell density was set to 2 × 10⁶. 6The cells were adjusted to the desired concentration per mL. Next, 100 μL / well (2 × 10⁶) of cells were added. 5 Cells were seeded in 96-well V plates (Corning, #3894) at a concentration of 100 μL / well, centrifuged at 4°C for 5 minutes, and the supernatant was discarded. Subsequently, gradient-diluted antibody molecules were added to the 96-well plates at a concentration of 100 μL / well and mixed uniformly. The antibody molecules were diluted at a total of 12 concentrations, starting from a maximum final concentration of 200 nM and progressing through a 3-fold concentration gradient. hIgG1iso (CrownBio, #C0001) was used as an isotyped control. The cells were placed at 4°C, protected from light, and incubated for 1 hour. Subsequently, the cells were rinsed twice with pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) at a concentration of 100 μL / well, centrifuged at 4°C for 5 minutes with 500 g of cells, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goathuman IgG(H+L)Alexa Fluor488 conjunction, Thermo, #A11013, 1:1000 dilution) was added, and the cells were incubated at 4°C for 1 hour in the dark. Then, the cells were rinsed twice with 200 μL / well of pre-cooled FACS buffer, followed by centrifugation of 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended with 200 μL / well of pre-cooled FACS buffer. The fluorescence emission signal values ​​were read using a BD FACS CANTO II flow cytometer.

[0236] Using the GraphPadPrism8 software, data processing and plotting analysis were performed, and nonlinear fitting of four parameters was used to obtain the antibody binding curve to target cells and parameters such as the EC50 value.

[0237] In this example, the positive control molecule is the anti-4-1BB monoclonal antibody Urelumab or tomilumab.

[0238] Example 4.3.1. Binding of human 4-1BB-highly expressing CHO-K1 cells to CHO-K1 / hu 4-1BB. As shown in Figure 25A, the IgG-VH (C-terminal) tetravalent symmetric biantibody molecules (PR003549, PR003550, PR003551) have a higher ability to bind human 4-1BB compared to the FIT-Ig biantibody molecules (PR000701, PR003052), and are superior to the positive control Urelumab in terms of maximum MFI.

[0239] As shown in Figure 25B, the biantibody molecule PR004270 with a Fab-HCAb symmetric structure and the biantibody molecule PR004268 with an IgG-VH (N-terminal) tetravalent symmetric structure exhibit superior ability to bind human 4-1BB compared to the positive controls Urelumab and Utomilumab in terms of maximum MFI.

[0240] As shown in Figures 25C to 25E, the binding of IgG-VH (C-terminal) tetravalent symmetric biantibody molecules (PR007130, PR007132, PR007133, PR007135, PR007136, PR007137, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) to human 4-1BB is superior to that of the positive control Urelumab in terms of maximum MFI.

[0241] Example 4.3.2. Binding of CHO-K1 cells highly expressing cynomolgus monkey 4-1BB to CHO-K1 / cyno4-1BB. As shown in Figures 26A and 26B, the biantibody molecules of this embodiment can bind to cynomolgus monkey 4-1BB, whereas Urelumab cannot. PR004270's ability to bind to cynomolgus monkey 4-1BB is slightly superior to Utomilumab's in terms of maximum MFI.

[0242] Example 4.4. Specific activation of T cells mediated by target cells that highly express PD-L1. This example aims to study how the presence of a PD-L1 × 4-1BB biantibody molecule in target cells activates T cell activity by binding to 4-1BB. Target cells may be cells expressing PD-L1 to varying degrees, such as CHO-K1 / hPDL1 (GENSCRIPT, M00543) which highly expresses human PD-L1, or MDA-MB-231 (ATCC, HTB-26) which highly expresses human PD-L1. Effector cells may be isolated human PBMCs or T cells.

