Anti-TNFR2 / anti-PD-1 or CTLA-4 bispecific antibody and application thereof

Abstract

Provided herein are anti-TNFR2 / anti-PD-1 or anti-CTLA-4 bispecific antibodies, and uses thereof. In particular, provided herein is a bispecific antibody (BsAb) against TNFR2 and against PD-1 or CTLA-4 comprising: (A) a TNFR2 binding domain comprising, for example, a binding fragment corresponding to an antigen binding fragment in an anti-TNFR2 monoclonal antibody P07262, P07263, P07268, P07282, P07291, P08940, or P07298, or a combination thereof, and (B) a PD-1 or CTLA-4 binding domain. Also provided are bispecific antibodies, coding sequences thereof, vectors and cells, related products and uses thereof (e.g., for cancer diagnosis, prevention and / or treatment).

Description

Anti-TNFR2 / anti-PD-1 or CTLA-4 bispecific antibodies and their applications Technical Field

[0001] The present invention belongs to the fields of biotechnology and medicine. Specifically, the present invention relates to anti-TNFR2 and anti-PD-1 or CTLA-4 bispecific antibodies, compositions or products comprising the antibodies, and uses thereof. Background Art

[0002] TNFRSF1B (TNF receptor superfamily member 1b or CD120b) is a member of the TNF receptor superfamily, also known as TNFR2. TNFR2 is a type I transmembrane glycoprotein composed of 439 amino acids (accession number UniProtKB-P01857), of which the first 235 amino acids constitute the extracellular region, forming four cysteine-rich domains (CRD). Each CRD contains 6 cysteines, forming three pairs of disulfide bonds. The unique spatial structure of CRD determines the specific binding between TNF and the receptor. CRD2 and CRD3 are the sites where the receptor binds to TNF (Mukai et al., Science Signaling 148: 1-10, 2010). Although CRD1 and CRD4 are not directly involved in the recognition and binding of TNF, their absence will lead to a significant decrease in the receptor's ability to bind to TNF.

[0003] TNFRSF1B's ligand, TNFα, primarily participates in apoptosis by recognizing TNFR1 and relies on TNFR2 to exercise functions related to immune system regulation. Studies have found that in mice, the loss of TNFR2 leads to reduced function of regulatory T cells (Tregs), thereby exacerbating autoimmune diseases and colitis. In humans, reduced TNFR2 expression or reduced binding to TNFα impairs TNFR2-mediated regulatory T cell signaling and is closely associated with autoimmune diseases such as systemic lupus erythematosus (SLE), Crohn's disease, and ulcerative colitis. In addition, TNFR2 is highly upregulated in cancer. Within the tumor microenvironment, TNFR2 is expressed on certain tumor cells and immune cells, including Tregs, CD8+ cytotoxic T cells, Bregs, NK cells, MDSCs (myeloid-derived suppressor cells), and other cell types, such as endothelial cells and their precursors, endothelial progenitor cells (ECs, EPCs), and mesenchymal stem cells (MSCs). These cells play important roles in tumor immune evasion, tumor growth, and tumor progression.

[0004] Currently, there are two major categories of anti-TNFR2 antibodies with therapeutic potential, namely TNFR2 antagonists and TNFR2 agonists, which have different properties and mechanisms of action. Existing preclinical data show that TNFR2 antagonists can be used to treat proliferative diseases, such as cancer or infectious diseases. WO2014 / 124134 discloses a TNFR2 antagonist that has the function of enriching lymphoid immune cells and specifically killing Treg cells in the tumor microenvironment. WO2017 / 197331 discloses an antagonistic TNFR2 antibody with a specific sequence of complementary determining region-heavy chain 3 (CDR-H3), which can reduce or inhibit Treg proliferation and / or promote the proliferation of T effector cells. On the other hand, agonistic TNFR2 antibodies can activate Treg cells and inhibit effector T cells, thereby helping to treat immune diseases. For example, WO2017 / 040312 discloses agonistic anti-TNFR2 antibodies that can promote TNFR2 signal transduction and promote the expansion or proliferation of Tregs, thereby treating autoimmune diseases and providing a new target for immunotherapy. In addition, studies have shown that agonistic TNFR2 antibodies can bind to any TNFR2-expressing immune cells, such as CD8-positive T cells.

[0005] Most of the TNFR2 antibody drugs currently under study are monoclonal antibodies. Combining them with other immune checkpoint inhibitors may solve the problems of off-target toxicity, low response rate and drug tolerance of monoclonal antibodies and achieve a clinical effect of 1+1>2. It has been reported that in mouse CT26 and MC38 tumor models, the anti-tumor effect of the combination of PD-1 antibody and TNFR2 antibody is better than that of PD-1 antibody or TNFR2 antibody alone. Its mechanism of action is to reduce Treg cells in the tumor microenvironment and increase the proportion of CD8+T cells (Katherine Case et al., J Leukoc Biol 2020 Jun, 107(6): 981-991). Similarly, in a mouse pancreatic cancer model, the combination of PD-L1 antibody and TNFR2 antibody can effectively treat pancreatic ductal adenocarcinoma (Zhang X et al., Journal for ImmunoTherapy of Cancer 2022;10:e003982.doi:10.1136 / jitc-2021-003982). BoInvent's TNFR2 antagonist antibody BI-1808 is currently in Phase I clinical trials. In addition to monotherapy, combination therapy with PD-1 inhibitors is also a primary development strategy. BeiGene's TNFR2 antagonist antibody BITR2101 has also initiated clinical trials in combination with the PD-1 antibody tislelizumab. Virbacter's TNFR2 antagonist antibody LBL-019, Simcere Pharmaceuticals' TNFR2 antagonist antibody SIM-235, and Arno Pharmaceuticals' TNFR2 antagonist antibody AN3025 have all demonstrated synergistic effects in combination with PD-1 antibodies in mouse tumor models, further enhancing the anti-tumor activity of TNFR2 antibodies. Immune checkpoint inhibition through PD-1 and CTLA-4 antibodies is one of the most widely used cancer immunotherapies. Similar to the combination of TNFR2 and PD-1 antibodies, the combination of TNFR2 and CTLA-4 antibodies may also achieve better results than monotherapy, making it a highly promising combination therapy.

[0006] Bispecific antibodies (BsAbs) are a type of genetically engineered antibody that can specifically bind to two antigens or two different epitopes of the same antigen. Currently, BsAbs have become a hot topic in the field of antibody engineering, with broad application prospects in the treatment of autoimmune diseases, tumors, and other diseases.

[0007] Currently, there are no reports of TNFR2 bispecific antibodies. However, the development of bispecific antibodies with highly effective anti-tumor biological activity has significant advantages in efficacy and cost compared to the combination therapy of monoclonal antibodies, and has significant clinical significance and broad market potential.

[0008] Summary of the Invention

[0009] The present invention provides a bispecific antibody that can simultaneously and specifically target TNFR2 and either PD-1 or CTLA-4. Its potent anti-tumor biological activity has been demonstrated through in vitro and in vivo experiments. Compared to combination therapy, bispecific antibodies offer significant advantages in both efficacy and cost, possessing significant clinical significance and promising a broad market potential.

[0010] In some aspects of the present invention, an anti-TNFR2 and anti-PD-1 or CTLA-4 bispecific antibody (BsAb) is provided, comprising:

[0011] (A) TNFR2 binding domain; and

[0012] (B) CTLA-4 binding domain or PD-1 binding domain.

[0013] In some embodiments, the TNFR binding domain in the anti-TNFR / CTLA-4 bispecific antibody utilizes an anti-TNFR monoclonal antibody disclosed herein or a functional fragment thereof (antigen-binding fragment). For example, the TNFR2 binding domain comprises any one of the VH CDRs, VL CDRs, VH, and VL selected from Tables 1, 2, and / or 3.

[0014] In some aspects of the present invention, a nucleic acid molecule or combination thereof is provided, which encodes the bispecific antibody of the present invention.

[0015] In some aspects of the present invention, a vector or cell is provided, comprising the bispecific antibody or fragment thereof and / or nucleic acid molecule or a combination thereof of the present invention, for example, the cell is a chimeric antigen receptor (CAR) cell.

[0016] In some aspects of the present invention, a product is provided, comprising the bispecific antibody or fragment thereof, nucleic acid molecule or combination thereof, vector or cell of the present invention. The product may be selected from: a pharmaceutical composition, a kit, a drug box. Alternatively, the product may be used for: the production of the bispecific antibody, the production of derivatives comprising the bispecific antibody (e.g., CAR cells), disease treatment, and / or disease detection.

[0017] In some aspects of the present invention, provided are uses of bispecific antibodies or fragments thereof, nucleic acid molecules or combinations thereof, vectors or cells or products as described herein for preparing drugs for preventing and / or treating diseases associated with TNFR2 and / or PD-1 or CTLA-4 overexpression (e.g., cancer, infectious diseases).

[0018] Those skilled in the art may arbitrarily combine the above technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention will be obvious to those skilled in the art due to the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below with reference to the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention and are not intended to limit the scope of the present invention.

[0020] Figure 1: Flow cytometric analysis of binding of anti-human TNFR2 mouse antibodies to membrane TNFR2.

[0021] Figure 2 shows flow cytometric analysis of the effect of anti-human TNFR2 mouse antibodies on the binding of recombinant human TNFa to cell membrane TNFR2.

[0022] Figure 3 shows flow cytometric analysis of the binding of anti-human TNFR2 humanized monoclonal antibodies to membrane TNFR2.

[0023] Figure 3A: Humanized antibody based on mouse anti-11G2;

[0024] Figure 3B: Humanized antibody based on mouse anti-12E12;

[0025] Figure 3C: Humanized antibody based on mouse anti-33C12.

[0026] Figure 4 shows the effect of anti-human TNFR2 humanized monoclonal antibody in inhibiting mouse tumor growth in vivo:

[0027] Figure 4A: Humanized antibody 11G2-P07263;

[0028] Figure 4B: Humanized antibody 33C12-P07298.

[0029] Figure 5: Schematic diagram of the structure of an exemplary anti-TNFR2 / anti-PD-1 or CTLA-4 bispecific antibody of the present application:

[0030] Figure 5A: Configuration 1 anti-TNFR2 / anti-PD-1 BsAb;

[0031] Figure 5B: Configuration 1 anti-TNFR2 / anti-CTLA-4 BsAb;

[0032] FIG5C : Configuration 2 anti-TNFR2 / anti-PD-1 or CTLA-4 BsAb.

[0033] FIG6 : SDS-PAGE method is used to identify the preparation of anti-TNFR2 / anti-PD-1 bispecific antibodies of configuration 1 and configuration 2.

