WO2021013142A1

WO2021013142A1 - Anti-4-1bb antibody, antigen-binding fragment thereof, and bispecific antibody

Info

Publication number: WO2021013142A1
Application number: PCT/CN2020/103192
Authority: WO
Inventors: 张伟; 陈思萌; 王蕾蕾; 姜福伟; 吴建权; 郭新; 杨翠青; 廖成; 林�源; 胡齐悦; 张连山
Original assignee: 江苏恒瑞医药股份有限公司
Priority date: 2019-07-22
Filing date: 2020-07-21
Publication date: 2021-01-28
Also published as: TW202118787A

Abstract

Provided are a 4-1BB antibody, an antigen-binding fragment thereof, a recombinant bispecific antibody against 4-1BB and PD-L1, a nucleic acid molecule encoding the 4-1BB antibody or an antigen-binding fragment thereof or the recombinant bispecific antibody, a vector and a host cell comprising the nucleic acid molecule, and a method for preparing the 4-1BB antibody or an antigen-binding fragment thereof or the recombinant bispecific antibody. Also provided are a pharmaceutical composition comprising the 4-1BB antibody or an antigen-binding fragment thereof or the recombinant bispecific antibody and a related pharmaceutical use thereof.

Description

Anti-4-1BB antibody, its antigen-binding fragment and bispecific antibody

Technical field

The present disclosure relates to the field of biomedicine, especially the field of prevention and treatment of diseases related to 4-1BB and/or PD-L1. Specifically, the present disclosure relates to 4-1BB antibodies and antigen-binding fragments thereof, bispecific antibodies that specifically bind 4-1BB and/or PD-L1, pharmaceutical compositions and related pharmaceutical applications thereof.

Background technique

4-1BB (CD137, TNFRSF9) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). 4-1BB is expressed in the form of monomer or dimer on the cell surface. After binding to its ligand (4-1BBL), it undergoes trimerization for signal transduction. It is CD8 ⁺ and CD4 ⁺ T cells, regulatory T cells ( Tregs), NK cells and NKT cells, B cells and neutrophils. On T cells, 4-1BB is not constitutively expressed, but is induced after T cell receptor (TCR) activation. It is stimulated by its natural ligand 4-1BBL or antibody agonists, and is stimulated by TNFR-related factors (TRAF) -2 and TRAF-1 conduct signal transduction. Early signal transduction of 4-1BB involves polyubiquitination of K-63, activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways, signal transduction leads to costimulation of T cells, cell proliferation, and cell proliferation. Factor production, maturation and prolonged survival of CD8 ⁺ T cells.

Studies have shown that antibody agonists against 4-1BB promote anti-tumor control of T cells in mice (Murillo et al., Clin Cancer Res. 2008; 14(21): 6895-906). Antibodies that activate 4-1BB can increase the expression of co-stimulatory molecules in many models, induce the survival and proliferation of T cells, thereby enhancing the anti-tumor immune response and causing anti-tumor efficacy. The existing 4-1BB activating antibodies in the prior art include urelumab from BMS, which is a human IgG4 antibody (WO2005035584); and utomilumab from Pfizer, which is a human IgG2 antibody (Fisher et al., Cancer Immunol. 2012; 61: 1721-1733). Other 4-1BB antibody related patents include WO2000029445, WO2012032433, WO2003049755, WO2017205745A and so on. Both BMS and Pfizer’s 4-1BB antibodies have entered phase I/II clinical trials, and similar products from Pieris are also in phase I clinical trials.

Programmed death-l (PD-l) belongs to the CD28 family and is mainly expressed on activated T cells, B cells and myeloid cells. PD-1 has two ligands, programmed death ligand 1 (PD-L1, CD274, B7H1) and programmed death ligand 2 (PD-L2). PD-L2 expression is more limited, mainly expressed on antigen presenting cells, such as activated macrophages and dendritic cells. PD-L1 belongs to the immunoglobulin (Ig) superfamily. It contains an Ig-like C2 type domain and an Ig-like V-type domain. It is a single type I transmembrane protein with three subtypes after variable cleavage. type. Freshly isolated T cells and B cells express negligible amounts of PD-L1, and some (about 16%) CD14 ⁺ monocytes express PD-L1 constitutively. It is known that interferon-γ (IFNγ) can up-regulate the PD-L1 of tumor cells, and PD-L1 can hinder anti-tumor immunity by binding to the receptor PD-1 on activated T cells to resist tumor reactivity T cells; make tumor cells resistant to CD8 ⁺ T cells, and through PD-L1 expressed by tumor cells, Fas ligand-mediated lysis through PD-1 signaling; CD80 (B7-1) expressed by T cells ), reverse signal transduction tolerant T cells; and, promote the development and maintenance of induced Tregs.

Studies have shown that high PD-L1 protein is detected in human tumor tissues such as breast cancer, lung cancer, stomach cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer. The expression of PD-L1 is closely related to the clinical and prognosis of patients. PD-L1 blocking antibodies have shown clinical activity in several cancers known to overexpress PD-L1 (including melanoma, NSCLC). For example, Roche's humanized IgG1 monoclonal antibody atezolizumab against PD-L1 is used in clinical trials as immunotherapy for several indications, including various types of solid tumors (see, for example, Rittmeyer et al., Lancet, 2017; 389: 255-265), and is approved for metastatic non-small cell lung cancer and metastatic bladder cancer. Another PD-L1 antibody, Merck Serono SA's avelumab, has been approved by the FDA for the treatment of Merkel cell carcinoma, bladder cancer, and metastatic renal cell carcinoma. It is currently in non-small cell lung cancer, ovarian cancer, and kidney cancer. Etc. (see, for example, the Cortellis database). And, MedImmune's PD-L1 antibody Durvalumab is approved for metastatic non-small cell lung cancer and metastatic bladder cancer, and is undergoing clinical development in a variety of solid tumors and blood cancers (see Cortellis database for example). PD-L1 antibody related patents include: WO0139722, WO2013173223, WO2014195852, WO2013181634, WO2015048520, WO2015036511, US2014335093, WO2014100079, WO2014055897, US6803192B1, WO2014022758, US8617546B2, and WO2010089411A2.

In the prior art, there are reports on the combined use of 4-1BB antibody and PD-L1 antibody. For example, the combination of 4-1BB antibody as an agonist and neutralizing PD-L1 antibody (Horton et al., J Immunother Cancer. 2015; 3( Suppl 2): O10). The combination therapy of utomilumab and avelumab is also being tested (Chen et al., J Clin Oncol 35, 2017suppl; abstr TPS7575, and clinical trial NCT02554812). Although the combination of the two antibodies may enhance the clinical anti-tumor activity and expand the indications. However, there is still a need in the art for bispecific and multispecific antibodies that can simultaneously bind PD-L1 and 4-1BB, because bispecific antibodies have active characteristics that the combination of two monoclonal antibodies does not possess. For example, bispecific antibodies can specifically activate 4-1BB at the tumor site and reduce the clinical hepatotoxicity caused by traditional activated monoclonal antibodies. Bispecific antibody (BsAb) is a kind of combination antibody with parental tropism, usually bivalent (also quadrivalent and hexavalent), that is, it has two antigen-binding arms and can bind two different specific antigens. Features. The antibody will simultaneously bind to antigen presenting cells (APC) or tumor cells expressing PD-L1 and T cells expressing 4-1BB, resulting in the conditional activation of cytotoxic T cells. The binding of PD-L1 to PD-1 expressed on activated T cells will result in T cell suppression. Patents related to the combination of 4-1BB antibody and PD-L1 antibody or bi-specific antibodies include: WO2019025545, WO2019104716, WO2018114754, WO2019072870, WO2019072869, WO2019072868, WO2019089753, WO2019005639. INHIBRx LLC's anti-4-1BB/PD-L1 bispecific antibody INBRX-105 is currently in non-small cell lung cancer, melanoma, head and neck cancer, renal cell carcinoma, gastric cancer, esophageal cancer, Hodgkin’s disease, non-Hodgkin’s Lymphoma, advanced solid tumor and other tumors or cancer phase I clinical stage. Genmab's anti-4-1BB/PD-L1 bispecific antibody GEN-1046 is currently in the phase II clinical stage of cervical tumors, endometrioid carcinoma, metastatic non-small cell lung cancer, advanced solid tumors, etc. Merus's anti-4-1BB/PD-L1 bispecific antibody MCLA-145 is also in the phase I clinical stage of advanced solid tumors and solid tumors.

In this field, there is still an unmet need for diseases such as tumors or autoimmune diseases, and it is still urgent and necessary to provide new structural, more effective, and less side effects treatment methods and drugs. The 4-1BB/PD-L1 bispecific antibody of the present disclosure retains the normal Fc segment, so the antibody has a longer half-life. In addition, the 4-1BB/PD-L1 bispecific antibody of the present disclosure has an affinity for human PD-L1 protein about 50 times higher than that of human 4-1BB protein, which greatly guarantees the effectiveness of the bispecific antibody at tumor sites. Enrichment, at the same time, greatly reduces the toxic and side effects that may be caused by the 4-1BB end, improves the safety of the drug, and has great clinical value.

In addition, this article incorporates related applications of 4-1BB antibody and PD-L1 antibody by way of full introduction, including PCT/CN2019/072484, CN201680027181.4, CN201710341680.7, CN201811248478.0, CN201811396143.3, CN201811526262.6.

Summary of the invention

The present disclosure provides an anti-4-1BB antibody and an antigen-binding fragment thereof, and a bispecific antibody capable of binding 4-1BB and PD-L1. The present disclosure also provides a nucleic acid molecule encoding the antibody, a vector containing the nucleic acid molecule, a method for preparing the antibody, a pharmaceutical composition containing the antibody, the use of the antibody in the preparation of medicine, and the antibody Use or method for treating/diagnosing diseases related to 4-1BB and/or PD-L1 (such as tumors or autoimmune diseases or infectious diseases), and kits containing the antibodies.

Anti4-1BB antibody and its antigen-binding fragment

The present disclosure provides an anti-4-1BB antibody and an antigen-binding fragment thereof, which comprises:

The heavy chain variable region includes at least one heavy chain complementarity determining region (HCDR) selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and/or

The light chain variable region includes at least one light chain complementarity determining region (LCDR) selected from the following sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

In some embodiments, the anti-4-1BB antibody and antigen-binding fragment thereof comprise:

The heavy chain variable region, which includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and/or

The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or the light chain variable region includes SEQ ID NO: 4. LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 7 and SEQ ID NO: 6.

In some embodiments, the anti-4-1BB antibody and antigen-binding fragments thereof are murine antibodies or fragments thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises a light chain framework region (FR region) or a light chain constant region of a murine kappa, lambda chain or a variant thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises a heavy chain framework region (FR region) or a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.

In some specific embodiments, the anti-4-1BB antibody and antigen-binding fragments thereof are chimeric antibodies or fragments thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.

In some specific embodiments, the anti-4-1BB antibody and antigen-binding fragments thereof are humanized antibodies, human antibodies or fragments thereof. In some embodiments, the heavy chain FR region is derived from the human germline heavy chain IGHV3-30 sequence or the human germline heavy chain IGHV1-46 sequence. In some embodiments, the light chain FR region is derived from the human germline light chain IGkV3-11 or the human germline light chain IGkV4-1 sequence. The amino acid sequence information of IGHV3-30, IGKV3-11, IGHV1-46, and IGKV4-1 can be shown in SEQ ID NO: 27-30.

