4-1BB binding protein and uses thereof

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 claims priority from Chinese patent application No. 202010619500.9 filed on June 30, 2020. This application cites said Chinese patent application in its entirety.

[0002] The present application relates to the biomedical field, specifically to 4-1BB binding proteins and their uses. [Background technology]

[0003] 4-1BB, also known as CD137, tumor necrosis factor receptor superfamily member 9 (TNFRSF 9), is an activation-induced costimulatory receptor molecule. 4-1BB is primarily expressed in lymphoid lineages, including activated T cells, activated spontaneously destroyed (NK) cells, activated thymocytes, and intradermal lymphocytes. 4-1BB is also expressed in dendritic cells, monocytes, neutrophils, and eosinophils. T cell activation requires costimulatory signals provided by the binding of costimulatory molecules on the surface of antigen-presenting cells (APCs) to the corresponding receptors on the surface of T cells, in addition to the specific binding of antigen-recognition receptors (TCRs) on the surface of T cells to MHC molecule antigen peptides. The binding of 4-1BB to its ligand 4-1BBL can provide costimulatory signals to activate T cells and enhance cytokines and immune function. 4-1BB has also been shown to promote central memory T cell responses, which may support treatment persistence and resistance to exhaustion of tumor-specific T cells in patients treated with 4-1BB agonists. [1,2] Overexpression of anti-4-1BB single-chain antibody fragment (scFv) on tumors promotes CD4+ T cell- and NK cell-dependent tumor elimination. [3、4、5] It has also been demonstrated that systemic injection of anti-4-1BB antibodies leads to delayed tumor growth in a mouse model. [6] .

[0004] It has been demonstrated that activating 4-1BB antibodies can activate the 4-1BB downstream pathway instead of its ligand. Currently, activating 4-1BB antibodies under clinical investigation include Urelumab (BMS-663513) and Utomilumab (PF-05082566). Utomilumab is a ligand-blocking IgG2 antibody, and Urelumab is a non-ligand-blocking IgG4 antibody. Utomilumab and Urelumab can enhance T cell function and promote antitumor activity both in vivo and in vitro. However, clinical trials have shown that some patients develop pneumonia toxicity when the dose of Urelumab is greater than 1 mg / kg (see NCT00309023, NCT00612664, NCT01471210). Compared with Urelumab, Utomilumab has a higher safety profile but a lower activity of activating 4-1BB. Therefore, the development of new, safer and more effective therapies targeting 4-1BB is urgently needed.

[0005] In order to solve the lack of anti-tumor therapeutic effects in the prior art and to solve technical problems such as safety, the present invention provides a fully human antibody targeting 4-1BB, and a bispecific antibody based on this antibody and a tumor-specific target and its use. The present application provides an anti-4-1BB antigen-binding protein having one or more of the following properties: 1) capable of binding human and monkey-derived 4-1BB protein; 2) capable of stimulating immune cells to secrete IFN-γ, IL2 and / or TNFα; 3) capable of suppressing tumor growth and / or tumor cell proliferation; 4) capable of activating the 4-1BB signal pathway. The present application also provides the use of the antigen-binding protein in the prevention and treatment of tumors. Summary of the Invention [Problem to be solved by the invention]

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of antibody drugs in the prior art, such as the lack of antitumor therapeutic effect and poor safety, by providing a binding protein targeting 4-1BB and uses thereof, in particular a fully human antibody targeting 4-1BB, and a bispecific antibody based on this antibody and a tumor-specific target and uses thereof. [Means for solving the problem]

[0007] A first aspect of the invention provides a 4-1BB binding protein, said 4-1BB binding protein comprising a heavy chain variable region; said heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the sequence as set forth in SEQ ID NO: 15 or variant 1 thereof, or SEQ ID NO: 16, said HCDR2 comprising the sequence as set forth in SEQ ID NO: 60 or variant 2 thereof, or SEQ ID NO: 50 or variant 4 thereof, and said HCDR3 comprising the sequence as 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; among them: The mutations of said variant 1 include one or more of T3I, S6N / G / R and Y7F; preferably, the sequence of said variant 1 is preferably 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 mutations of said variant 2 include 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 any of the sequences shown in 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 of said variant 3 include one or more of 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 said variant 3 is preferably any of the sequences shown in SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 104-106, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 112-115 in the sequence listing; The mutation of said variant 4 includes N1I or N1Q; the amino acid sequence of said variant 4 is preferably shown in SEQ ID NO: 53 or 54 in the sequence listing; The mutation of said variant 5 includes E4A / P and / or N13Y; the amino acid sequence of said variant 5 is preferably shown in SEQ ID NO: 327-330 in the sequence listing; Mutant 1, Mutant 2, Mutant 3, Mutant 5 and Mutant 4 each contain at least the function of the sequence before mutation.

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

[0009] [Table A]

[0010] The gene encoding the FR of the heavy chain variable region is preferably derived from the germline V gene IGHV4-34, IGHV3-23, IGHV3-11 or IGHV3-74; among them: HFWR1 preferably comprises any of the amino acid sequences 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 any of the amino acid sequences 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 any of the amino acid sequences 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 amino acid sequence of HFWR1 The amino acid sequence of HFWR2 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 the HFWR2 is shown in SEQ ID NO: 36, the amino acid sequence of the HFWR3 is shown in SEQ ID NO: 80, and the amino acid sequence of the HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of the HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of the HFWR2 is shown in SEQ ID NO: 38, the amino acid sequence of the HFWR3 is shown in SEQ ID NO: 80, and the amino acid sequence of the HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of the HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of the HFWR2 is shown in SEQ ID NO: 39, and the amino acid sequence of the HFWR3 is shown in SEQ ID NO: 8 0, 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: 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, 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: 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: 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 shown in SEQ ID NO: 119. The amino acid sequence is shown in SEQ ID NO: 80, and the amino acid sequence of the HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of the HFWR1 is shown in SEQ ID NO: 8, the amino acid sequence of the HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of the HFWR3 is shown in SEQ ID NO: 88, and the amino acid sequence of the HFWR4 is shown in SEQ ID NO: 119, the amino acid sequence of the HFWR1 is shown in SEQ ID NO: 12, the amino acid sequence of the HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of the HFWR3 is shown in SEQ ID NO: 89, and the amino acid sequence of the HFWR4 is shown in SEQ ID NO: 123;The amino acid sequence of the HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of the HFWR2 is shown in SEQ ID NO: 34, the amino acid sequence of the HFWR3 is shown in SEQ ID NO: 88, and the amino acid sequence of the HFWR4 is shown in SEQ ID NO: 119. The amino acid sequence of the HFWR1 is shown in SEQ ID NO: 6, the amino acid sequence of the HFWR2 is shown in SEQ ID NO: 43, the amino acid sequence of the HFWR3 is shown in SEQ ID NO: 90, and the amino acid sequence of the HFWR4 is shown in SEQ ID NO: 119. The amino acid sequence of the HFWR1 is shown in SEQ ID NO: 6, and the amino acid sequence of the HFWR2 is shown in SEQ ID NO: 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 amino acid sequence of HFWR The amino acid sequence of HFWR1 is set forth in SEQ ID NO: 119, or the amino acid sequence of HFWR1 is set forth in SEQ ID NO: 1, the amino acid sequence of HFWR2 is set forth in SEQ ID NO: 46, the amino acid sequence of HFWR3 is set forth in SEQ ID NO: 92, and the amino acid sequence of HFWR4 is set forth in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is set forth in SEQ ID NO: 294, the amino acid sequence of HFWR2 is set forth in SEQ ID NO: 301, the amino acid sequence of HFWR3 is set forth in SEQ ID NO: 318, and the amino acid sequence of HFWR4 is set forth in SEQ ID NO: 335, or The amino acid sequence of HFWR1 is set forth in SEQ ID NO: 295, the amino acid sequence of HFWR2 is set forth in SEQ ID NO: 302, the amino acid sequence of HFWR3 is set forth in SEQ ID NO: 319, and the amino acid sequence of HFWR4 is set forth in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is set forth in SEQ ID NO: 294, the amino acid sequence of HFWR2 is set forth in SEQ ID NO: 302, the amino acid sequence of HFWR3 is set forth in SEQ ID NO: 320, and the amino acid sequence of HFWR4 is set forth in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is set forth in SEQ ID NO: 294,The amino acid sequence of the HFWR2 is set forth in SEQ ID NO: 302, the amino acid sequence of the HFWR3 is set forth in SEQ ID NO: 321, and the amino acid sequence of the HFWR4 is set forth in SEQ ID NO: 335; or the amino acid sequence of the HFWR1 is set forth in SEQ ID NO: 296, the amino acid sequence of the HFWR2 is set forth in SEQ ID NO: 302, the amino acid sequence of the HFWR3 is set forth in SEQ ID NO: 322, and the amino acid sequence of the HFWR4 is set forth in SEQ ID NO: 335; or the amino acid sequence of the HFWR1 is set forth in SEQ ID NO: 297, and the amino acid sequence of the HFWR2 is set forth in SEQ ID NO: 303; The amino acid sequence of the HFWR3 is set forth in SEQ ID NO: 318, and the amino acid sequence of the HFWR4 is set forth in SEQ ID NO: 335, or the amino acid sequence of the HFWR1 is set forth in SEQ ID NO: 294, the amino acid sequence of the HFWR2 is set forth in SEQ ID NO: 301, the amino acid sequence of the HFWR3 is set forth in SEQ ID NO: 322, and the amino acid sequence of the HFWR4 is set forth in SEQ ID NO: 118, or the amino acid sequence of the HFWR1 is set forth in SEQ ID NO: 294, the amino acid sequence of the HFWR2 is set forth in SEQ ID NO: 302, the amino acid sequence of the HFWR3 is set forth in SEQ ID NO: 323, 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: 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, or or the amino acid sequence of HFWR1 is set forth in SEQ ID NO: 298, the amino acid sequence of HFWR2 is set forth in SEQ ID NO: 302, the amino acid sequence of HFWR3 is set forth in SEQ ID NO: 322, and the amino acid sequence of HFWR4 is set forth in SEQ ID NO: 335, or the amino acid sequence of HFWR1 is set forth in SEQ ID NO: 294, the amino acid sequence of HFWR2 is set forth in SEQ ID NO: 301, the amino acid sequence of HFWR3 is set forth in SEQ ID NO: 318, and the amino acid sequence of HFWR4 is set forth in SEQ ID NO: 118, or the amino acid sequence of HFWR1 is set forth 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 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 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 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. For details, see Table b below. ,

[0011] [Table B]

[0012] In the present invention, the heavy chain variable region preferably comprises 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 mentioned above, the 4-1BB binding protein according to the present invention further comprises a light chain variable region, said light chain variable region comprising LCDR1, LCDR2 and LCDR3, said LCDR1 comprising the sequence shown in SEQ ID NO: 133, said LCDR2 comprising the sequence shown in SEQ ID NO: 145 and said LCDR3 comprising the sequence shown in SEQ ID NO: 158. Preferably, the gene encoding the FR of said light chain variable region is derived from the germline 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, said light chain variable region comprises the sequence shown in SEQ ID NO: 201 in the sequence listing or a variant thereof, said variant being based on the occurrence of one or more amino acid mutations 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 invention, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 168 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 201; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 171 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 204; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 172 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 204. For details, see Table c below.

[0015] [Table C]

[0016] Furthermore, the 4-1BB binding protein according to the present invention may comprise a heavy chain constant region and / or a light chain constant region, preferably the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4 or a mutant thereof and the light chain constant region is selected from a κ chain or a λ chain or a mutant thereof.

[0017] Among them, the mutation of said 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 of said hIgG4 variant is preferably S228P.

[0018] In a preferred embodiment of the present invention, the heavy chain of the 4-1BB binding protein comprises any of the sequences shown in SEQ ID NO: 209, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 216-222, SEQ ID NO: 224-238, SEQ ID NO: 353-368 and SEQ ID NO: 240 in the sequence listing, and / or the light chain comprises the sequence shown in SEQ ID NO: 246 or SEQ ID NO: 243 in the sequence listing. For example: the amino acid sequence of the heavy chain is shown in SEQ ID NO: 209, and the amino acid sequence of the light chain is shown in SEQ ID NO: 243; or the amino acid sequence of the heavy chain is shown in SEQ ID NO: 212, and the amino acid sequence of the light chain is shown in SEQ ID NO: 246; or the amino acid sequence of the heavy chain is shown in SEQ ID NO: 213, and the amino acid sequence of the light chain is shown in SEQ ID NO: 246; or the amino acid sequence of the heavy chain is shown in SEQ ID NO: 216, and the amino acid sequence of the light chain is shown in SEQ ID NO: 246. As shown in Table d below.

[0019] [Table D]

[0020] The 4-1BB binding protein of 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 may be a monoclonal or multiclonal antibody derived from said antibody.

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

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

[0023] In particular, the first protein functional region or the second protein functional region may be in the form of an scFv, VHH, immunoglobulin, Fab, Fab', F(ab')2 or heavy chain variable region, e.g., the first protein functional region is a Fab and the second protein functional region is a VHH; or, the first protein functional region is a Fab and the second protein functional region is an immunoglobulin; or, the first protein functional region is an immunoglobulin and the second protein functional region is 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 linker, and the amino acid sequence of the linker is shown in GS or in any of SEQ ID NOs: 273-293 in the sequence listing.

[0025] In a particular embodiment of the 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 said polypeptide chain 1 is set forth in SEQ ID NO:244, and the amino acid sequence of said polypeptide chain 2 is set forth in any of SEQ ID NOs:251-265; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:271; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:249, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:272; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:270; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:269; or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of polypeptide chain 2 is set forth in SEQ ID NO:268; or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of polypeptide chain 2 is set forth in any of SEQ ID NOs:369-382; or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:267, the amino acid sequence of polypeptide chain 2 is set forth in SEQ ID NO:266, and the amino acid sequence of polypeptide chain 3 is set forth in SEQ ID NO:246; or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:250, the amino acid sequence of polypeptide chain 2 is set forth in SEQ ID NO:249, and the amino acid sequence of polypeptide chain 3 is set forth in SEQ ID NO:246.

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

[0027] A fourth aspect of the invention provides an expression vector comprising the isolated nucleic acid according to the third aspect.

[0028] A fifth aspect of the invention provides a host cell comprising an expression vector according to the fourth aspect of the invention; preferably, said host cell comprises a prokaryotic or eukaryotic cell, wherein said eukaryotic cell is preferably a mammalian cell.

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

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

[0031] An eighth aspect of the invention provides an antibody drug conjugate comprising a cytotoxic agent and a 4-1BB binding protein according to the first aspect of the invention or a bispecific antibody according to the second aspect of the invention.

[0032] The cytotoxic agent is preferably a cytotoxin, a chemotherapeutic agent, a radioisotope, a therapeutic nucleic acid, an immunomodulatory agent, an anti-hemotubule agent, an anti-proliferative apoptotic agent or a cell-soluble enzyme. More preferably, the cytotoxic agent is a tubulin synthesis enzyme inhibitor, methylolestatin F (MMAF) or methylolestatin E (MMAE).