[0034] FIG6A shows configuration 1 anti-TNFR2 / anti-PD-1 BsAb; wherein lanes 1 and 2 show non-reduced and reduced configuration 1 anti-TNFR2 / anti-PD-1 (pembrolizumab) BsAb, respectively;

[0035] Figure 6B: Configuration 2 anti-TNFR2 / anti-PD-1 BsAb; lanes 1 and 2 represent non-reduced and reduced Configuration 2 anti-TNFR2 / anti-PD-1 (pembrolizumab) BsAb, respectively; lanes 3 and 4 represent non-reduced and reduced Configuration 2 anti-TNFR2 / anti-PD-1 (nivolumab) BsAb, respectively.

[0036] FIG7 : SDS-PAGE method is used to identify the preparation of anti-TNFR2 / anti-CTLA-4 bispecific antibodies of configuration 1 and configuration 2.

[0037] Figure 7A: Configuration 1 anti-TNFR2 / anti-CTLA-4 BsAb;

[0038] Figure 7B: Configuration 2 anti-TNFR2 / anti-CTLA-4 BsAb,

[0039] Lane 1: human IgG1 (non-reduced); Lane 2: human IgG1 (reduced); Lane 3: anti-TNFR2 / anti-CTLA-4 BsAb (non-reduced); Lane 4: anti-TNFR2 / anti-CTLA-4 BsAb (reduced).

[0040] Figure 8: ELISA study showing the antigen binding activity of anti-TNFR2 / anti-PD-1 BsAb of Configuration 1

[0041] Figure 8A: Binding activity study with TNFR2;

[0042] Figure 8B: Binding activity study with PD-1.

[0043] Figure 9: ELISA study showing the antigen binding activity of anti-TNFR2 / anti-PD-1 BsAb of configuration 2

[0044] Figure 9A: Binding activity study with TNFR2;

[0045] Figure 9B: Binding activity study with PD-1.

[0046] Figure 10: ELISA study showing the binding activity of anti-TNFR2 / anti-CTLA-4 BsAb of Configuration 1 to CTLA-4

[0047] Figure 11: ELISA study showing the antigen binding activity of anti-TNFR2 / anti-CTLA-4 BsAb of Configuration 2

[0048] Figure 11A: Binding activity study with TNFR2;

[0049] FIG11B : Binding activity study with CTLA-4.

[0050] Figure 12: Sandwich ELISA study showing the antigen binding activity of anti-TNFR2 / anti-PD-1 BsAbs of configurations 1 and 2

[0051] Figure 12A: Study on the bispecific antibody binding activity of configuration 1;

[0052] FIG12B : Study on the binding activity of the bispecific antibody of configuration 2.

[0053] FIG13 shows a sandwich ELISA study of antigen binding activity of configuration 1 anti-TNFR2 / anti-CTLA-4 BsAb.

[0054] Figure 14: Flow cytometric analysis of binding of anti-TNFR2 / anti-PD-1 BsAbs of configurations 1 and 2 to membrane TNFR2.

[0055] Figure 15 shows flow cytometric analysis of binding of configuration 2 anti-TNFR2 / anti-CTLA-4 BsAb to membrane TNFR2 and membrane CTLA-4.

[0056] Figure 15A: Cell membrane TNFR2;

[0057] Figure 15B: Cell membrane CTLA-4.

[0058] Figure 16 shows the effects of TNFR2 / PD-1 BsAbs of configurations 1 and 2 on IFNγ production by exhausted human T lymphocytes, and a comparison of the effects with TNFR2 mAb alone, PD-1 mAb alone, and the combination of TNFR2 mAb + PD-1 mAb. DETAILED DESCRIPTION

[0059] The present disclosure provides bispecific antibodies, antibody fragments, and related compositions, molecules, and products that can simultaneously and specifically bind to TNFR2 and PDv1 or CTLA-4. Specifically, the present disclosure first constructs an anti-TNFR2 monoclonal antibody with high specificity, high affinity, and efficient inhibitory effect on TNFR2. The antigen-binding active fragment of the anti-TNFR2 monoclonal antibody is used to form bispecific antibodies of different configurations with the antigen-binding active fragment of the anti-PD-1 or CTLA-4 antibody through genetic engineering. The bispecific antibodies of the present disclosure have high specificity and high affinity for both TNFR2 and PD-1 or TNFR2 and CTLA-4, and can effectively block the binding of TNFR2 to its ligand CD155, as well as the binding of PD-1 to receptors such as PDL-1 or CTLA-4 to the B7 ligand, thereby effectively enhancing the release of cytokines from immune cells, increasing immune cell activity, and enhancing immune responses. The efficient anti-tumor biological activity of the bispecific antibodies of the present disclosure has been verified by in vivo and in vitro experiments, and in some embodiments, the bispecific antibodies also produce additive or synergistic effects. In addition, compared with combination therapy, bispecific antibodies have greater advantages in efficacy and cost, and have significant clinical significance and broad market space.

[0060] Thus, the present disclosure provides methods for preventing and / or treating diseases related to the expression or overexpression of TNFR2 and PD-1 or CTLA-4, such as tumors, infections, or infectious diseases. The bispecific antibodies of the present disclosure can also reduce or eliminate cells (e.g., Treg cells) expressing TNFR2 and / or PD-1 or CTLA-4 or their activity.

[0061] The present disclosure also provides a method for enhancing immune response, preventing and / or treating tumors, infections or infectious diseases, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of the bispecific antibody or derivative thereof (e.g., a chimeric antigen receptor drug) described herein.

[0062] All numerical ranges provided herein are intended to expressly include all values ​​falling between the endpoints of the ranges and ranges therebetween. Features described herein or in the embodiments may be combined. All features disclosed herein may be used in any combination, and any feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are intended only to be general examples of equivalent or similar features.

[0063] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0064] Monoclonal antibodies, functional fragments thereof and their preparation

[0065] The term "monoclonal antibody (mAb)" as used herein refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody.

[0066] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0067] As used herein, the term "variable" refers to certain portions of the variable region in an antibody that differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-sheet structure. The CDRs in each chain are closely together through the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0068] As used herein, the term "target molecule" refers to a molecule that can be specifically bound by an antibody or its targeting domain. Herein, the target molecule may be located on cells that express or overexpress TNFR2, CTLA-4, or both. Preferably, the target molecule is exposed to the exterior of the target cell or is enriched thereon. In some embodiments, the target molecule is expressed on cancer cells or infected cells. In some embodiments, the target molecule is TNFR2, CTLA-4, or both.

[0069] As used herein, the term "sequence identity" or "% identity" refers to the percentage of identical residues (e.g., amino acids or nucleic acids) between a candidate sequence and a reference sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity. For example, as used herein, "at least 70% sequence identity" means that the sequence identity between the candidate sequence and the reference sequence is greater than 70%, such as 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any numerical value therein.

[0070] The present disclosure provides monoclonal antibodies, preferably humanized antibodies, that specifically bind to human TNFR2. In the present disclosure, unless otherwise specified, "m" indicates a murine portion and "h" indicates a human portion. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs), and a light chain variable region (VL) comprising three CDRs.

[0071] In some embodiments, the anti-TNFR2 monoclonal antibody comprises any one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, VLCDR3, a combination of VH CDR1-3 thereof (3CDR combination), a combination of VL CDR1-3 (3CDR combination), or a combination of VH CDR1-3 and VL CDR1-3 (i.e., 6CDR combination), VL, VH, or a combination of VL and VH selected from Table 1, Table 2, or Table 3.

[0072] In some embodiments, the anti-TNFR2 monoclonal antibody comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NO: 16, 20, 24, 36, 40, 44, or 56, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the anti-TNFR2 monoclonal antibody comprises a light chain variable region (VL) selected from the group consisting of SEQ ID NO: 28, 32, 48, 52, or 60, or an amino acid sequence having at least 80% sequence identity thereto.

[0073] In some embodiments, the anti-TNFR2 monoclonal antibody comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

[0074] (a) a combination of SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO: 28 or an amino acid sequence having at least 80% sequence identity thereto;

[0075] (b) a combination of SEQ ID NO: 20 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO: 28 or an amino acid sequence having at least 80% sequence identity thereto;

[0076] (c) a combination of SEQ ID NO: 24 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO: 32 or an amino acid sequence having at least 80% sequence identity thereto;

[0077] (d) a combination of SEQ ID NO: 36 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO: 48 or an amino acid sequence having at least 80% sequence identity thereto;

[0078] (e) a combination of SEQ ID NO:40 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO:52 or an amino acid sequence having at least 80% sequence identity thereto;

[0079] (f) a combination of SEQ ID NO: 44 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO: 52 or an amino acid sequence having at least 80% sequence identity thereto;

[0080] (g) A combination of SEQ ID NO: 56 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO: 60 or an amino acid sequence having at least 80% sequence identity thereto.

[0081] The novel anti-TNFR2 antibodies provided herein can stimulate signals transduced by TNFR2 and modulate the activity of cells expressing TNFR2. Some antibodies can significantly promote TNFα-induced apoptosis in TNFR2-expressing cells, while others have a significant protective effect against TNF-induced apoptosis. The antibodies of the present invention can also enhance cytokine release from immune cells, increasing immune cell activity and enhancing immune responses. In vivo anti-tumor MC-38 studies in mice demonstrated that the antibodies of the present invention have a significant inhibitory effect on tumors, demonstrating their potential for therapeutic use.

[0082] Various anti-PD-1 monoclonal antibodies or active fragments thereof may be used in this application. In some embodiments, exemplary anti-PD-1 monoclonal antibodies may include, but are not limited to, commercially available monoclonal antibodies, such as pembrolizumab or nivolumab, or active fragments thereof. In some embodiments, for example, the anti-PD-1 monoclonal antibody may comprise a VL CDR, VH CDR, VH, and / or VL selected from pembrolizumab or nivolumab, or a fragment or combination thereof having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity with the aforementioned sequences and having PD-1 binding activity.

[0083] Various anti-CTLA-4 monoclonal antibodies or active fragments thereof can be used in this application. In some embodiments, exemplary anti-CTLA-4 monoclonal antibodies comprise one or more fragments selected from the group consisting of: the CTLA-4 binding domain comprises one or more fragments selected from Table 9, or fragments having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity to the aforementioned sequences and having CTLA-4 binding activity, or a combination thereof. In some embodiments, the anti-CTLA-4 monoclonal antibody or active fragment thereof comprises a sequence selected from any one of SEQ ID NOs: 64-71, or any combination thereof.

[0084] Bispecific antibodies

[0085] As used herein, the term "bispecific" refers to an antibody that has the ability to specifically interact with two different ligands simultaneously.

[0086] After obtaining the high-affinity, high-specificity, and high-efficiency anti-TNFR2 monoclonal antibodies and antigen-binding fragments thereof described herein (e.g., as described in the previous section), they can be combined with anti-CTLA-4 antibodies or antigen-binding fragments thereof using known bispecific antibody construction methods and configurations to construct the bispecific antibodies herein.