In some embodiments, anti-4-1BB antibodies and antigen-binding fragments thereof according to the present disclosure comprise:

The heavy chain variable region, which includes the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3; and the light chain variable region, which includes the light chain variable region, respectively, as shown in SEQ ID NO : 4, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 7 and SEQ ID NO: 6.

In some embodiments, the heavy chain variable region of the anti-4-1BB antibody and antigen-binding fragment thereof according to the present disclosure comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; and/or, the light chain variable region Contains or consists of the amino acid sequence shown in SEQ ID NO: 9.

In some embodiments, the heavy chain of the anti-4-1BB antibody and antigen-binding fragment thereof according to the present disclosure comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; and/or, the light chain comprises or consists of SEQ ID NO. : Composition of the amino acid sequence shown in 11.

In some embodiments, the heavy chain variable region of an anti-4-1BB antibody or antigen-binding fragment thereof according to the present disclosure has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes; 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the light chain variable region. In some specific embodiments, the amino acid changes are conservative substitutions, substitutions or modifications, and/or deletions or additions that do not affect function.

In some specific embodiments, the amino acid sequence of the heavy chain variable region (VH) of the anti-4-1BB antibody or antigen-binding fragment thereof according to the present disclosure is the same as the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 8 At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100 % Sequence identity; and/or, the amino acid sequence of the light chain variable region (VL) of the antibody has at least 80%, at least 85% of the amino acid sequence of the light chain variable region shown in SEQ ID NO: 9 , At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In some specific embodiments, the amino acid sequence of the heavy chain of the anti-4-1BB antibody or antigen-binding fragment thereof according to the present disclosure has at least 70%, at least 75%, at least 80%, at least the sequence shown in SEQ ID NO: 10 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity And/or, the light chain amino acid sequence of the antibody has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least the amino acid sequence shown in SEQ ID NO: 11 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

Bispecific antibody

The present disclosure provides a bispecific antibody that binds the PD-L1 antigen and the 4-1BB antigen.

In some specific embodiments, the bispecific antibody comprises:

1) The first antibody or antigen-binding fragment thereof that specifically binds to the first antigen; and

2) A second antibody or antigen-binding fragment thereof that specifically binds to the second antigen;

The first antigen is 4-1BB, and the second antigen is PD-L1; or, the first antigen is PD-L1, and the second antigen is 4-1BB;

When the first antigen or the second antigen is 4-1BB, the first antibody, the second antibody, or the antigen-binding fragments of the first antibody and the second antibody include those shown in SEQ ID NOs: 1, 2 and 3, respectively. HCDR1, HCDR2, and HCDR3 as shown; and/or LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, 7 and 6, respectively.

In some embodiments, the first antibody or antigen-binding fragment thereof of the above-mentioned bispecific antibody comprises a heavy chain and a light chain; and/or the second antibody or antigen-binding fragment thereof comprises an scFv;

Wherein, the scFv is connected to the N-terminal or C-terminal of the heavy chain or the light chain of the first antibody or its antigen-binding fragment;

Preferably, the first antibody or antigen-binding fragment thereof includes two heavy chains and two light chains, wherein the heavy chain variable region of one heavy chain of the first antibody or antigen-binding fragment thereof is The light chain variable region forms the antigen binding site, and the heavy chain variable region of another heavy chain and the light chain variable region of the other light chain form the antigen binding site.

In some embodiments, the bispecific antibodies of the present disclosure comprise:

1) A first antibody or antigen-binding fragment thereof that specifically binds to a first antigen, the first antibody or antigen-binding fragment thereof including a heavy chain (HC) and a light chain (LC); and

2) An antibody fragment containing a heavy chain variable region (VH) and a light chain variable region (VL) that specifically binds to the second antigen;

Wherein, the antibody fragment is connected to the N-terminal or C-terminal of the heavy chain or light chain of the first antibody;

The first antigen is 4-1BB, and the second antigen is PD-L1; or, the first antigen is PD-L1, and the second antigen is 4-1BB.

In some embodiments, the antibody fragment is Fv, scFv or di-scFv. In some specific embodiments, the antibody fragment is a scFv.

In some embodiments, the bispecific antibody molecule includes one of the first antibody or antigen-binding fragment thereof and two of the scFv; and, the first antibody or antigen-binding fragment thereof includes two HCs and Two LCs, where the heavy chain variable region (VH) of one HC and the light chain variable region (VL) of one LC of the first antibody or its antigen-binding fragment form the antigen binding site, and the heavy chain of the other HC The variable region (VH) and the light chain variable region (VL) of another LC form an antigen binding site.

In some embodiments, the first antibody or antigen-binding fragment thereof is an IgG type antibody, more preferably, an IgG1, IgG2, IgG3, IgG4 type antibody, and more preferably, an IgG1 type antibody.

In some embodiments, one of the scFv is connected to the N-terminus of a heavy chain or light chain of the first antibody or antigen-binding fragment thereof, and the other scFv is connected to the first antibody or antigen-binding fragment thereof The C-terminus of the other heavy chain or light chain.

In some embodiments, the two scFvs are respectively connected to the N-terminus or C-terminus of the two heavy chains or the two light chains of the first antibody or antigen-binding fragment thereof. In some specific embodiments, two of the scFv are respectively connected to the N-terminus of the two heavy chains of the first antibody or antigen-binding fragment thereof. In some specific embodiments, each of the scFv is respectively connected to the C-terminus of the two heavy chains of the first antibody or antigen-binding fragment thereof.

In some specific embodiments, the first antigen is PD-L1, the second antigen is 4-1BB, and the scFv that binds to the second antigen is connected to the N-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof Or C-terminal. In some specific embodiments, the first antigen is 4-1BB, the second antigen is PD-L1, and the scFv that binds the second antigen is connected to the N-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof Or C-terminal.

In some embodiments, two of the scFv are respectively attached to the N-terminus of the two light chains of the first antibody or antigen-binding fragment thereof. In some specific embodiments, two of the scFv are respectively connected to the C-terminus of the two light chains of the first antibody or antigen-binding fragment thereof. In some specific embodiments, one of said scFv is connected to the N-terminus of a heavy chain or light chain of said first antibody or its antigen-binding fragment, and the other said scFv is connected to said first antibody or its antigen binding fragment. The C-terminus of the other heavy or light chain of the fragment.

In some embodiments, when the first antigen or the second antigen is 4-1BB, the first antibody or scFv includes the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. HCDR1, HCDR2, and HCDR3 shown as shown; and/or LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.

In some specific embodiments, the bispecific antibody according to the present disclosure comprises:

1) A first antibody or antigen-binding fragment thereof that specifically binds to a first antigen, the first antibody comprising a heavy chain (HC) and a light chain (LC); and

2) scFv that specifically binds to a second antigen; the bispecific antibody includes one of the first antibody or antigen-binding fragment thereof and two of the scFv; and, the first antibody or antigen-binding fragment thereof includes Two HCs and two LCs, wherein the VH of one HC and the VL of one LC of the first antibody or its antigen-binding fragment form the antigen binding site, and the VH of the other HC and the VL of the other LC form the antigen binding site; The two scFvs are respectively connected to the N-terminals of the two heavy chains of the first antibody or antigen-binding fragment thereof; or, the two scFvs are respectively connected to the two heavy chains of the first antibody or the antigen-binding fragment thereof. The C-terminus of the heavy chain; the first antigen is 4-1BB and the second antigen is PD-L1; or the first antigen is PD-L1 and the second antigen is 4-1BB.

In some specific embodiments, the VH and VL of the scFv are connected via linker 1. In some specific embodiments, the two scFvs are respectively connected to the N-terminus or C-terminus of the two heavy chains of the first antibody or antigen-binding fragment thereof through a linker 2. In some specific embodiments, the structure of the scFv is NH ₂ -VL-linker 1-VH-COOH or NH ₂ -VH-linker 1-VL-COOH.

In some specific embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a CH1 domain, and the light chain comprises a light chain variable region (VL) and a light chain Chain constant region (CL). The antigen-binding fragment of the first antibody may be a Fab fragment, a Fab' fragment or a F(ab')2 fragment. In some specific embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region ( VL) and light chain constant region (CL). The first antibody may be a full-length antibody.

In some specific embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof is of the IgG isotype, such as IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof is of human IgG1 or IgG2 isotype. In some specific embodiments, the light chain of the first antibody or antigen-binding fragment thereof is of Kappa isotype.

In some specific embodiments, the two HCs of the first antibody or antigen-binding fragment thereof comprise the same CDR; and/or, the two LCs of the first antibody or the antigen-binding fragment thereof comprise the same CDR.

In some specific embodiments, the two HCs of the first antibody or antigen-binding fragment thereof comprise the same VH; and/or, the two LCs of the first antibody or antigen-binding fragment thereof comprise the same VL.

In some specific embodiments, the two HCs of the first antibody or antigen-binding fragment thereof have the same amino acid sequence; and/or, the two LCs of the first antibody or the antigen-binding fragment thereof have the same amino acid sequence .

In some specific embodiments, two of the scFvs have the same or different amino acid sequences. In some specific embodiments, two of the scFv have the same amino acid sequence.

In some embodiments, the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein:

a) the two first polypeptide chains each independently comprise the heavy chain (HC) of the first antibody or antigen-binding fragment thereof and the scFv; and

b) The two second polypeptide chains each independently comprise the light chain (LC) of the first antibody or antigen-binding fragment thereof;

Wherein, the scFv is connected to the N-terminus or C-terminus of the HC of the first antibody or its antigen-binding fragment through a linker 2.

In some specific embodiments, the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein:

i) the two first polypeptide chains each independently comprise the light chain (LC) of the first antibody or antigen-binding fragment thereof and the scFv; and

ii) The two second polypeptide chains each independently comprise the heavy chain (HC) of the first antibody or antigen-binding fragment thereof;

Wherein, the scFv is connected to the N-terminus or C-terminus of the LC of the first antibody or its antigen-binding fragment through a linker 2.

In some specific embodiments, the scFv has the structure: NH ₂ -VH-linker 1-VL-COOH or NH ₂ -VL-linker 1-VH-COOH.

In some specific embodiments, the linker 1 and/or linker 2 is a peptide linker, for example, having an amino acid sequence as shown in (G _m S _n ) _x , wherein m and n are each independently selected from 1-8 (E.g., 1, 2, 3, 4, 5, 6, 7 or 8), x is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some specific embodiments, the linker 1 and/or linker 2 has an amino acid sequence as shown in (G ₄ S) _x , and x is independently selected from an integer of 1-6.

In some specific embodiments, the amino acid sequence of the linker 1 is (G ₄ S) ₃ (ie "GGGGSGGGGSGGGGS"), and/or the amino acid sequence of the linker 2 is (G ₄ S) ₄ (ie "GGGGSGGGGSGGGGSGGGGS" ).

In some specific embodiments, there is a disulfide bond between the VH and VL of the scFv. The method of introducing disulfide bonds between the VH and VL of an antibody is well known in the art (see, for example, US5747654; Rajagopal et al., Prot. Engin. 10 (1997) 1453-1459; Reiter et al., Nature Biotechnology. 14 (1996) 1239). -1245; Reiter et al., Protein Engineering. 8 (1995) 1323-1331; Webber et al., Molecular Immunology. 32 (1995) 249-258; Reiter et al., Immunity. 2 (1995) 281-287; Reiter et al., JBC.269 (1994) 18327-18331; Reiter et al., Inter. J. of Cancer. 58 (1994) 142-149; Reiter et al., Cancer Res. 54 (1994) 2714-2718, which is incorporated herein by reference in its entirety).

