Combination of antibodies specifically recognizing tnfr2 and pd-l1 or pd-1

Abstract

The present application provides an anti-TNFR2 antibody, a pharmaceutical composition or combination comprising an anti-TNFR2 antibody and an anti-PD-L1 antibody or an anti-PD-1 antibody, a method of treating cancer or an infectious disease using a pharmaceutical composition or combination comprising an anti-TNFR2 antibody and an anti-PD-L1 antibody or an anti-PD-1 antibody.

Description

Submit sequence listing

[0001] The contents of the electronic sequence listing (Combination Therapy SEQ.xml; size: 44 kb; and record date: 2023.01.06) are incorporated herein by reference in their entirety. Technical Field

[0002] The present invention relates to a combination therapy of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-1 or PD-L1, and methods for preparing and using the same, including methods for treating and preventing cancer or infectious diseases. Background Art

[0003] Tumor necrosis factor receptor 2 (TNFR2), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) and CD120b, is a membrane receptor that binds to its cognate ligands, TNFα and lymphotoxin-α (LTα). Unlike TNFR1, which possesses a death domain (DD) in its cytoplasm and can activate caspase-dependent and NFκB pathways, TNFR2 lacks a DD but can recruit the adaptor proteins TNF receptor-associated factor 2 (TRAF2) and TRAF3 and activate the non-canonical NFκB and MAP kinase pathways (Brenner et al., 2015). TNFR2 is expressed on immune cells and some non-immune cells, including endothelial cells, cardiomyocytes, and astrocytes (Ward-Kavanagh et al., 2016). Although early studies demonstrated that TNFR2 co-stimulates naive T cell function, it was later shown that TNFR2 also limits CD8+ T cell-mediated viral clearance and anti-tumor immunity by inducing rapid CD8+ T cell contraction (Bertrand et al., 2015; DeBerge et al., 2015; Kim et al., 2009; Wortzman et al., 2013b). Several studies have shown that TNFR2 expression is higher in regulatory T cells (Treg cells) than in naive T cells, and that TNFR2 signaling is crucial for the development, proliferation, and survival of Treg cells (Chen et al., 2013; Horwitz et al., 2013; Mahmud et al., 2014). Therefore, TNFR2 signaling plays a key role in regulating immune responses. Furthermore, within the tumor microenvironment, TNFR2 is highly expressed in Treg cells and myeloid-derived suppressor cells (MDSCs), suggesting a potential role for TNFR2 in tumor immunity (Chen et al., 2013; Hu et al., 2014). Indeed, several studies have reported anti-tumor effects of anti-mouse TNFR2 antibodies, although the underlying mechanism remains unclear (Case et al., 2020; Nie et al., 2016; Tam et al., 2019; Williams et al., 2018). Furthermore, a known mutation in TNFR2 is associated with T-cell lymphomas, including mycosis fungoides and Sézary syndrome, suggesting that it may function as an oncogene (Ungewickell et al., 2015). TNFR2 has also been reported to be upregulated in certain cancer types and associated with poor prognosis (Yang et al., 2017; Zhang et al., 2018).

[0004] Programmed death protein 1 (PD-1) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). PD-1 was discovered by screening for differential expression in apoptotic cells (Ishida et al. (1992) EMBO J 11:3887-95).

[0005] PD-L1 is a member of the B7 family and is expressed on a variety of cells, including APCs and activated T cells (Yamazaki et al. (2002) J. Immunol. 169:5538). PD-L1 binds to both PD-1 and B7-1. Both PD-L1 binding to B7-1 expressed on T cells and B7-1 binding to PD-L1 expressed on T cells lead to T cell inhibition (Butte et al. (2007) Immunity 27:111). There is also evidence that, like other B7 family members, PD-L1 can provide co-stimulatory signals to T cells (Subudhi et al. (2004) J. Clin. Invest. 113:694; Tamura et al. (2001) Blood 97:1809). PD-L1 is a ligand for PD-1 and is expressed in antigen-presenting cells (APCs) such as activated monocytes and dendritic cells (Journal of Experimental Medicine (2000), vol. 19, issue 7, p1027-1034). These cells present interacting molecules that can induce various immune-inducing signals to T lymphocytes, and PD-L1 is one of these molecules that induces inhibitory signals through PD-1. It has been revealed that PD-L1 ligand stimulation inhibits the activation of T lymphocytes expressing PD-1 (cell proliferation and induction of various cytokine production). The expression of PD-L1 has been confirmed not only in immunocompetent cells, but also in certain tumor cell lines (cell lines derived from monocytic leukemia, cell lines derived from mast cells, cell lines derived from liver cancer, cell lines derived from neuroblastoma, and cell lines derived from breast cancer) (Nature Immunology (2001), vol. 2, issue 3, p. 261-267).