[0033] The antibody-drug conjugate may be prepared by any method known in the art, preferably the method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124. Preferably, the method produces an antibody-drug conjugate with a minimum low conjugate fraction (LCF) of less than 10%.

[0034] The antibody drug conjugate can be in any physical form known in the art, preferably a clear solution.

[0035] A ninth aspect of the invention provides a drug composition comprising a 4-1BB binding protein according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention and / or an antibody-drug conjugate according to the eighth aspect of the invention, and a pharma- ceutically acceptable vector.

[0036] The pharmaceutical composition preferably comprises other anti-tumor antibodies as active ingredients, and / or further comprises one or more of the group consisting of hormonal 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.

[0037] The pharma- ceutically acceptable vector may be a vector conventional in the art, and the vector may be any suitable physiologically or pharma- ceutical acceptable drug additive. The drug additive is a conventional drug additive in the art, and preferably includes a pharma- ceutical acceptable excipient, filler, stabilizer or diluent, etc. More preferably, the drug composition comprises 0.01-99.99% of the protein and / or the antibody-drug conjugate and 0.01-99.99% of the drug vector, the percentage being the mass percentage in the drug composition.

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

[0039] The administration route of the pharmaceutical composition according to the present invention is preferably enterogastric administration, injection administration or oral administration. The injection administration preferably includes routes such as intravenous injection, intramuscular injection, peritoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition may be in various dosage forms conventional in the art, preferably in solid, semisolid or liquid form, i.e., aqueous, non-aqueous or suspension, more preferably, tablet, capsule, pellet, injection or infusion, etc. More preferably, it is administered via intravascular, subcutaneous, intraperitoneal or intramuscular. Preferably, the pharmaceutical composition may be administered as an aerosol or coarse spray, i.e., intranasally, or may be administered intrathecally, intramedullary or intraventricularly. More preferably, the pharmaceutical composition may be administered transdermally, transdermally, topically, intestinally, 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 the composition to achieve the desired diagnostic or therapeutic result. The administration scheme can be a single injection or multiple injections, or can be adjusted. The selected dose level and scheme can be reasonably adjusted depending on various factors, including the activity and stability (i.e., half-life) of the drug composition, the formulation, the route of administration, the combination with other drugs or treatments, the disease or disorder to be detected and / or treated, and the health condition and previous medical history of the subject to be treated, etc.

[0041] The therapeutically effective dose of the pharmaceutical composition of the present invention can be estimated first in cell culture experiments or animal models, such as rodents, rabbits, dogs, pigs and / or primates.Animal models can also be used to determine the appropriate concentration range and route of administration.Then, it can be used to determine the useful dose and route of administration to humans.In general, the determination and adjustment of the effective amount or dose to be administered, and the evaluation of the timing and method of such adjustment are known to those skilled in the art.

[0042] In combination therapy, the 4-1BB binding protein, the antibody drug conjugate, and / or the additional therapeutic or diagnostic agent can each be used as a single agent for any time range suitable for carrying out the desired treatment or diagnosis. Thus, these single agents can be administered substantially simultaneously (i.e., as a single formulation, or within minutes or hours), or sequentially in sequence. For example, these single agents can be administered within 1 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, methods of administration, and measurable therapeutic outcomes, see such works as Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey, Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

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

[0045] In some aspects, 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 to a subject a pharma- ceutical effective amount of said 4-1BB binding protein or containing said pharmaceutical composition or said nucleic acid molecule, and treating a disease associated with one or more of 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 one or more abnormal expression of HER2, PD-L1 and 4-1BB, as commonly known in the art, such as squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, hepatic lymphoma, non-hepatic lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), primary mediastinal large B-cell lymphoma, theatric cell lymphoma (MCL), Small lymphocytic lymphoma (SLL), large B-cell lymphoma with T-cell / tissue cell enriched, multiple myeloma, medullary cell leukemia-1 protein (Mcl-1), myeloproliferative syndrome (MDS), gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, pharyngeal cancer, hepatobiliary cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloid degeneration, lymphocytic lymphomalymphoma), myeloproliferative syndromes, myeloproliferative neoplasms, neuroendocrine tumors, Meckel cell carcinoma, testicular cancer and skin cancer, PD-L1 positive squamocellular carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamocellular carcinoma (HNSCC), glioma, myeloma lymphoma, non-myeloma lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), primary mediastinal large B-cell lymphoma, theatric cell lymphoma (MCL), small lymphocytic Lymphoma (SLL), large B-cell lymphoma enriched for T-cells / histocells, multiple myeloma, medullary cell leukemia-1 protein (Mcl-1), myeloproliferative syndrome (MDS), gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, hepatic sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear 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, lymphoplasmacytic lymphoma, myeloproliferative syndrome, myeloproliferative neoplasm, neuroendocrine tumor, 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 according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention, a chimeric antigen receptor according to the seventh aspect of the invention, an antibody-drug conjugate according to the eighth aspect of the invention and / or a drug composition according to the ninth aspect of the invention.

[0048] Preferably, the reagent kit further comprises: (i) a device for administering the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate or drug composition; and / or (ii) instructions for use.

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

[0050] In order to solve the above technical problems, the 4-1BB binding protein according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the chimeric antigen receptor according to the seventh aspect of the present invention, the antibody-drug conjugate according to the eighth aspect of the present invention and / or the pharmaceutical composition according to the ninth aspect of the present invention may further be administered in combination with other drugs, for example, hormonal preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, tumor lytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, etc. and / or other anti-tumor antibodies (or pharmaceutical compositions comprising said other anti-tumor antibodies).

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

[0052] Preferably, said disease or disorder is a tumor, preferably a 4-1BB positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal 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 invention provides a method for immunodetection or measurement of 4-1BB, the method comprising using a 4-1BB binding protein according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention, a chimeric antigen receptor according to the seventh aspect of the invention, an antibody drug conjugate according to the eighth aspect of the invention or a drug composition according to the ninth aspect of the invention; preferably, said detection is not for diagnostic and / or therapeutic purposes.

[0054] A fifteenth aspect of the present invention provides a combination therapy comprising administering to a patient in need thereof a 4-1BB binding protein according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention, a chimeric antigen receptor according to the seventh aspect of the invention, an antibody drug conjugate according to the eighth aspect of the invention or a pharmaceutical composition according to the ninth aspect of the invention, and a second therapeutic agent, preferably comprising another anti-tumour antibody or a pharmaceutical composition comprising said other anti-tumour antibody, and / or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, lytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules and vaccines.

[0055] The above-mentioned preferred conditions may be arbitrarily combined in accordance with common knowledge in the art, that is, to obtain preferred embodiments of the present invention.

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

[0057] The positive and improved effects of the present invention are as follows: 1. The present application provides a fully human antibody of anti-4-1BB binding protein having at least one of the following properties: 1) The fully human antibody of anti-4-1BB binding protein of the present invention includes a full-length antibody and a completely new fully human antibody containing only a "heavy chain", and has the activity of binding to human 4-1BB and cynomolgus monkey 4-1BB; in which, the size of the heavy chain antibody of the 4-1BB binding protein is only half that of a conventional IgG antibody, and does not contain a light chain, so that this antibody can be used for bispecific antibodies, and the problems of light chain mismatch and heterodimerization are solved. It can bind human and monkey-derived 4-1BB proteins; 2) The binding epitope is different from that of Urelumab; 3) It can activate the 4-1BB signal pathway, stimulate the secretion of IFNγ, IL2 and / or TNFα in immune cells, and suppress tumor growth and / or tumor cell proliferation; the activity is 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. First, the PD-L1×4-1BB bispecific antibody can activate T cells by blocking the PD-1 / PD-L1 signal pathway. Second, the PD-L1 molecule highly expressed on the surface of tumor cells can promote the cross-linking and trimerization of 4-1BB molecules on the surface of T cells using the bispecific antibody molecule, activating downstream signal transduction pathways, and further promoting T cell activation and proliferation, and its ability to activate T cells is superior to that of Urelumab. Third, the activating effect of the PD-L1×4-1BB bispecific antibody on T cells is specifically PD-L1-dependent expression, and the T cell activation mediated by the bispecific antibody molecule is limited within the tumor microenvironment. The present invention demonstrates that, compared with the prior art HCAb monoclonal antibodies that rely on cross-linked anti-4-1BB and cannot directly activate T cells, the PD-L1×4-1BB bispecific antibody constructed using HCAb monoclonal antibodies can specifically activate T cells in the presence of cells highly expressing PD-L1, thereby avoiding the toxic side effects caused by monoclonal antibodies such as Urelumab excessively activating T cells in normal tissues. Fourthly, this embodiment uses 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 to construct anti-PD-L1×4-1BB bispecific antibody molecules with various structures. It shows the flexibility of constructing the molecular structure of bispecific antibodies based on HCAb, and adjusts the functional activity of activating T cells according to parameters such as different structural types, relative positions, and binding valencies. The HCAb-based dual antibody structures, especially the dual antibody molecules with IgG-VH tetravalent symmetric structure and the dual antibody molecules with Fab-HCAb structure, retained the activity of the PD-L1 end and demonstrated stronger T cell activation ability than the corresponding anti-PD-L1 parent monoclonal antibodies in MLR experiments; meanwhile, PD-L1 molecules highly expressed on target cells can mediate the cross-linking and trimerization of 4-1BB to transmit T cell activation signals, and its ability to activate T cells is superior to that of Urelumab.Furthermore, the dual antibody molecules with an IgG-VH tetravalent symmetric structure and a Fab-HCAb symmetric structure exhibit stronger T cell activation ability than the dual antibody molecule with an 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. First, the HER2×4-1BB bispecific antibody retains the mechanism of action of conventional HER2 inhibitors (blocking HER2 dimerization, promoting HER2 internalization and degradation, and suppressing downstream phosphorylation signals). Second, the highly expressed HER2 molecules on the surface of tumor cells promote the cross-linking and trimerization of 4-1BB molecules on the surface of T cells, activating downstream signal transduction pathways, and further promoting T cell activation and proliferation, and its ability to activate T cells is superior to that of Urelumab. Third, the activation effect of the HER2×4-1BB bispecific antibody on T cells is specifically dependent on the expression of HER2, and T cell activation mediated by the bispecific antibody molecule is limited within the tumor microenvironment. Compared with the prior art HCAb monoclonal antibodies that rely on cross-linked anti-4-1BB, which cannot directly activate T cells, the HER2×4-1BB biantibody constructed using the HCAb monoclonal antibody of the present invention can specifically activate T cells in the presence of cells highly expressing HER2, thereby avoiding the toxic side effects caused by monoclonal antibodies such as Urelumab overactivating T cells in normal tissues. Fourth, this example constructs anti-HER2×4-1BB bispecific antibody molecules with two structures, IgG-VH and IgG-scFv, to adjust the functional activity of activating T cells according to parameters such as different structural types, relative positions, and binding valencies. At the same time, the flexibility of constructing bispecific antibody molecular structures based on HCAb is demonstrated. [Brief description of the drawings]

[0060] [Figure 1A] 1A-1B: FACS detection of binding of 4-1BB H2L2 antibody to CHO-K1 cells overexpressing human or cynomolgus 4-1BB. [Figure 1B] Same as above. [Diagram 2] FIG. 2: FACS detection of 4-1BB H2L2 antibody blocking binding of 4-1BB ligand to CHO-K1 / human 4-1BB cells. [Figure 3A] Figures 3A-B: Activation of the 4-1BB signal pathway by 4-1BB H2L2 antibody was detected using HEK293 / 4-1BB / NF-kb reporter gene cells. [Figure 3B] Same as above. [Figure 4] Figure 4: 4-1BB H2L2 antibody activates the 4-1BB pathway in vitro and induces and activates IFN-γ secretion in T cells. [Figure 5A] FIG. 5A-B: 4-1BB H2L2 antibody activates the 4-1BB pathway in vitro and induces and activates IFN-γ secretion in CD8+ and CD4+ T cells. [Figure 5B] Same as above. [Figure 6A] Figures 6A-C: Tumor growth inhibitory activity of 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-C: PR000980 antibody activates the 4-1BB pathway in vitro and induces secretion of IFN-γ, TNF-α and IL-2 in activated T cells. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] Figures 9A-D: BIACORE detection of the affinity of 4-1BB H2L2 antibody with human or monkey 4-1BB protein. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A] Figures 10A-B: Tumor growth inhibitory activity of 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-L: FACS detection of binding of 4-1BB HCAb antibody to CHO-K1 cells overexpressing cynomolgus 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-J: Activation of the 4-1BB signaling pathway by 4-1BB HCAb antibody is detected using HEK293 / 4-1BB / NF-kb 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-K: FACS detection of 4-1BB HCAb antibody blocking binding of 4-1BB ligand to 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-D: 4-1BB HCAb antibody activates the 4-1BB pathway in vitro and induces and activates 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-B: 4-1BB HCAb antibody, with or without CHO-K1 / CD32b cross-linking, activates the 4-1BB pathway in vitro and induces and activates IFN-γ secretion in T cells. [Figure 16B] Same as above. [Figure 17A] Figures 17A-D: 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-C: Structural schematic of the HER2x4-1BB bispecific antibody. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19A] Figures 19A-C: FACS detection of binding of HER2x4-1BB 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 HER2x4-1BB bispecific antibody to CHO-K1 cells overexpressing human 4-1BB. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 20E] Same as above. [Figure 21A] Figures 21A-B: FACS detection of binding of HER2x4-1BB bispecific antibody to CHO-K1 cells overexpressing cynomolgus 4-1BB. [Figure 21B] Same as above. [Figure 22A]Figures 22A-D: HER2x4-1BB bispecific antibody activates the 4-1BB pathway in vitro upon crosslinking of SK-BR-3 cells and induces and activates 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: Structural schematic of PD-L1x4-1BB bispecific antibody. [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-L1x4-1BB 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 x 4-1BB bispecific antibody to CHO-K1 cells overexpressing human 4-1BB. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above. [Figure 26A] Figures 26A-B: FACS detection of binding of PD-L1x4-1BB bispecific antibody to CHO-K1 cells overexpressing cynomolgus 4-1BB. [Figure 26B] Same as above. [Figure 27A]Figure 27A-I: PD-L1 x 4-1BB bispecific antibody activates the 4-1BB pathway in vitro by crosslinking CHO-K1 / hPD-L1 or MDA-MB-231 cells and induces and activates 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. [Fig. 27H] Same as above. [Figure 27I] Same as above. [Figure 28A] Figure 28A-Figure 28K: Mixed lymphocyte reaction (MLR) is used to study the activation effect of PD-L1x4-1BB biantibody molecule on T cells. [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. [Fig. 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 x C57BL / 6J mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Certain technical and scientific terms are specifically defined below: Unless otherwise clearly defined in the present specification, all technical and scientific terms used in the present specification have the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs.

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

[0063] The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence order of the heavy chain constant region of immunoglobulins are different, so their antigenicity is also different. This allows immunoglobulins to be classified into five types or homogenies of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, whose corresponding heavy chains are μ, δ, γ, α and ε chains, respectively. The same type of Ig can be divided into different subtypes according to the difference in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chains are divided into κ or λ chains according to the difference in the constant region. Of the five types of Ig, each type of Ig may have κ or λ chains.