In some specific embodiments, when the first antibody or scFv binds to 4-1BB, it includes HCDR1 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. HCDR2 and HCDR3; and/or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.

In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds 4-1BB, and the scFv specifically binds PD-L1, wherein:

The first antibody or antigen-binding fragment thereof comprises:

HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 1, 2, 3; LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5 or 7; and, as SEQ ID NO: LCDR3 shown in 6.

In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds PD-L1, and the scFv specifically binds to 4-1BB, wherein the first antibody or antigen-binding fragment thereof comprises:

HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 19, 20, 21; LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 22, 23, 24.

In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds 4-1BB, and the scFv specifically binds PD-L1, wherein the scFv comprises:

In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds PD-L1, and the scFv specifically binds 4-1BB, wherein the scFv comprises:

HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3; LCDR1 shown in SEQ ID NO: 4, such as SEQ ID NO: 5 Or LCDR2 shown in 7; and LCDR3 shown in SEQ ID NO: 6.

In some specific embodiments, the first antibody or antigen-binding fragment thereof specifically binds to 4-1BB, and the scFv specifically binds to PD-L1, wherein the first antibody or antigen-binding fragment thereof comprises:

HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 1, 2, 3; LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 4, 7, 6; and, the scFv includes:

In some specific embodiments, the amino acid sequence of the heavy chain variable region (VH) of the first antibody or antigen-binding fragment thereof has at least 80% of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 8 , At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and The amino acid sequence of the light chain variable region (VL) of the first antibody or its antigen-binding fragment has at least 80%, at least 90%, or at least the amino acid sequence of the light chain variable region shown in SEQ ID NO: 9 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the weight of the scFv The amino acid sequence of the variable chain region (VH) is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, and at least 94 as shown in SEQ ID NO: 25. %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the amino acid sequence of the light chain variable region (VL) of the scFv is the same as SEQ ID NO: The amino acid sequence of the light chain variable region shown by 26 has at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 99%, or 100% sequence identity.

In some embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 8, and/or the light chain variable region is selected from SEQ ID NO: 9 The amino acid sequence shown. In some embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 25, and/or the light chain variable region is selected from SEQ ID NO: 26 The amino acid sequence shown.

In some specific embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 9 The light chain variable region; and, the heavy chain variable region of the scFv is selected from the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region is selected from the light chain variable region shown in SEQ ID NO: 26.

In some specific embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 26 The light chain variable region; and, the heavy chain variable region of the scFv is selected from the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region is selected from the light chain variable region shown in SEQ ID NO: 9.

In some specific embodiments, the bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.

In some specific embodiments, when the first antibody or scFv binds to PD-L1, the first antibody or scFv may be an existing PD-L1 antibody or scFv fragments thereof, biosimilars thereof, and Its antigen-binding fragment. For example, atezolizumab (trade name TECENTRIQTM) is a fully humanized Fc-modified monoclonal antibody of the IgG1 isotype of PD-L1; MDX-1105, which is a fully human binding to PD-L1 Monoclonal antibody; Avirulumab (MSB0010718C, Merck KGaA, Darmstadt, Germany & Pfizer), which is a fully human monoclonal PD-L1 antibody of isotype IgG1; Duvazumab (MedImmune/AstraZeneca), which is Fc optimized Of anti-PD-L1mAb.

In some specific embodiments, the first polypeptide chain has an amino acid sequence selected from the following: SEQ ID NOs: an amino acid sequence shown in any one of 12-13 and 15-17; and/or, the first polypeptide chain The two polypeptide chains have an amino acid sequence selected from the following: SEQ ID NOs: the amino acid sequence shown in any one of 14 and 18.

In some specific embodiments, the bispecific antibody comprises:

(1) The first polypeptide chain shown in SEQ ID NO: 12 and the second polypeptide chain shown in SEQ ID NO: 14; or

(2) The first polypeptide chain as shown in SEQ ID NO: 13 and the second polypeptide chain as shown in SEQ ID NO: 14; or

(3) The first polypeptide chain shown in SEQ ID NO: 15 and the second polypeptide chain shown in SEQ ID NO: 18; or

(4) The first polypeptide chain as shown in SEQ ID NO: 16 and the second polypeptide chain as shown in SEQ ID NO: 18; or

(5) The first polypeptide chain as shown in SEQ ID NO: 17 and the second polypeptide chain as shown in SEQ ID NO: 18.

In some embodiments, the bispecific antibodies of the present disclosure have antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or have complement-dependent cytotoxicity (CDC) activity. In some embodiments, the bispecific antibodies of the present disclosure have reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or have reduced complement-dependent cytotoxicity (CDC) activity. In some specific embodiments, the reduced ADCC and/or CDC activity is caused by mutations in the first antibody CH of the bispecific antibody. Fc mutations of IgG1 such as D265A, N297A, L234A/L235A, L234F/L235A, P329G, etc. can reduce ADCC, and mutations in or near P331S can reduce CDC. IgG2 mutations and IgG2/4 Fc hybrid antibodies can also reduce ADCC and CDC. The core hinge region of IgG4 contains the S228P mutation, which can strengthen the disulfide bond connection in the core hinge region, thereby preventing the exchange of IgG4Fab arms, greatly reducing the formation of half-molecule antibodies, and further introducing F234A and L235A mutations. This form of IgG4 mutant antibody Change the CH2 domain to reduce the interaction with Fc receptors to achieve the effect of reducing ADCC activity.

In some embodiments, the bispecific antibodies of the present disclosure have affinity for 4-1BB and PD-L1. In some specific embodiments, the bispecific antibodies of the present disclosure have the same affinity for 4-1BB and PD-L1 as compared to their respective parent antibodies. In some embodiments, the bispecific antibody of the present disclosure has the same affinity for PD-L1 as compared to its parent antibody. In some specific embodiments, the bispecific antibody of the present disclosure has the same affinity for 4-1BB as compared to its parent antibody. In some specific embodiments, the bispecific antibody of the present disclosure has a weaker affinity for 4-1BB than its parent antibody. In some specific embodiments, the bispecific antibody of the present disclosure has the same affinity for PD-L1 as compared to its parent antibody, and has a weaker affinity for 4-1BB compared to its parent antibody.

On the other hand, the bispecific antibody of the present disclosure has good thermal stability. In some specific embodiments, the bispecific antibodies of the present disclosure have substantially the same thermal stability as the parent antibody.

Expression of anti-4-1BB antibody, its antigen-binding fragment and bispecific antibody

The present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the anti-4-1BB antibody and antigen-binding fragment thereof of the present disclosure.

The present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of the present disclosure that specifically binds 4-1BB and PD-L1.

In some specific embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of the present disclosure, and/or comprises a nucleotide sequence encoding the second polypeptide chain of the present disclosure. In some specific embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of the present disclosure and a nucleotide sequence encoding the second polypeptide chain of the present disclosure.

In another aspect, the present disclosure provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present disclosure. In some specific embodiments, the vector comprises a nucleotide sequence encoding the first polypeptide chain and/or the second polypeptide chain of the present disclosure.

In some specific embodiments, the vectors of the present disclosure are, for example, plasmids, cosmids, bacteriophages and the like. In some specific embodiments, the vector can express the anti-4-1BB antibody and antigen-binding fragments thereof, bispecific antibody, first polypeptide chain or The second polypeptide chain.

In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid molecule of the present disclosure or the vector of the present disclosure. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some specific embodiments, the host cell of the present disclosure is a mammalian cell, such as CHO (e.g. CHO-K1, CHO-S, CHO DG44) or HEK, HEK293, HEK-293F, Expi293F, PER.C6, NSO cell or Lymphocytes.

In another aspect, the present disclosure provides a method for preparing the anti-4-1BB antibody and the antigen-binding fragment thereof of the present disclosure, or the bispecific antibody of the present disclosure, which includes: Fragment, or the bispecific antibody of the present disclosure, culture the host cell of the present disclosure, and recover the anti-4-1BB antibody and its antigen-binding fragment, or the bispecific antibody of the present disclosure from the cultured host cell Specific antibodies.

In some specific embodiments, the method for an anti-4-1BB antibody or an antigen-binding fragment thereof includes constructing an expression vector containing a nucleotide sequence encoding an anti-4-1BB antibody and an antigen-binding fragment thereof, transforming it into a host cell and culturing , Recover the antibody or its antigen-binding fragment from the culture.

In some specific embodiments, the method of preparing the bispecific antibody of the present disclosure includes:

(1) Construct an expression vector comprising a nucleotide sequence encoding the first polypeptide chain and a nucleotide sequence encoding the second polypeptide chain;

(2) Transform the expression vector described in step (1) into a host cell;

(3) Culture the host cell described in step (2) under conditions that allow the expression of the bispecific antibody of the present disclosure; and

(4) Recover the bispecific antibody from the cultured host cell culture.

Optionally, the method for preparing the bispecific antibody of the present disclosure includes:

(1) Constructing a first expression vector containing a nucleotide sequence encoding the first polypeptide chain and a second expression vector containing a nucleotide sequence encoding the second polypeptide chain;

(2) Transform the first expression vector and the second expression vector described in step (1) into host cells;

(4) Recover the bispecific antibody from the cultured host cell culture.

Treatment method and pharmaceutical composition

The present disclosure provides a pharmaceutical composition, which contains the above-mentioned anti-4-1BB antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient, dilution or carrier.

The present disclosure provides a method for treating or preventing diseases, including administering to a subject an effective amount of the aforementioned anti-4-1BB antibody or antigen-binding fragment thereof, or pharmaceutical composition, or antibody-drug conjugate. In some embodiments, the disease is cancer. In some specific solutions, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, Esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

On the other hand, the present disclosure provides the use of the aforementioned anti-4-1BB antibody or antigen-binding fragment thereof in the preparation of a medicine; in some specific embodiments, the medicine is used to treat cancer. In other specific embodiments, the drug is used to treat melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, head and neck cancer, and thyroid cancer. Cancer, esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

The bispecific antibody of the present disclosure can be used for inhibiting the activity of 4-1BB and PD-L1 in vitro or in a subject, blocking the 4-1BB and/or PD-L1 signaling pathway, and for prevention and/or treatment Diseases related to 4-1BB and/or PD-L1 (eg autoimmune diseases or tumors or infectious diseases).

Therefore, in another aspect, the present disclosure provides a pharmaceutical composition, which contains the bispecific antibody of the present disclosure, and a pharmaceutically acceptable carrier and/or excipient. In certain specific embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents. In certain specific embodiments, the additional pharmaceutically active agent is a drug for the prevention and/or treatment of diseases related to 4-1BB and/or PD-L1 (such as autoimmune diseases or tumors or infectious diseases) .

In another aspect, the present disclosure provides the use of the bispecific antibody of the present disclosure or the pharmaceutical composition of the present disclosure in the preparation of a medicament for the treatment of 4-1BB and 4-1BB in a subject (such as a human) / Or PD-L1 related diseases (such as autoimmune diseases or tumors or infectious diseases), and/or used to inhibit the activity of 4-1BB and/or PD-L1 in vitro or in a subject (such as a human).

In another aspect, the present disclosure provides a method for preventing and/or treating 4-1BB and/or PD-L1 related diseases (such as autoimmune diseases or tumors or infectious diseases) in subjects (such as humans). Disease), and/or a method for inhibiting the activity of 4-1BB and/or PD-L1 in vitro or in a subject (such as a human), wherein the method includes administering an effective method to a subject in need An amount of a bispecific antibody of the present disclosure, or a pharmaceutical composition of the present disclosure.