[0087] The terms "antigen-binding domain" and "antigen-binding portion" of an antibody are used interchangeably and refer to one or more portions of an antibody, as used herein, that have specific binding affinity for TNFR2 or CTLA-4. Portions of intact antibodies have been shown to be capable of performing the antigen-binding function of an antibody. Examples of antigen-binding portions include, but are not limited to: (i) a Fab portion, a monovalent portion, comprising the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 portion, a bivalent portion, comprising two Fab portions connected to each other by a disulfide bridge at the hinge region; (iii) an Fd portion, comprising the VH and CH1 domains; (iv) an Fv portion, comprising the FL and VH domains of a single arm of an antibody; (v) a dAb portion, consisting of the VH domain or consisting of VH, CH1, CH2, DH3 or VH, CH2, CH3 (dAbs, or single-domain antibodies, contain only the VL domain that have also been shown to specifically bind to a target epitope).

[0088] In some embodiments, the TNFR2 binding domain and / or PD-1 or CTLA-4 binding domain is an antibody fragment selected from the group consisting of: a Fab fragment; a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment; a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; a Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment; isolated CDRs; and scFv.

[0089] According to the number of antigen binding sites in BsAb, BsAb can be bivalent, trivalent, tetravalent or even more valent. In terms of the format of bispecific antibodies, they can be mainly divided into three categories: (1) Fusion proteins assembled from antigen binding regions (Fab), which can avoid problems such as light and heavy chain mispairing. At the same time, since they do not contain glycosylated Fc segments, they can be produced using relatively simple prokaryotic or low-level mammalian cells, with high yield and low cost; (2) Symmetric BsAb, which fuses antibody fragments on the basis of naked antibodies, and generally adopts tetravalent 2+2; (3) Asymmetric BsAb, whose structure mimics natural antibodies, lacks non-natural antibody domains or linkers, and is considered to be the lowest immunogenic structure. Generally, asymmetric bispecific antibodies are bivalent 1+1, that is, the antibody binds to two antigens in a monovalent form. It is also the structure that pharmaceutical companies currently use more in development to retain the properties of natural antibodies as much as possible.

[0090] Although the two domains of the Fv portion (i.e., VL and VH) are encoded by different genes, they can be synthesized using a synthetic linker (e.g., poly-G4S amino acid sequence (GGGGS) n , n = any integer from 1 to 10) and recombinant methods to link them to each other, making it possible to prepare them as one protein chain in which the VL and VH domains combine to form a monovalent molecule (called single-chain Fv (ScFv)). The term "antigen-binding portion" of an antibody also encompasses such single-chain antibodies.

[0091] Other forms of single-chain antibodies, such as "diabodies," are also encompassed herein. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to combine the two domains on the same chain, thereby forcing the domains to pair with complementary domains from different chains to form two antigen-binding sites. An immunoglobulin constant region refers to a heavy or light chain constant region. The amino acid sequences of the human IgG heavy and light chain constant regions are known in the art.

[0092] In some embodiments, the bispecific antibodies herein may not contain an Fc segment (non-IgG-like structure). Such bispecific antibodies have the advantages of simple production, high yield, and low cost. For example, the bispecific antibodies herein may be formed by two single-chain antibodies ScFv connected by a short peptide chain, and may be antibody dimers (diabodies) composed of two cross-linked single-chain antibodies ScFv. Bispecific antibodies of this structure may be further modified to improve their stability, for example, by introducing cysteine ​​at the C-terminus of their peptide chains to form a disulfide bond between VH and VL.

[0093] In some embodiments, the bispecific antibodies herein comprise an Fc fragment, i.e., bispecific antibodies with an IgG-like structure. Such bispecific antibodies retain the corresponding functions of the Fc fragment and have higher stability. Such bispecific antibodies may include, but are not limited to, knob-into-hole (KIH), scFv2-Fc, triomabs, dual-variable domain immunoglobulins (DVD-Ig), and the like.

[0094] In some embodiments, the bispecific antibodies herein employ a knob-and-hole structure, wherein mutations (e.g., S354C and T366W mutations) are introduced into the heavy chain CH3 region of one of the two antibodies, the anti-TNFR2 antibody and the anti-PD-1 or CTLA-4 antibody, and the smaller amino acids are replaced with larger amino acids to form a knob structure. Mutations are then introduced into the heavy chain CH3 region of the other antibody, and the larger amino acids are replaced with smaller amino acids to form a hole structure. The spatial structural complementarity of the hole-like structure is then utilized to achieve the correct assembly of the two antibodies.

[0095] In some embodiments, the bispecific antibodies herein have a knob-in-hole configuration 1 (see Figures 5A and 5B ), with an engineered Fc and a CrossMab. Specifically, exemplary configuration 1 bispecific antibodies have the following structural features (all mutations are numbered using EU numbering):

[0096] ——The anti-PD-1 or CTLA-4 antibody heavy chain variable domain is fused to the human antibody light chain constant lambda domain and the human IgG1 hinge domain -CH2-CH3 (knob heavy chain) containing the mutations S354C and T366W (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3), and the anti-PD-1 or CTLA-4 antibody light chain variable domain is fused to the CH1 of human IgG1 (SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6) through two amino acids (-Ser-Ser-), to form the antigen binding domain of one of the antibodies of the bispecific antibody in the form of a crossmab.

[0097] The other half of the bispecific antibody is composed of an anti-TNFR2 antibody heavy chain (hole heavy chain) containing IgG1 Fc mutations Y349C, T366S, L368A, and Y407V (SEQ ID NO: 7 or SEQ ID NO: 8) and an anti-TNFR2 antibody light chain (SEQ ID NO: 9).

[0098] The two amino acids L234 / L235 in the CH1 region of the two heavy chains of TNFR2 / PD-1 BsAbs were replaced by the amino acid Ala (LALA variant), abolishing the antibody's ADCC / CDC function;

[0099] ——The knob heavy chain and the hole heavy chain form heterodimers, thereby forming anti-TNFR2 / anti-PD-1 or CTLA-4 BsAbs.

[0100] In some embodiments, the bispecific antibodies herein have a configuration 2 of an IgG-scFv structure (or scFv-IgG) (see Figure 5C ). Specifically, exemplary configuration 2 bispecific antibodies have the following structural features:

[0101] - The C-termini of both heavy chains of an anti-PD-1 antibody (IgG4) (SEQ ID NO: 10 or SEQ ID NO: 11) or both heavy chains of a CTLA-4 antibody (IgG1) (SEQ ID NO: 12) are linked to an anti-TNFR2 antibody scFv fragment;

[0102] The heavy chain and scFv, and the VH and VL of scFv are connected by n GGGGS repeating (n is an integer) sequence flexible peptides;

[0103] These heavy chains form homodimeric bispecific antibodies with the anti-PD-1 antibody light chain (SEQ ID NO: 13 or SEQ ID NO: 14) or the CTLA-4 antibody light chain (SEQ ID NO: 15), respectively.

[0104] The bispecific antibodies herein can be identified by conventional means. For example, the binding specificity of the bispecific antibodies can be determined by flow cytometry, immunoprecipitation, in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)), or biofilm interferometry analysis (BLI).

[0105] The bispecific antibodies herein can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0106] Bispecific antibody or fragment encoding molecule, expression vector containing the molecule and host cell

[0107] Also provided herein are nucleic acid molecules encoding the anti-TNFR2 / anti-PD-1 or CTLA-4 bispecific antibodies or fragments thereof. The sequences of these nucleic acid molecules can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies. Also disclosed herein are expression vectors containing the aforementioned nucleic acid molecules, such as pcDNA3.4 and pcDNA3.1; and host cells transformed with the aforementioned expression vectors, such as CHO cells and COS cells.

[0108] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0109] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0110] Currently, it is possible to obtain nucleic acid sequences encoding the bispecific antibodies (or fragments thereof, or derivatives thereof) described herein entirely by chemical synthesis. This sequence can then be introduced into various existing molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences described herein by chemical synthesis.

[0111] This document also relates to vectors comprising the above-mentioned coding sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express the protein. The host cells can be prokaryotes, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, for example, CHO cells, COS cells, etc. Mammalian cell lines are commonly used as host cells for expressing eukaryotic cell-derived polypeptides. The propagation of mammalian cells in culture is well known in the art. See "Tissue Culture", Academic Press, edited by Kruse and Patterson (1973), which is incorporated herein by reference. Preferred mammalian cells are the numerous commercially available immortalized cell lines. These immortalized cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells, Vero cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (such as Hep G2), and many other cell lines. They provide post-translational modifications to protein molecules, including correct folding, correct disulfide bond formation, and glycosylation at the correct sites.

[0112] There are many methods for transforming host cells with expression vectors, and the transformation procedure used depends on the host to be transformed. The method of importing heterologous polynucleotides into mammalian cells is known in the art, and includes dextran-mediated transfection, calcium phosphate precipitation, Polybrene (1,5-dimethyl-1,5-diazoundecamethylene polybromide) mediated transfection, protoplast fusion, electroporation, liposome-mediated transfection, and direct microinjection of DNA into the nucleus. In this article, preferred methods are electroporation or liposome-mediated transfection. For example, the liposome-mediated transfection kit of Invitrogen can be used to transfect host cells such as COS and CHO cells.

[0113] The transformed host cells are cultured under conditions suitable for expression of the bispecific antibodies herein. The antibodies herein are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0114] Conjugate, chimeric antigen receptor, and immune cell expressing chimeric antigen receptor

[0115] The present application also provides conjugates comprising the antibodies or antigen-binding fragments thereof herein, chimeric antigen receptors, and immune cells expressing the chimeric antigen receptors. Due to the specificity of the antibodies or antigen-binding fragments thereof of the present application for TNFR2 and PD-1 or CTLA-4, the conjugates, chimeric antigen receptors, and immune cells expressing the chimeric antigen receptors can target cells or tissues expressing or overexpressing TNFR2 and / or PD-1 or CTLA-4, and preferably produce additive or synergistic effects. Furthermore, since the antibodies or antigen-binding fragments thereof of the present application themselves have biological activity (e.g., immunomodulatory activity), the antibodies or antigen-binding fragments thereof in the conjugates, chimeric antigen receptors, and immune cells expressing chimeric antigen receptors can also play a corresponding role.

[0116] In some embodiments, an antibody conjugate may comprise: an antibody or antigen-binding fragment thereof as described herein; a conjugated moiety, such as a drug, toxin, cytokine, radionuclide, or enzyme; and optionally, a linker. In some embodiments, the conjugation is achieved by enzymatic conjugation, chemical conjugation, fusion, or the like. The linker may be a "cleavable linker" that facilitates release of the conjugated moiety in cells, such as an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, and a disulfide-containing linker.