[0006] Over the past few years, tumor immunotherapy, as a novel treatment modality, has become a major focus in the field of cancer treatment. Antagonistic antibodies targeting immune checkpoint proteins, such as anti-PD-1 and anti-PD-L1 antibodies, have been used to treat a variety of cancers, achieving revolutionary results and significantly extending the survival of patients with malignant tumors.

[0007] However, because tumors often utilize multiple, overlapping, and redundant mechanisms to block anti-tumor immune responses, combination therapy may be required to achieve sustained efficacy across a wide range of tumor types. Therefore, new combination therapies are needed to improve the treatment of all cancers. The present invention prepares anti-TNFR2 antibodies and combines them with existing anti-PD-L1 or anti-PD-1 antibodies to achieve enhanced anti-tumor efficacy and sustained immune memory.

[0008] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. Application Overview

[0009] In one aspect, a method of treating cancer or an infectious disease in an individual in need thereof is provided, comprising administering to the individual an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1. In some embodiments, the antibody that specifically recognizes human TNFR2 and the antibody that specifically recognizes human PD-L1 or PD-1 are administered to the individual in need thereof simultaneously or sequentially.

[0010] In one aspect, an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 are provided for use in treating cancer or infectious diseases in individuals in need thereof. In another aspect, an antibody that specifically recognizes human TNFR2 is provided for use in combination with an antibody that specifically recognizes human PD-L1 or PD-1 to treat cancer or infectious diseases in individuals in need thereof. In some embodiments, the antibody that specifically recognizes human TNFR2 and the antibody that specifically recognizes human PD-L1 or PD-1 are administered to an individual in need thereof simultaneously or sequentially.

[0011] In some embodiments, the method or the antibody for the above use, wherein the antibody that specifically recognizes human TNFR2 is administered to the individual during the treatment with the antibody that specifically recognizes human PD-L1 or PD-1.

[0012] In some embodiments, the method or the antibody for the above use, wherein the individual is administered an antibody that specifically recognizes human PD-L1 or PD-1 during the treatment with the antibody that specifically recognizes human TNFR2.

[0013] On the other hand, the present application provides a pharmaceutical composition comprising an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

[0014] In another aspect, the present application provides a combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

[0015] In some embodiments, methods are provided for treating cancer or an infectious disease in an individual in need thereof, comprising administering to the individual the pharmaceutical composition or combination described above.

[0016] In some embodiments, pharmaceutical compositions are provided for treating cancer or infectious diseases in an individual in need thereof.

[0017] In some embodiments, a method of treating cancer or an infectious disease in an individual in need thereof is provided, comprising administering to the individual the above-mentioned combination, wherein the antibody that specifically recognizes human TNFR2 and the antibody that specifically recognizes human PD-L1 or PD-1 are administered to the individual in need thereof simultaneously or sequentially.

[0018] In some embodiments, the invention provides a combination for treating cancer or infectious diseases in an individual in need thereof, wherein the antibody that specifically recognizes human TNFR2 and the antibody that specifically recognizes human PD-L1 or PD-1 are administered to the individual in need thereof simultaneously or sequentially.

[0019] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0020] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Comprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 7-15 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V LComprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 16-20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0021] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7-15 or a variant thereof, wherein the variant has at least about 80% sequence identity with any one of SEQ ID NOs: 7-15; and V L , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 16-20 or a variant thereof, wherein the variant has at least about 80% sequence identity with any one of the amino acid sequences in SEQ ID NOs: 16-20.

[0022] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0023] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0024] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 18.

[0025] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 19.

[0026] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0027] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 10; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0028] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0029] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 12; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0030] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 13; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0031] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 14; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0032] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 15; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0033] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L, comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 37.

[0034] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 38.

[0035] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 30; and V L , comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 39.

[0036] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 30; and V L , comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0037] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 30; and V L , comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 38.

[0038] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 31; and V L , comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 41.

[0039] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 32; and V L , comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 41.

[0040] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 33; and V L , comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 41.

[0041] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 34; and V L , comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41.

[0042] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 35; and V L , comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41.

[0043] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 36; and V L , comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41.

[0044] In some embodiments, the antibody that specifically recognizes human PD-L1 is selected from the group consisting of avelumab, durvalumab, atezolizumab, KN035, CK-301, ADG-104, BCD-135, garivulimab, CBT-502, HLX-20, IMC-001, tagitanlimab, LAE-005, LP-002, MSB-2311, adebrelimab, sugemalimab, and socazolimab.