In the present disclosure, the diseases related to 4-1BB and/or PD-L1 include, but are not limited to, autoimmune diseases or tumors or infectious diseases, such as tumors, including but not limited to: adenocarcinoma, leukemia, lymph Tumor, melanoma, sarcoma, or including but not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, Tumors related to prostate, skin, salivary glands, spleen, testes, thymus, thyroid and uterus.

The anti-4-1BB antibody or its antigen-binding fragment, bispecific antibody or pharmaceutical composition of the present disclosure can be formulated into any dosage form known in the medical field, preferably injections (including injections, sterile powder for injection and injections). Use concentrated solutions), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present disclosure should be sterile and stable under production and storage conditions. One specific dosage form is injection. The injection may contain a buffer, and its concentration may be 1-100 mM. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

In addition, the anti-4-1BB antibody or its antigen-binding fragment or bispecific antibody of the present disclosure may be present in a pharmaceutical composition in a unit dosage form for easy administration. In some embodiments, the unit dose may be 0.1-2000mg, 1-500mg, preferably 1-200mg, in the case of the pharmaceutical composition liquid, it may contain a concentration of 1-500mg, preferably 1-200mg Active ingredients. In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the 4-1BB antibody, antigen-binding fragment or bispecific antibody of the present disclosure in the unit measurement.

The anti-4-1BB antibody or its antigen-binding fragment, bispecific antibody or pharmaceutical composition of the present disclosure can be administered by any suitable method known in the art, preferably the route/mode of administration is parenteral administration ( Such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled person should understand that the route and/or manner of administration will vary according to the intended purpose. In a specific embodiment, the bispecific antibody or pharmaceutical composition of the present disclosure is administered by intravenous infusion or injection.

In the present disclosure, the dosage regimen can be adjusted to obtain the best targeted response (e.g., therapeutic or preventive response). For example, it can be administered in a single dose, can be administered multiple times over a period of time, or the dose can be reduced or increased proportionally to the urgency of the treatment situation.

In the present disclosure, the subject may be a mammal, such as a human.

Testing/diagnostic methods and kits

The bispecific antibody of the present disclosure can specifically bind to 4-1BB and/or PD-L1, so that it can be used to detect the presence or level of 4-1BB and/or PD-L1 in a sample, and to diagnose whether a subject is suffering from There are diseases related to 4-1BB and/or PD-L1 (such as autoimmune diseases or tumors or infectious diseases).

In another aspect, the present disclosure provides a kit, which includes the 4-1BB antibody of the present disclosure or an antigen-binding fragment thereof, or a bispecific antibody. In some embodiments, the 4-1BB antibody or antigen-binding fragment thereof, or bispecific antibody bears a detectable label. In some specific embodiments, the kit containing the aforementioned bispecific antibody further includes a second antibody, which specifically recognizes the first antibody or scFv of the bispecific antibody of the present disclosure. In some specific embodiments, the second antibody further includes a detectable label. The detectable label may be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means. Such labels can be applied to immunological detection (for example, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g. , 3H, 125I, 35S, 14C or 32P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g. Cy7, Alexa750)), acridine ester compounds, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., poly Styrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (for example, streptavidin) modified by the above-mentioned label. The markers covered in this disclosure can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation calculator, and fluorescent labels can be detected using a light detector to detect the emitted light. Enzyme markers are generally detected by providing a substrate to the enzyme and detecting reaction products produced by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing colored markers. In some embodiments, the detectable label as described above can be attached to the bispecific antibody of the present disclosure through linkers of different lengths to reduce potential steric hindrance.

In another aspect, the present disclosure provides a method for detecting the presence or level of 4-1BB and/or PD-L1 in a sample, which includes using the 4-1BB antibody of the present disclosure or an antigen-binding fragment thereof, a bispecific antibody A step of. In a preferred embodiment, the 4-1BB antibody or antigen-binding fragment or bispecific antibody thereof of the present disclosure also carries a detectable label. In another preferred embodiment, the method further comprises using a reagent with a detectable label to detect the 4-1BB antibody or antigen-binding fragment or bispecific antibody fragment thereof of the present disclosure. The method can be used for diagnostic purposes, or for non-diagnostic purposes (for example, the sample is a cell sample, not a sample from a patient).

In another aspect, the present disclosure provides a method for diagnosing whether a subject has a disease related to 4-1BB and/or PD-L1 (such as an autoimmune disease or tumor or infectious disease), which includes: using the present disclosure The bispecific antibody detects the presence or level of 4-1BB and/or PD-L1 in a sample from the subject. In a preferred embodiment, the bispecific antibody of the present disclosure also bears a detectable label. In another preferred embodiment, the method further includes the step of using a reagent with a detectable label to detect the 4-1BB antibody or its antigen-binding fragment or bispecific antibody of the present disclosure.

In another aspect, there is provided the use of the 4-1BB antibody or its antigen-binding fragment or bispecific antibody of the present disclosure in the preparation of a kit for detecting 4-1BB and/or PD-L1 in a sample The presence or level of, or used to diagnose whether the subject has a disease related to 4-1BB and/or PD-L1 (such as autoimmune disease or tumor or infectious disease).

Definition of Terms

In order to make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs.

The three-letter codes and one-letter codes of amino acids used in the present disclosure are as described in J. biool. chem, 243, p3558 (1968).

The "antibody" described in the present disclosure refers to an immunoglobulin, which is usually a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE. The corresponding heavy chains are μ chain, δ chain, and γ chain. , Α chain, and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference of the constant region. Each of the five types of Ig can have κ (kappa) chain or λ (lambda) chain.

In the present disclosure, the antibody light chain variable region described in the present disclosure may further comprise a light chain constant region, and the light chain constant region comprises human or murine κ, λ chains or variants thereof.

In the present disclosure, the antibody heavy chain variable region described in the present disclosure may further include a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions. The sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

In the present disclosure, "PD-L1" is also referred to as "Programmed death-ligand 1", "Programmed cell death ligand 1", and "Protein PD-L1". L1", "PD-L1", "PDL1", "PDCDL1", "hPD-L1", "hPD-LI", "CD274" and "B7-H1", and can be used interchangeably. Human, rhesus monkey (cynomolgus monkey), African elephant, wild boar and mouse PD-L1 sequences can be found through Genbank accession numbers, which are NP_054862.1, XP_005581836, XP_003413533, XP_005665023 and NP_068693, respectively.

The term "antibody" is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies can be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

The antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

The term "murine antibody" in the present disclosure is a monoclonal antibody to human PD-L1 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with PD-L1 antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated. In a preferred embodiment of the present disclosure, the murine PD-L1 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine κ, λ chain or a variant thereof, or further comprise murine IgG1 , IgG2, IgG3 or its variant heavy chain constant region.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed, and convert the mouse variable region The gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. In one embodiment, the aforementioned bispecific antibody of 4-1BB/PD-L1 contains human IgG1, or there is no ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity after amino acid mutation). ) Toxic IgG4.

The term "humanized antibody", also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germlines The antibody produced in the antibody framework sequence. It can overcome the strong variable antibody response induced by the chimeric antibody due to the large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase ), and in Kabat, EA, etc. Human, 1991 Sequences of Proteins of Immunological Interest, found in the 5th edition. In order to avoid the decrease of immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation for CDR by phage display.

The "antigen-binding fragment" in the present disclosure refers to Fab, Fv, sFv, F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody peptibody with antigen-binding activity , Domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv). In some embodiments, the "antigen-binding fragment" includes one or more CDR regions selected from SEQ ID NO: 1, 2, 3, 4, 5, and 6 of the antibody described in the present disclosure; or includes one or more CDR regions selected from SEQ ID NO : One or more CDR regions of 1, 2, 3, 4, 7, and 6; or comprising one or more CDR regions selected from SEQ ID NO: 19, 20, 21, 22, 23, 24.

"Fv fragment" refers to the smallest antibody fragment that contains the variable region of the antibody heavy chain and the variable region of the light chain, but does not have the constant region, and has all the antigen binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies into a polypeptide chain, which is called a single chain antibody or single chain Fv (scFv).

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) . Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996) , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

The term "binding to PD-L1" refers to the ability to interact with human PD-L1. The term "binding to 4-1BB" refers to the ability to interact with human 4-1BB. The term "antigen-binding site" in the present disclosure refers to a discrete three-dimensional site on the antigen that is recognized by the antibody of the present disclosure or an antigen-binding fragment thereof.

The term "parent antibody" refers to the anti-PD-L1 antibody or 4-1BB antibody used to prepare the bispecific antibody of the present disclosure. The amino acid sequence of the antibody can be used for example by amino acid substitution or structural change. The first antibody or scFv contained in the bispecific antibody of the present disclosure is prepared.

The CDR, VH, VL, CH, CL, HC, and LC contained in the bispecific antibody of the present disclosure can also be derived from other antibodies or antibody fragments thereof known in the art that can specifically bind PD-L1 or 4-1BB Alternative, or with the above known antibody, its antibody fragment or its CDR, VH, VL, CH, CL, HC, LC has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

"Linker" refers to a linear polypeptide formed by connecting multiple amino acid residues by peptide bonds. The linker of the present disclosure may be an artificially synthesized amino acid sequence, or a naturally-occurring polypeptide sequence, such as a polypeptide having a hinge region function. Such linker polypeptides are well known in the art (see, for example, Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA. 90: 6444-6448; Poljak RJ et al. (1994) Structure 2: 1121-1123) .

"Specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody has a concentration of less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, An affinity (K _D ) of 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less binds the antigen. In the present disclosure, the term "K _D "refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Generally, an antibody (for example, an antibody of the present disclosure) is less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less The dissociation equilibrium constant (K _D ) binds to an antigen (e.g., HBsAg), for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR). The term "k _d "(sec ^-1 ) refers to the dissociation rate constant of a specific antibody-antigen interaction, also known as the k off value.

"ADCC" stands for antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, which means that cells expressing Fc receptors directly kill target cells coated with antibodies by recognizing the Fc segment of antibodies. The ADCC effect function of antibodies can be reduced or eliminated by modifying the Fc section of IgG. The modification refers to mutations in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A, and P329G selected from IgG1; IgG2/4chimera, and F234A/L235A mutations of IgG4.

"CDC" stands for Complement-dependent cytotoxicity. Complement-dependent cytotoxicity refers to a form of cytotoxicity that activates the complement cascade by binding complement component C1q to antibody Fc. Methods for detecting ADCC and CDC activities of antibodies are known in the art. For example, CDC can be evaluated by measuring the binding activity between the antibody to be tested and the Fc receptor (for example, C1q).

The methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as Cold Spring Harbor’s Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human PD-L1, 4-1BB or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The disclosed antibodies or antigen-binding fragments thereof are genetically engineered to add one or more human FR regions to non-human CDR regions. The human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.

In the present disclosure, the engineered antibody or antigen-binding fragment thereof can be prepared and purified by conventional methods. For example, the cDNA sequence encoding the heavy chain and/or light chain can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region. Positive clones stably expressing antibodies are expanded in the serum-free medium of the bioreactor to produce antibodies. The antibody-secreted culture medium can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the PH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions that interact with animals, humans, and recipients. Contact with subjects, cells, tissues, organs or biological fluids. "Administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells. "Administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as a composition containing any one of the combination compounds of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have Therapeutic effect. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, in order to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can assess whether the symptoms of the disease have been alleviated. Although the embodiments of the present disclosure (such as treatment methods or products) may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.