[0117] In some embodiments, the conjugate moiety attached to the antibody may be, for example, a bioactive agent, which may be any synthetic, recombinantly produced, or naturally occurring substance that enhances and / or modulates a desired biological effect. In some embodiments, the bioactive agent may provide effects including, but not limited to, regulating, stimulating, and / or inhibiting growth signals; regulating, stimulating, and / or inhibiting anti-apoptotic signals; regulating, stimulating, and / or inhibiting apoptotic or necrosis signals; regulating, stimulating, and / or inhibiting ADCC cascades; regulating, stimulating, and / or inhibiting CDC cascades, etc. For example, it may be a chemotherapeutic agent, a toxin, a cytokine, an isotope, an enzyme, etc.

[0118] Also provided in the present application is a chimeric antigen receptor (CAR), which includes an extracellular domain and an intracellular domain, wherein the extracellular domain includes an antibody or its antigen-binding fragment of the present application. In some embodiments, the CAR of the present application also includes a transmembrane domain connecting the extracellular domain and the intracellular domain, such as the transmembrane portion of CD28. The CAR of the present application may be a first generation, second generation, third generation or fourth generation CAR.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof in the present application CAR is scFv or VH sdAb, or is composed thereof.In some embodiments, the intracellular domain of CAR includes a signal that simulates or approximates a natural antigen receptor, a signal by such receptors in combination with a costimulatory receptor, and / or a signal by a separate costimulatory receptor.

[0120] In some embodiments, the intracellular domain includes one or more costimulatory domains and activation domains, such as one or more selected from the group consisting of an ITAM domain, CD3ζ, CD28, 4-1BB, OX40, CD27, ICOS, or a combination thereof, such as (CD28+CD3ζ), (CD28+CD27+CD3ζ), (CD28+OX40+CD3ζ), (CD28+4-1BB+CD3ζ), (CD28+CD27+OX40+CD3ζ), (CD28+4-1BB+CD27+CD3ζ), (CD28+4-1BB+OX40+CD3ζ), (4-1BB+CD3ζ), (4-1BB+OX40+CD3ζ), (4-1BB+CD27+CD3ζ), (CD27+CD3ζ), (CD27+OX 40+CD3ζ), (CD28Δ+CD3ζ), (CD28Δ+CD27+CD3ζ), (CD28Δ+OX40+CD3ζ), (CD28Δ+4-1BB+CD3ζ), ( CD28Δ+4-1BB+OX40+CD3ζ), (CD28Δ+CD27+OX40+CD3ζ), (CD28Δ+4-1BB+CD27+CD3ζ)), (4-1BB+ ICOS+CD3ζ), (CD28+ICOS+CD3ζ), (ICOS+CD3ζ).

[0121] In some embodiments, the extracellular domain of CAR further comprises a hinge region, for example, the hinge region is derived from the hinge of IgG or the extracellular region of CD8α / CD28, such as selected from: IgG4 FcΔEQ, IgG4 FcΔQ, (t-12AA+t-20AA), mKate, phiLov, dsRed, Venus, eGFP, CH3 HA, (CD8α+t-20AA), double t-20AA, (t-20AA+CD8α), (CD8α+leucine zipper Basep1), (CD8α+leucine zipper Acid1), 2D3, CD8α or IgG4 Fc.

[0122] Also provided herein is a transformed immune cell expressing the chimeric antigen receptor of the present application, for example, the immune cell is selected from a T cell, such as an αβT cell, a γδT cell, or a NK T cell, or a T cell derived from a pluripotent cell. The immune cell may be from an individual to be treated or from other sources. The transformed (optionally expanded) immune cell may be administered to the individual to be treated, for example, for adoptive therapy.

[0123] In addition, this application also includes CAR constructs, expression vectors, preparation methods and applications for transforming immune cells.

[0124] Pharmaceutical composition

[0125] Also provided herein is a pharmaceutical composition comprising a pharmaceutically effective amount of the antibody or immunoconjugate thereof and a pharmaceutically acceptable carrier.

[0126] As used herein, the term "pharmaceutically acceptable" means that when the molecular entities and compositions are properly administered to animals or humans, they do not produce adverse, allergic or other untoward reactions. As used herein, a "pharmaceutically acceptable carrier" should be compatible with the active substance herein, i.e., it can be blended with it without significantly reducing the effectiveness of the pharmaceutical composition under normal circumstances. Such carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0127] Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof. Additionally, auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, and the like, may also be present in these carriers.

[0128] The compositions herein can be administered orally, intravenously, intramuscularly, or subcutaneously; preferably, they can be administered orally or by intravenous injection. The pharmaceutical compositions herein can be prepared into various dosage forms as needed, and a physician can determine the dosage that is beneficial to the patient based on factors such as the patient's type, age, weight, general condition, and route of administration.

[0129] When using a pharmaceutical composition, a safe and effective amount of the antibody or immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally about 0.1 μg to 5 mg / kg body weight, and in most cases does not exceed about 3 mg / kg body weight. Preferably, the dose is about 1-10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0130] As used herein, the term "unit dosage form" refers to a dosage form in which the composition of the present disclosure is prepared for single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release dosage forms.

[0131] In another preferred embodiment of the present disclosure, the composition is in unit dosage form or multiple dosage form, and the active substance content is 0.01 to 2000 mg / dose, preferably 0.1 to 1500 mg / dose, and more preferably 1 to 1000 mg / dose. In another preferred embodiment of the present disclosure, 1 to 6 doses of the composition of the present disclosure are administered daily, preferably 1 to 3 doses; most preferably, the daily dose is 1 dose.

[0132] The bispecific antibodies or immunoconjugates described herein can effectively block the binding of TNFR2 to its ligand CD155, and can also effectively block the binding of PD-1 to PD-L1 or CTLA-4 to the B7 ligand, thereby enhancing the release of cytokines from immune cells, increasing the activity of immune cells, and enhancing the immune response, and are used to prevent and / or treat tumors, infections, or infectious diseases. The active substances herein can be used to treat various types of tumors, such as (including but not limited to): adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma; tumor tissue from the adrenal gland, gall bladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary gland, spleen, testicle, thymus, thyroid or uterus; tumors of the central nervous system, such as glioma, or astrocytoma; eye tumors (e.g., basal cell carcinoma, squamous cell carcinoma or melanoma), endocrine gland tumors, neuroendocrine system tumors, gastrointestinal pancreatic endocrine system tumors, reproductive system tumors or head and neck tumors.

[0133] Detection kit

[0134] Also provided herein is a kit for detecting TNFR2 and / or PD-1 or CTLA-4, comprising the bispecific antibody, active fragment thereof, or immunoconjugate described herein. The kit herein can be used to detect the presence or absence of TNFR2, PD-1, or CTLA-4, or the simultaneous presence or amount of TNFR2, PD-1, or CTLA-4 in a biological sample. The detection method comprises the steps of: (a) contacting the sample with the bispecific antibody or immunoconjugate described herein; and (b) detecting the formation of a specific antigen-antibody complex (e.g., a TNFR2 antigen-antibody complex, a PD-1 antigen-antibody complex, a CTLA-4 antigen-antibody complex, a TNFR2 / PD-1 antigen-antibody dual complex, and / or a TNFR2 / CTLA-4 antigen-antibody dual complex), wherein the formation of a complex indicates the presence of TNFR2 and / or PD-1 or CTLA-4 in the sample, or quantitatively detecting the amount of the formed antigen-antibody complex reflects the amount of TNFR2 and / or PD-1 or CTLA-4 in the sample. Particularly preferred is the use of the kit herein to detect the simultaneous presence of TNFR2 and PD-1 or CTLA-4. The sample to be tested may or may not have been pretreated, for example, it may have been extracted, purified or concentrated.

[0135] The kit comprises a container, a bispecific antibody described herein, or a detection plate containing the antibody, located within the container, and instructions for use. The kit may also contain other reagents required for detection, such as a buffer, an indicator, and the like. Those skilled in the art may adjust the contents of the kit according to specific needs.

[0136] Example

[0137] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make appropriate modifications and variations to the present invention, and these modifications and variations are within the scope of the present invention.

[0138] For experimental procedures in the following examples where specific conditions are not specified, conventional methods in the art may be employed, for example, as described in Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, New York, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and are also available from commercial companies.

[0139] Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0140] Example 1. Acquisition and performance determination of anti-TNFR2 monoclonal antibodies and their coding sequences

[0141] 1.1 Obtaining anti-TNFR2 monoclonal antibodies and their coding sequences

[0142] The anti-TNFR2 antibodies are multiple anti-TNFR2 humanized monoclonal antibodies developed and prepared by the applicant. The preparation method and amino acid sequence of the antibodies can be found in the Chinese invention patent application (CN202111105848.7, "Anti-TNFR2 monoclonal antibodies and their applications", application date September 22, 2021).

[0143] Specifically, according to conventional methods, 10 Balb / c mice (6-8 weeks old, weighing about 18 g, female, purchased from the Experimental Animal Management Department of the Family Planning Science Research Institute) were treated with 50 μg of recombinant human TNFR2-Fc (Shanghai Saijin Biopharmaceutical Co., Ltd. The protein (batch number A100114U, the same below) was emulsified in complete Freund's adjuvant (CFA) at 100 μL of protein and 100 μL of CFA, and multiple immunizations were performed intraperitoneally and subcutaneously (50 μL per injection). Immunizations were also performed in incomplete Freund's adjuvant (IFA) at weeks 2, 3, 4, and 5, with 100 μL of protein emulsified and 100 μL of IFA.

[0144] The spleen cells and lymphocytes of immunized mice were fused with mouse myeloma cells (purchased from the cell bank of the Chinese Academy of Sciences, product number TCM18). The specific steps are as follows: 72 hours after the sprint immunization (week 6, subcutaneous injection of 25μg TNFR2-Fc fusion protein), the mouse eyeballs were bled, the neck was stretched and killed, and disinfected. The spleen was removed aseptically, the surrounding connective tissue was peeled off, and the spleen cells were washed. The spleen cells were cut into pieces and ground, blown away, filtered, centrifuged, resuspended, and counted. The myeloma cells passaged 24 hours ago were mixed with the mouse spleen cells, centrifuged, the cell supernatant was discarded, and preheated in a 37°C water bath. 1ml of PEG preheated to 37°C was added dropwise to the centrifuge tube within 60s, gently stirred for 1min, and the culture medium was added. Let it stand for 10min and centrifuged. The supernatant was discarded, and the cells were resuspended in IMDM medium containing 20% ​​FBS (containing 1× HAT (Sigma, H0262-10VL) and 1× double antibody (Gibco, 15140122)). The cells were aliquoted into 25 96-well cell culture plates and incubated (200 μl / well, 5% CO2, 37°C). On days 5 to 10 after fusion, the medium was completely replaced with IMDM medium containing 10% FBS (containing 1× HAT and 1× double antibody) according to the cell growth density.