[0064] Antibody Light Chain The antibody can further comprise a light chain constant region, which can comprise a kappa, lambda chain or a variant thereof of human origin.

[0065] Antibody Heavy Chain The antibody can further comprise a heavy chain constant region, which can comprise an IgG1, 2, 3, 4 of human origin or a variant thereof.

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

[0067] In this application, the term "fully human antibody" generally refers to an antibody in which all of the genes encoding a human antibody are transferred to a genetically engineered antibody gene-deficient animal and expressed in the animal. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce immune side reactions in the human body caused by foreign antibodies. Methods for obtaining fully human antibodies in the 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 variable domains of the heavy (VH) and light (VL) chains of an antibody are linked by a connecting peptide, and is the minimum antibody fragment containing an intact antigen-binding site.

[0069] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or non-adjacent amino acids juxtaposed by a triple fold of a protein. Epitopes formed from adjacent amino acids are usually retained after exposure to denaturing solvents, whereas epitopes formed by triple folds are usually lost after treatment with denaturing solvents. Epitopes typically contain at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to a particular antibody are well known in the art and include immunoimprinting and immunoprecipitation detection assays. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as x-ray crystallography and 2D nuclear magnetic resonance.

[0070] The term "nucleic acid" refers to DNA molecules and RNA molecules. It may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operatively linked" when it places it into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively connected to a coding sequence if it affects the transcription of 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 such binding requires a certain degree of complementarity between the antigen-binding region and the epitope. According to this definition, an antibody is said to "specifically bind" to an antigen if it is more likely to bind to an epitope via its antigen-binding region than it would bind to a random, unrelated epitope.

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

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

[0074] Example 1. Obtaining fully human 4-1BB H2L2 antibody Example 1.1. Production of monoclonal antibodies Harbour H2L2 mice (Harbour Antibodies BV) are transgenic mice with a human immunoglobulin immune library, and the antibodies produced by them have a complete human antibody variable domain and a rat constant domain. Harbour H2L2 mice were subjected to multiple rounds of immunization with soluble recombinant human 4-1BB-Fc fusion protein. After detecting that the 4-1BB-specific antibody titer in the mouse serum reached a certain level, the spleen cells of the mice were taken out and fused with a myeloma cell line to obtain hybridoma cells; after multiple rounds of screening and cloning of the hybridoma cells, two hybridoma cell lines expressing anti-4-1BB monoclonal antibody molecules, 65D4G5G11 and 79B10G8D4, were isolated. The nucleotide sequences encoding the antibody molecule variable domains 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 the immunized Harbour H2L2 mice are human-derived antibody sequences. The CDR sequences of antibody variable domains can be analyzed by Kabat, Chothia, or a combined definition rule that combines the Kabat and Chothia definitions. In an embodiment of the present invention, the CDR sequences are classified according to the Chothia definition rule.

[0075] Example 1.2. Production of recombinant fully human antibodies After obtaining light and heavy chain variable domain sequences encoding an antibody molecule, conventional recombinant DNA technology can be used to fuse and express the light and heavy chain variable domain sequences with corresponding human antibody light and heavy chain constant domain sequences to obtain a recombinant antibody molecule.

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

[0077] At the same time, the present application prepares the anti-4-1BB positive control antibody Urelumab and Utomilumab analogues, the antibody number corresponding to Urelumab is PR000628, and the antibody number corresponding to Utomilumab is PR000483. The corresponding amino acid sequences are derived from the IMGT database.

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

[0079] A plasmid encoding an antibody heavy chain and a plasmid encoding an antibody light chain can be co-transfected into a mammalian host cell such as human embryonic kidney cell HEK293, and a purified recombinant antibody in which the light chain and the heavy chain are correctly assembled can be obtained using conventional recombinant protein expression and purification techniques. (商標) The cells were cultured in F17 Expression Medium (Thermo, A1383504). Before the start of instantaneous transfection, the cell concentration was adjusted to 6–8 × 10 5 The cells were adjusted to 1.2 × 10 cells / ml and cultured at 37°C in an 8% CO2 rocker for 24 hours. 6 The concentration is cells / ml. 30 ml of cultured cells are prepared. A total of 30 μg of the plasmids encoding the antibody heavy chain and the plasmids encoding the antibody light chain were mixed in a ratio of 2:3, dissolved in 1.5 ml of Opti-MEM serum reduced medium (Thermo, 31985088), and filtered through a 0.22 μm filter membrane to sterilize. Further, 1.5 ml of Opti-MEM was dissolved in 120 μl of 1 mg / ml PEI (Polysciences Inc, 23966-2) and left to stand for 5 minutes. PEI was slowly added to the plasmid, incubated at room temperature for 10 minutes, and the plasmid-PEI mixed solution was slowly dropped while rocking the culture bottle, and cultured at 37 ° C for 5 days on a rocking bed with 8% CO2. After 5 days, the cell viability was observed. The culture was collected, centrifuged at 3300 G for 10 minutes, and the supernatant was collected. The supernatant was then centrifuged at high speed to remove impurities. MabSelect (商標)A gravity column (Bio-Rad, 7311550) containing 100% glycine (GE Healthcare Life Science, 71-5020-91 AE) was equilibrated and washed with 2-5 column volumes. The supernatant sample was passed through the column. The column was washed with 5-10 column volumes of PBS. The target protein was eluted with 0.1 M glycine at pH 3.5, then neutralized with Tris-HCl at pH 8.0, and finally concentrated and exchanged into PBS buffer in an ultrafiltration tube (Millipore, UFC 901024) to obtain the purified antibody solution. Then, the purified antibody solution was filtered using a NanoDrop (Thermo Scientific) (商標) NanoDrop (商標) Measure the concentration using a 100% ethanol solution, divide it into portions, and store it in 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 gene V, D, and J gene fragments of the heavy chain gene group on a chromosome and high frequency somatic mutation; the light chain variable domain sequence is derived from events such as genetic recombination of germline gene V, J gene fragments of the kappa or lambda light chain gene group and high frequency somatic mutation. Genetic recombination and high frequency somatic mutation 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 there is a high degree of 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 estimate the 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 gene V gene fragments of the heavy chain variable domain (VH) and light chain variable domain (VL) are shown in Table 1.

[0081] When a protein or polypeptide amino acid chain is translated and synthesized in a cell, chemical modifications called post-translational modifications (PTMs) may be introduced. For antibodies, some PTM sites are very conservative. For example, asparagine Asn, a conservative amino acid at position 297 (EU numbering) of the constant domain of human IgG1 antibodies, is usually modified by glycosylation to form a glycan, and this glycan structure is extremely important for antibody structure and associated effector functions. However, when PTMs are present in the variable domain of an antibody, especially in the antigen-binding region such as the CDR, the presence of these PTMs may have a significant effect on antigen binding and may also cause changes in the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, oxidation, etc., can 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 very important for the development of therapeutic antibodies. With the accumulation of experience, it has been discovered that some PTMs are highly associated with the composition of the amino acid sequence, especially the "pattern" of the adjacent amino acid composition, which allows us to predict 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 causing PTM may be derived from the germline gene sequence, for example, the human germline gene fragment IGHV3-33 naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic high frequency mutation. Table 1 shows the predicted PTMs of the variable domains VH and VL of the antibody of Example 1.1. Specifically, NHS may be a glycosylation site, and NG may be a deamidation site.

[0082] [Table 1]

[0083] Amino acid mutations can disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of certain PTMs. There are different mutation design methods depending on the antibody sequence and PTM sequence pattern. One method is to replace the "hot spot" amino acid (e.g., N or S in NS mode) with a physicochemically similar amino acid (such as mutating N to Q). If the PTM sequence pattern is derived from somatic high frequency mutations and does not exist in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence. In practical operations, multiple mutation design methods can be adopted for the same PTM sequence pattern.

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

[0085] It was also found in the present application that this E345R mutation can effectively increase the activation effect of agonistic 4-1BB antibody. In this example, the sequence of the anti-4-1BB hybridoma monoclonal antibody in Table 1 was optimized by IgG subtype conversion, variable region mutation, and Fc mutation to obtain a series of recombinant antibodies or their mutants (see Table 2). Tables 3.1, 3.2, and Table A show the amino acid sequences of the light and heavy chain variable domains, the full-length light chain amino acid sequences, the full-length heavy chain amino acid sequences, and the amino acid sequences of the CDRs 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 Binding of H2L2 Antibody to 4-1BB on the Cell Surface CHO-K1 cells expressing human 4-1BB and CHO-K1 cells expressing cynomolgus monkey 4-1BB were expanded and cultured, washed three times with PBS, and then incubated at 3 × 10 5Add 100μl of the antigen-binding protein or positive control antibody Utomilumab or Urelumab to the wells of a 96-well plate (Corning, #3799) at 1 / well. Add 100μl of the antigen-binding protein or positive control antibody Utomilumab or Urelumab to a maximum concentration of 200nM, dilute 5-fold, and incubate at 4°C for 1 hour. Wash twice with FACS buffer, add AF488-conjugated sheep anti-human IgG antibody (Life Technologies, #A11013) diluted 1:1000, and incubate at 4°C for 1 hour. Wash twice with FACS buffer, resuspend in 100μl of PBS, perform FACS (BD Biosciences, Canto II) test, and read the fluorescent signal. From the measurement results, the half-maximal binding concentration (EC50) was calculated as the basis for evaluating the 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 proteins with human 4-1BB, and Figure 1B shows the binding activity of the antigen-binding proteins with monkey 4-1BB. Both PR000447 and PR000448 can bind human and cynomolgus monkey 4-1BB, and their binding activity is superior to that of the positive control Urelumab. Urelumab cannot cross-link cynomolgus monkey 4-1BB.

[0091] [Table 4]

[0092] Example 1.5. 4-1BB H2L2 antibody blocks binding of 4-1BB ligand to 4-1BB To study the activity of human 4-1BB binding protein in blocking the binding between human 4-1BB and human 4-1BBL in vitro, human 4-1BB / human 4-1BBL binding blocking experiments were performed at the cellular level using a CHO-K1 cell line overexpressing human 4-1BB (CHO-K1 / hu 4-1BB). Briefly, CHO-K1 / hu 4-1BB cells were digested and resuspended in F-12K complete medium at a cell density of 1 × 10 6Adjust to cells / mL. 100μL cells / well were seeded into a 96-well V-plate (Corning, #3894), then 100μL / well of the antigen-binding protein to be measured, diluted in a 3-fold concentration gradient of 2x the final concentration, was added and mixed uniformly, of which the highest final concentration of the antigen-binding protein was 100nM, there were a total of 8 concentrations, and hIgG1 was used as a control. The cells were placed at 4°C and incubated for 1 hour in the dark. Then, the cells were centrifuged at 4°C for 5 minutes, the supernatant was discarded, and then 50μL / well of biotin-labeled human 4-1BBL protein (Acro, #41L-H82F9) at a concentration of 1μg / mL was added and incubated at 4°C for 30 minutes in the dark. 100μL / well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500g, 4°C for 5 minutes, and the supernatant was discarded. Fluorescent secondary antibody (PE Streptavidin, BD, #554061, 1:200) was added at 100 μL / well and incubated for 30 min at 4 °C in the dark. The cells were washed twice with 200 μL / well pre-chilled PBS, centrifuged at 500g, 4 °C for 5 min, and the supernatant was discarded. Finally, the cells were resuspended with 200 μL / well pre-chilled PBS, and the fluorescence signal value was read using a BD FACS CANTOII to calculate the IC50, where % inhibition = 1-MFI(4-1BB Ab) / MFI(iso).

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

[0094] Example 1.6. Detection of stimulatory effects on the 4-1BB signal pathway using a reporter gene cell line CD32b-expressing CHO-K1 cells, CHO-K1 / CD32b (Genscript, #M00587) or CHO-K1 (ATCC, #CCL-61), were plated in a 96-well plate (Perkin Elmer, #6005225) at a cell volume of 1.5 × 10 4 / well, 100μL / well. Incubate at 37°C overnight in 5% CO2. Remove the supernatant and add 40μL / well of 2x dilution of antigen-binding protein to be measured, with an initial concentration of 200nM and 5x dilution. hlgG1 was the control. 4.5×10 4 Add 40μL / well of HEK293 reporter cells (HEK293 / 4-1BB / NF-kb reporter cells, BPS Biosciences, #79289) carrying a luciferase reporter gene capable of continuously expressing 4-1BB and NF-kb response elements. Incubate at 37°C in a 5% CO2 environment for 6 hours. TM Luciferase reagent (Promega, #E6110) was added, incubated at room temperature for 5 minutes, and luminescence was measured using a microplate reader.

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

[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 (CHO-K1 cells overexpressing human CD32b) cells were treated with 10 μg / ml mitomycin (Ruitaibio, 10107409001) and left at 37 °C for 30 min. Then, they were washed four times with F-12K medium containing 10% FBS. The treated cells were placed in a 96-well plate, and 1.5 × 10 cells were placed in each well. 4 The cells were cultured overnight in a 37°C incubator. The next day, human CD3-positive T cells were isolated from human PBMCs using a MACS kit (Miltenyi Biotec, #130-096-535). First, the cell number was confirmed, and then MACS buffer and Pan-T cell biotin antibody were added according to the cell number, mixed, and left at 4°C for 5 minutes. Then, a corresponding amount of microbeads was added and left at 4°C for 10 minutes. CD3-positive T cells were passed through the LS column. The culture medium of the 96-well plate from the previous day was washed, and purified T cells were added, at 1 × 10 per well. 5 Then, the corresponding concentration of 4-1BB antigen binding protein or control antibody Utomilumab, PR000196 was added, and OKT3 (eBiosciences, #16-0037-85) was added to a final concentration of 0.3 μg / ml. The plates were incubated for 72 hours in a 37°C incubator. After 72 hours, the supernatant was collected and the content of IFN-γ was detected using an ELISA kit (Invitrogen, #88-7316-88). Coating antibody was added to a 96-bottom 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 for 2 hours at room temperature. The plate was washed twice, 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. TMB substrate was then added, and later ELISA stop solution (BBI, #E661006-0200) was added. The absorbance values ​​at 450 nm and 570 nm were read using a microplate reader (Perkin Elemer Enspire), and the IFN-γ concentration was calculated using OD450-OD570.

[0098] As a result, as shown in Figure 4, PR000197, PR000447 and PR000448 antibodies can all activate the 4-1BB pathway, activate T cells and induce IFN-γ secretion, and their activation effects are stronger than those of Utomilumab and PR000196.

[0099] Example 1.7.2.4-1BB H2L2 Antibody Induces CD4+ and CD8+ T Cell Activation According to the method of Example 1.7.1, CHO-K1 or CHO-K1 / CD32b cells were treated, plated, and cultured overnight in a 37°C incubator. The next day, selection reagents (Miltenyi Biotec, #130-096-495, #130-096-533) were used to select CD8+ and CD4+ T cells. Then, the corresponding concentrations of 4-1BB antibody or control antibody were added, and OKT3 (eBiosciences, #16-0037-85) was added to a final concentration of 0.3 μg / ml. The cells were cultured for 72 hours in a 37°C incubator. 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 FIG. 5A, and CD4+ T cell induction is shown in FIG. 5B.