"Conservative modification" or "conservative substitution or substitution" means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein. Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, the substitution of amino acids with similar structure or function is unlikely to disrupt biological activity.

"Effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.

"Exogenous" refers to substances produced outside organisms, cells, or humans according to circumstances. "Endogenous" refers to substances produced in cells, organisms, or human bodies according to circumstances.

"Homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position . The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent homology.

The expressions "cell," "cell line," and "cell culture" are used interchangeably herein, and all such names include progeny. Therefore, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.

"Polymerase chain reaction" or "PCR" as used herein refers to a procedure or technique in which a small amount of a specific portion of nucleic acid, RNA, and/or DNA is amplified as described in, for example, U.S. Patent No. 4,683,195. Generally speaking, it is necessary to obtain sequence information from the end or outside of the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5'terminal nucleotides of the two primers can be identical to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; Erlich edited, (1989) PCR Technology (Stockton Press, N.Y.). PCR used herein is regarded as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, and the method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Produce specific parts of nucleic acid.

"Optional" or "optionally" means that the event or environment described later can but does not have to occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specific sequence may but need not be present.

"Pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art, including But not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffer. Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like. Agents for maintaining osmotic pressure include but are not limited to sugar, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredients and thus the biological activity.

In the present disclosure, "subject" refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the subject (e.g., human) has a disease related to 4-1BB and/or PD-L1, or is at risk of suffering from the above-mentioned diseases. Generally speaking, this type of disease or disease state is characterized in that it will benefit from a reduction in 4-1BB and/or PD-L1 levels or inhibition of 4-1BB and/or PD-L1 activity to be alleviated or cured.

Compared with the prior art, the technical solution of the present disclosure has at least the following beneficial effects:

In the present disclosure, the 4-1BB antibody B1E7 introduces the N53Q mutation to obtain the antibody HR137-07, which reduces the affinity of the antibody by about 2.8 times, reduces the affinity of the antigen and the antibody, and enhances the activation activity of H137-07.

In addition, the bispecific antibody of the present disclosure can not only specifically recognize/bind 4-1BB and PD-L1, but also has a weaker affinity for 4-1BB than its parent antibody, and can significantly and simultaneously activate 4- 1BB and inhibit the activity of PD-L1, activate 4-1BB and block the PD-L1 signal pathway. Moreover, the affinity of the double antibody to human PD-L1 protein is about 50 times higher than that of human 4-1BB protein, which greatly guarantees the effective enrichment of the double antibody at the tumor site, and also greatly reduces the 4-1BB end. Possible side effects. Since 9EN-FM has PD-L1-dependent 4-1BB target cross-linking, the bispecific antibody of the present disclosure even exhibits significantly better activity and reduced activity than its parental antibody combination drug in cell level and in vivo experiments. Antibody toxicity reaction improves the safety of the drug. Therefore, the bispecific antibody of the present disclosure has the potential to treat diseases related to 4-1BB and PD-L1 (such as autoimmune diseases or tumors or infectious diseases) and has great clinical value.

Description of the drawings

Figure 1: The results of the 4-1BB/NF-κB signal pathway activation experiment of antibody HR137-07.

Figures 2A to 2D: schematic diagrams of the structure of the anti-4-1BB/PD-L1 bispecific antibody. Figure 2A is a schematic diagram of the antibody structure of 8E; Figure 2B is a schematic diagram of the antibody structure of 8EN; Figure 2C is a schematic diagram of the antibody structure of 9E-FM; Figure 2D is a schematic diagram of the antibody structure of 9EN-FM and 9EN-FM-1.

Figure 3: SDS-PAGE electropherogram of the bispecific antibody 9EN-FM.

Figure 4A is a graph of the binding ELISA detection result of the bispecific antibody 9EN-FM and PD-L1 protein; Figure 4B is a graph of the binding ELISA detection result of the bispecific antibody 9EN-FM and 4-1BB protein.

Figure 5A is a graph of the SPR detection result of the affinity measurement of the bispecific antibody 9EN-FM and 4-1BB protein; Figure 5B is a graph of the SPR detection result of the affinity measurement of 9EN-FM and PD-L1 protein.

Figure 6: The results of the luciferase reporter gene system detection in the 41BB-NFκB signal pathway activation experiment of the bispecific antibody 9EN-FM.

Figure 7: Experimental results of the luciferase reporter system detecting the blocking effect of the bispecific antibody 9EN-FM on the PD-1/PD-L1 signaling pathway.

Figure 8: The experimental result of detecting the activation of T lymphocytes by the bispecific antibody 9EM-FM.

Figure 9: Results of anti-tumor activity of 9EN-FM-1 on Balbc PD-L1 and 4-1BB double-derived mice.

Detailed ways

The following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure.

The experimental methods without specific conditions in the embodiments or test examples of the present disclosure usually follow conventional conditions or the conditions recommended by the raw material or commodity manufacturers. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents without specific sources are conventional reagents purchased on the market.

Example 1. Preparation of anti-4-1BB antibody

The N53Q mutation was introduced into the CDR2 part of the light chain of the anti-4-1BB antibody B1E7 (derived from PCT/CN2019/072484) sequence using the method of full gene synthesis to obtain the sequence of the antibody HR137-07.

The preparation process of antibody HR137-07 includes:

The heavy chain vector is designed as follows: signal peptide + mutant heavy chain variable region sequence + human IgG1 constant region sequence. The light chain vector is designed as follows: signal peptide+mutated light chain variable region sequence+human Kappa constant region sequence. The above sequences were inserted into the pCEP4 vector. Ask a third-party gene synthesis company to synthesize the expression vector according to the above design, and send the vector plasmid for sequencing verification. The qualified plasmid was transfected into human 293F cells with PEI and cultured continuously, and the 293F cells were cultured with serum-free medium (Shanghai Optima Biotech, OPM-293CD03) to the logarithmic growth phase for cell transfection. Dissolve 21.4 μg of humanized antibody light chain plasmid and 23.6 μ of humanized antibody heavy chain plasmid in 10 mL

Mix well in I Reduced Serum Medium (GIBCO, 31985-070), then add 200ug PEI, mix well, incubate at room temperature for 15 minutes, and add to 50mL cells. The cell culture conditions are: 5% CO2, 37°C, 125 rpm/min. During the culture period, feed was added on the 1st and 3rd day until the cell viability was less than 70%, and the cell supernatant was collected and centrifuged. Load the cell culture fluid after centrifugation and filtration on the antibody purification affinity column, wash the column with phosphate buffer, elute with glycine hydrochloric acid buffer (pH2.70.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH9.0, and Phosphate buffer solution was dialyzed to obtain purified antibody. SDS-PAGE and SEC-HPLC detection confirmed that the antibody is HR137-07.

The CDR sequence of B1E7 is shown in Table 1:

Table 1. CDR sequence of B1E7

名称name	序列sequence	编号Numbering
HCDR1HCDR1	GYTFTSYGLNGYTFTSYGLN	SEQ ID NO：1SEQ ID NO: 1

HCDR2HCDR2	YINPGSGYTKYNEKFEGYINPGSGYTKYNEKFEG	SEQ ID NO：2SEQ ID NO: 2
HCDR3HCDR3	WGLGRNWNFAVWGLGRNWNFAV	SEQ ID NO：3SEQ ID NO: 3
LCDR1LCDR1	RASESVDSYGNTFMHRASESVDSYGNTFMH	SEQ ID NO：4SEQ ID NO: 4
LCDR2LCDR2	RASNLESRASNLES	SEQ ID NO：5SEQ ID NO: 5
LCDR3LCDR3	QQSNEDPLTQQSNEDPLT	SEQ ID NO：6SEQ ID NO: 6

Among them, HCDR is the heavy chain CDR, and LCDR is the light chain CDR. Among them, the number and position of CDR amino acid residues comply with the known Kabat numbering rules (LCDR1-3, HCDR2-3), or the numbering rules of Kabat and chothia (HCDR1). The numbering rules of CDRs in Table 2 are the same as those in Table 1. .

The CDR sequence of HR137-07 is shown in Table 2:

Table 2. CDR sequence of HR137-07

名称name	序列sequence	编号Numbering
HCDR1HCDR1	GYTFTSYGLNGYTFTSYGLN	SEQ ID NO：1SEQ ID NO: 1
HCDR2HCDR2	YINPGSGYTKYNEKFEGYINPGSGYTKYNEKFEG	SEQ ID NO：2SEQ ID NO: 2
HCDR3HCDR3	WGLGRNWNFAVWGLGRNWNFAV	SEQ ID NO：3SEQ ID NO: 3
LCDR1LCDR1	RASESVDSYGNTFMHRASESVDSYGNTFMH	SEQ ID NO：4SEQ ID NO: 4
LCDR2LCDR2	RASQLESRASQLES	SEQ ID NO：7SEQ ID NO: 7
LCDR3LCDR3	QQSNEDPLTQQSNEDPLT	SEQ ID NO：6SEQ ID NO: 6

The heavy chain variable region sequence of HR137-07 is shown below (CDR is underlined):

The light chain variable region sequence of HR137-07 is shown below (CDR is underlined):

The heavy chain sequence of HR137-07 is shown below:

The light chain sequence of HR137-07 is as follows:

The only difference between antibody HR137-07 and B1E7 is that HR137-07 has N53Q mutation in LCDR2.

Example 2. Antigen binding affinity detection of antibody HR137-07

Octet was used to detect the affinity between the anti-4-1BB antibody HR137-07 and its antigen human 4-1BB protein. First, immerse the avidin probe in buffer PBS for 10 minutes to reach equilibrium. Then, immerse in 20 μg/mL biotinylated antigen human 4-1BB protein for 360 seconds to immobilize the antigen on the probe, and immerse in the buffer for 360 seconds to wash away the excess antigen. The antibodies to be tested were diluted to 100nM, 33nM, 10nM, 3.3nM and 1nM, respectively, and added to the black 96-well plate with a volume of 200μL/well, and the 96-well plate was placed into the Octet instrument. Place the probes in each well containing the antibody and bind for 120 seconds. Then put it in the buffer and dissociate for 360 seconds. The obtained affinity is shown in Table 3. The result shows that the affinity of HR137-07 to the antigen human 4-1BB is 11.4 nM. Compared with B1E7, the affinity of HR137-07 with 4-1BB is about 2.8 times lower.