[0045] In some embodiments, the above-mentioned antibody that specifically recognizes human PD-1 is selected from cemiplimab, nivolumab, pembrolizumab, sintilimab, camrelizumab, toripalimab, tislelizumab, zimberelimab, prolgolimab, dostarlimab, AMG-404, balstilimab, budigalimab, cililimab, The group consisted of etrelimab, ezabenlimab, genolimzumab, LZM-009, nofazinlimab, penpulimab, pimivalimab, pucotenlimab, QL-1604, retifanlimab, rulonilimab, sasanlimab, SCT-I10A, serplulimab, SG-001, spartalizumab, SYSA-1802, and TY-101.

[0046] In some embodiments, an isolated nucleic acid molecule is provided, encoding any one of the antibodies that recognize human TNFR2, or antibodies that recognize human PD-L1 or PD-1. In some embodiments, a vector is provided, comprising any one of the nucleic acid molecules described above. In some embodiments, a host cell is provided, expressing any one of the antibodies, nucleic acid molecules, or vectors described above. In some embodiments, a method for producing an anti-TNFR2, anti-PD-L1, or anti-PD-1 antibody is provided, comprising: a) culturing any one of the host cells described above under conditions effective for expressing the anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody; and b) obtaining the expressed anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody from the host cell.

[0047] Also provided are pharmaceutical compositions, kits, and articles of manufacture comprising any one or more of the anti-TNFR2 and / or anti-PD-L1 or anti-PD-1 antibodies, nucleic acids, vectors, and isolated host cells described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figures 1A-1B show the results of epitope elucidation of an exemplary humanized antibody SB1901-76 using alanine scanning.

[0049] Figure 2A Shown are the results of binding assays of exemplary anti-TNFR2 antibodies analyzed by FACS. Figure 2B Shown are the results of ligand blocking assays of exemplary anti-TNFR2 antibodies analyzed by FACS.

[0050] Figure 3 Shown are the results of an in vitro human primary Treg cell proliferation assay using exemplary humanized anti-TNFR2 antibodies.

[0051] Figure 4A Shown are the tumor growth curves of monotherapy or combination therapy with anti-PD-L1 antibody. The mean tumor volume change in each group is shown in Figure 4B shown.

[0052] Figure 4C Shown are the tumor growth curves of monotherapy or combination therapy with anti-PD-1 antibody. The mean tumor volume change in each group is shown in Figure 4D shown. Detailed Description of the Invention definition

[0053] As described herein, " treatment (treatment) " or " treatment (treating) " is a method for obtaining beneficial or desired results, including clinical results. In view of the purpose of the application, the beneficial or desired clinical results, including but not limited to the following one or more: alleviate one or more symptoms caused by the disease, alleviate the degree of disease, stabilize the disease (for example, prevent or delay disease worsening), prevent or delay the spread of the disease (for example, the systemic spread of pathogens), prevent or delay disease recurrence, delay or slow down disease progression, improve the disease state, alleviate the disease (partial or complete), reduce the dosage of one or more other drugs required for the treatment of the disease, delay disease progression, improve or improve quality of life, gain weight, and / or prolong life. At the same time, " treatment " also includes the minimizing (for example, host cell lysis or necrosis) of the pathological results of infection. The method of the present application takes into account any one or more aspects of these treatments.

[0054] The term "prevent" and similar words, such as "prevented," "preventing," "prevention," or "prophylactic," refer to methods of preventing, inhibiting, or reducing the likelihood of a disease or condition (e.g., a pathogen infection) from occurring or recurring. It also refers to delaying the occurrence or recurrence of a disease or condition or delaying the occurrence or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it recurs. As used herein, "prevention" and similar words also include reducing the risk and susceptibility to the occurrence or recurrence of a disease or condition (e.g., a pathogen infection).

[0055] As described herein, the term "antibody" is used in its broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, as long as they exhibit desired antigen-binding activity. A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains typically include three highly variable loops, known as complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified or typed based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, which are characterized by having α, δ, ε, γ and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0056] As used herein, the term "antigen-binding fragment" includes antibody fragments, including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain Fv (scFv), scFv dimers (divalent diabodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, divalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure. Antigen-binding fragments also include fusion proteins comprising antibody fragments as described above. Antigen-binding fragments also include fusion proteins comprising antibody fragments as described above. Antigen-binding fragments can bind to the same antigen as a parent antibody or parent antibody fragment (such as a parent scFv). In some embodiments, an antigen-binding fragment may include one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0057] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they likely bind to the same epitope on the antigen.