[0101] The results showed that PR000447 and PR000448 could significantly activate IFN-γ secretion in CD8+ T cells, but had no significant activation function on CD4+ T cells, which may be due to the higher expression of 4-1BB in CD8+ T cells than in CD4+ T cells.

[0102] Example 1.8.4-1BB H2L2 Antibody Inhibits Tumor Growth in Vivo A B6-h4-1BB transgenic mouse (Biocytogen Pharmaceuticals (Beijing) Co., Ltd, #11004) MC38 subcutaneous colon cancer model was established. 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. MC38 cells in logarithmic growth phase (record the passage number) were collected, the culture medium was removed, and the cells were washed twice with PBS before inoculation (pre-tumor cell viability: 98.9%, post-tumor cell viability: 92%). Inoculation amount: 5 × 10 5 / 100μl / mouse (without matrix adhesive), inoculation location: right subcutaneous. According to the grouping criteria, the mice were divided into 5 groups, each group had 6 mice, and the average value of each group was close, with the average value ranging from 80 to 120mm. 3 The inclusion criteria for each group were: the volume of the single mouse tumor in each group should be 150 mm 3 The day of grouping was defined as day 0, and administration was started on day 0, the day of grouping.

[0103] The samples to be measured were prepared in PBS, and the administration method was intraperitoneal injection, with a dosage volume of 10 μl / g, a dose of 5 mg / kg, twice a week, and a period of 3 weeks, for a total of 6 administrations. On the day of administration, the body weight and tumor volume of the mice were measured, and the average tumor volume was 700-800 mm. 3 The tumors were measured twice weekly until they reached a mean volume of 700–800 mm 3 When the tumor volume exceeded 100 mm , the tumor volume was measured three times a week. The tumor volume was calculated as follows: tumor volume (mm ); 3 ) = 0.5 × tumor long axis × tumor short axis 2 .

[0104] Mouse weight monitoring is shown in FIG. 6A, tumor volume monitoring is shown in FIG. 6B, after 27 days the mice were euthanized, the tumors were removed and photographed, and the tumor size is shown in FIG. 6C.

[0105] The results showed that PR000448 had obvious tumor-inhibiting effect, and the body weight of each group increased during the experiment, indicating that the animals had good tolerance to the test product, and there was no obvious toxic effect on the animals, with relatively good safety.

[0106] Example 1.9. Pharmacokinetic Study The pharmacokinetics of PR000448 was evaluated in female C57BL / 6J mice. PR000448 and Utomilumab were administered intravenously at a single dose of 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 15,000 rpm for 15 minutes at 4°C to obtain plasma, which was then stored in a refrigerator at -20°C or lower. The concentration of the antibody in the plasma was measured using the ELISA method. The plasma concentration changes of PR000448 and Utomilumab are shown in Figure 7. The plasma concentration change data obtained were analyzed using the pharmacokinetic analysis software WinNolin, and the drug half-life (t 1 / 2 ) was calculated. l / 2 was calculated from the final three points or the terminal plasma concentration automatically set by the software. 1 / 2 This is shown in Figure 7 and Table 6 below.

[0107] The results showed that PR000448 has a longer blood half-life than Utomilumab.

[0108] [Table 6]

[0109] Example 1.10. In vitro activation function of Fc point mutant 4-1BB H2L2 antibody To evaluate the activation effect of Fc mutant antibody PR000980 on the 4-1BB pathway, the contents of IFN-γ, TNF-α and IL-2 were 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] The results, as shown in Figures 8A, 8B and 8C, show that all of the antigen-binding proteins tested can stimulate cytokine secretion from activated T cells, more potently than Utomilumab, and PR000980 after Fc mutation has the ability to stimulate T cell cytokine (IFN-γ, TNF-α, IL-2) secretion as the parent antibody PR000448.

[0111] Example 1.11. Epitope Identification of Antigen-Binding Proteins Using the ForteBioOctet platform, the epitopes of the 4-1BB H2L2 antibody (PR000448 and PR000980) obtained in Example 1, Utomilumab, and Urelumab were identified. Here, Urelumab is Bristol-MyersSquibb's anti-4-1BB antibody BMS-663513 (CAS: 934823-49-1), which was obtained by expression and purification according to the method of Example 1.2. In the first step, the histidine-tagged 4-1BB antigen was captured by the sensor, and then the sensor was immersed in the antibody to obtain 100% signal of the antibody. In the second step, the first antibody was loaded onto multiple AHC tips, and then a 60-second baseline was obtained in the measurement of a buffer solution pH 7.5. The tip was then exposed to histidine-tagged 4-1BB for 180 seconds to allow antigen binding. The tip was transferred to a well containing a second antibody in assay buffer for 90 seconds, and the final signal was recorded as the signal of the second antibody. The inhibition rate was calculated by the following formula: inhibition rate (%) = (AB) / A*100, where A is the 100% signal of a certain antibody (obtained from the first step), and B is the signal of this antibody as the second antibody (obtained from the second step).

[0112] If the second antibody shows significant binding (i.e., inhibition rate is less than 40%), it is considered a non-competitor (i.e., in a different epitope interval than the first antibody). If the second antibody shows no significant binding (i.e., inhibition rate is 40% or more), it is considered a competitor (i.e., in the same epitope interval as the first antibody). Binding measurements were performed by comparing the binding of the second antibody to 4-1BB in the presence of the first antibody with the blocking of the first antibody itself.

[0113] As a result, as shown in Table 7 below, PR000448 and PR000980 have high overlap with Utomilumab at the 4-1BB binding site, but low overlap with Urelumab's binding site.

[0114] [Table 7]

[0115] Example 1.12. Affinity Testing of 4-1BB H2L2 Antibody with Human or Monkey 4-1BB Protein The 4-1BB H2L2 antibody (PR000448 and PR000980) obtained in Example 1, Utomilumab, and Urelumab were identified by affinity using the Biacore platform. For the test, HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P20, pH 7.4, GE Healthcare, BR-1006-69) was used as the running buffer, 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 to the four channels of CM5 by the following procedure: 1) Set the injection time to 800 s, and freshly mix 50 mM NHS and 200 mM EDC in a 1:1 volume ratio, then inject into the four channels; 2) Dilute Protein A to 20 μg / ml in sodium acetate pH 4.5 (GE Healthcare, BR-1003-50) and inject into each channel for 800 s; 3) Inject 1 M pH 8.5 ethanolamine for 800 s to seal the remaining active carboxyl groups on the chip surface. After sealing, the device was equilibrated with 1×HBS-EP+ buffer for 2 h, and the final conjugation amount of Protein A was about 2000 RU. In addition, a multi-cycle kinetic mode was set to perform affinity measurements of the antibody and protein, with each cycle including antibody capture, analyte binding, and chip regeneration. All antibodies were diluted to 1μg / ml and injected into channels 2, 3, and 4 for 30s at a flow rate of 10μl / min, and each antibody was captured by pre-conjugated Protein A, with a capture capacity of approximately 120RU. Human 4-1BB or cynomolgus 4-1BB was injected into the four channels with a concentration gradient of 0nM, 0.391nM, 0.781nM, 1.5625nM, 3.125nM, 6.25nM, 12.5nM, and 25nM, respectively, at a flow rate of 30μl / min, with dissociation times of 600s for PR000448 and PR000980, and 80s for Uremulab and Utomilumab, and injection times of 120s for both.Finally, to remove the test antibody from the surface, 10 mM glycine-HCl pH 1.5 (GE Life Sciences, BR-1003-54) was injected at the same flow rate for 30 s to regenerate the chip. The experimental results were analyzed using Biacore T200 analysis software 2.0, one channel was subtracted as the reference channel, and the analysis model was selected as the 1:1 kinetic fitting model.

[0116] The results are shown in Table 8 and Figures 9A-9D, which show the affinity of the 4-1BB H2L2 antibody with histidine-tagged human 4-1BB protein and histidine-tagged monkey 4-1BB protein. The results showed that, regardless of the binding affinity to human or monkey 4-1BB protein, the binding affinity of the antibodies PR000448 and PR000980 was higher than that of Uremulab and Utomilumab, and Urelumab did not bind to monkey 4-1BB protein.

[0117] [Table 8]

[0118] Example 1.13. In vivo tumor growth suppression activity of Fc point mutant antibodies According to the method of Example 5, a B6-h4-1BB transgenic mouse MC38 subcutaneous colon cancer model was established. The mean tumor volume was 99.14 mm 3 When tumor volume reached 100 mm, the mice were randomly grouped based on tumor volume, with 6 mice in each group. The day of grouping was defined as day 0, and administration began on day 0, the day of grouping. Samples to be measured were prepared in PBS, and the administration method was intraperitoneal injection, with a dose of 2 mg / kg. After administration began, the mice were weighed twice a week. Tumor volumes were measured twice a week, and tumor volumes (mm 3 ) = 0.5 × tumor long axis × tumor short axis 2 .

[0119] As shown in Figures 10A and 10B, the changes in mouse body weight and tumor volume were measured 29 days after administration, and it was found that the Fc variant PR000980 of PR000448 had stronger tumor growth inhibitory activity.

[0120] summary The present application provides antigen-binding proteins having one or more of the following properties: 1) capable of binding 4-1BB proteins derived from humans and monkeys; 2) capable of stimulating the secretion of IFN-γ, IL2 and / or TNFα from immune cells; 3) capable of suppressing tumor growth and / or proliferation of tumor cells; 4) capable of activating the 4-1BB signal pathway. The present application also provides uses of said antigen-binding proteins in the prevention and treatment of tumors.

[0121] Example 2. Obtaining fully human 4-1BB HCAB antibody Example 2.1. Immunization of all human-derived HCAb mice and acquisition of 4-1BB antibodies The Harbour HCAb mouse (HarbourAntibodiesBV, WO2002 / 085945A3) is a transgenic mouse with a human immunoglobulin immune bank, which is only half the size of a conventional IgG antibody and can produce an antibody consisting of a completely new "heavy chain". The produced antibody has only a human antibody "heavy chain" variable domain and a mouse Fc constant domain. Due to the characteristic of not containing a light chain, this antibody almost completely solves the problems of light chain mismatch and heterodimerization, and this technology platform can develop products that are difficult to realize with conventional antibody platforms.

[0122] Example 2.1.1. Immunized HCAb Mice Harbour human-derived antibody transgenic mice aged 6 to 8 weeks were subjected to multiple rounds of immunization with Harbour HCAb mice using two groups of immunization schemes. Immunization scheme 1 is immunization with recombinant human 4-1BB-ECD-Fc (ChemPartner, Shanghai) antigen protein. The total injection volume each mouse receives is 100 microliters by subcutaneous inguinal or intraperitoneal injection for each immunization. In the first immunization, each mouse was immunized with an immunogen reagent made by mixing 50 micrograms of antigen protein with complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of boosting, each mouse was immunized with an immunogen reagent made by mixing 25 micrograms of antigen protein with Ribi adjuvant (Sigma Adjuvant System, Sigma, #S6322). Immunization scheme 2 uses NIH3T3-h4-1BB (ChemPartner, Shanghai) overexpressing human 4-1BB to stabilize the cell line and immunize each mouse with 2 × 10 6 The cell suspension was injected intraperitoneally. The interval between boosting rounds was at least 2 weeks, and usually did not exceed 5 rounds. The immunization times were 0, 14, 28, 42, 56, and 70 days, and the mouse serum antibody titers were measured on days 49 and 77. Five days before performing HCAb mouse splenic B cell isolation, each mouse was given a final boost with a dose of 25 micrograms of antigen protein.

[0123] Example 2.1.2. Obtaining anti-4-1BB HCAb antibody sequence The blood of the mice was collected, and six concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:100000, 1:1000000) were collected by 10-fold dilution, and ELISA detection was performed on ELISA plates coated with human 4-1BB-ECD-Fc (Chempartner, Shanghai) to determine the antibody titer of anti-human 4-1BB in the mouse blood, and 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 mother cells was measured by flow cytometry. The blank control group (PB) was the serum of pre-immune mice. After detecting that the 4-1BB-specific antibody titer in mouse serum reached a certain level, mouse spleen cells were removed to separate B cells, and CD138-positive plasma cells and human 4-1BB antigen-positive B cell populations were sorted using BD FACS AriaIII CellSorter. B cell RNA was extracted, and cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, 18091200) was reverse transcribed, and then human VH genes were amplified using specific primer PCR. PCR primers 5'-GGTCCCAGTGTSAGAGAGTGTG-3', 5'-AATCCCTGGCACTGAGACTGACC-3'. The amplified VH gene fragment was constructed into a mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc domain sequence.

[0124] The constructed plasmid was transfected into mammalian host cells (e.g. human embryonic kidney cells HEK293) for expression, and the antibody of HCAb was obtained. The binding of the supernatant expressing HCAb with the stable cell line CHO-K1 / hu 4-1BB (ChemPartner, Shanghai) overexpressing human 4-1BB was detected, and at the same time, Acumen screening was performed using CHO-K1 cells as a negative control. The obtained positive monoclonal antibodies were further detected for their binding and cross-linking activity with the stable cell line CHO-K1 / cyno4-1BB (ChemPartner, Shanghai) overexpressing cynomolgus monkey 4-1BB. The expression supernatants of 683 monoclonals binding CHO-K1 / hu 4-1BB and CHO-K1 / cyno4-1BB were obtained and subjected to NF-kb function test, and at the same time, the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence were obtained by conventional sequence confirmation means. After removing the repetitive sequences, we obtained 323 functional, unique sequence-specific, all-human 4-1BB monoclonal antibodies that simultaneously bound CHO-K1 / hu 4-1BB and CHO-K1 / cyno4-1BB. Based on the results of human monkey cell binding ability and NF-Kb function test, the overall top 38 antibodies were selected for recombinant expression.

[0125] [Table 9]

[0126] For one antibody with a potential PTM site from Example 2.1.2, new antibody molecules (called PTM mutants) were obtained by amino acid mutations.