[0145] On the 10th to 14th day after fusion, ELISA test was performed according to the cell growth density. The cells in the positive wells were subcloned for the first and second time. The ELISA test steps are as follows: coated with TNFR2-His (Sino Biological, 10417-H08H, Accession# NP_001057.1.Metl-Asp257, C-terminal expression of polyhistidine tag), the ELISA plate was blocked, and the hybridoma supernatant was incubated (100 μl / well added to the ELISA plate, sealed with a sealing film and placed at 37°C for 1 hour), and then incubated with goat anti-mouse HRP secondary antibody (Jackson Immuno Research, 115-035-164; diluted with binding solution 1:3000, 100 μl / well added to the ELISA plate, 37°C for 1 hour), TMB color development, and detection with an enzyme reader: dual wavelength 450nm / 655nm detection, the results are expressed as OD 450 -OD 655 calculate.

[0146] After two rounds of subcloning, multiple antibodies that bind to hTNFR2 were screened, and three of them were sequenced. These three antibodies are 11G2, 12E12, and 33C12.

[0147] Total RNA was extracted from hybridoma cells (11G2, 12E12, and 33C12) and reverse transcribed. The resulting cDNA was used as a template to amplify the VH and VL genes of the hybridoma antibodies and sequence the VH and VL sequences of the TNFR2 mouse monoclonal antibody. Prior to humanization, we constructed a recombinant human-mouse chimeric antibody expression plasmid and incorporated the hybridoma antibody V region into a chimeric antibody expression vector (pcDNA3.1). Specifically, the V region sequence was linked to an expression vector containing either human IgG1 or human λ / κ constant regions. The chimeric antibodies were transiently expressed and purified in HEK293F cells.

[0148] Design humanized antibody sequences based on the mouse parent antibody sequence. The specific steps are as follows: First, use Discovery Studio and Antibody Modeling uses homology modeling to construct a three-dimensional molecular model of the variable region. Next, by comparing existing antibody structures in the database, structural simulations are performed on the variable region and CDR of the parent antibody. At the same time, cDNA-derived human germline sequences with high homology to the murine parent antibody VH and VL are selected for comparison. For the heavy chain VH of 11G2, IGHV1 with the highest homology was selected as the humanization design template, and the sequence was designed; for the light chain VL, IGKV1 was selected as the humanization design template, and the sequence was designed. For the heavy chain VH of 12E12, IGHV1 with the highest homology was selected as the humanization design template, and the sequence was designed; for the light chain VL, IGLV7 and IGKV3 were selected as the humanization design template, and the sequence was designed. For the heavy chain VH of 33C12, IGHV3 with the highest homology was selected as the humanization design template, and the sequence was designed; for the light chain VL, IGKV3 was selected as the humanization design template, and the sequence was designed.

[0149] The humanized sequences are shown in Tables 1 to 3:

[0150] Table 1. VH and VL sequences of humanized antibodies based on 11G2

[0151] Table 2. VH and VL sequences of humanized antibodies based on 12E12

[0152] Table 3. VH and VL sequences of humanized antibodies based on 33C12

[0153] The above humanized VH and VL were combined to obtain the corresponding humanized antibodies:

[0154] Table 4. Combinations of different heavy and light chains of humanized antibodies and their corresponding numbers

[0155] Based on the humanized design results, the sequence was constructed into the pcDNA3.4 vector. Plasmids were obtained after PCR, enzyme digestion, ligation, transformation, identification, sequencing, alignment, and extraction. Based on the humanized expression profile, the plasmids were expressed in ExpiCHO-S cells using the ExpiCHO-S Expression System (Thermo Fisher, A29133) for 7 days as required. Expression levels were measured by HPLC on day 6 of expression. Finally, the antibody protein was purified by Protein A affinity chromatography.

[0156] 1.2 Flow cytometry was used to study the binding of human-mouse chimeric monoclonal antibodies to TNFR2 protein on the cell membrane

[0157] In order to determine whether the human-mouse chimeric TNFR2 monoclonal antibody of the present invention can bind to the TNFR2 protein on the cell membrane, an in vitro test was performed.

[0158] Purified anti-human TNFR2 human-mouse monoclonal chimeric antibodies were co-incubated with TNFR2-positive Jurkat cells (Jurkat cells were transfected with the pcDNA3.4-TNFR2 plasmid using conventional methods, and a Jurkat cell line with stable and high TNFR2 expression was obtained after screening). Antibody binding strength was analyzed by flow cytometry. The specific steps were as follows: Jurkat-TNFR2 cells were counted, centrifuged, resuspended, and added to a 96-well conical-bottom plate. The supernatant was discarded. The anti-TNFR2 antibody was serially diluted and added to a 96-well conical-bottom plate. After incubation and washing, a secondary antibody, FITC-anti-human IgG (Abcam: 6854), was added. Samples were detected using a Beckman CytoFLEX.

[0159] Experimental results and analysis:

[0160] The results are shown in Figure 1. The experimental results show that the human-mouse chimeric TNFR2 monoclonal antibodies 11G2-hFc, 12E12-hFc and 33C12-hFc can specifically bind to TNFR2 on the cell membrane and bind to active EC 50 They are: 0.022 μg / mL (0.148 nM), 0.012 μg / mL (0.083 nM), and 0.66 μg / mL (4.4 nM) respectively.

[0161] The results show that the TNFR2 monoclonal antibody of the present invention can specifically bind to TNFR2 on the cell membrane.

[0162] 1.3 Flow cytometry study of the effect of human-mouse chimeric TNFR2 monoclonal antibody on the binding of TNFα to TNFR2 on the cell membrane

[0163] In order to detect whether the human-mouse chimeric TNFR2 monoclonal antibody of the present invention can inhibit the binding of TNFα to TNFR2, we used flow cytometry to detect the effect of the antibody on the binding of TNFα to TNFR2 on the cell membrane.

[0164] Purified anti-human TNFR2 human and mouse monoclonal antibodies were co-incubated with Jurkat cells transfected with TNFR2 (Jurkat cells were transfected with the pcDNA3.4-TNFR2 plasmid using conventional methods, and a Jurkat cell line with stable and high TNFR2 expression was obtained after screening). Antibody binding strength was analyzed by flow cytometry. The specific steps were as follows: cells were counted, centrifuged, resuspended, plated into a 96-well conical-bottom plate, and the supernatant discarded. After incubation and washing with serially diluted anti-TNFR2 antibodies and a fixed concentration of TNFα fusion protein, the cells were then incubated with a secondary antibody, PE-streptavidin (Biolegend, 405203). Samples were detected using a Beckman CytoFLEX.

[0165] Experimental results and analysis:

[0166] As shown in FIG2 , the experimental results show that the human-mouse chimeric TNFR2 monoclonal antibodies 11G2-Fc, 12E12-Fc and 33C12-Fc did not block the binding of TNFα to TNFR2 on the cell membrane.

[0167] The results showed that the binding of the TNFR2 monoclonal antibody of the present invention to TNFR2 did not block the binding of TNFR2 to TNFα.

[0168] 1.4 Biofilm Interferometry (BLI) to study the binding activity of antibodies

[0169] The dissociation constant (Kd) of chimeric and humanized TNFR2 monoclonal antibodies binding to recombinant human TNFR2 was determined using a GATOR (ProbeLife) instrument and HFC (LOT#2007065 Tray 2) Human Antibody Capture probe. Monoclonal antibodies were diluted to 30 nM in binding buffer (Q buffer [PBS (10 mM pH 7.4) + 0.02% Tween 20 + 0.2% BSA]. Recombinant human TNFR2 protein (His tag) was serially diluted two-fold in Q buffer to varying concentrations ranging from 40 nM to 10 nM. Kinetic association assays were initiated by placing the antibody capture sensor in the serially diluted antigen solution.

[0170] Table 5 shows the binding kinetic parameters of humanized antibodies designed based on the murine parent antibody 11G2.

[0171] Table 5 Summary of binding kinetic parameters of humanized antibodies designed based on the murine parent antibody 11G2

[0172] Table 6 shows the binding kinetic parameters of humanized antibodies designed from the murine parent antibody 12E12.

[0173] Table 6 Summary of binding kinetic parameters of humanized antibodies designed based on the murine parent antibody 12E12

[0174] Table 7 shows the binding kinetic parameters of humanized antibodies designed from the murine parent antibody 33C12.

[0175] Table 7 Summary of binding kinetic parameters of humanized antibodies designed based on the murine parent antibody 33C12

[0176] The experimental results show that the correlation coefficient R of all antibodies in the Global fitting mode 2 They are all greater than 0.95, which meets the system adaptability requirements and the results are reliable.

[0177] 1.5 Flow cytometry to study the binding of human-mouse chimeric and humanized monoclonal antibodies to TNFR2 protein on the cell membrane

[0178] In order to confirm that the humanized TNFR2 monoclonal antibody disclosed herein can bind to the TNFR2 protein on the cell membrane, an in vitro test was performed, and a human-mouse chimeric antibody was used as a positive control.

[0179] Purified humanized TNFR2 antibodies were co-incubated with CHO-K cells transfected with a human TNFR2 expression plasmid (CHO-K cells were transfected with the pcDNA3.1-TNFR2 plasmid using conventional methods, and a CHO-K cell line stably expressing TNFR2 was obtained after screening). Antibody binding strength was analyzed by flow cytometry. The specific steps were as follows: CHO-TNFR2 cells were counted, centrifuged, resuspended, and added to a 96-well conical-bottom plate. The supernatant was discarded. Anti-TNFR2 antibodies were serially diluted and added to a 96-well conical-bottom plate. After incubation and washing, a secondary antibody, FITC-anti-human IgG (Abcam: 6854), was added. Samples were detected using a Beckman CytoFLEX.

[0180] Experimental results and analysis

[0181] As shown in Figure 3A, the 11G2 anti-human TNFR2 humanized monoclonal antibody can bind to CHO-TNFR2. The human-mouse chimeric 11G2 antibody was used as a control and bound to CHO-TNFR2. 50 The EC binding activity of humanized antibodies P07262, P07263 and P07268 to CHO-TNFR2 was 0.186nM. 50 0.006nM, 0.153nM and 0.200nM respectively.

[0182] As shown in Figure 3B , the 12E12 anti-human TNFR2 humanized monoclonal antibody bound well to CHO-TNFR2. The human-mouse chimeric 12E12 antibody was used as a control. 50 The concentration of humanized antibodies P07282, P07291 and P08940 bound to the EC on the CHO-TNFR2 cell membrane. 50 They are: 0.136nM, 0.144nM and 0.073nM respectively.

[0183] As shown in Figure 3C, the humanized monoclonal antibody 33C12 against human TNFR2 can bind to CHO-TNFR2. The human-mouse chimeric 33C12 antibody, as a control, binds to CHO-TNFR2. 50 The humanized 33C12 antibody P07298 binds to CHO-TNFR2 with an EC value of 0.951 nM. 50 is: 0.448nM.

[0184] The above results indicate that the expressed recombinant humanized antibody can bind to the cell membrane antigen TNFR2, and the binding activity is comparable to that of the human-mouse chimeric antibody.