[0243] Specifically, first, 0.3 μg / mL of anti-CD3 antibody OKT3 (Thermo, #16-0037-81) was encapsulated in 100 μL / well of a 96-well plate (Corning, #3599). Next, the density of human T cells (obtained by isolation from human PBMCs using a T cell sorting kit (Miltenyi, #130-096-535)) was set to 2 × 10⁻⁶. 6 Adjust to cells / mL and set the target cell density to 3 × 10⁶ 5 After adjusting to cells / mL, two cell suspensions were inoculated into 96-well plates at 50 μL / well each, with a final target ratio of 20:3. Subsequently, antibody molecules of different concentrations were added at 100 μL / well, with final antibody concentrations of (10 nM, 1 nM), 20 nM, or a total of eight concentrations obtained by diluting from the highest final concentration of 20 nM with a 5-fold concentration gradient, and these were added to two combined wells; hIgG1 iso(CrownBio,#C0001) and hIgG4 iso(CrownBio,#C0045) were used as controls. The 96-well plates were incubated in a 37°C, 5% CO2 culture box for 3 days. The supernatants were collected after 48 hours and 72 hours of incubation, respectively. The IL-2 concentration in the supernatant after 48 hours was detected using an IL-2 ELISA kit (Thermo, #88-7025-88), and the IFN-γ concentration in the supernatant after 72 hours was detected using an IFN-γ ELISA kit (Thermo, #88-7316-77). Refer to the operating instructions for the relevant kits for the ELISA detection method.

[0244] Data processing and plotting / analysis are performed using the application software GraphPad Prism 8. In this example, the positive control molecule is the anti-4-1BB monoclonal antibody Urelumab.

[0245] Example 4.4.1. CHO-K1 / hPDL1-mediated T cell-specific activation As shown in Figures 27A-27D, in a system mixing target cells CHO-K1 / hPDL1 with T cells, the crosslink-independent anti-4-1BB monoclonal antibody Urelumab can activate IFN-γ release from T cells, while the crosslink-dependent anti-4-1BB monoclonal antibodies (PR000448, PR001758, PR001760, PR001836) can hardly activate T cells. The FIT-Ig biantibody molecules (PR003052, PR000701), the IgG-VH tetravalent symmetric biantibody molecules (PR003549, PR003550, PR003551, PR004268, PR007130, PR007132, PR007133, PR007135, PR007136, PR007137, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149), and the Fab-HCAb biantibody molecule (PR004270) can all activate T cells and release cytokines. This indicates that the activation of biantibody molecules by T cells depends on the specific activation of target cells. Furthermore, the IgG-VH tetravalent symmetrical biantibody molecules (PR003549, PR003550, PR007130, PR007132, PR007133, PR007135, PR007136, PR007137, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) and the Fab-HCAb biantibody molecule (PR004270) can induce higher cytokine release levels and exhibit stronger T cell activation ability compared to the FIT-Ig biantibody molecule (PR003052, PR000701), making them superior to Urelumab.

[0246] Example 4.4.2. MDA-MB-231-mediated T cell-specific activation As shown in Figure 27E, in a system mixing target cells MDA-MB-231 with T cells, the IgG-VH tetravalent symmetric biantibody molecule (PR003549) and the Fab-HCAb biantibody molecule (PR004270) exhibited similar T cell activation capabilities and were superior to the FIT-Ig biantibody molecules (PR003052, PR000701).

[0247] As shown in Figures 27F and 27G, in a system of mixed target cells MDA-MB-231 and T cells, the IgG-VH tetravalent symmetric biantibody molecule (PR003549) exhibits stronger T cell activation ability and better promotes the production of IFN-γ and IL-2 than Urelumab.

[0248] As shown in Figures 27H and 27I, in a system in which target cells MDA-MB-231 and T cells are mixed, the IgG-VH tetravalent symmetric biantibody molecules (PR007130, PR007132, PR007133, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) exhibit significantly greater T cell activation ability than urelumab.

[0249] Example 4.5. Mixed lymphocyte reaction (MLR) This example studies the activating effect of the PD-L1×4-1BB biantibody molecule on T cells using a mixed lymphocyte reaction (MLR).