[0127] [Table 10]

[0128] [Table 11]

[0129] Example 2.1.3. Preparation of anti-4-1BB fully human recombinant antibody The plasmid encoding the HCAb antibody can be transfected into mammalian host cells (e.g., human embryonic kidney cells HEK293) and purified anti-4-1BB recombinant heavy chain antibody can be obtained using conventional recombinant protein expression and purification techniques. (商標) The cells were cultured in F17 Expression Medium (Thermo, A1383504). The cell concentration was 6 × 10 5 The cells were adjusted to 1.2 × 10 cells / ml and cultured at 37°C in an 8% CO2 rocker for 24 hours. 6 The concentration was 1000 cells / ml. 30 ml of cultured cells were prepared, and 30 μg of the plasmid encoding the HCAb heavy chain was dissolved in 1.5 ml Opti-MEM serum reduced medium (Thermo, #31985088), and 1.5 ml Opti-MEM was further dissolved in 120 μl of 1 mg / ml PEI (Polysciences, Inc, #23966-2) and left to stand for 5 minutes. PEI was slowly added to the plasmid, incubated at room temperature for 10 minutes, and the plasmid-PEI mixed solution was slowly dropped while rocking the culture bottle, and cultured for 5 days at 37 ° C and 8% CO2 rocking bed. After 5 days, the cell viability was observed. The culture was collected and centrifuged at a rotation speed of 3300 G for 10 minutes, and the supernatant was collected. The supernatant was then centrifuged at high speed to remove impurities. MabSelect (商標)A gravity column (Bio-Rad, #7311550) containing 100% glycerol (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 with an ultrafiltration tube (Millipore, UFC901024) to obtain the purified anti-4-1BB heavy chain antibody solution. The antibody concentration was obtained by measuring the 280 nm absorbance with NanoDrop, and the purity of the antibody was obtained by measuring with SEC-HPLC and SDS-PAGE.

[0130] Example 2.1.4. Analysis of Protein Purity and Multimerization Using HPLC-SEC Analytical size exclusion chromatography (SEC) was used to analyze the purity and multimeric form 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) (model number: Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg, sample concentration adjusted to 1 mg / ml) was filtered through a 0.22 μm filtration membrane and then injected into the system, and the HPLC program was set: run the sample through the column with PBS (pH 7.4) buffer at a flow rate of 1.0 ml / min, the maximum time was 20 min; the detection wavelength was 280 nm. After collection, the chromatogram was integrated using ChemStation software, the relevant data was calculated, and an analytical report was generated to report the retention times of different molecular size components in the sample.

[0131] Example 2.1.5. Analysis of Protein Purity and Hydrophobicity Using HPLC-HIC Analytical hydrophobic interaction chromatography (HIC) was used to analyze the purity and hydrophobicity of protein samples. An analytical column, TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6 mm × 3.5 cm), was connected to a high pressure liquid chromatography (HPLC) (model number: Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 h. The setup method was 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 min, the flow rate was set to 0.7 ml / min, the protein sample concentration was 1 mg / ml, the injection volume was 20 μl, and the measurement wavelength was 280 nm. After collection, ChemStation software was used to integrate the chromatograms, calculate the relevant data, generate analytical reports, and report the retention times of different molecular size components within the sample.

[0132] Example 2.1.6. Measurement of Thermal Stability of Antibody Molecules Using DSF Differential Scanning Fluorimetry (DSF) is a common high-flux method for measuring the thermal stability of proteins. Using a real-time fluorescent quantitative PCR device, the change in the fluorescence intensity of the dye bound to the unfolded protein molecule is monitored to reflect the process of protein denaturation and reflect the thermal stability of the protein molecule. This example uses the DSF method to measure the thermal denaturation temperature (Tm) of the protein molecule. 10 μg of protein was added to a 96-well PCR plate (Thermo, AB-0700 / W), then 2 μl 100X diluted dye SYPROTM (Invitrogen, 2008138) was added, and then buffer was added to make the final volume 40 μl / well. The PCR plate was sealed and placed in a real-time fluorescent quantitative PCR instrument (Bio-Rad CFX96 PCR System) and incubated at 25°C for 5 min, followed by a gradual increase in temperature from 25°C to 95°C at a gradient of 0.2°C / 0.2 min, at which point the temperature was reduced to 25°C. Data analysis was performed using Bio-Rad CFX Maestro software using the FRET scan mode to calculate the Tm of the samples.

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

[0134] Example 2.2.4-1BB Binding of HCAb Antibody to Cell Surface 4-1BB This example is to study the in vitro binding activity of anti-human 4-1BB HCAb monoclonal antibody to human and cynomolgus 4-1BB. Antibody binding experiments at the cellular level were performed using a CHO-K1 cell line overexpressing human 4-1BB (CHO-K1 / hu 4-1BB, Genescript) and a CHO-K1 cell line overexpressing cynomolgus 4-1BB (CHO-K1 / cyno4-1BB, Genescript). Briefly, CHO-K1 / hu 4-1BB and CHO-K1 / cyno4-1BB cells were digested and resuspended in F12K complete medium to a cell density of 1 × 10 6 The solution was adjusted to cells / ml. 100 μL cells / well were seeded into a 96-well V-plate (Corning, #3894), followed by the addition of 100 μl / well of the antibody to be measured, diluted in a 3-fold gradient at 2x the final concentration. The cells were placed at 4°C and incubated for 1 h in the dark. The cells were then rinsed twice with 100 μl / well of pre-chilled PBS, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. 100 μl / well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-06, diluted 1:500) was added and incubated at 4°C for 30 min in the dark. The cells were washed twice with 100 μl / well pre-chilled PBS, centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. Finally, the cells were resuspended with 200 μl / well pre-chilled PBS and the fluorescence signal was read using a BD FACS CANTOII.

[0135] As shown in Figure 11A to Figure 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.Compared to the reference antibodies (Urelumab and Utomilumab), these antibodies can bind human 4-1BB more avidly at lower concentrations, where PR001758-PR001760, PR001764, PR001830, PR001831, PR001833, PR001836, and PR001838 are the best, with EC50s all below 0.3nM, which are equivalent to the EC50s of the reference antibodies Utomilumab and Urelumab. The fluorescence maxima of binding of PR007287, PR007292, PR007293, PR007294 and PR007298 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, and the detected antibody binding ability increases in a positive correlation with the antibody concentration. These antibodies can bind to monkey 4-1BB at lower concentrations compared to the reference antibody Tab (Utomilumab), and the EC50 is equivalent to or superior to that of the reference antibody Utomilumab. The fluorescence maxima of PR007287, PR007292, PR007293, PR007294, and PR007298 binding to CHO-K1 / hu 4-1BB cells are higher than those of the reference antibodies Utomilumab and Urelumab. Meanwhile, the reference antibody Urelumab has no activity of cross-binding to monkey 4-1BB.

[0137] Example 2.3. Detection of stimulatory effects on the 4-1BB signal pathway using a reporter gene cell line CHO-K1 cells expressing CD32b (CHO-K1 / CD32b) were plated in a 96-well plate (Perkin Elmer, #6005225) at a cell volume of 1.5 × 10 4 / well, 100μL / well. Incubate overnight at 37°C in 5% CO2. Remove the supernatant and add 40μL / well of 2x dilution of antigen-binding protein to the plate, with hlgG1 as the control, at 200nM initial concentration, 3x dilution or 30nM initial concentration, 5x dilution. 4 HEK293 reporter cells (HEK293 / 4-1BB / NF-kb reporter cells, BPS Biosciences, #79289) with a luciferase reporter gene capable of continuously expressing 4-1BB and NF-kb response elements were added at 40 μL / well. The cells were cultured at 37°C in a 5% CO2 environment for 6 hours. TM Luciferase reagent (Promega, #E6110) was added, incubated at room temperature for 5 minutes, and luminescence was measured using a microplate reader.

[0138] The results, as shown in Figures 13A to 13I, show that under CHO-K1 / CD32b cross-linking, the promoting effect of most of the 4-1BB antigen-binding proteins of the present application on the 4-1BB-mediated NF-Kb signal pathway increases in a positive correlation with their concentrations. Compared with the reference antibody Tab (Utomilumab), the EC50 is equivalent to or lower than that of the reference antibody, and the maximum fluorescence value is also equivalent to or higher than that of the reference antibody, indicating that these antibodies can promote the activation of NF-Kb at lower concentrations, where PR00758, PR001759 and PR001760, PR007286, PR007291, PR007295, PR007296, PR007297, PR007299, PR007300 are the best, and their EC50s are all less than 0.8 nM, which is equivalent to the EC50 of the reference antibody.

[0139] The results, as shown in Figure 13J, show that without CHO-K1 / CD32b cross-linking, all 4-1BB antigen-binding proteins described in this application had no activation of the 4-1BB-mediated NF-KB signaling pathway, while the reference antibody Tab (Urelumab) still had a strong activation effect.

[0140] Example 2.4. Antigen-binding proteins block the binding of 4-1BB ligand to 4-1BB To study the activity of human 4-1BB binding protein in blocking the binding between human 4-1BB and human 4-1BBL in vitro, human 4-1BB / human 4-1BBL binding blocking experiments were performed at the cellular level using a CHO-K1 cell line overexpressing human 4-1BB (CHO-K1 / hu 4-1BB). Briefly, CHO-K1 / hu 4-1BB cells were digested and resuspended in F-12K complete medium at a cell density of 1 × 10 6 The cells were adjusted to 100 μL / mL. 100 μL cells / well were seeded into a 96-well V-plate (Corning, #3894), followed by adding 100 μL / well of the antigen-binding protein diluted in a 3-fold gradient of 2x the final concentration, and mixing evenly, of which the highest final concentration of the antigen-binding protein was 100 nM, there were eight concentrations, and hIgG1 was used as a control. The cells were placed at 4 °C and incubated for 1 h in the dark. Then, the cells were centrifuged at 4 °C for 5 min, the supernatant was 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, and the cells were incubated at 4 °C for 30 min in the dark. 100 μL / well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g, 4 °C for 5 min, and the supernatant was discarded. Fluorescent secondary antibody (PEStreptavidin, BD, #554061, 1:200) was added at 100 μL / well and incubated at 4 °C in the dark for 30 min. Cells were washed twice with pre-chilled PBS at 200 μL / well, centrifuged at 500 g, 4 °C for 5 min, and the supernatant was discarded. Finally, cells were resuspended with pre-chilled PBS at 200 μL / well, and the fluorescence emission signal value was read using BD FACS CANTOII, and the IC50 was calculated as inhibition %=1-MFI(4-1BBAb) / MFI(iso).

[0141] The results are shown in Figures 14A to 14K. The 4-1BB antigen-binding proteins of the present application are classified into two types. Class 1 was able to block the binding of human 4-1BBL to human 4-1BB on the cell surface, and showed blocking effects equivalent to those of 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 class is similar to the reference antibody (Urelumab) and provides weak or no blocking effect (PR001763, PR001767, PR001768, PR001771, PR001774, PR001780, PR001781, PR001840, PR001842, PR007286, PR007297).

[0142] Example 2.5. Antigen binding proteins 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 Co., Ltd., #10107409001) for 30 min at 37 °C. Then, they were washed four times with F-12K medium containing 10% FBS. The treated cells were placed in a 96-well plate, with 1.5 × 10 cells per well. 4The cells were cultured overnight in a 37°C incubator. The next day, human CD3-positive T cells were isolated from human PBMCs using a MACS kit (Miltenyi Biotec, #130-096-535). First, the cell number was confirmed, and then MACS buffer and Pan-T cell biotin antibody were added according to the cell number, mixed, and left at 4°C for 5 minutes. Then, a corresponding amount of microbeads was added and left at 4°C for 10 minutes. CD3-positive T cells were passed through the LS column. The culture medium of the 96-well plate from the previous day was washed, and purified T cells were added at 1 × 10 per well. 5 Then, the corresponding concentration of 4-1BB antibody or control antibody was added, and OKT3 (eBiosciences, #16-0037-85) was added to a final concentration of 0.3 μg / ml. The plates were incubated for 72 hours in a 37 °C incubator. After 72 hours, the supernatant was collected and the content of IFN-γ was detected using an ELISA kit (Invitrogen, #88-7316-88). Coating antibody was added to a 96-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 for 2 hours at room temperature. The plate was washed twice, 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. TMB substrate was then added, and later ELISA stop solution (BBI, E661006-0200) was added. The optical density at 450 nm (OD450) was read using a microplate reader (Perkin Elemer Enspire) and the IFN-γ concentration was calculated.

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

[0144] As shown in Figures 16A to 16B, the results show that PR0001758, PR001759 and PR001760 antibodies all activate the 4-1BB pathway dependent on CD32b crosslinking and induce the function of activated T cells. In CHO-K1 / CD32b cell crosslinking, their activation effects are all stronger than Utomilumab and slightly weaker than Urelumab. In the absence of CHO-K1 / CD32b cell crosslinking, the HCAb antibody of this example cannot activate the function of T cells, while Urelumab can activate T cells, which is also considered to be the cause of Urelumab toxicity. The activation effect increases in a positive correlation with the antibody concentration, and the EC50 is all smaller than that of the reference antibody Utomilumab, indicating that the 4-1BB pathway can be activated at a low concentration, and the maximum value of cytokine interferon gamma release is 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 operation and method provided by the manufacturer. Specifically, human 4-1BB protein, His tag (Acrobiosystem, #41B-H5227) or cynomolgus monkey 4-1BB protein, His tag (Acrobiosystem, #41B-C52H4) were diluted to 400 nM using 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 bearing human 4-1BB protein or monkey 4-1BB protein at 30 °C for 3 min. The reaction mixture was incubated at 30°C for 5 min in PBS buffer (pH 7.4) containing 0.1% (v / w) BSA and 0.02% (v / v) TWEEN 20. OctetRed96 records the binding and dissociation signals of 4-1BB antibody and 4-1BB protein in real time. The affinity, correlation and dissociation constants were confirmed by Octet software, and the results are shown in Table 13 and Table 14.

[0146] As a result, the KD values ​​of the PR001836 and PR001838 antibodies were somewhat lower than those of the antibodies detected, regardless of whether they were binding to human 4-1BB or cynomolgus monkey 4-1BB, and they showed stronger 4-1BB binding affinity.

[0147] [Table 13]

[0148] [Table 14]

[0149] Example 2.7. Epitope binning of antigen-binding proteins The ForteBio Octet platform was used to identify the epitopes of the antigen-binding proteins obtained in Example 1, utomilab, and urelumab. Briefly, the first antibody was loaded onto multiple AHC tips, and then a 60-second baseline was obtained in a measurement of pH 7.5 buffer. The tips were then exposed to histidine-labeled 4-1BB for 180 seconds to allow antigen binding. The tips were then transferred to wells containing the second antibody in measurement buffer for 90 seconds. If the second antibody shows clear binding, it is considered a non-competitor (i.e., a different epitope section from the first antibody). If the second antibody does not show clear binding, it is considered a competitor (i.e., the same epitope section 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 first antibody blockade itself. The inhibition rate was calculated by the formula: inhibition rate (%) = (AB) / A * 100 (Note: A: 100% signal of a certain antibody, B: this antibody is the signal of the second antibody).

[0150] An inhibition rate of over 80% means that the two antibodies have very close epitopes, an inhibition rate between 40% and 80% means that the two antibodies have relatively close but not completely overlapping epitopes, and an inhibition rate of less than 40% means that the two antibodies have non-overlapping epitopes.

[0151] The results, as shown in Table 15 below, show that PR001760, PR001779, PR001838 and PR001840 have high overlap with Utomiluab but low overlap with Urelumab at the binding site for 4-1BB, while the binding site of PR001767 has different specificity from Utomiluab and Urelumab.

[0152] [Table 15]

[0153] Example 2.8. Specific binding of antigen-binding proteins 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 have been identified as immune common stimulators that play important roles, including CD40, OX40, 41BB, CD27, GITR, and CD30. Similarly, inducible T cell costimulator (ICOS) is another receptor that plays a key role in the function and survival of activated or memory T cells.