[0185] 1.6 Effects of humanized anti-human TNFR2 monoclonal antibodies on T cell activation

[0186] Resuscitate commercially available frozen PBMC cells (Oricells) and adjust the cell density to 5×10 6 / mL. Dilute PHA to 2μg / mL with RPMI1640 + 10% FBS medium. Then, gradiently dilute the test antibody to 20μg / mL using the aforementioned PHA working solution and gently mix. Add 100μL of antibody to each well of a 96-well plate, followed by 100μL of cell suspension to the corresponding well. After gentle mixing, incubate at 37°C, 5% CO2 in a humidified incubator for 3 days. After 3 days, aspirate 100μL of the supernatant and assay for IFNγ (Ecosai, Cat. No. EH008).

[0187] For the determination of CD25 expression and CD8+ / CD4+ cell ratio, cells were collected, resuspended and washed with PBS, and stained with FITC-anti-human-CD4 (Biolegend), PE-anti-human CD8a (Biolegend), and APC-anti-huamn-CD25 (Biolegend, 302610). After incubation at 4°C for 30 minutes, cells were washed, resuspended in PBS, and detected by the analyzer.

[0188] Experimental results and analysis

[0189] The experimental results are summarized in Table 8-1 and Table 8-2.

[0190] Table 8-1. Effects of humanized antibodies based on 11G2 and 33C12 on T cell activation

[0191] Table 8-2. Effects of 12E12-based humanized antibodies on T cell activation

[0192] The experimental results showed that humanized monoclonal antibodies based on the parental antibody strains 11G2, 33C12 and 12E12, as well as human-mouse chimeric antibodies, can promote PHA-activated PBMCs to produce more IFNγ. Compared with the control IgG1 group, the IFNγ content in the cell group treated with the antibody of the present invention was significantly increased, with the increase percentage reaching hundreds or even thousands of percent.

[0193] CD25 Expression: A hallmark of T cell activation is high cell surface expression of CD25 molecules, such as CD25. We analyzed CD25 expression in cells treated for 72 hours. The results are shown in the table above (Table 8-1). Compared to the negative control IgG1, CD25 expression was significantly increased in cells treated with the anti-TNFR2 chimeric antibody (11G2-hFc) and the humanized antibody, indicating that T cells were activated by the TNFR2 antibody.

[0194] CD8+ / CD4+ Cell Ratio: We also analyzed the percentage of CD8+ and CD4+ cells in the total cell population at 72 hours and calculated the ratio between the two. The results showed that at 72 hours, the CD8+ / CD4+ ratio in the anti-TNFR2 antibody-treated group was higher than that in the control group, indicating that the antibodies of the present invention primarily promote the proliferation of CD8+ T cells.

[0195] Cytokine IFNγ: Upon activation, T cells release a large number of cytokines, such as IFNγ. We measured IFNγ levels in the cell supernatant after 72 hours. The results showed that IFNγ levels were significantly elevated in cells treated with the antibody of this invention compared to the control group, with increases ranging from 173% to 556%.

[0196] The above test results show that the anti-TNFR2 chimeric antibody and the humanized antibody of the present invention can significantly promote the activation of T cells.

[0197] 1.7 Functional study of humanized TNFR2 monoclonal antibody inhibiting mouse tumor growth in vivo

[0198] Since the anti-TNFR2 antibodies of the present invention do not bind to mouse TNFR2, we used human TNFR2 knock-in mice (C57BL / 6) to study the in vivo anti-tumor effects of the antibodies. Human TNFR2 knock-in mice were housed in an SPF-grade environment. In this example, the mouse tumor model was a syngeneic transplant mouse model, and the tumor cells were mouse colon cancer cells MC38 (Shanghai South Model Organisms Science Co., Ltd.). The method is briefly described as follows: 8-10 week-old human TNFR2 knock-in C57 / B6 mice were subcutaneously injected with 1×10 MC38 cells in the mouse armpit. 6 On the 7th day of inoculation (D7), the mice were randomly divided into multiple groups according to the size of the tumor, with 8 mice in each group (half male and half female). Each group of mice was given PBS (control group) and 1-10 mg / kg of TNFR2 humanized antibody by intraperitoneal injection. The drug was administered twice a week for a total of 4 times. The tumor volume (i.e., long diameter × short diameter) was measured at each administration. 2 The mice were weighed. The experiment was terminated 3 days after the fourth dose. The mice were sacrificed by cervical dislocation, and the tumors were removed and weighed.

[0199] The experimental results are shown in Figure 4. Figure 4A shows the effect of the TNFR2 humanized antibody 11G2-P07263 on mouse tumor growth. The inhibitory effect of 11G2-P07263 on MC38 tumors has a dose-response relationship. The antibody at a dose of 10 mg / kg can significantly inhibit the growth of MC38 tumors, with a significant difference compared to the PBS group (control group) (p < 0.05). Figure 4B shows the effect of the TNFR2 humanized antibody 33C12-P07298 on mouse tumor growth. The inhibitory effect of 33C12-P07298 on MC38 tumors has a dose-response relationship. The antibody at a dose of 3 mg / kg can significantly inhibit the growth of MC38 tumors, with a significant difference compared to the PBS group (control group) (p < 0.05).

[0200] Example 2. Preparation of anti-TNFR2 / anti-PD-1 or CTLA-4 bispecific antibodies

[0201] 2.1 Introduction

[0202] PD-1 and CTLA-4 are inhibitory immune checkpoint receptors expressed on immune cells. When the interaction between these receptors and their corresponding ligands is blocked by anti-PD-1 or CTLA-4 antibodies, the exhaustion of immune cells can be suppressed and the functional activity of immune cells can be enhanced. TNFR2 is one of the co-stimulatory molecules for the activation of immune cells, and when combined with anti-TNFR2 agonistic antibodies, the functional activity of immune cells can be enhanced. The combination of anti-TNFR2 and anti-PD-1 or CTLA-4 antibodies showed a synergistic or cumulative effect in inhibiting tumor growth in a mouse tumor model. Here, we expressed and prepared anti-TNFR2 / anti-PD-1 or CTLA-4 bispecific antibodies, characterized them, and demonstrated that the bispecific antibodies retained binding affinity for the corresponding target antigens.

[0203] 2.2 Plan

[0204] Anti-TNFR2 / anti-PD-1 or CTLA-4 bispecific construct design: The bispecific antibodies of the present invention were generated using the following two different configurations.

[0205] Configuration 1 (Figures 5A and 5B): Knob-in-hole (KIH) Fc engineering plus CrossMab, that is, the heavy chain variable domain of the anti-PD-1 or CTLA-4 antibody is fused to the human antibody light chain constant lambda domain and the human IgG1 hinge domain -CH2-CH3 (knob heavy chain) containing mutations S354C and T366W (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3), and the light chain variable domain of the anti-PD-1 or CTLA-4 antibody is fused to the CH1 of human IgG1 (SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6) through two amino acids (-Ser-Ser-), to form the antigen-binding domain of one of the antibodies of the bispecific antibody in the form of a crossmab. The other half of the bispecific antibody, the TNFR2 antibody, is composed of an anti-TNFR2 antibody heavy chain (hole heavy chain) containing the IgG1 Fc mutations Y349C, T366S, L368A, and Y407V (SEQ ID NO: 7 or SEQ ID NO: 8) and an anti-TNFR2 antibody light chain (SEQ ID NO: 9). The two amino acids L234 / L235 in the CH1 region of both heavy chains of the TNFR2 / PD-1 BsAb are substituted with Ala (LALA variant), abolishing the antibody's ADCC / CDC function. The knob heavy chain and hole heavy chain form a heterodimer, thereby forming an anti-TNFR2 / anti-PD-1 or anti-CTLA-4 BsAb.

[0206] Configuration 2 ( FIG5C ): IgG-scFv format, i.e., the C-termini of both heavy chains of anti-PD-1 (IgG4) (SEQ ID NO: 10 or SEQ ID NO: 11) or both heavy chains of CTLA-4 (IgG1) (SEQ ID NO: 12) are linked to the scFv fragment of the anti-TNFR2 antibody, wherein the heavy chain and the scFv, and the VH and VL of the scFv are connected by flexible peptides with n GGGGS repeats (n is an integer). These heavy chains form homodimeric bispecific antibodies with the light chain of the anti-PD-1 antibody (SEQ ID NO: 13 or SEQ ID NO: 14) or the light chain of the CTLA-4 antibody (SEQ ID NO: 15).

[0207] The anti-CTLA-4 antibody used to construct anti-TNFR2 / CTLA-4 BsAbs is the KD6001 anti-CTLA-4 monoclonal antibody, and the amino acid sequence of its active fragment is shown in Table 9:

[0208] Table 9. Amino acid sequences of active fragments of anti-CTLA-4 monoclonal antibodies

[0209] 2.3 Expression and Purification of Anti-TNFR2 / Anti-PD-1 or Anti-CTLA-4 Bispecific Antibodies

[0210] The DNA encoding the heavy and light chains of bispecific antibody configuration 1 was cloned into the expression vector pcDNA3.4 (ThermoFisher) to generate four independent vectors, or the DNA encoding the heavy and light chains of bispecific antibody configuration 2 was cloned into the expression vector pcDNA3.4 (ThermoFisher) to generate two independent vectors. The recombinant plasmids were then extracted. The four plasmids of configuration 1 or the two plasmids of configuration 2 were co-transfected into 293F cells using sinofection (SinoBiological). After 7 days of cell culture, the culture medium was centrifuged at high speed, and the supernatant was purified using a Protein A affinity chromatography column to obtain TNFR2 / PD-1 or TNFR2 / CTLA-4 bsAbs.

[0211] The purified samples were added into reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer respectively, boiled and then subjected to SDS-PAGE electrophoresis detection.

[0212] The electrophoretic patterns of anti-TNFR2 / anti-PD-1 bispecific antibodies of configuration 1 and configuration 2 are shown in Figures 6A and 6B. Configuration 1 bispecific antibody is a heterodimer, and the theoretical molecular weights of the two heavy chains are not much different, about 50 kDa, and the theoretical molecular weights of the two light chains are also not much different (653 Da difference), about 23 kDa, both of which are close to the molecular weight of normal human immunoglobulin (IgG1) (Figure 6A). Configuration 2 bispecific antibody is a homodimer, and the theoretical molecular weight of the non-reduced bispecific antibody is 186 kD. Due to glycosylation, the actual molecular weight is greater than the theoretical molecular weight. The theoretical molecular weights of the heavy chain and light chain of the reduced bispecific antibody are 76.7 kDa and 23.6 kDa, respectively (Figure 6B, indicated by arrows).