[0250] In the first step, mononuclear cells (monocytes) were isolated from the first donor PBMC cells (MT-BIO) using CD14 beads (Meltenyi, #130-050-201); refer to the instructions of the relevant kit for specific procedures. Subsequently, 50 ng / mL recombinant human IL-4 (PeproTech, #200-02-A) and 100 ng / mL recombinant human GM-CSF (PeproTech, #300-03-A) were added, and the cells were induced at 37°C for 7 days to obtain immature dendritic cells (iDC cells). Subsequently, 1 μg / mL of lipopolysaccharide (LPS, Sigma, #L 6529) was added, and the cells were induced for 24 hours to obtain mature dendritic cells (mDC cells). In the second step, T lymphocytes were isolated from the second donor PBMC cells (MT-BIO) using a T cell isolation kit (Meltenyi, #130-096-535). In the third step, the obtained T cells and mDC cells were seeded in a 5:1 ratio in a 96-well plate (1 × 10⁻¹⁶). 5 / well T cells and 2×10 4 mDC cells were added to two combined wells. Then, 50 μL / well of antibody molecules at different concentrations were added, resulting in eight final antibody concentrations (10 nM, 1 nM), or a total of eight concentrations obtained by diluting from the highest final concentration of 50 nM with a 3-fold concentration gradient. hIgG1 iso (CrownBio, #C0001) or a blank well was used as a control. The mixture was incubated at 37°C in a 5% CO2 culture box for 5 days. In the fourth step, the supernatants were collected on days 3 and 5, respectively. IL-2 concentration in the day 3 supernatant was detected using an IL-2 ELISA kit (Thermo, #88-7025-88), and IFN-γ concentration in the day 5 supernatant was detected using an IFN-γ ELISA kit (Thermo, #88-7316-77). Refer to the operating instructions for the relevant kit for ELISA detection methods.

[0251] As shown in Figure 28, in multiple independent MLR experiments (different donor pairs), the activating effect of anti-4-1BB monoclonal antibodies on T cells was limited, and their ability to produce cytokines was weak; anti-PD-L1 monoclonal antibodies showed a clear activating effect. Biantibody molecules can further enhance T cell function.

[0252] As shown in Figures 28A to 28F, the IgG-VH tetravalent symmetric biantibody molecules (PR003549, PR003550, PR003551, PR007130, PR007132, PR007133, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) induce higher cytokine release levels than the maternal PD-L1 antibody PR000265, demonstrating stronger T cell activation ability and indicating that the biantibody molecules are superior to the anti-PD-L1 monoclonal antibody.

[0253] As shown in Figures 28A and 28D, the IgG-VH tetravalent symmetric biantibody molecules (PR003549, PR003550, PR003551) induce higher cytokine release levels and exhibit stronger T cell activation ability than the FIT-Ig biantibody molecules (PR003052, PR000701); and the biantibody molecules are also superior to PD-L1 monoclonal antibodies.

[0254] As shown in Figures 28G and 28H, the IgG-VH tetravalent symmetric biantibody molecules (PR003549, PR003550) and the Fab-HCAb biantibody molecule (PR004270) can induce higher cytokine release levels than the FIT-Ig biantibody molecules (PR003052, PR000701), demonstrating stronger T cell activation ability.

[0255] As shown in Figures 28I to 28K, the IgG-VH tetravalent symmetric biantibody molecules (PR003549, PR003550, PR003551) and the Fab-HCAb biantibody molecule (PR004270) exhibit strong T cell activation capabilities.

[0256] Example 4.6. Pharmacokinetic Studies This example investigated the pharmacokinetic properties of the PD-L1×4-1BB biantibody molecule PR004270, which possesses a Fab-HCAb symmetric structure, in the mouse body.