[0154] This example investigates the in vitro binding specificity of anti-human 4-1BB with HCAb monoclonal antibody against three receptors of the TNF tumor necrosis factor receptor superfamily and ICOS by flow cytometry detection. Antibody binding experiments at the cellular level were performed using CHO-K1 cell line overexpressing human 4-1BB (CHO-K1 / hu 4-1BB, Genescript), CHO-K1 cell line overexpressing human CD40 (CHO-K1 / hu CD40, Beijing Kongyuan Boxuang, #KC-1286), CHO-K1 cell line overexpressing human OX40 (CHO-K1 / hu OX40, Genescript, #M00561), and HEK293 cell line overexpressing human ICOS (HEK293T / ICOS, Genescript, #KC-0210). Briefly, these cells were digested and resuspended in F12K or DMEM complete medium at a cell density of 1 × 10 6 The cells were adjusted to cells / ml. 100 μL cells / well were seeded into a 96-well V-plate (Corning, #3894), followed by the addition of 100 μl / well of the antibody to be measured, diluted in a 3-fold gradient at 2x the final concentration. The cells were placed at 4°C and incubated for 1 h in the dark. The cells were then rinsed twice with 100 μl / well of pre-chilled PBS, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. 100 μl / well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific, Jackson, #109-545-06, diluted 1:1000) was added and incubated for 30 min at 4°C in the dark. The cells were washed twice with 100 μl / well pre-chilled PBS, centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. Finally, the cells were resuspended with 200 μl / well pre-chilled PBS and the fluorescence signal was read using a BD FACS CANTOII.

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

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

[0157] Example 3. HER2x4-1BB 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 ErbB2 or neu in rats, is a member of the ErbB protein family, usually 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 involved in signaling pathways that normally 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, communication of neuromuscular junctions, and regulation of cell growth and differentiation.

[0158] The HER2×4-1BB dual antibody aims to promote 4-1BB aggregation by conjugating T cells with HER2-positive tumor cells, provide effective costimulatory signals to tumor antigen-specific T cells, further enhance T cell receptor (TCR)-mediated activity, and induce tumor dissection. Therefore, HER2×4-1BB-mediated 4-1BB activation is biased toward colocalization of T cells and tumor cells in vivo, for example, between primary tumors and tumor-infiltrating lymphocytes (TILs) or lymph nodes containing tumor metastases.

[0159] In this example, a bispecific antibody that simultaneously targets HER2 and 4-1BB is constructed, aiming to enhance antitumor efficacy and safety through one or more mechanisms of action. First, HER2×4-1BB bispecific antibody is enriched in tumor tissues with high HER2 expression, and T cells complex with HER2-positive tumor cells to promote 4-1BB aggregation, provide effective costimulatory signals to tumor antigen-specific T cells, and further enhance T cell receptor (TCR)-mediated T cell activity and enhance antitumor activity. Therefore, HER2×4-1BB-mediated 4-1BB activation is biased toward colocalization of T cells and tumor cells in vivo, such as primary tumors and lymph nodes containing tumor-infiltrating lymphocytes (TILs) or tumor metastases. Second, the function of the anti-4-1BB agonist antibody used in this example relies on molecular bridging, which can only mediate T cell activation using target cells in the tumor microenvironment, avoiding the toxic side effects caused by monoclonal antibodies such as Urelumab excessively activating T cells in normal tissues.

[0160] Example 3.1. Structure and Design of HER2x4-1BB Bispecific Antibody In this example, the anti-HER2 IgG antibody trastuzumab was used, whose corresponding amino acid sequence was derived from the IMGT database. The antibody number produced was PR000210.

[0161] [Table 16]

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

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

[0164] In this and subsequent examples, the positive control molecule is the anti-HER2 IgG monoclonal antibody PR000210 (a trastuzumab analog), the parent monoclonal antibody at the HER2 end of the HER2x4-1BB biantibody molecule.

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

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

[0167] In one embodiment, CH3 of polypeptide chain 2 is directly fusion linked to VH_B, i.e., the length of L is 0. In another embodiment, CH3 of polypeptide chain 2 is linked to VH_B via a connecting peptide L; L may be a sequence listed in Table 17.

[0168] [Table 17]

[0169] Using anti-HER2 IgG antibody and anti-4-1BB HCAb heavy chain antibody, HER2x4-1BB dual antibody molecules with IgG-VH tetravalent symmetric structure were designed and summarized in Table 18; physicochemical property analysis of the prepared dual antibody molecule samples is summarized in Table 19.

[0170] [Table 18]

[0171] [Table 19]

[0172] Example 3.1.2. Construction of a bispecific antibody with a tetravalent symmetrical IgG-scFv 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 FIG. 18B) comprises two polypeptide chains: polypeptide chain 1, also called the short chain, which comprises from the amino terminus to the carboxyl terminus VL_A-CL; and polypeptide chain 2, also called the long chain, which comprises from the amino terminus to the carboxyl terminus VH_A-CH1-h-CH2-CH3-L1-VH_BL2-VL_B. or

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

[0174] The connecting peptide L1 and the connecting peptide L2 of the polypeptide chain 2 can be the sequences listed in Table 17.

[0175] An IgG-scFv tetravalent symmetric HER2×4-1BB bibody molecule was designed using anti-HER2 IgG antibody and anti-4-1BB H2L2 antibody, and is summarized in Table 20; the physicochemical property analysis of the prepared bibody molecule samples is summarized in Table 21.

[0176] [Table 20]

[0177] [Table 21]

[0178] [Table 22]

[0179] Example 3.2. FACS detection of the binding ability of HER2x4-1BB dual antibody to SK-BR-3 cells This example is to study the binding activity of 4-1BB bispecific antibody with SK-BR-3 in vitro. SK-BR-3 is a breast cancer cell line that highly expresses HER2, and antibody binding experiments were performed at the cell level using SK-BR-3. Briefly, SK-BR-3 cells were digested and resuspended in complete medium at a cell density of 1 × 10 6 The antibody to be measured was diluted in a 3-fold gradient to 2x the final concentration and adjusted to cells / ml. After seeding 100 μL cells / well into a 96-well V-plate (Corning, #3894), 100 μL / well of the antibody to be measured was added at 100 μL / well. The cells were placed at 4°C and incubated for 1 hour in the dark. After that, 100 μL / well of pre-chilled PBS was added to rinse the cells twice, and the cells were centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. In addition, 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 Immunoreserach, #109-605-006, 1:1000 dilution) was added and incubated at 4°C for 30 min in the dark. The cells were washed twice with 100 μl / well pre-chilled PBS, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. Finally, the cells were resuspended with 200 μl / well pre-chilled PBS, and the fluorescence signal was read using a BD FACS CANTOII.

[0180] As shown in Figure 19, all of the HER2x4-1BB bispecific antibodies of the present invention can bind to human SK-BR-3, and the detected antibody binding ability increases in a positive correlation with the antibody concentration. Compared with the reference antibody (Trastuzumab), at the same concentration, PR002813 shows a lower EC50 than the reference antibody, and the other antibodies correspond to the reference antibody.

[0181] Example 3.3. FACS detection of the binding ability of HER2x4-1BB double antibody to CHO-K1 / hu 4-1BB cells This example is to study the in vitro binding activity of 4-1BB bispecific antibodies. A CHO-K1 cell line expressing human 4-1BB (CHO-K1 / hu 4-1BB, Genescript) was used to perform antibody binding experiments at the cellular level. Briefly, the cells CHO-K1 / hu 4-1BB cells were digested and resuspended in F12K complete medium at a cell density of 1 × 10 6 The antibody to be measured was diluted in a 3-fold gradient at 2x the final concentration at 100 μl / well. The cells were placed at 4°C and incubated for 1 h in the dark. After that, the cells were rinsed twice by adding 100 μl / well of pre-chilled PBS, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. A fluorescent secondary antibody (Alexa Fluor ... (登録商標)647 Affini Pure Goat Anti-Human IgG, F(ab')2 fragment specific, Jackson Immunoreserach, #109-605-006, 1:1000 dilution) was added and incubated at 4°C for 30 min in the dark. The cells were washed twice with 100 μl / well pre-chilled PBS, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. Finally, the cells were resuspended with 200 μl / well pre-chilled PBS, and the fluorescence signal was read using a NovoCyte flow cytometer (ACEA Biosciences).

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

[0183] Example 3.4. FACS detection of the binding ability of HER2x4-1BB double antibody to CHO-K1 / cyno4-1BB cells This example is to study the in vitro binding activity of 4-1BB bispecific antibodies. Antibody binding experiments at the cellular level were carried out using a CHO-K1 cell line (CHO-K1 / cyno4-1BB, Genescript) expressing cynomolgus monkey 4-1BB. Briefly, the cells CHO-K1 / cyno4-1BB cells were digested and resuspended in F12K complete medium at a cell density of 1 × 10 6The antibody to be measured was diluted in a 3-fold gradient at 2x the final concentration at 100 μl / well. The cells were placed at 4°C and incubated for 1 h in the dark. After that, the cells were rinsed twice by adding 100 μl / well of pre-chilled PBS, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. A fluorescent secondary antibody (Alexa Fluor ... (登録商標) 647 Affini Pure Goat Anti-Human IgG, F(ab')2 fragment specific, Jackson Immunoreserach, #109-605-006, 1:1000 dilution) was added and incubated at 4°C for 30 min in the dark. The cells were washed twice with 100 μl / well pre-chilled PBS, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. Finally, the cells were resuspended with 200 μl / well pre-chilled PBS, and the fluorescence signal was read using a NovoCyte flow cytometer (ACEA Biosciences).

[0184] As shown in Figure 21, all of the HER2x4-1BB biantibodies of the present invention can bind well to cynomolgus monkey 4-1BB, and the detected antibody binding ability increases in a positive correlation with the antibody concentration. Compared with the reference antibody (Utomilumab), PR001212, PR002824, and PR002826-PR002829 have equivalent binding to cynomolgus monkey 4-1BB.

[0185] Example 3.5. HER2 / 4-1BB dual antibody can activate T cell pathways in vitro 1 mg / ml OKT3 (eBiosciences, #16-0037-85) was diluted in PBS and 100 μL of 0.08 μg / ml OKT3 was placed in a 96-well plate (Corning, #3599) per well to a final concentration of 10 μg / ml. The plate was coated overnight at 4 °C. The next day, SK-BR-3 cells were digested and resuspended in complete medium to a cell density of 4 × 10 5The cells were adjusted to 1000 cells / ml and kept as a reserve. Human CD3-positive T cells were isolated from human PBMCs using a MACS kit (Miltenyi Biotec, #130-096-535). First, the cell number was confirmed, and then MACS buffer and Pan-T cell biotin antibody were added according to the cell number, mixed, and left at 4°C for 5 minutes. Then, a corresponding amount of microbeads was added and left at 4°C for 10 minutes. CD3-positive T cells were passed through the LS column. The OKT3 that had been coated on the 96-well plate from the previous day was washed away, and 50 μL of purified T cells were added, at 1 × 10 per well. 5 Further, 50 μL of SK-BR-3 cells were added to a 96-well plate, and 2 × 10 cells were added per well. 4 Then, the corresponding concentrations of HER2 / 4-1BB bispecific antibody or control monoclonal antibody were added and incubated at 37°C in a 5% CO2 culture box. After 72 hours of incubation, the supernatant was collected and the IFN-γ content was detected using an ELISA kit (Invitrogen, #88-7316). ELISA tests were performed according to the manufacturer's instructions. Briefly, coating antibodies were added to a 96-bottom plate and left at 4°C overnight. 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, detection antibodies were 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. TMB substrate was then added and ELISA stop solution (BBI life sciences, #E661006-0200) was added for 10-30 minutes. The 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-D, both of the HER2x4-1BB dual antibodies of the present invention have the ability to activate the 4-1BB-mediated T cell pathway upon crosslinking of tumor cells SK-BR-3, where PR002813 and PR002827 show low EC50 and high IFN-gamma release values.

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

[0188] The two structures of anti-HER2x4-1BB bispecific antibody molecules regulate the functional activity of activating T cells by parameters such as different structural types, relative positions, and binding valency, and also demonstrated the flexibility of constructing bispecific antibody molecular structures based on HCAbs.

[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 bibody on T cell activation depends specifically on the expression of HER2. In the presence of cells with high HER2 expression, HER2×4-1BB bibody can specifically activate T cells.

[0190] As described above, in this Example, a HER2x4-1BB bispecific antibody molecule with outstanding functional activity and excellent molecular stability was constructed.

[0191] Example 4. PD-L1x4-1BB Bispecific Antibody Programmed death receptor 1 (PD-1) is mainly expressed on immune cells such as T cells, and it has two ligands, namely programmed death ligand 1 (PD-L1) and PD-L2. PD-L1 is mainly expressed on antigen-presenting cells and some tumor cells. The interaction between PD-L1 and PD-1 reduces the activity of T cells, weakens the secretion of cytokines, and plays an immunosuppressive role. The expression of PD-L1 protein has been detected in many human tumor tissues, and the microenvironment at the tumor site can induce the expression of PD-L1 on tumor cells. The expressed PD-L1 is favorable for the development and growth of tumors, and can induce the apoptosis of antitumor T cells, further protecting 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 a multifunctional regulator of immune activity. It is induced and expressed in immune cells such as activated 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 the function of suppressing tumors, and the first 4-1BB fully human monoclonal antibody to enter clinical trials was Urelumab (BMS-663513) from Bristol-Myers Squibb (BMS). The first clinical results of Urelumab were published in 2008, and encouraging therapeutic effects were observed in some patients, but the data showed that Urelumab caused liver toxicity, which was related to the target and dose. In addition, two patients died of liver toxicity in clinical trials, and related clinical trials were discontinued.

[0193] In this example, a bispecific antibody was constructed that simultaneously targets PD-L1 and 4-1BB, improving antitumor efficacy and safety through one or more mechanisms of action. First, the PD-L1×4-1BB bispecific antibody can activate T cells by blocking the PD-1 / PD-L1 signal pathway. Second, the PD-L1 molecule, which is highly expressed on the surface of tumor cells, can promote the cross-linking and trimerization of 4-1BB molecules on the surface of T cells by using the bispecific antibody molecule, activating downstream signal transduction pathways, and further promoting the activation and proliferation of T cells. Third, the bispecific antibody molecule-mediated T cell activation is limited within the tumor microcirculation, which can avoid the toxic side effects caused by monoclonal antibodies such as Urelumab excessively activating T cells in normal tissues.

[0194] Example 4.1. Structure and design of PD-L1x4-1BB bispecific antibodies This example uses the anti-PD-L1 IgG antibody PR000151 (an analogue of Atesolizumab), whose corresponding amino acid sequence is from the IMGT database.