[0213] The electrophoretic patterns of the anti-TNFR2 / anti-CTLA-4 bispecific antibodies of configurations 1 and 2 are shown in Figures 7A and 7B . Configuration 1 bispecific antibody is a heterodimer, with the theoretical molecular weights of its two heavy chains approximately 50 kDa apart, and the theoretical molecular weights of its two light chains differing by 1207 Da, with two light chain bands visible on the SDS-PAGE gel ( Figure 7A ). Configuration 2 bispecific antibody is a homodimer, with the theoretical molecular weight of the non-reduced bispecific antibody being 198 kDa, and the theoretical molecular weights of the heavy and light chains of the reduced bispecific antibody being 76.5 kDa and 23.7 kDa, respectively ( Figure 7B ).

[0214] The results showed that the constructs produced antibodies with the correct molecular weight and composition.

[0215] Example 3. ELISA study of binding of anti-TNFR2 / anti-PD-1 or CTLA-4 BsAbs to antigens

[0216] The in vitro binding activity of bispecific antibodies to their antigens was assessed using an ELISA. The method was briefly described as follows: Plates were coated with 50 μl of NaHCO₃ (pH 9.6) buffer containing 1 μL / mL of recombinant human TNFR2, PD-1, or CTLA-4 overnight at 4°C. The following day, after washing twice with PBS, the plates were blocked with 3% BSA, incubated at 37°C for 1.5 hours, and washed four times with PBST. TNFR2 / PD-1 or CTLA-4 BsAbs were serially diluted in binding solution (TBST containing 1% BSA) and added to the blocked and washed plates (50 μl per well) for 2 hours at 37°C. After washing four times with PBST, a secondary antibody alkaline phosphatase-labeled anti-hFc (Jackson Immuno Research) diluted in a certain proportion was added to each well and incubated at 37°C for 1.5 h. Finally, after washing four times with PBST, PNPP color development solution was added and incubated at 37°C for 10-15 min. The wells were read using a microplate reader (wavelengths of 405 nm and 490 nm).

[0217] Figure 8 shows the in vitro binding activity of the anti-TNFR2 / anti-PD-1 BsAb of configuration 1 to recombinant human TNFR2 and PD-1. The experimental results showed that the anti-TNFR2 / anti-PD-1 BsAb of configuration 1 can specifically bind to TNFR2 (Figure 8A) and PD-1 (Figure 8B).

[0218] Figure 9 shows the in vitro binding activity of the anti-TNFR2 / anti-PD-1 BsAb of Configuration 2 to recombinant human TNFR2 and PD-1. The experimental results showed that the anti-TNFR2 / anti-PD-1 BsAb of Configuration 2 can specifically bind to TNFR2 (Figure 9A) and PD-1 (Figure 9B).

[0219] Figure 10 shows the in vitro binding activity of the anti-TNFR2 / anti-CTLA-4 BsAb of Configuration 1 to recombinant human CTLA-4. The experimental results showed that the anti-TNFR2 / anti-CTLA-4 BsAb of Configuration 1 can specifically bind to CTLA-4 (Figure 10).

[0220] Figure 11 shows the in vitro binding activity of the anti-TNFR2 / anti-CTLA-4 BsAb of Configuration 2 to recombinant human TNFR2 and CTLA-4. The experimental results showed that the anti-TNFR2 / anti-CTLA-4 BsAb of Configuration 2 can specifically bind to TNFR2 (Figure 11A) and CTLA-4 (Figure 11B).

[0221] Example 4. ELISA study of dual binding of anti-TNFR2 / anti-PD-1 or CTLA-4 BsAbs to antigens

[0222] A sandwich ELISA assay was used to test the ability of anti-TNFR2 / anti-PD-1 or CTLA-4 BsAbs to simultaneously bind to TNFR2 and PD-1 or CTLA-4. To prepare ELISA plates for analyzing bispecific antibody binding, the plates were coated overnight at 4°C with 50 μl of 1 μL / mL recombinant human PD-1-His or CTLA-4-His in NaHCO₃ (pH 9.6). The next day, after washing twice with PBS, the plates were blocked with 3% BSA, and 100 μL per well was incubated at 37°C for 1.5 hours. The plates were then washed four times with PBST. A serial dilution of the anti-TNFR2 / anti-PD-1 or CTLA-4 BsAbs was added to the blocked and washed plates (50 μl per well) in binding solution (TBST containing 1% BSA) and incubated at 37°C for 2 hours. After washing four times with PBST, biotin-conjugated recombinant human TNFR2-Fc diluted in binding solution was added to each well and incubated at 37°C for 2 hours. The cells were washed four times with PBST, and a certain proportion of alkaline phosphatase-labeled streptavidin (Jackson Immuno Research) was added and incubated at 37°C for 1.5 h. Finally, after washing four times with PBST, PNPP colorimetric solution was added, and the cells were incubated at 37°C for 10-15 min before reading using a microplate reader (wavelengths of 405 nm and 490 nm).

[0223] Experimental results and analysis

[0224] Figure 12 shows a sandwich ELISA study of antigen binding of anti-TNFR2 / anti-PD-1 dual antibodies of configuration 1 (Figure 12A) and configuration 2 (Figure 12B). The results show that both anti-TNFR2 / anti-PD-1 dual antibodies of these two configurations can bind to TNFR2 and PD-1 simultaneously.

[0225] Figure 13 shows a sandwich ELISA study of antigen binding of the anti-TNFR2 / anti-CTLA-4 dual antibody of Configuration 1. The results show that the anti-TNFR2 / anti-CTLA-4 dual antibody of Configuration 1 can bind to TNFR2 and CTLA-4 simultaneously.

[0226] Example 5. Flow cytometry study of the binding of anti-TNFR2 / anti-PD-1 or CTLA-4 BsAbs to TNFR2 and PD-1 or CTLA-4 proteins on the cell membrane

[0227] A stable cell line expressing on the membrane was constructed using full-length human TNFR2 and PD-1 cDNAs, while a stable cell line expressing on the membrane was constructed using human-mouse chimeric CTLA-4 cDNA. Because the intracellular sequence of human CTLA-4 limits the transfer of CTLA-4 from the cytoplasm to the cell membrane, in order to increase the expression of CTLA-4 on the membrane, most of the sequence of the intracellular segment of CTLA-4 was replaced with the sequence of the intracellular segment of the mouse membrane receptor protein CD28. The expressed CTLA-4 is a human-mouse chimeric protein, that is, the human CTLA-4 amino acid sequence 1-190 (including the extracellular segment, the transmembrane segment and part of the intracellular segment (8 amino acids)) is connected to the intracellular end amino acid sequence 178-218 of the mouse membrane receptor protein CD28.

[0228] The cDNA encoding the desired antigens was inserted into the mammalian cell expression vector pcDNA3.1 (ThermoFisher). The expression plasmid was then transfected into Jurkat cells using electroporation. The cells were cultured in G418-containing medium for two weeks. Flow cytometry was then used to isolate single cell lines that showed high expression of TNFR2, PD-1, or CTLA-4. The single cell lines were cultured in 96-well plates and expanded in T25 flasks. Flow cytometry was then used to analyze membrane protein expression, identifying stable Jurkat cell lines with high expression.

[0229] The flow cytometric method for studying the binding of anti-TNFR2 / anti-PD-1 or anti-CTLA-4 BsAbs to cell membrane TNFR2, PD-1, or CTLA-4 is briefly described as follows: First, prepare FACS buffer (1× PBS + 0.5% BSA solution). Take the required number of cells constructed as described above (approximately 1×10^5 to 5×10^5 cells per sample). Dilute the bispecific antibody to be tested in FACS buffer at a specific ratio and mix with the cells in a volume of approximately 100 μL. Incubate at 4°C for 30 minutes. Wash the cells once with FACS buffer and resuspend them in FITC-anti-human IgG (Abcam: 6854) diluted in FACS buffer. Incubate at 4°C for 30 minutes. Finally, wash the cells with FACS buffer and resuspend them in 200 μL of FACS buffer before analysis.

[0230] Experimental results and analysis

[0231] The experimental results showed (Figure 14) that the anti-TNFR2 / anti-PD-1 BsAbs of configurations 1 and 2 can bind to TNFR2 on the cell membrane with high affinity. The anti-TNFR2 / anti-CTLA-4 BsAb of configuration 2 binds to TNFR2 (Figure 15A) and CTLA-4 (Figure 15B) on the cell membrane with high affinity.

[0232] Example 6. TNFR2 / PD-1 BsAbs enhance INFγ production in human PBMCs in vitro

[0233] Upon activation, T lymphocytes increase their surface expression of PD-1, which induces activated T cell exhaustion and reduces immune cell activity. To test whether TNFR2 / PD-1 BsAbs could inhibit activated T cell exhaustion, enhance activated T cell activity, and increase the secretion of the cytokine IFNγ, we conducted the following experiment. The method is briefly described as follows: frozen human peripheral blood mononuclear cells (PBMCs) were thawed and adjusted to a cell density of 5 × 10^6 cells / mL. The cells were cultured for 3 days in a medium containing 1.0 μg / mL of PHA to activate the lymphocytes. PHA-activated PBMCs were washed with PBS and cultured in a medium containing 1.0 μg / mL PHA (T lymphocytes are in the post-activation exhaustion stage). TNFR2 / PD-1 BsAbs of configurations 1 and 2 were added to the culture medium at concentrations of 2 μg / mL and 1 μg / mL, respectively, as well as IgG1 (negative control), TNFR2 monoclonal antibody 11G2-P07263, PD-1 monoclonal antibody nivolumab, and 11G2-P07263+nivolumab (antibody concentrations were all 1.0 μg / mL) and cultured for 3 days. The IFNγ content in the supernatant was then detected.

[0234] The experimental results (Figure 16) show that compared to the negative control (IgG1), the PD-1 monoclonal antibody nivolumab enhanced IFNγ production, with the IFNγ concentration being 2.77 times that of the negative control. However, the TNFR2 monoclonal antibody 11G2-P07263 did not increase IFNγ production. The IFNγ content produced by the TNFR2 monoclonal antibody 11G2-P07263 combined with the PD-1 monoclonal antibody nivolumab (11G2-P07263+nivolumab) (2.43 times that of the negative control) was no different from that of the PD-1 monoclonal antibody nivolumab, indicating that the PD-1 monoclonal antibody can inhibit the attenuation of T lymphocytes, while the TNFR2 monoclonal antibody 11G2-P07263 has no effect on T lymphocyte exhaustion. Notably, both TNFR2 / PD-1 BsAbs with different configurations in the present invention significantly enhanced IFNγ production, with the IFNγ levels produced not only being 4.25-fold (Configuration 1) and 4.50-fold (Configuration 2) higher than the negative control, but also exceeding that of the PD-1 monoclonal antibody nivolumab, reaching 1.54-fold (Configuration 1) and 1.63-fold (Configuration 2) of the IFNγ levels produced by nivolumab. More importantly, the ability of the two TNFR2 / PD-1 BsAbs with different configurations to stimulate activated T lymphocytes was stronger than the combined effect of the TNFR2 monoclonal antibody 11G2-P07263 and the PD-1 monoclonal antibody nivolumab.