[0257] Procedure: For each test antibody molecule, six female BALB / c mice weighing 18-22 grams were selected and administered a bispecific antibody at a dose of 5 mg / kg by intravenous injection. Whole blood samples were collected from three mice in one group before administration and at 15 minutes, 24 hours (1 day), 4 days, and 10 days after administration. Whole blood samples were collected from three mice in another group before administration and at 5 hours, 2 days, 7 days, and 14 days after administration. The whole blood was allowed to coagulate for 30 minutes, then centrifuged, and the separated serum samples were frozen and stored at -80°C until analysis. In this example, the drug concentration in mouse serum was quantitatively measured using two different ELISA methods. ELISA method 1, i.e., Fc-end detection (whole detection) method, involves capturing human Fc-containing antibodies in mouse serum using goat anti-human Fc polyclonal antibody encapsulated in a 96-well plate, followed by detection with HRP-labeled goat anti-human Fc 2 antibody; ELISA method 2, i.e., PD-L1-end detection (functional domain detection) method, involves capturing antibodies that specifically recognize PD-L1 in mouse serum using human PD-L1 protein encapsulated in a 96-well plate, followed by detection with HRP-labeled goat anti-human Fc 2 antibody. Pharmacokinetic parameters were analyzed using Phoenix WinNonlin software version 8.2, selecting a non-atrioventricular (NCA) model.

[0258] As shown in Figure 29 and Table 32, the Fab-HCAb biantibody molecule PR004270 exhibits a serum half-life similar to that of conventional IgG antibodies. 1 / 2 The functional domain detection method has a value, and its t 1 / 2The value indicated that it exceeded 10 days.

[0259] [Table 32]

[0260] summary This example demonstrates the construction of various structures of anti-PD-L1 × 4-1BB bispecific antibody molecules using the antigen-binding domain Fab of an anti-PD-L1 IgG antibody and the antigen-binding domain VH of an anti-4-1BB HCAb antibody. It shows flexibility in constructing bispecific antibody molecular structures based on HCAb, and modulates T cell activation activity through different structural types, relative positions, and binding valencies.

[0261] The lack of target specificity in urelumab's activation of T cells is one of the causes of clinical toxic side effects. The activating effect of PD-L1×4-1BB bi-antibodies on T cells is specifically dependent on PD-L1 expression. While cross-linked anti-4-1BB dependent HCAb monoclonal antibodies cannot directly activate T cells, PD-L1×4-1BB bi-antibodies constructed using these HCAb monoclonal antibodies can specifically activate T cells in the presence of cells that highly express PD-L1.

[0262] HCAb-based dual antibody structures, particularly the IgG-VH tetravalent symmetric structure and the Fab-HCAb structure, retained PD-L1 terminal activity and exhibited stronger T cell activation ability than the corresponding anti-PD-L1 parental monoclonal antibody in MLR experiments. On the other hand, PD-L1 molecules highly expressed on target cells can transmit T cell activation signals by mediating 4-1BB crosslinking and trimerization, and their T cell activation ability is superior to that of urelumab. Furthermore, the IgG-VH tetravalent symmetric structure and Fab-HCAb symmetric structure dual antibody molecules showed stronger T cell activation ability than the FIT-Ig structure dual antibody molecule.

[0263] Based on the above, this embodiment constructed a PD-L1×4-1BB bispecific antibody molecule that exhibits good safety, outstanding functional activity, and good molecular stability.

[0264] References: 1. Bertram EM, Lau P, Watts TH. Temporal segregation of4-1BB versus CD28-mediated costimulation:4-1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection. Journal of immunology 2002;168: 3777-85. 2. Kawalekar OU, O'Connor RS, Fraietta JA, et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. Immunity 2016;44: 380-90. 3. Ye Z, Hellstrom I, Hayden-Ledbetter M, Dahlin A, Ledbetter JA, Hellstrom KE.Gene therapy for cancer using single-chain Fv fragments specific for4-1BB. Nature medicine 2002;8: 343-8. 4. Zhang H, Knutson KL, Hellstrom KE, Disis ML, Hellstrom I. Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv-expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion. Molecular cancer therapeutics 2006;5: 149-55. 5 Yang Y, Yang S, Ye Z, et al. Tumor cells expressing anti-CD137 scFv induce a tumor-destructive environment. Cancer research 2007;67: 2339-44. 6. Martinet O, Divino CM, Zang Y, et al. T cell activation with systemic agonistic antibody versus local 4- 1BB ligand gene delivery combined with interleukin-12eradicate liver metastases of breast cancer.Gene therapy2002;9: 786-92。

Claims

1. A 4-1BB binding protein comprising a heavy chain variable region capable of binding to 4-1BB, wherein the 4-1BB binding protein does not contain a light chain or a light chain variable region. A 4-1BB-binding protein in which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 each contain the sequences shown in SEQ ID NO: 20, SEQ ID NO: 71, and SEQ ID NO: 115, respectively.