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

[0196] Harbour H2L2 mice (HarbourAntibodiesBV) are transgenic mice with a human immunoglobulin immune library, and the antibodies produced by them have a complete human antibody variable domain and a rat constant domain. Harbour H2L2 mice were subjected to multiple rounds of immunization with soluble recombinant human PD-L1 protein (NovoProtein, #C764). After detecting that the PD-L1-specific antibody titer in the mouse serum reached a certain level, the mouse spleen cells were taken out and fused with a myeloma cell line to obtain hybridoma cells. After multiple rounds of screening and cloning of the hybridoma cells, several monoclonal antibody molecules that can specifically identify PD-L1 were identified. These monoclonal antibodies were further identified, and several preferred candidate antibody molecules were selected based on parameters such as the binding ability to human PD-L1, the binding ability to cynomolgus monkey PD-L1, and the inhibitory ability to the binding of PD-L1 and PD-1. The candidate antibody molecules were then sequence analyzed and optimized, and several mutant sequences were obtained. The antibody VL and VH sequences were fused with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules. The anti-PD-L1 recombinant fully human IgG antibodies are listed in Table 23.

[0197] [Table 23]

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

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

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

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

[0202] Example 4.1.1. Constructing a bispecific antibody molecule with FIT-Ig structure using anti-PD-L1 IgG antibody and anti-4-1BB IgG antibody In this example, the antigen-binding domain Fab of anti-PD-L1 IgG antibody PR000265 or PR000151 (atezolizumab analogue) and the antigen-binding domain Fab of anti-4-1BB IgG antibody PR000197 or PR000448 are used to construct an anti-PD-L1 x 4-1BB bispecific antibody molecule with a FIT-Ig structure. 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. Antibody molecule samples were then prepared and analyzed according to the methods described in Examples 1 and 2, and are summarized in Table 25. Table 26 shows the SEQ ID NOs corresponding to the polypeptide chain sequences of the dual antibody molecules of 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 In this example, 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 antibodies PR001758, PR001760, or PR001836 were used to construct anti-PD-L1 x 4-1BB bispecific antibody molecules with multiple structures.

[0208] In this and subsequent examples, the positive control molecule was the anti-PD-L1 IgG monoclonal antibody PR000265, the parent monoclonal antibody at the PD-L1 end of the PD-L1x4-1BB biantibody molecule.

[0209] In this and subsequent examples, the positive control molecules are the anti-4-1BB IgG monoclonal antibodies urelumab (IgG4) or utomilumab (IgG2).

[0210] As shown in Figure 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 connecting peptides, respectively.

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

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

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

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

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

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

[0217] Using anti-PD-L1 IgG antibody and anti-4-1BB heavy chain antibody, a Fab-HCAb symmetrical PD-L1×4-1BB bibody molecule was designed and summarized in Table 27; the physicochemical property analysis of the constructed bibody molecule is summarized in Table 28.

[0218] [Table 27]

[0219] [Table 28]

[0220] Example 4.1.3. Construction of IgG-VH tetravalent symmetrical molecule Using anti-PD-L1 IgG antibody and anti-4-1BB heavy chain antibody, a PD-L1×4-1BB bibody 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 connected to the N-terminus of the heavy chain of the PD-L1 IgG antibody via a connecting peptide (as shown in Figure 23E). The binding protein in the structure of Figure 23E comprises two distinct polypeptide chains: polypeptide chain 1, also referred to as the short chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2, also referred to as the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L-VH_A-CH1-h-CH2-CH3. In one embodiment, the 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, the VH_B of polypeptide chain 2 is linked to VH_A via a connecting peptide L; L may be a sequence listed in Table 17.

[0222] Using anti-PD-L1 IgG antibody and anti-4-1BB heavy chain antibody, an IgG-VH symmetric PD-L1×4-1BB bibody molecule was designed and summarized in Table 29; the physicochemical property analysis of the constructed bibody molecule is summarized in Table 30.

[0223] [Table 29]

[0224] [Table 30]

[0225] Example 4.1.4. Sequence listing of PD-L1x4-1BB biantibody molecule and control molecule Table 31 shows the SEQ ID NOs corresponding to the sequences of the PD-L1x4-1BB biantibody molecules constructed in this embodiment.

[0226] [Table 31]

[0227] Example 4.2. Binding to cells highly expressing human PD-L1 This example was designed to investigate the binding activity of the PD-L1x4-1BB dual antibody molecule to PD-L1.

[0228] The binding ability of the antibody molecules to the CHO-K1 cell line CHO-K1 / hPDL1 (Nanjing Jinsirui, M00543) that highly expresses human PD-L1 or MDA-MB-231 (ATCC, HTB-26) that highly expresses human PD-L1 was tested using flow cytometry cell method FACS. Specifically, CHO-K1 / hPDL1 cells or MDA-MB-231 were digested and resuspended in complete medium; the cell density was 1×10 6The concentration of antibody molecules was adjusted to 100 cells / mL. The cells were then seeded into a 96-well V-plate (Corning, #3894) at 100 μL / well, centrifuged at 4 °C for 5 min, and the supernatant was discarded. Gradient-diluted antibody molecules were then added to the 96-well plate at 100 μL / well and mixed evenly, with the antibody molecules diluted in a 3-fold concentration gradient from the highest final concentration of 200 nM to a total of 12 concentrations, and hIgG1iso (CrownBio, #C0001) was used as the homogenous control. The cells were placed at 4 °C and incubated for 1 h in the dark. After that, the cells were rinsed twice by adding pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) at 100 μL / well, centrifuged at 500 g for 5 min at 4 °C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goat human lgG (H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added and incubated at 4 °C for 1 h in the dark. After that, 200 μL / well of pre-chilled FACS buffer was added to rinse the cells twice, and then the cells were centrifuged at 500 g for 5 min at 4 °C and the supernatant was discarded. Finally, 200 μL / well of pre-chilled FACS buffer was added to resuspend the cells. Fluorescence signal values ​​were read using a BD FACS CANTOII flow cytometer.

[0229] Data processing and graph analysis were performed using the software GraphPad Prism 8, and parameters such as the binding curve of the antibody to the target cells and the EC50 value were obtained by four-parameter nonlinear fitting.

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

[0231] As shown in Figure 24A, the PD-L1 binding abilities of the IgG-VH (C-terminus) tetravalent symmetric bibody molecules (PR003549, PR003550, PR003551) are similar to that of the parent monoclonal antibody PR000265, and the PD-L1 binding EC50 values ​​and maximum MFI values ​​are slightly better than those of the FIT-Ig bibody molecules (PR000701, PR003052).

[0232] As shown in Figure 24B, the PD-L1 binding abilities of the Fab-HCAb symmetrical bibody molecule PR004270 and the IgG-VH (N-terminal) tetravalent symmetrical bibody molecule PR004268 are similar to those of the parent monoclonal antibodies, with their PD-L1 binding EC50 values ​​being slightly weaker than those of the parent monoclonal antibodies, but their maximum binding MFIs being higher than those of the parent monoclonal antibodies.

[0233] As shown in Figures 24C-E, the ability of the IgG-VH (C-terminus) tetravalent symmetrical bibody molecules (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 monoclonal antibody PR000265.

[0234] Example 4.3.4-Binding to CHO-K1 cells overexpressing 1BB This example was designed to investigate the binding activity of the PD-L1×4-1BB dual antibody molecule to 4-1BB.

[0235] The binding ability of the antibody molecules to the CHO-K1 cell line CHO-K1 / hu 4-1BB (Nanjing Jinsirui, M00538) that highly expresses human 4-1BB and the CHO-K1 cell line CHO-K1 / cyno4-1BB (Nanjing Jinsirui, M00569) that highly expresses cynomolgus monkey 4-1BB were tested using flow cytometry (FACS). Specifically, the cells were digested and resuspended in complete medium; the cell density was adjusted to 2 × 10 6The cells were then plated at 100 μL / well (2 × 10 5 The cells were seeded at 100 μL / well into a 96-well V-plate (Corning, #3894), centrifuged at 4 °C for 5 min, and the supernatant was discarded. Then, the gradient-diluted antibody molecules were added to the 96-well plate at 100 μL / well and mixed evenly. The antibody molecules were diluted in a 3-fold concentration gradient from the highest final concentration of 200 nM to a total of 12 concentrations, and hIgG1iso (CrownBio, #C0001) was used as an isotype control. The cells were placed at 4 °C and incubated for 1 h in the dark. Then, the cells were rinsed twice by adding pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) at 100 μL / well, centrifuged at 500 g for 5 min at 4 °C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goat human lgG (H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added and incubated at 4 °C for 1 h in the dark. After that, 200 μL / well of pre-chilled FACS buffer was added to rinse the cells twice, followed by centrifugation at 500 g for 5 min at 4 °C and discarding the supernatant. Finally, 200 μL / well of pre-chilled FACS buffer was added to resuspend the cells. Fluorescence signal values ​​were read using a BD FACS CANTOII flow cytometer.

[0236] Data processing and graph analysis were performed using the software GraphPad Prism 8, and parameters such as the binding curve of the antibody to the target cells and the EC50 value were obtained by four-parameter nonlinear fitting.

[0237] In this example, the positive control molecules are the anti-4-1BB monoclonal antibodies Urelumab or tomilumab.

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

[0239] As shown in Figure 25B, the ability of the Fab-HCAb symmetrical dual antibody molecule PR004270 and the IgG-VH (N-terminal) tetravalent symmetrical dual antibody molecule PR004268 to bind human 4-1BB is superior to that of 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-terminus) tetravalent symmetrical structured dual antibody molecules (PR007130, PR007132, PR007133, PR007135, PR007136, PR007137, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) to human 4-1BB is superior to the positive control Urelumab in terms of maximum MFI.

[0241] Example 4.3.2. Binding to CHO-K1 cells CHO-K1 / cyno4-1BB that highly express cynomolgus monkey 4-1BB As shown in Figures 26A and 26B, the bibody molecule of this example can bind to cynomolgus monkey 4-1BB, whereas Urelumab cannot. The ability of PR004270 to bind to cynomolgus monkey 4-1BB is slightly superior to Utomilumab in terms of maximum MFI.

[0242] Example 4.4. Specific activation of T cells mediated by target cells highly expressing PD-L1 This example is to study that the presence of PD-L1×4-1BB dual antibody molecule in target cells activates the activity of T cells by binding 4-1BB. The target cells can be cells expressing PD-L1 to different degrees, such as CHO-K1 / hPDL1 (GENSCRIPT, M00543) that highly expresses human PD-L1, or MDA-MB-231 (ATCC, HTB-26) that highly expresses human PD-L1. The effector cells can be isolated human PBMCs or T cells.

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

[0244] Data processing and plot analysis were 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 in which target cells CHO-K1 / hPDL1 and T cells were mixed, the anti-4-1BB monoclonal antibody Urelumab, which is not dependent on cross-linking, was able to activate IFN-γ release from T cells, whereas the anti-4-1BB monoclonal antibodies (PR000448, PR001758, PR001760, PR001836) which are dependent on cross-linking were barely able to activate T cells. The FIT-Ig structured bibody molecule (PR003052, PR000701), the IgG-VH tetravalent symmetric structured bibody molecule (PR003549, PR003550, PR003551, PR004268, PR007130, PR007132, PR007133, PR007135, PR007136, PR007137, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) and the Fab-HCAb structured bibody molecule (PR004270) can all activate T cells and release cytokines, indicating that the activation of the bibody molecule against T cells depends on the specific activation of the target cells. In addition, the IgG-VH tetravalent symmetrical antibody molecules (PR003549, PR003550, PR007130, PR007132, PR007133, PR007135, PR007136, PR007137, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) and the Fab-HCAb antibody molecule (PR004270) were able to induce higher cytokine release levels and showed stronger T cell activation abilities than the FIT-Ig antibody molecules (PR003052, PR000701), and were 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 and T cells, the IgG-VH tetravalent symmetrical bibody molecule (PR003549) and the Fab-HCAb bibody molecule (PR004270) had similar T cell activation ability and were superior to the FIT-Ig bibody molecules (PR003052, PR000701).

[0247] As shown in Figures 27F and 27G, in a system mixing target cells MDA-MB-231 and T cells, the IgG-VH tetravalent symmetrical dual antibody molecule (PR003549) has stronger T cell activation ability than Urelumab and can better promote the production of IFN-γ and IL-2.

[0248] As shown in Figures 27H and 27I, in a system where target cells MDA-MB-231 and T cells were mixed, the IgG-VH tetravalent symmetrical dual antibody molecules (PR007130, PR007132, PR007133, PR007138, PR007139, PR007141, PR007142, PR007143, PR007145, PR007146, PR007149) had significantly greater T cell activation ability than Urelumab.

[0249] Example 4.5. Mixed Lymphocyte Reaction (MLR) This example uses a mixed lymphocyte reaction (MLR) to study the activating effect of the PD-L1x4-1BB bibody molecule on T cells.

[0250] In the first step, monocytes were isolated from the primary donor PBMC cells (MT-BIO) using CD14 beads (Meltenyi, #130-050-201); for specific operations, please refer to the instructions of the relevant kit. Then, 50ng / mL recombinant human IL-4 (PeproTech, #200-02-A) and 100ng / mL recombinant human GM-CSF (PeproTech, #300-03-A) were added, and after induction at 37°C for 7 days, immature dendritic cells (iDC cells) were obtained. Then, 1μg / mL lipopolysaccharide (LPS, Sigma, #L 6529) was added, and after induction for 24 hours, mature dendritic cells (mDC cells) were obtained. 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 96-well plate at a ratio of 5:1 (1 × 10 5 / well with T cells 2 x 10 4 mDC cells per well). Then, antibody molecules of different concentrations were added at 50μL / well, the final antibody concentration could be (10nM, 1nM), or a total of 8 concentrations diluted in a 3-fold concentration gradient from the highest final concentration of 50nM, and added to two combined wells; hIgG1 iso (CrownBio, #C0001) or blank wells served as controls. Incubated at 37°C, 5% CO2 culture box for 5 days. In the fourth step, the supernatants on the 3rd and 5th days were collected, respectively, and the IL-2 concentration in the supernatant on the 3rd day was detected by IL-2 ELISA kit (Thermo, #88-7025-88) and the IFN-γ concentration in the supernatant on the 5th day was detected by IFN-γ ELISA kit (Thermo, #88-7316-77). For the ELISA detection method, please refer to the operation instructions of the related kit.

[0251] As shown in Figure 28, in multiple independent MLR experiments (different donor pairs), anti-4-1BB monoclonal antibody has limited activation effect on T cells and weak ability to produce cytokines; anti-PD-L1 monoclonal antibody has obvious activation effect. Dual antibody molecules can further improve the function of T cells.

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

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

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

[0255] As shown in Figures 28I to 28K, the IgG-VH tetravalent symmetrical dual antibody molecules (PR003549, PR003550, PR003551) and the Fab-HCAb dual antibody molecule (PR004270) show strong T cell activation ability.

[0256] Example 4.6. Pharmacokinetic Studies This example investigated the pharmacokinetic properties of the PD-L1×4-1BB dual antibody molecule PR004270 with a Fab-HCAb symmetric structure in mice.