[0235] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0236] Attachment: Sequence information

[0237] SEQ ID NO: 1: PD-1 antibody (pembrolizumab) heavy chain variable domain fused to a human antibody light chain constant lambda domain and a human IgG1 hinge domain-CH2-CH3 (knob heavy chain 1) containing the LALA variant and the S354C and T366W variants

[0238] SEQ ID NO: 2: PD-1 antibody (nivolumab) heavy chain variable domain fused to a human antibody light chain constant lambda domain and a human IgG1 hinge domain containing the LALA variant and the S354C and T366W variants - CH2-CH3 (knob heavy chain 2)

[0239] SEQ ID NO: 3: CTLA-4 antibody (KD6001) heavy chain variable domain fused to a human antibody light chain constant lambda domain and a human IgG1 hinge domain containing S354C and T366W mutations - CH2-CH3 (knob heavy chain 3)

[0240] SEQ ID NO: 4: PD-1 antibody (pembrolizumab) light chain variable domain fused to CH1 of human IgG1

[0241] SEQ ID NO: 5: PD-1 antibody (nivolumab) light chain variable domain fused to CH1 of human IgG1

[0242] SEQ ID NO: 6: Anti-CTLA-4 antibody light chain variable domain fused to CH1 of human IgG1

[0243] SEQ ID NO: 7: Heavy chain variable domain of TNFR2 antibody (P07263) fused to IgG1 Fc (hole heavy chain 1) containing the LALA variant and the Y349C, T366S, L368A, and Y407V variants, heavy chain of TNFR2 / PD-1 BsAb

[0244] SEQ ID NO: 8: Heavy chain variable domain of TNFR2 antibody (P07263) fused to IgG1 Fc containing Y349C, T366S, L368A, and Y407V variants (hole heavy chain 2), heavy chain of TNFR2 / CTLA-4 BsAb

[0245] SEQ ID NO: 9: TNFR2 antibody (P07263) light chain

[0246] SEQ ID NO: 10: scFv fragment of the heavy chain of PD-1 antibody (pembrolizumab) linked to the C-terminus of TNFR2 antibody (P07263)

[0247] SEQ ID NO: 11: scFv fragment of the PD-1 antibody (nivolumab) heavy chain linked to the C-terminus of the TNFR2 antibody (P07263)

[0248] SEQ ID NO: 12: scFv fragment of the heavy chain of CTLA-4 antibody (KD6001) linked to the C-terminus of TNFR2 antibody (P07263)

[0249] SEQ ID NO: 13: Anti-PD-1 antibody (pembrolizumab) light chain

[0250] SEQ ID NO: 14: Anti-PD-1 antibody (nivolumab) light chain

[0251] SEQ ID NO: 15: Anti-CTLA-4 antibody light chain

[0252] SEQ ID NOs: 16-35: VH, VH CDR1-3, VL, and VL CDR1-3 of 11G2-based humanized anti-TNFR2 antibody

[0253] See Table 1

[0254] SEQ ID NOs: 36-55: VH, VH CDR1-3, VL, and VL CDR1-3 of a humanized anti-TNFR2 antibody based on 12E12

[0255] See Table 2

[0256] SEQ ID NOs: 56-63: VH, VH CDR1-3, VL, and VL CDR1-3 of a humanized anti-TNFR2 antibody based on 33C12

[0257] See Table 3

[0258] SEQ ID NOs: 64-71: VH, VH CDR1-3, VL, and VL CDR1-3 of anti-CTLA-4 monoclonal antibody KD6001

[0259] See Table 9

Claims

1. A bispecific antibody (BsAb) against TNFR2 and PD-1 or CTLA-4, comprising: (A) A TNFR2 binding domain comprising heavy chain complementary determining regions (VH CDR) 1-3 and / or light chain complementary determining regions (VL CDR) 1-3 selected from the group consisting of: (i) the VH CDR1 is any VH CDR1 selected from Table 1, Table 2 or Table 3; (ii) the VH CDR2 is any VH CDR2 selected from Table 1, Table 2 or Table 3; (iii) the VH CDR3 is any VH CDR3 selected from Table 1, Table 2 or Table 3; (iv) the VL CDR1 is any VL CDR1 selected from Table 1, Table 2 or Table 3; (v) the VL CDR2 is any VL CDR2 selected from Table 1, Table 2 or Table 3; (vi) the VL CDR3 is any VL CDR3 selected from Table 1, Table 2 or Table 3; and (B) CTLA-4 binding domain or PD-1 binding domain.

2. The bispecific antibody according to claim 1, wherein The TNFR2 binding domain comprises a combination of any VH CDR1-3 selected from Table 1, Table 2 or Table 3; and / or, The TNFR2 binding domain comprises a combination of any VL CDR1-3 selected from Table 1, Table 2 or Table 3; (For example, the TNFR2 binding domain comprises: a combination of VH CDR1-3 and VL CDR1-3 selected from Table 1, a combination of VH CDR1-3 and VL CDR1-3 selected from Table 2, or a combination of VH CDR1-3 and VL CDR1-3 selected from Table 3; For example, the TNFR2 binding domain comprises: a combination of VH CDR1-3 and VL CDR1-3 of a heavy chain and a light chain combination as shown in Table 4).

3. The bispecific antibody according to claim 1, wherein The TNFR2 binding domain comprises any heavy chain variable region (VH) selected from Table 1, Table 2 or Table 3 or an amino acid sequence having at least 80% sequence identity thereto; and / or The TNFR2 binding domain comprises any light chain variable region (VL) selected from Table 1, Table 2 or Table 3 or an amino acid sequence having at least 80% sequence identity thereto, (For example, the TNFR2 binding domain comprises a combination of a heavy chain variable region (VH) and a light chain variable region selected from the group consisting of a VH selected from Table 1 or an amino acid sequence having at least 80% sequence identity thereto and a VL selected from Table 1 or an amino acid sequence having at least 80% sequence identity thereto; a VH selected from Table 2 or an amino acid sequence having at least 80% sequence identity thereto and a VL selected from Table 2 or an amino acid sequence having at least 80% sequence identity thereto; or a VH selected from Table 3 or an amino acid sequence having at least 80% sequence identity thereto and a VL selected from Table 3 or an amino acid sequence having at least 80% sequence identity thereto, For example, the TNFR2 binding domain comprises: a VH and a VL combination as shown in Table 4).

4. The bispecific antibody according to claim 1, wherein The TNFR2 binding domain comprises one or more fragments selected from the group consisting of fragments shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or fragments having at least 90% (eg, 95%) sequence identity with the aforementioned sequences and having TNFR2 binding activity.

5. The bispecific antibody according to claim 1, wherein The CTLA-4 binding domain comprises the antigen binding domain of an anti-CTLA-4 antibody; For example, the CTLA-4 binding domain comprises one or more fragments selected from SEQ ID NOs: 64 to 71, or fragments thereof having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity to the foregoing sequences and having CTLA-4 binding activity, or a combination thereof; For example, the CTLA-4 binding domain comprises one or more fragments selected from SEQ ID NO: 3, 6, 12 or 16, or fragments thereof having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity to the foregoing sequences and having CTLA-4 binding activity, or a combination thereof.

6. The bispecific antibody according to claim 1, wherein The PD-1 binding domain comprises the antigen binding domain of an anti-PD-1 antibody; For example, the PD-1 binding domain comprises VL CDR, VH CDR, VH and / or VL selected from pembrolizumab or nivolumab, or a fragment thereof having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity with the aforementioned sequences and having PD-1 binding activity, or a combination thereof; and / or For example, the PD-1 binding domain comprises one or more fragments selected from SEQ ID NO: 1, 2, 4, 5, 10, 11, 13 or 14, or fragments thereof having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity with the aforementioned sequences and having PD-1 binding activity, or a combination thereof.

7. The bispecific antibody according to claim 1, wherein The TNFR2 binding domain, PD-1 and / or CTLA-4 binding domain is an antibody fragment selected from the group consisting of: a Fab fragment; a monovalent fragment consisting of VL, VH, CL and CH1 domains; a F(ab)2 fragment; a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; a Fd fragment consisting of VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single antibody arm; a dAb fragment; an isolated CDR; and a scFv; and / or The form of the bispecific antibody is selected from the following groups: 1) a fusion protein assembled from the antigen binding region (Fab); (2) a symmetric BsAb; (3) an asymmetric BsAb; and / or The bispecific antibody is a multivalent antibody, such as a 2-valent, 3-valent, 4-valent, 5-valent, 6-valent or higher valent antibody; and / or The bispecific antibody has a knob-in-hole (KIH) configuration or an IgG-scFv configuration with a scFv fragment linked to the C-terminus of the heavy chain, (For example, the bispecific antibody has a KIH configuration comprising the following fragments: an antigen binding domain comprising a heavy chain selected from SEQ ID NO: 1, 2 or 3 and a light chain selected from SEQ ID NO: 4, 5 or 6 linked thereto; and a heavy chain selected from SEQ ID NO: 7 or 8 and a light chain selected from SEQ ID NO: 9 linked thereto; or, For example, the bispecific antibody has an IgG-scFv configuration comprising the following fragments: a heavy chain connected to a scFv comprising SEQ ID NO: 10, 11 or 12 and a light chain comprising SEQ ID NO: 13, 14 or 15; and / or The bispecific antibody is a homodimer or multimer, or a heterodimer or multimer.

8. A nucleic acid molecule or a combination thereof, encoding the bispecific antibody according to any one of claims 1 to 7.

9. A vector or cell comprising the bispecific antibody or fragment thereof as described in any one of claims 1 to 7 and / or the nucleic acid molecule or a combination thereof as described in claim 8, for example, the cell is a chimeric antigen receptor (CAR) cell.

10. A product comprising the bispecific antibody or fragment thereof according to any one of claims 1 to 7, the nucleic acid molecule or a combination thereof according to claim 8, the vector or cell according to claim 9, For example, the product is selected from: a pharmaceutical composition, a kit, a medicine box; and / or, For example, the product is used for: production of the bispecific antibody, production of derivatives (such as CAR cells) containing the bispecific antibody, disease treatment and / or disease detection.

11. Use of the bispecific antibody or fragment thereof according to any one of claims 1 to 7, the nucleic acid molecule or a combination thereof according to claim 8, the vector or cell according to claim 9, or the product according to claim 10 for preparing a drug for preventing and / or treating diseases related to overexpression of TNFR2 and / or PD-1 or CTLA-4, For example, the overexpression-related disease is selected from: cancer (such as adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma; tumor tissue from the adrenal gland, gall bladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary gland, spleen, testis, thymus, thyroid or uterus; tumors of the central nervous system, such as glioma, or astrocytoma; eye tumors (such as basal cell carcinoma, squamous cell carcinoma or melanoma), endocrine gland tumors, neuroendocrine system tumors, gastrointestinal pancreatic endocrine system tumors, reproductive system tumors or head and neck tumors); and infection or infectious diseases.

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