Table 3. Affinity results of B1E7 and HR137-07 with 4-1BB measured by Octet

抗体antibody	KD(M)KD(M)	K _on(1/Ms) K _on (1/Ms)	K _off(1/s) K _off (1/s)
HR137-07HR137-07	1.14E-081.14E-08	1.25E+051.25E+05	1.42E-031.42E-03
B1E7B1E7	4.09E-094.09E-09	1.66E+051.66E+05	6.79E-046.79E-04

Example 3. 4-1BB/NF-κB luciferase reporter gene detection experiment of antibody HR137-07

The full-length human 4-1BB gene (Genbank Accession NO: NM_001561.5) was stably transfected into HEK293 cell line (Thermo fisher scientific) to make HEK293 cell line HEK293F-h4-1BB positive for human 4-1BB protein. The NF-κB luciferase reporter gene plasmid was stably transfected into the HEK293F cell line positive for human 4-1BB protein to make a stable cell line HEK293F- which is positive for human 4-1BB protein and has the NF-κB luciferase reporter gene. h4-1BB-NFκB. Inoculate 50 μL of 4-1BB-NFκB HEK293 cells (5×10 ⁵ /mL) in a 96-well plate. The antibody to be tested was cross-linked by anti-human IgG F(ab) ₂ (Jackson ImmunoResearch). The specific cross-linking method is to incubate with anti-human IgG F(ab)2 (Jackson Immuno Research) at a molar concentration ratio of 1:1.5 at 37°C for 30 minutes before the antibody is added to the cells. And add to the cells and incubate for 5 hours. Add ONE-GLUTM luciferase assay reagent (Promega), and read the fluorescence value with Envision (PerkinElmer, 2150). The result is shown in Figure 1. The results showed that HR137-07 showed stronger activation activity than B1E7, indicating that N53Q reduced the affinity of the antibody to 4-1BB and at the same time increased the antibody's agonistic activity to 4-1BB. Similar to the phenomenon that reduced affinity improves antibody activation activity has also been reported in Fas receptor agonists in the same TNFR family (Cell Death and Differentiation (2012) 19, 1187-1195). N53Q improves the antibody's agonistic activity to 4-1BB, can enhance the antibody's activation of T cells, and enhance the anti-tumor effect.

Example 4. Blood concentration of anti-4-1BB antibody HR137-07 in cynomolgus monkey

Three cynomolgus monkeys (general grade, animal source: Guangxi Guidong Primate Development and Experiment Co., Ltd., Guangzhou Xiangguan Biological Technology Co., Ltd.) were used and divided into two groups. The first group of two, one male and one female, were injected intravenously with B1E7-IgG1. One male in the second group received HR137-07 intravenously. It is administered once a week, the dose is set at 5 mg/kg, and the administration volume is 10 mL/kg (diluted with 0.9% sodium chloride injection). Blood was collected at the following time points: 0h, 0.08 or 2h, 4h or 6h or 8h, 24h, 48h, 96h after the first administration; 0h on the 8th day, 2h after the second administration; 0h on 15 days, 2h after the third administration; 0h on the 21st day, 0h, 0.08h, 0.08 or 2h, 4h or 6h or 8h, 24h, 48h, 96h, 168h after the fourth administration. The blood concentration was detected by ELISA method. The plates were coated with biotin-labeled 4-1BB and incubated at 25°C for 1 hour. After washing with 300μL PBS for three times, add the test serum and incubate at 25°C for 1 hour. After washing with 300 μL PBS for three times, add 100 μL goat anti-human IgG fab solution to each well, seal the plate with sealing film, and incubate at 25°C for 1 hour. The detection concentration of the secondary antibody is 1:30000, and the dilution is 1% BSA-PBST+1% monkey serum.

The results showed that in the first week of administration, the blood concentration trends of B1E7 and HR137-07 were basically the same. However, after the fourth week of administration, the blood concentration of B1E7 decreased rapidly over time, and the blood concentration of HR137-07 decreased slowly. The blood concentration of HR137-07 8h after administration was the blood concentration of B1E7 6h after administration. 43.8-113.8 times of HR137-07, showing that the half-life of HR137-07 is significantly prolonged, see Table 4.

Table 4. Plasma concentration of anti-4-1BB antibody in cynomolgus monkey

Example 5. Preparation of bispecific antibodies that bind 4-1BB/PD-L1

1. The structure of the bispecific antibody that binds to 4-1BB/PD-L1

The bispecific antibodies 8E, 8EN, 9E-FM, 9EN-FM and 9EN-FM-1 that bind 4-1BB/PD-L1 were designed and prepared as follows, all of which are IgG-scFv type double antibodies.

The sequence structure of linker-1 (linker-1) and linker-2 (linker-2) used in the double antibody is:

Linker 1 is three G4S repeats (ie, GGGGSGGGGSGGGGS (SEQ ID NO: 38));

Linker 2 is 4 G4S repeats (ie, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 39)).

Among them, the Fc part of 8E and 8EN are wild-type human IgG1, the first polypeptide chain sequence of 8E and 8EN antibodies are shown in SEQ ID NO: 12 and 13, respectively, and the second polypeptide chain sequence is shown in SEQ ID NO: 14. Show. The Fc parts of 9E-FM, 9EN-FM and 9EN-FM-1 were all mutated to eliminate Fc-mediated cell killing. Among them, the mutations of 9E-FM and 9EN-FM are: L234A, L235A and P329G. The mutations of 9EN-FM-1 are: L234A, L235A. The sequences of the first polypeptide chain of the antibodies of 9E-FM, 9EN-FM, and 9EN-FM-1 are shown in SEQ ID NO: 15, 16 and 17, respectively, and the sequence of the second polypeptide chain is shown in SEQ ID NO: 18 .

The antibody structure of 8E is shown in Figure 2A. The IgG part is anti-human PD-L1 antibody (anti-PD-L1IgG), and the scFv part is composed of the heavy chain variable region (VH) and light chain variable region (VL) of the anti-human 4-1BB antibody (anti-4 -1BB scFv), the heavy chain variable region (VH) and the light chain variable region (VL) are connected by linker 1, and the anti-4-1BB scFv is connected by linker 2 to the C-terminus of anti-PD-L1IgG.

The antibody structure of 8EN is shown in Figure 2B. The difference from 8E is that anti-4-1BB scFv is connected to the N-terminus of anti-PD-L1IgG by linker 2.

The first polypeptide chain of antibody 8E is shown below (the linker is underlined):

The first polypeptide chain of antibody 8EN is as follows:

The second polypeptide chains of antibodies 8E and 8EN are as follows:

The antibody structure of 9E-FM is shown in Figure 2C. The IgG part is anti-human 4-1BB antibody (anti-4-1BB IgG), and the scFv part is composed of the heavy chain variable region (VH) and light chain variable region (VL) of the anti-human PD-L1 antibody (anti- PD-L1scFv), the heavy chain variable region (VH) and the light chain variable region (VL) are connected by linker 1, and the anti-PD-L1 scFv is connected by linker 2 to the C-terminus of anti-4-1BB IgG.

The antibody structures of 9EN-FM and 9EN-FM-1 are shown in Figure 2D. The difference from 9E-FM is that anti-PD-L1 scFv is connected to the N-terminus of anti-4-1BB IgG by linker 2.

The first polypeptide chain of antibody 9E-FM is shown below (linkers are underlined, and L234A, L235A and P329G mutations are in gray):

The first polypeptide chain of antibody 9EN-FM is shown below (linker is underlined, L234A, L235A and P329G mutations are in gray):

The first polypeptide chain of antibody 9EN-FM-1 is shown below (linker is underlined, and L234A and L235A mutations are in gray):

The second polypeptide chains of antibodies 9E-FM, 9EN-FM, and 9EN-FM-1 are as follows:

The CDR sequence of PD-L1 antibody (HRP00052) is shown in Table 5:

Table 5. PD-L1 antibody (HRP00052)

名称name	序列sequence	编号Numbering
HCDR1HCDR1	SYWMHSYWMH	SEQ ID NO：19SEQ ID NO: 19
HCDR2HCDR2	RIGPNSGFTSYNEKFKNRIGPNSGFTSYNEKFKN	SEQ ID NO：20SEQ ID NO: 20
HCDR3HCDR3	GGSSYDYFDYGGSSYDYFDY	SEQ ID NO：21SEQ ID NO: 21
LCDR1LCDR1	RASESVSIHGTHLMHRASESVSIHGTHLMH	SEQ ID NO：22SEQ ID NO: 22
LCDR2LCDR2	AASNLESAASNLES	SEQ ID NO：23SEQ ID NO: 23
LCDR3LCDR3	QQSFEDPLTQQSFEDPLT	SEQ ID NO：24SEQ ID NO: 24

Among them, the number and position of CDR amino acid residues comply with the known Kabat numbering rules.

The light chain variable region sequence of PD-L1 antibody (HRP00052) (CDR is underlined):

2. Preparation of bispecific antibodies that bind 4-1BB/PD-L1

Use CHO-S cells (purchased from Thermo Company, Catalog No. A29133) in a good growth state in the logarithmic growth phase, centrifuge and inoculate 250 mL at 6×10 ⁶ cells/mL. Add solution 2 (dilute 800ul transfection reagent with 9.2mL culture medium, mix well) into solution 1 (dilute 250ug plasmid with 10mL culture medium, mix well), the total volume is 20mL, mix gently and incubate at room temperature for 1-5 minutes , Do not exceed 5 minutes, add the mixed transfection solution dropwise to the cell fluid, and add while shaking. Then place the culture flask on a 5% CO ₂ , 32°C shaker, add 16 mL of supplementary feed (purchased from Thermo Company, product number A29133) for 18-22 hours, and enhancer Enhancer (purchased from Thermo Company, product number A29133) 0.6 mL. On the fifth day, 16 mL of supplementary feed (purchased from Thermo Company, article number A29133) was added, cultured at 123 rpm, 5% CO ₂ , and 32° C. The supernatant was collected by centrifugation on day 12-14. The recombinant antibody was purified by two-step method of affinity chromatography (Protein A) and ion exchange. The media used in the purification are MabSelect SuRe column (GE, 17-5438) and HiTrap Q HP column (GE, 17515601) produced by GE. The PD-L1/4-1BB bispecific antibody 9EN-FM was prepared and detected by 10% SDS-PAGE gel electrophoresis, as shown in Figure 3.

The results show that the theoretical molecular weight of the double antibody molecule is 200kDa, the first polypeptide chain is 75kDa, and the second polypeptide chain is 25kDa. It is judged by the size of the non-reduced band and the size of the reduced band of SDS-PAGE gel electrophoresis. The band size is in line with expectations, indicating that the recombinant bispecific antibody can be assembled and expressed correctly without significant aggregation and degradation.

Example 6. Detection of antigen binding activity of anti-PD-L1/4-1BB bispecific antibody

The binding activity of PD-L1/4-1BB bispecific antibody 9EN-FM with PD-L1 protein and 4-1BB protein was detected by ELISA method. Before the start of the experiment, the 96-well plate was labeled accordingly, and coated with an antigen concentration of 1ug/mL, 50ul per well, and overnight at 4°C in a refrigerator. The next day, take out the coated antigen plate the day before, and wash it with a plate washer (washing solution: 1x PBST). After washing, block with 1% BSA blocking solution configured with 1x PBST at 37°C for 1 hour. After washing the plate with 1x PBST cleaning solution for 3 times, add different dilution concentrations of the double antibody 9EN-FM to be tested, and incubate for 1 hour at 37°C. After washing the plate 3 times with 1x PBST cleaning solution, add 100ul of 1:5000 diluted goat anti-human IgG secondary antibody, incubate at 37°C for 0.5 hours. After washing the plate, take TMB chromogenic solution A and B and mix 1:1 After color development. The color reaction was terminated with 1M hydrochloric acid in 15 minutes. On the SpectraMax M5 multi-function plate reader, the fluorescence value of 450nm was detected. The results are shown in Figure 4A and Figure 4B.

The results showed that the binding activity of the PD-L1/4-1BB bispecific antibody 9EN-FM to the PD-L1 protein and 4-1BB protein respectively maintained the same binding activity as the original parental monoclonal antibody.