[0257] Methodology: For each antibody molecule tested, six female BALB / c mice weighing 18-22 grams were selected and administered the bispecific antibody via intravenous injection at a dose of 5 mg / kg. One group of three mice had their whole blood collected before and 15 minutes, 24 hours (day 1), day 4, and day 10 after administration, and another group of three mice had their whole blood collected before and 5 hours, day 2, day 7, and day 14 after administration. The whole blood was left to clot for 30 minutes, then centrifuged, and the separated serum samples were frozen and stored at -80°C until analysis. This example used two types of ELISA methods to quantitatively measure drug concentrations in mouse serum. ELISA method 1, Fc end detection (total detection), captures human Fc-containing antibodies in mouse serum with goat anti-human Fc polyclonal antibody encapsulated in a 96-well plate, and then detects by adding HRP-labeled goat anti-human Fc secondary antibody; ELISA method 2, PD-L1 end detection (functional domain detection), captures antibodies that specifically identify PD-L1 in mouse serum with human PD-L1 protein encapsulated in a 96-well plate, and then detects by adding HRP-labeled goat anti-human Fc secondary antibody. Pharmacokinetic parameters were analyzed by selecting the non-atrium model (NCA) using Phoenix WinNonlin software version 8.2.

[0258] As shown in FIG. 29 and Table 32, the Fab-HCAb dual antibody molecule PR004270 has a serum half-life t 1 / 2 value, and the functional domain detection method 1 / 2values ​​were shown to exceed 10 days.

[0259] [Table 32]

[0260] summary In this example, anti-PD-L1×4-1BB bispecific antibody molecules with various structures were constructed 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, demonstrating the flexibility of constructing bispecific antibody molecular structures based on HCAbs, and adjusting the functional activity of T cell activation through parameters such as different structural types, relative positions, and binding valencies.

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

[0262] The HCAb-based dual antibody structures, especially the IgG-VH tetravalent symmetric dual antibody molecules and the Fab-HCAb dual antibody molecules, retain the activity of the PD-L1 end and demonstrate stronger T cell activation ability than the corresponding anti-PD-L1 parent monoclonal antibodies in MLR experiments; meanwhile, the highly expressed PD-L1 molecules on target cells can mediate the cross-linking and trimerization of 4-1BB to transmit T cell activation signals, and their T cell activation ability is superior to that of Urelumab. In addition, the IgG-VH tetravalent symmetric dual antibody molecules and the Fab-HCAb symmetric dual antibody molecules demonstrate stronger T cell activation ability than the FIT-Ig dual antibody molecules.

[0263] In conclusion, this example constructed a PD-L1×4-1BB bispecific antibody molecule with 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. comprising a heavy chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence set forth in SEQ ID NO: 15 or variant 1 thereof, or SEQ ID NO: 16, the 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 the 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; among them: The mutations of said variant 1 include one or more of T3I, S6N / G / R and Y7F; preferably, the sequence of said variant 1 is preferably 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 mutations of said variant 2 include 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 any of the sequences shown in 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 of said variant 3 include one or more of 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 said variant 3 is preferably any of the sequences shown in SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:104-106, SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:112-115 in the sequence listing; The mutation of said variant 4 includes N1I or N1Q; the amino acid sequence of said variant 4 is preferably shown in SEQ ID NO: 53 or 54 in the sequence listing; The mutation of said variant 5 includes E4A / P and / or N13Y; the amino acid sequence of said variant 5 is preferably shown in SEQ ID NO: 327-330 in the sequence listing; The mutant 1, mutant 2, mutant 3, mutant 5 and mutant 4 are 4-1BB binding proteins that contain at least the function of the sequence before the mutation.

2. The HCDR1, HCDR2 and HCDR3 are the sequences shown in SEQ ID NO: 16, SEQ ID NO: 50 and SEQ ID NO: 96 in the sequence listing, respectively, or the sequences shown in SEQ ID NO: 16, SEQ ID NO: 53 and SEQ ID NO: 96, respectively, or the sequences shown in SEQ ID NO: 16, SEQ ID NO: 54 and SEQ ID NO: 96, respectively, or the sequences shown in SEQ ID NO: 19, SEQ ID NO: 57 and SEQ ID NO: 101, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 58 and SEQ ID NO: 102, respectively, or the sequences shown in SEQ ID NO: 20, SEQ ID NO: 59 and SEQ ID NO: 103, respectively, or the sequences shown in SEQ ID NO: 20, SEQ ID NO: 60 and SEQ ID NO: 104, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 61 and SEQ ID NO: 105, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 60 and SEQ ID NO: 106, respectively, or the sequences shown in SEQ ID NO: 20, SEQ ID NO: 63 and SEQ ID NO: 108, respectively, or the sequences shown in SEQ ID NO: 20, SEQ ID NO: 60 and SEQ ID NO: 108, respectively, or the sequences shown in SEQ ID NO: 22, SEQ ID NO: 64 and SEQ ID NO: 109, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 60 and SEQ ID NO: 110, respectively, or the sequences shown in SEQ ID NO: 22, SEQ ID NO: 64 and SEQ ID NO: 109, respectively, or the sequences shown in or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 65 and SEQ ID NO: 111, or the sequences shown in SEQ ID NO: 22, SEQ ID NO: 66 and SEQ ID NO: 112, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 49 and SEQ ID NO: 113, respectively, or the sequences shown in SEQ ID NO: 20, SEQ ID NO: 60 and SEQ ID NO: 103, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 63 and SEQ ID NO: 104, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 60 and SEQ ID NO: 114, respectively, or the sequences shown in SEQ ID NO: 23, SEQ ID NO: 67 and SEQ ID NO: 105, respectively, or the sequences shown in SEQ ID NO: 24, SEQ ID NO: 68 and SEQ ID NO: 103, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 60 and SEQ ID NO: 105, respectively, or the sequences shown in SEQ ID NO: 20, SEQ ID NO: 69 and SEQ ID NO: 115, respectively, or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 70 and SEQ ID NO: 116, respectively, or the sequences shown in SEQ ID NO: 20, SEQ ID NO: 71 and SEQ ID NO: 115, respectively, or the sequences shown in SEQ ID NO: 300, SEQ ID NO: 308 and SEQ ID NO: 326, respectively, or the sequences shown in SEQ ID NO: 300, SEQ ID NO: 309 and SEQ ID NO: 327, respectively,or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:310 and SEQ ID NO:328, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:308 and SEQ ID NO:329, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:311 and SEQ ID NO:330, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:312 and SEQ ID NO:331, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:308 and SEQ ID NO:332, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:313 and SEQ ID NO:330, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:314 and SEQ ID NO:329, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:315 and SEQ ID NO:331, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:314 and SEQ ID NO:327, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:316 and SEQ ID NO:331, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:308 and SEQ ID NO:333, respectively, or the sequences as set forth in SEQ ID NO:300, SEQ ID NO:317 and SEQ ID NO:334, respectively; Preferably, the gene encoding the FR of the heavy chain variable region is derived from an embryonic V gene IGHV4-34, IGHV3-23, IGHV3-11 or IGHV3-74; More preferably, the heavy chain variable region comprises any of the 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.

3. the 4-1BB binding protein further comprises a light chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the sequence set forth in SEQ ID NO:133, the LCDR2 comprising the sequence set forth in SEQ ID NO:145 and the LCDR3 comprising the sequence set forth in SEQ ID NO:158; Preferably, the gene encoding the FR of the light chain variable region is derived from the germline V gene IGKV3-15; among them: 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 the occurrence of one or more amino acid residue mutations in the sequence shown in SEQ ID NO: 201; the sequence of the light chain variable region obtained after mutation is preferably shown in SEQ ID NO: 204; Even more preferably, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 168 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 201; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 171 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 204; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 172 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

204.

4. Said 4-1BB binding protein further comprises a heavy chain constant region and / or a light chain constant region; preferably, said heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4 or variants thereof and said light chain constant region is selected from a kappa chain or a lambda chain or variants thereof; wherein: The 4-1BB binding protein of any one of claims 1-3, wherein the mutation of 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; and the mutation of the hIgG4 variant is preferably S228P.

5. The 4-1BB binding protein comprises a heavy chain and a light chain, the heavy chain comprising any of the sequences shown in SEQ ID NO:209, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:216-222, SEQ ID NO:224-238, SEQ ID NO:353-368, and SEQ ID NO:240 in the sequence listing; and / or the light chain comprising the sequence shown in SEQ ID NO:246 or SEQ ID NO:243 in the sequence listing; Preferably, the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 209, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 243; or the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 212, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 246; or the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 213, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 246; or the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 216, and the amino acid sequence of the light chain is set forth in SEQ ID NO:

246. The 4-1BB binding protein of claim 4.

6. Full-length antibody, Fab, Fab', F(ab') ２ , Fv, scFv, bispecific, multispecific, heavy chain, single domain or single domain antibody forms, or monoclonal or multiclonal antibodies derived from said antibodies; Preferably, the heavy chain of the full length antibody comprises the sequence shown in SEQ ID NO: 209, and the light chain comprises the sequence shown in SEQ ID NO: 243; or comprises the sequence shown in SEQ ID NO: 212, and the light chain comprises the sequence shown in SEQ ID NO: 246, or comprises the sequence shown in SEQ ID NO: 213, and the light chain comprises the sequence shown in SEQ ID NO: 246, or comprises the sequence shown in SEQ ID NO: 216, and the light chain comprises the sequence shown in SEQ ID NO:

246. A 4-1BB binding protein according to any one of claims 1 to 5.

7. The bispecific antibody comprises a first protein functional domain and a second protein functional domain, wherein the first protein functional domain is a 4-1BB binding protein according to any one of claims 1-6, and the second protein functional domain targets a tumor antigen; preferably a HER2 antibody or a PD-L1 antibody; wherein the HER2 antibody is preferably trastuzumab or pertuzumab, and the PD-L1 antibody is preferably atezolizumab or PR000265, the heavy chain of PR000265 being set forth in SEQ ID NO:211, and the light chain of PR000265 being set forth in SEQ ID NO:

245.

8. The first protein functional region or the second protein functional region is selected from the group consisting of scFv, VHH, immunoglobulin, Fab, Fab', F(ab') ２ or a heavy chain variable region; for example, the first protein functional region is a Fab and the second protein functional region is a VHH; or the first protein functional region is a Fab and the second protein functional region is an immunoglobulin; or the first protein functional region is an immunoglobulin and the second protein functional region is a heavy chain variable region.

9. The bispecific antibody according to claim 8, wherein 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 binder, and the amino acid sequence of the binder is GS or is set forth in any of SEQ ID NOs: 273-293 in the Sequence Listing.

10. The bispecific antibody comprises polypeptide chain 1 and polypeptide chain 2, and preferably further comprises polypeptide chain 3; Preferred: the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:244, and the amino acid sequence of said polypeptide chain 2 is set forth in any of SEQ ID NOs:251-265; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:271; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:249, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:272; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:270; or the amino acid sequence of said polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of said polypeptide chain 2 is set forth in SEQ ID NO:269; or or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of polypeptide chain 2 is set forth in SEQ ID NO:268; or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:245, and the amino acid sequence of polypeptide chain 2 is set forth in any of SEQ ID NOs:369-382; or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:267, the amino acid sequence of polypeptide chain 2 is set forth in SEQ ID NO:266, and the amino acid sequence of polypeptide chain 3 is set forth in SEQ ID NO:246; or the amino acid sequence of polypeptide chain 1 is set forth in SEQ ID NO:250, and the amino acid sequence of polypeptide chain 2 is set forth in SEQ ID NO:249, and the amino acid sequence of polypeptide chain 3 is set forth in SEQ ID NO:

246.

11. An isolated nucleic acid encoding a 4-1BB binding protein according to any one of claims 1-6 or a bispecific antibody according to any one of claims 7-10.

12. An expression vector comprising the isolated nucleic acid of claim 11.

13. A host cell comprising the expression vector of claim 12; preferably a prokaryotic or eukaryotic cell.

14. A method for producing a 4-1BB binding protein, comprising culturing the host cell of claim 13 and obtaining the 4-1BB binding protein from the culture.

15. A chimeric antigen receptor comprising a 4-1BB binding protein according to any one of claims 1-6 or a bispecific antibody according to any one of claims 7-10.

16. An antibody drug conjugate comprising a cytotoxic agent and a 4-1BB binding protein according to any one of claims 1-6 or a bispecific antibody according to any one of claims 7-10; preferably, said cytotoxic agent comprises MMAF or MMAE.

17. A drug composition comprising a 4-1BB binding protein according to any one of claims 1-6, a bispecific antibody according to any one of claims 7-10 and / or an antibody-drug conjugate according to claim 16.

18. Use of a 4-1BB binding protein according to any one of claims 1 to 6, a bispecific antibody according to any one of claims 7 to 10 and a pharmaceutical composition according to claim 17 in the manufacture of a drug for treating and / or preventing cancer, comprising: 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 tumors or colorectal cancer.

19. comprising a 4-1BB binding protein according to any one of claims 1-6, a bispecific antibody according to any one of claims 7-10, the chimeric antigen receptor according to claim 15, an antibody-drug conjugate according to claim 16 and / or a drug composition according to claim 17; Preferably, the reagent kit further comprises: (i) a device for administering the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate or drug composition; and / or (ii) instructions for use.

20. A drug cassette A and a drug cassette B, in which: said drug cassette A comprising a 4-1BB binding protein according to any one of claims 1-6, a bispecific antibody according to any one of claims 7-10, a chimeric antigen receptor according to claim 15, an antibody-drug conjugate according to claim 16 and / or a drug composition according to claim 17; The drug cassette B includes another anti-tumor antibody or a drug composition containing the other anti-tumor antibody, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, tumor lytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

21. A method for diagnosing, treating and / or preventing a 4-1BB mediated disease or disorder, comprising: The method comprises administering to a patient in need thereof a therapeutically effective amount of a 4-1BB binding protein according to any one of claims 1-6, a bispecific antibody according to any one of claims 7-10, a chimeric antigen receptor according to claim 15, an antibody-drug conjugate according to claim 16 or a pharmaceutical composition according to claim 17, or treating a patient in need thereof with a pharmaceutical kit according to claim 20.

22. The method of claim 21, wherein the disease or disorder is a tumor, preferably a 4-1BB positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and / or leukemia.

23. A method for immunodetecting or measuring 4-1BB, comprising:

17. A method comprising using a 4-1BB binding protein according to any one of claims 1-6, a bispecific antibody according to any one of claims 7-10, a chimeric antigen receptor according to claim 15, an antibody-drug conjugate according to claim 16 or a drug composition according to claim 17; preferably, said detection is not for diagnostic and / or therapeutic purposes.

24. Administering to a patient in need thereof a 4-1BB binding protein according to any one of claims 1-6, a bispecific antibody according to any one of claims 7-10, a chimeric antigen receptor according to claim 15, an antibody-drug conjugate according to claim 16 or a pharmaceutical composition according to claim 17, and a second therapeutic agent, said second therapeutic agent preferably comprising another anti-tumor antibody or a pharmaceutical composition comprising said other anti-tumor antibody, and / or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, protease inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, lytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules and vaccines.

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