2. The 4-1BB binding protein according to claim 1, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 198 or SEQ ID NO:

352.

3. The 4-1BB binding protein according to claim 1, wherein the 4-1BB binding protein further comprises a heavy chain constant region.

4. The 4-1BB binding protein according to claim 3, wherein the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3, or hIgG4, or a variant thereof.

5. The 4-1BB binding protein according to claim 4, wherein the IgG1 variant comprises one or more mutations from L234A, L235A, E345R, and P329G; and the IgG4 variant comprises the S228P mutation.

6. The 4-1BB binding protein according to claim 1, wherein the 4-1BB binding protein comprises a heavy chain, and the heavy chain comprises the sequence shown in SEQ ID NO: 240 or SEQ ID NO:

368.

7. The 4-1BB-binding protein according to claim 1, wherein the 4-1BB-binding protein is in the form of a heavy chain antibody or a single-domain antibody.

8. A bispecific antibody comprising a first protein functional domain and a second protein functional domain, wherein the first protein functional domain is the 4-1BB binding protein described in claim 1, and the second protein functional domain targets a tumor antigen.

9. The bispecific antibody according to claim 8, wherein the second protein functional region is a HER2 antibody or a PD-L1 antibody.

10. The bispecific antibody according to claim 9, wherein the HER2 antibody is trastuzumab or pertuzumab, the PD-L1 antibody is atezolizumab, or the PD-L1 antibody comprises the heavy chain shown in SEQ ID NO: 211 and the light chain shown in SEQ ID NO:

245.

11. The second protein functional region is scFv, VHH, immunoglobulin, Fab, Fab', F(ab') ２ The bispecific antibody according to claim 8, or in the form of a heavy chain variable region.

12. A bispecific antibody comprising polypeptide chain 1 and polypeptide chain 2, A bispecific antibody wherein the amino acid sequence of polypeptide chain 1 is shown in SEQ ID NO: 245, and the amino acid sequence of polypeptide chain 2 is shown in SEQ ID NO:

369.

13. An isolated nucleic acid encoding a 4-1BB binding protein according to any one of claims 1 to 7 or a bispecific antibody according to any one of claims 8 to 12.

14. An expression vector comprising the isolated nucleic acid described in claim 13.

15. A host cell comprising the expression vector described in claim 14.

16. A method for producing a protein capable of binding to 4-1BB, comprising culturing host cells and obtaining from the culture a 4-1BB binding protein according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-12, wherein the host cells comprise an expression vector comprising an isolated nucleic acid encoding the 4-1BB binding protein according to any one of claims 1-7 or the bispecific antibody according to any one of claims 8-12.

17. A chimeric antigen receptor comprising a 4-1BB binding protein according to any one of claims 1 to 7 or a bispecific antibody according to any one of claims 8 to 12.

18. An antibody-drug conjugate comprising a cytotoxic agent and a 4-1BB binding protein according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-12.

19. A composition comprising the 4-1BB binding protein according to any one of claims 1 to 7 and / or the bispecific antibody according to any one of claims 8 to 12.

20. A drug composition comprising a 4-1BB binding protein according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-12, for use in treating and / or preventing a 4-1BB-mediated disease or disorder in a patient in need.

21. The drug composition according to claim 20, wherein the disease or disorder is a tumor.

22. The drug composition according to claim 21, wherein the tumor is a 4-1BB-positive tumor.

23. The drug composition according to claim 21, wherein the tumor is gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma, or leukemia.

24. An in vitro method for immunodetecting or measuring 4-1BB, A method comprising using a 4-1BB binding protein according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-12, wherein the detection is not for diagnostic or therapeutic purposes.

25. A drug composition comprising a 4-1BB binding protein according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-12, for use in combination therapy with a second therapeutic agent in patients requiring it.

26. The drug composition according to claim 25, wherein the second therapeutic agent comprises one or more from the group consisting of other antitumor antibodies, and / or hormone preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, lysogenic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

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