Example 7. Detection of antigen binding affinity of anti-PD-L1/4-1BB bispecific antibody

Surface plasmon resonance (SPR) technology was used to detect the affinity between the PD-L1/4-1BB bispecific antibody 9EN-FM and its antigen human PD-L1 protein and human 4-1BB protein (instrument Biacore, . Fix the antigen human PD-L1 protein and human 4-1BB protein to the CM5 chip respectively. The coupling level is set at 100RU, and the instrument Biacore is used for detection (T200, GE Healthcare, BIAC-B20-03). The running buffer is HBS-EP+(10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). The diluted antibody (6.25, 12.5, 25, 50, 100nM) flows through the experimental channel and the control channel at a flow rate of 30μl/min (100nM antigen human PD-L1 protein/antigen human 4-1BB) for 3 minutes, dissociate for 5 minutes. Then use regeneration buffer 10mM glycine pH1.5 (GE Healthcare, BR-1003-54) at a flow rate of 30μl/min Run for 30 seconds. The data is analyzed with Biacore 8K evaluation software. The results are shown in Figure 5A and Figure 5B.

The results show that the affinity of the bispecific antibody to human 4-1BB protein is 3.2nM, and the affinity to human PD-L1 protein is 0.07nM. The affinity of the bispecific antibody to human PD-L1 protein is comparable to that of human 4-1BB The protein is about 50 times higher, which greatly ensures the effective enrichment of the double antibody at the tumor site, and also greatly reduces the toxic side effects that may be caused by the 4-1BB end.

Example 8. Detection of PD-L1-dependent 4-1BB signal activation by anti-PD-L1/4-1BB bispecific antibody in NF-κB luciferase reporter gene experiment

In the HEK293 cell line positive for the human 4-1BB protein used in Example 3, the NF-κB luciferase reporter gene plasmid (Promega, pGL4.32[luc2P/NF-κB-RE/Hygro]Vector ), a stable cell line 4-1BB/NF-κB HEK293 with NF-κB luciferase reporter gene positive for human 4-1BB protein. Co-culture the 4-1BB/NF-κB HEK293 cells with different concentrations of the antibody to be tested for 6 hours, add ONE-GLUTM luciferase assay reagent (Promega), and read the fluorescence value with Envision (PerkinElmer, 2150) . In order to detect PD-L1 mediated activation of 4-1BB, the Hep3B cell line stably and highly expressing PD-L1 was incubated with 4-1BB/NF-κB HEK293. 9EM-FM has a strong activation activity on the 4-1BB signaling pathway in the presence of PD-L1 positive cells, as shown in Figure 6. In the absence of PD-L1 positive cells, 9EN-FM has no activation effect on the 4-1BB signal. The 4-1BB monoclonal antibody HR137-07 used in the bispecific antibody can activate the 4-1BB signal pathway after exogenous cross-linking. The specific cross-linking method is to contact the anti-human IgG F before the antibody is added to the cell. (ab) ₂ (Jackson ImmunoResearch) was incubated at 37°C for 30 minutes at a molar concentration ratio of 1:1.5. Urelumab (BMS-663513, fully human IgG4) can activate the 4-1BB signaling pathway alone, and the degree of activation is enhanced after the addition of exogenous crosslinking.

The above results show that when 9EN-FM is used in vivo, only in areas with high PD-L1 expression, such as tumor tissues, can the activity of 4-1BB be activated.

Example 9. Blocking experiment of anti-PD-L1/4-1BB bispecific antibody on PD-1/PD-L1 signaling pathway

Stable transfection of PD-1 and NFAT luciferase reporter gene plasmids (Promega, pGL4.30[luc2P/NFAT-RE/Hygro]Vector) into Jurkat cells to produce PD-1 expression with NFAT luciferin Stable cell line of enzyme reporter gene. At the same time, a Hep3B cell line with stable and high expression of PD-L1 was added. After adding different concentrations of the antibody to be tested, incubate for 6 hours, add luciferase detection reagent ONE-GLUTM luciferase detection reagent (Promega), and read the fluorescence value with Envision (PerkinElmer, 2150). The result is shown in FIG. 7.

The results showed that 9EN-FM can relieve the inhibition of NFAT fluorescence signal mediated by PD-1/PD-L1 signaling pathway in a dose-dependent manner. The inhibitory activity of 9EN-FM is slightly lower than that of PD-L1 monoclonal antibody (HRP00052) used in bispecific antibodies.

Example 10. T lymphocyte activation experiment

The human peripheral blood mononuclear lymphocytes (StemExpress) were added with 100ng/mL SEB (Kangbo Benin), and the antibody to be tested was added at the same time. In order to verify the PD-L1-mediated activation of 4-1BB, HEK293 cells with high PD-L1 expression were added to peripheral blood mononuclear lymphocytes. HEK293 cells were transiently transfected with PD-L1 plasmid (Lipofectemine 2000, Invitrogen), and treated with 10g/mL mitomycin C (TGI) for 1 hour 24 hours after transfection. After washing three times with PBS, human peripheral blood mononuclear lymphocytes stimulated with SEB were added. After 72 hours of co-cultivation, the secretion of IL-2 (human IL-2kit, cisbio) in the supernatant was detected by ELISA. The result is shown in Figure 8.

The results showed that when HEK293 cells with high PD-L1 expression were not added, 9EM-FM activated T cells stronger than the two monoclonal antibodies HR137-07, PD-L1 monoclonal antibody (HRP00052) alone and the combination of two monoclonal antibodies . When HEK-293 cells with high PD-L1 expression are added to the SEB experiment, the activity of 9EN-FM on T cells will be further enhanced. It further shows that 9EN-FM mediates PD-L1-dependent T cell activation.

Example 11. Anti-tumor activity in 4-1BB/PD-L1 double antibody mice

MC38-hPD-L1 cells were inoculated subcutaneously on the right side of Balbc-hPD-L1/h4-1BB double-derived mice (provided by Biocytometer) at a concentration of 5×10 ⁵ cells/0.1mL, and the tumor grew to approximately At 120mm ³ , 42 mice were randomly selected according to the tumor volume, 6 mice in each group, 7 groups in total: human IgG (7.5mg/kg), HRP00052 (2.25mg/kg), HR137-07 (0.225mg/kg) , HPR00052(2.25mg/kg)+HR137-07(0.225mg/kg), 9EN-FM-1(0.3mg/kg), 9EN-FM-1(3mg/kg) and 9EN-FM-1(10mg/kg) kg). The administration was administered twice a week, and the weight and tumor volume of the mice were measured twice a week during the administration and observation period, as shown in Figure 9, and the measured values were recorded.

To calculate the T/C value, the formula is as follows:

T/C%= average tumor volume of the drug administration group/average tumor volume of the control group%;

CR% (proportion of complete tumor regression) = number of mice with complete tumor regression/number of mice enrolled in the group.

The results showed that 9EN-FM had dose-dependent anti-tumor activity, and the T/C% values of tumors in the 0.3 mpk, 3 mpk and 10 mpk groups were 132.2, 69.9 and 10.7, respectively (see Table 6). The anti-tumor T/C in the high-dose 10mpk administration group was similar to the T/C of the two-drug combination group, 10.7 and 19.6, respectively, but in terms of the rate of complete tumor regression (CR%), 9EN-FM-1 The 10mpk group was 83.3%. It shows that 9EN-FM-1 has better anti-tumor activity than HR137-07 and HRP00052 in combination.

Table 6. Anti-tumor activity of 4-1BB/PD-L1 double antibody and monoclonal antibody combination

Example 12. Single-dose pharmacokinetic study of 4-1BB/PD-L1 bispecific antibody in cynomolgus monkeys

Three male cynomolgus monkeys aged 2-4.5 (general grade, animal source: Guangxi Guidong Primate Development and Experiment Co., Ltd., Guangzhou Xiangguan Biotechnology Co., Ltd.) were used to inject 9EN-FM intravenously. For single administration, the dose is set to 5 mg/kg, and the administration volume is 10 mL/kg (diluted with 0.9% sodium chloride injection). Blood was collected at the following time points: 0h, 0.25h, 2h, 8h, 24h, 48h, 96h, 168 hours, 240 hours, 336 hours, 504 hours, 672 hours after the first administration. The blood concentration was detected by ELISA method. The plates were coated with biotin-labeled 4-1BB and incubated at 25°C for 1 hour. After washing with 300μL PBS for three times, add the test serum and incubate at 25°C for 1 hour. After washing with 300 μL PBS for three times, add 100 μL goat anti-human IgG fab solution to each well, seal the plate with sealing film, and incubate at 25°C for 1 hour. The detection concentration of the secondary antibody is 1:30000, and the dilution is 1% BSA-PBST+1% monkey serum.

The results show that the half-life of 9EN-FM-1 at a dose of 5mpk is 37.3 hours, which is similar to the antibody half-life at the same dose of HRP00052. See Table 7, suggesting that this is mainly due to the condition that the antibody does not reach the target saturation concentration. The distribution of antibodies mediated by this end of L1.

Table 7. Pharmacokinetic parameters of 9EN-FM in cynomolgus monkeys

Other sequence information is as follows:

Human germline heavy chain template IGHV3-30 sequence:

Human germline light chain template IGKV3-11 sequence:

Human germline heavy chain template IGHV1-46 sequence:

Human germline light chain template IGKV4-1 sequence:

The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 8E:

The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 8EN:

The coding nucleic acid sequence of the amino acid sequence of the second polypeptide chain of antibody 8E and antibody 8EN:

The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 9E-FM:

The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 9EN-FM:

The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 9EN-FM-1:

The coding nucleic acid sequence of the amino acid sequence of the second polypeptide chain of antibody 9E, antibody 9EN-FM, and antibody 9EN-FM-1:

Claims

An anti-4-1BB antibody or antigen-binding fragment thereof, comprising:

The heavy chain variable region, which includes the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and/or

The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.
The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 1, which is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody or a fragment thereof.
The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein:

The heavy chain framework region of the antibody or its antigen-binding fragment is derived from the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 29; and/or, the light chain framework region is derived from SEQ ID NO: 28 or SEQ ID NO: 30 amino acid sequence.
The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region of the humanized antibody comprises the heavy chain framework region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof ；

Preferably, it comprises a heavy chain framework region of human IgG1, IgG2 or IgG4;

More preferably, it comprises the heavy chain framework region of human IgG1.
The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 1, wherein:

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and/or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9.
The anti-4-1BB antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein:

1) The anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 10, and/or a light chain as shown in SEQ ID NO: 11;

II) The anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain as shown in a variant of SEQ ID NO: 10, and/or a light chain as shown in a variant of SEQ ID NO: 11; preferably The variant of SEQ ID NO: 10 refers to a variant of the heavy chain variable region shown in SEQ ID NO: 8 that contains 1 to 10 amino acid changes, and/or the variant of SEQ ID NO: 11 It means that the light chain variable region shown in SEQ ID NO: 9 contains 1 to 10 amino acid changes, and the amino acid changes are conservative modifications, substitutions or substitutions;

III) The anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 10, and/or the sequence shown in SEQ ID NO: 11 A light chain with at least 90% sequence identity;

IV) The anti-4-1BB antibody or antigen-binding fragment thereof has a reduced or eliminated ADCC effect; preferably, the antibody has one or more of D265A, N297A, L234A or F/L235A, P329G in the Fc region Mutant IgG1 type antibody, or IgG2/4 Fc region hybrid antibody, or IgG4 type antibody with one or more of S128P, F234A/L235A mutation; or

V) The anti-4-1BB antibody has a reduced or eliminated CDC effect; preferably, the antibody has a P331S mutation in the Fc of IgG1, or is a hybrid antibody to the Fc region of IgG2/4.
An anti-4-1BB antibody or antigen-binding fragment thereof, which competes with the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1-6 to bind to the same epitope.
A polynucleotide encoding the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.
An expression vector containing the polynucleotide of claim 8.
A host cell transformed with the expression vector of claim 9; preferably, the host cell is selected from bacteria, yeast and mammalian cells; more preferably, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.
A method for preparing an anti-4-1BB antibody or an antigen-binding fragment thereof, including the steps:

The expression vector of claim 9 is expressed in the host cell of claim 10, and the anti-4-1BB antibody or antigen-binding fragment thereof isolated and expressed from the host cell.
A pharmaceutical composition comprising the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable excipient, diluent or carrier.
The use of the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the preparation of a medicine or a pharmaceutical composition for the treatment of diseases;

Preferably, the disease is cancer;

More preferably, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer , Cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.
A method of treating diseases, the method comprising:

Administering to a subject a therapeutically effective amount of the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 12;

Preferably, the disease is cancer;

More preferably, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, Cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.
A bispecific antibody comprising:

1) The first antibody or antigen-binding fragment thereof that specifically binds to the first antigen; and

2) A second antibody or antigen-binding fragment thereof that specifically binds to the second antigen;

The first antigen is 4-1BB, and the second antigen is PD-L1; or, the first antigen is PD-L1, and the second antigen is 4-1BB;

When the first antigen or the second antigen is 4-1BB, the first antibody, the second antibody or the antigen-binding fragments of the first antibody and the second antibody include SEQ ID NO:1 and SEQ ID NO: 2 and HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 3; and/or LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.
The bispecific antibody of claim 15, wherein:

The first antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; and

The second antibody or antigen-binding fragment thereof comprises scFv;

Wherein, the scFv is connected to the N-terminal or C-terminal of the heavy chain or the light chain of the first antibody or its antigen-binding fragment;

Preferably, the first antibody or antigen-binding fragment thereof includes two heavy chains and two light chains, wherein the heavy chain variable region of one heavy chain of the first antibody or antigen-binding fragment thereof is The light chain variable region forms the antigen binding site, and the heavy chain variable region of another heavy chain and the light chain variable region of the other light chain form the antigen binding site.
The bispecific antibody according to claim 15 or 16, wherein each bispecific antibody molecule comprises one of the first antibody or antigen-binding fragment thereof and two of the scFv;

In addition, one of the scFv is connected to the N-terminus of the heavy or light chain of the first antibody or its antigen-binding fragment, and the other scFv is connected to the heavy or light chain of the first antibody or its antigen-binding fragment. Or, the two scFvs are respectively connected to the N-terminus of the two heavy chains or the two light chains of the first antibody or its antigen-binding fragment; or, the two scFvs are respectively connected to the The C-terminus of the two heavy chains or the two light chains of the first antibody or antigen-binding fragment thereof.
The bispecific antibody of any one of claims 15-17, wherein:

The variable region of the heavy chain and the variable region of the light chain of the scFv are connected by linker 1, and/or the two scFvs are connected to the two heavy chains of the first antibody or antigen-binding fragment thereof by linker 2 respectively The N terminal or C terminal connection;

More preferably, the structure of the scFv is NH2-VL-linker 1-VH-COOH or NH2-VH-linker 1-VL-COOH, and the linker 1 and the linker 2 may be the same or different.
The bispecific antibody according to any one of claims 15-18, wherein the first antibody or antigen-binding fragment thereof is of IgG isotype, such as IgG1, IgG2, IgG3, or IgG4, preferably, of IgG1 isotype Type; and/or, the light chain of the first antibody or antigen-binding fragment thereof is of Kappa isotype.
The bispecific antibody of any one of claims 15-19, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein:

a) the two first polypeptide chains each independently comprise the heavy chain of the first antibody or antigen-binding fragment thereof and the scFv; and

b) the two second polypeptide chains each independently comprise the light chain of the first antibody or antigen-binding fragment thereof;

Wherein, the scFv is connected to the N-terminus or C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof through linker 2;

or,

i) the two first polypeptide chains each independently comprise the light chain of the first antibody or antigen-binding fragment thereof and the scFv; and

ii) The two second polypeptide chains each independently comprise the heavy chain of the first antibody or antigen-binding fragment thereof;

Wherein, the scFv is connected to the N-terminus or C-terminus of the light chain of the first antibody or its antigen-binding fragment through linker 2;

Preferably, the scFv has the structure: NH2-VH-linker 1-VL-COOH or NH2-VL-linker 1-VH-COOH;

Preferably, the bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains, and the linker 1 and the linker 2 may be the same or different.
The bispecific antibody according to claim 15-20, which has the same or weaker activity of binding to the first antigen than the parent antibody of the first antibody or antigen-binding fragment thereof;

Preferably, the bispecific antibody has the same or weaker activity of binding to the second antigen than the parent antibody of the scFv;

More preferably, the bispecific antibody has the same or weaker activity of binding to the first antigen than that of the parent antibody of the first antibody or antigen-binding fragment thereof, and has the same or weaker activity as the parent antibody of the scFv Compared with the same or weaker binding activity to the second antigen;

More preferably, the bispecific antibody has the same binding activity as that of the parent antibody that binds PD-L1;

More preferably, the bispecific antibody has weaker binding activity than the parent antibody that binds to 4-1BB;

More preferably, the bispecific antibody has the same binding activity as the parent antibody that binds PD-L1, and has weaker binding activity than the parent antibody that binds 4-1BB.
The bispecific antibody according to any one of claims 15-21, wherein the linker 1 and/or the linker 2 is a peptide linker, preferably, the linker 1 and/or the linker 2 has a linker such as ( G m S n ) the amino acid sequence represented by x , wherein m and n are each independently an integer selected from 1-8, and x is an integer selected from 1-20;

Preferably, the linker 1 and/or the linker 2 has an amino acid sequence as shown in (G 4 S) x , and x is an integer selected from 1-6;

Preferably, the linker 1 and/or the linker 2 is (G 4 S) 3 or (G 4 S) 4 ;

Preferably, the linker 1 is (G 4 S) 3 and the linker 2 is (G 4 S) 4 .
The bispecific antibody of any one of claims 15-22, wherein there is a disulfide bond between the VH and VL of the scFv.
The bispecific antibody according to any one of claims 15-23, wherein the first antibody or antigen-binding fragment thereof specifically binds 4-1BB, and the scFv specifically binds PD-L1, wherein The first antibody or antigen-binding fragment thereof includes: HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 1, 2, 3; and/or LCDR1, LCDR2 shown in SEQ ID NO: 4, 7, 6 , LCDR3; and, the scFv includes: HCDR, HCDR2, HCDR3 shown in SEQ ID NO: 19, 20, 21, and/or LCDR1, shown in SEQ ID NO: 22, 23, 24, respectively LCDR2, LCDR3.
The bispecific antibody of any one of claims 15-23, wherein the first antibody or antigen-binding fragment thereof specifically binds PD-L1, and the scFv specifically binds 4-1BB, wherein

The first antibody or antigen-binding fragment thereof includes: HCDR, HCDR2, HCDR3 shown in SEQ ID NO: 19, 20, 21, and/or, respectively, shown in SEQ ID NO: 22, 23, 24 LCDR1, LCDR2, LCDR3; and, the scFv includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 1, 2, 3; and/or, LCDR1, as shown in SEQ ID NO: 4, 7, 6 LCDR2, LCDR3.
The bispecific antibody of any one of claims 15-23, wherein the scFv comprises:

I) VH as shown in SEQ ID NO: 25, and/or VL as shown in SEQ ID NO: 26; or

II) VH as shown in SEQ ID NO: 8, and/or VL as shown in SEQ ID NO: 9.
The bispecific antibody of any one of claims 15-23, wherein:

I) The first antibody or antigen-binding fragment thereof includes the VH shown in SEQ ID NO: 8 and the VL shown in SEQ ID NO: 9; and, the scFv includes the VH shown in SEQ ID NO: 25 VH and VL as shown in SEQ ID NO: 26; or

II) The first antibody or antigen-binding fragment thereof includes the VH shown in SEQ ID NO: 25 and the VL shown in SEQ ID NO: 26; and, the scFv includes the VH shown in SEQ ID NO: 8 VH and VL as shown in SEQ ID NO: 9.
The bispecific antibody according to any one of claims 15-27, having reduced ADCC activity, and/or reduced CDC activity.
The bispecific antibody of any one of claims 15-28, wherein the Fc region of the first antibody contains mutations;

Preferably, the Fc region of the first antibody or antigen-binding fragment thereof has one or more of the following mutations: mutation at position 265 to A, mutation at position 297 to A, mutation at position 234 to A or F, Mutation at position 235 to A or E, mutation at position 329 to G;

More preferably, the Fc region of the first antibody or antigen-binding fragment thereof has one or more of the following mutations: D265A, N297A, L234A or L234F, L235A or L235E, P329G;

More preferably, the Fc region of the first antibody or antigen-binding fragment thereof has the following mutations: L234A/L235A.
The bispecific antibody of any one of claims 15-29, wherein the bispecific antibody comprises:

1) The first polypeptide chain as shown in SEQ ID NO: 12 and the second polypeptide chain as shown in SEQ ID NO: 14; or

II) The first polypeptide chain as shown in SEQ ID NO: 13 and the second polypeptide chain as shown in SEQ ID NO: 14; or

III) The first polypeptide chain as shown in SEQ ID NO: 15 and the second polypeptide chain as shown in SEQ ID NO: 18; or

IV) The first polypeptide chain shown in SEQ ID NO: 16 and the second polypeptide chain shown in SEQ ID NO: 18; or

V) The first polypeptide chain shown in SEQ ID NO: 17 and the second polypeptide chain shown in SEQ ID NO: 18.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 15-30;

Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of the bispecific antibody of any one of claims 20-30, and/or encoding any one of claims 20-30 The nucleotide sequence of the second polypeptide chain of the bispecific antibody; more preferably, the isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO: 31-32, 34-36; and/or SEQ ID NO: the sequence shown by 33 and 37.
A vector comprising the isolated nucleic acid molecule of claim 31.
A host cell comprising the isolated nucleic acid molecule of claim 31 or the vector of claim 32.
A method for preparing the bispecific antibody according to any one of claims 15-30, which comprises culturing the host cell according to claim 33 under conditions that allow the expression of the bispecific antibody, and obtaining from the cultured host The bispecific antibody is recovered from the cell culture.
A pharmaceutical composition comprising the bispecific antibody according to any one of claims 15-30, and a pharmaceutically acceptable carrier and/or excipient.
Use of the bispecific antibody according to any one of claims 15-30 or the pharmaceutical composition according to claim 35 in the preparation of a medicament for the treatment of diseases in a subject;

Preferably, the disease is an autoimmune disease or tumor or infectious disease, such as tumor, including but not limited to solid tumor or blood cancer, or including but not limited to adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, or including But not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, skin, salivary gland, spleen, testis , Thymus, thyroid and uterine related tumors, infectious diseases include but not limited to hepatitis B, hepatitis A, HIV;

Preferably, the subject may be a mammal, such as a human.
A method for treating a disease in a subject, wherein the method comprises administering an effective amount of the bispecific antibody of any one of claims 15 to 30 to a subject in need thereof, or claims The pharmaceutical composition described in 35;

Preferably, the disease is an autoimmune disease or tumor or infectious disease, such as tumor, including but not limited to solid tumor or blood cancer, or including but not limited to adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, or including But not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, skin, salivary gland, spleen, testis , Thymus, thyroid and uterine related tumors, infectious diseases including but not limited to hepatitis B, hepatitis A, HIV;

Preferably, the subject may be a mammal, and more preferably, the subject is a human.