[0058] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as the binding of an antibody to a target that can determine the presence of the target in a heterogeneous population of molecules, including biomolecules. For example, an antibody can specifically recognize a target (which can be an epitope) if the antibody binds to the target with greater affinity, avidity, more readily, and / or more prolonged than it binds to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its binding affinity to other targets.

[0059] As used herein, an "isolated" antibody is one that (1) is not related to naturally occurring proteins, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not occur in nature.

[0060] As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA, or synthetic origin, or a combination thereof. Depending on its origin, the "isolated nucleic acid" is (1) unrelated to all or part of a polynucleotide found in nature, (2) operably linked to a polynucleotide to which it is not naturally associated, or (3) not found in nature as part of a longer sequence.

[0061] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable domains of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions are described in, for example, Desmond et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlap or subsets of amino acid residues when compared to one another. However, any definition used to designate a CDR of an antibody, grafted antibody, or variant thereof is included within the scope of the term as defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the various references cited above for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and in one or more claims that may be included in this article. Table 1: CDR Definition 1The amino acid residue numbers refer to the nomenclature of Kabat et al. 2 The amino acid residue numbers refer to the nomenclature of Chothia et al. 3 The amino acid residue numbers refer to the nomenclature of MacCallum et al. 4 The amino acid residue numbers refer to the nomenclature of Lefranc et al. 5 The amino acid residue numbers refer to the nomenclature of Honegger and Plückthun.

[0062] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0063] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable domain and a light chain variable domain tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and confer specificity for binding to the antibody. However, even a single variable domain (or half of an Fv fragment, which only contains 3 CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site.

[0064] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a Fv that contains V molecules linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V LThe linker polypeptide between the domains allows the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0065] The term "diabodies" refers to double-chain antibodies H and V L A small antibody fragment prepared by constructing scFv fragments (see above) with short linkers (e.g., 5-10 residues) between the domains, which allows the variable domains to pair between chains rather than within the chain, resulting in a bivalent fragment, that is, a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the V domains of the two antibodies are H and V L The domains are located on different polypeptide chains. Diabodies are fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0066] The "humanized" form of a non-human (such as a rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced by hypervariable region residues from a non-human species such as mouse, rat, rabbit or non-human primate with ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region (FR) are replaced by corresponding non-human residues. In addition, a humanized antibody can include residues that are not present in either the recipient antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody essentially comprises at least one, usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. The human antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0067] The "percent (%) amino acid sequence identity" or "homology" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues between a candidate sequence and the polypeptide sequence being compared, when conservative substitutions are considered part of the sequence identity. Percent amino acid sequence identity can be determined by a variety of alignment methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to maximize alignment over the full length of the compared sequences. However, for the purposes of this article, percent amino acid sequence identity values ​​are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0068] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term FcR in this application encompasses other types of FcRs, including those identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0069] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2 microglobulin. The various functions of the neonatal Fc receptor FcRn are described in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulins (IgGs) from mother to newborn and in regulating serum IgG levels. As a salvage receptor, FcRn can bind and transport endocytosed IgGs in an intact form within and between cells, protecting them from the default degradation pathway.

[0070] The "CH1 domain" of a human IgG Fc region typically stretches from amino acid 118 to amino acid 215 (EU numbering system).

[0071] The "hinge region" is generally defined as stretching from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence.

[0072] The "CH2 domain" of the human IgG Fc region typically extends from amino acids 231 to 340. The CH2 domain is unique in that it does not closely pair with another domain. Instead, two N-terminally linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is hypothesized that carbohydrates may serve as a substitute for domain-to-domain pairing, helping to stabilize the CH2 domain structure. Burton, Molec Immunol. 22:161-206 (1985).

[0073] The "CH3" domain includes the CH2 domain extending from the C-terminal residue within the Fc region (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 in IgG).

[0074] A "functional Fc fragment" possesses the "effector functions" of a native Fc region sequence. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable region) and can be assessed using a variety of experimental methods known in the art.

[0075] IgG Fc variant antibodies with "altered" FcR binding affinity or ADCC activity have increased or decreased FcR binding activity (e.g., FcγR or FcRn) and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence. Fc variants that exhibit "enhanced binding" to FcRs have a higher binding affinity (e.g., lower apparent Kd or IC) to at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence. 50In some embodiments, the binding capacity is enhanced by 3-fold, e.g., 5-, 10-, 25-, 50-, 60-, 100-, 150-, 200-, or even up to 500-fold, or the binding capacity is increased by 25% to 1000%, compared to the parent polypeptide. An Fc variant that exhibits "reduced binding" to an FcR has a lower affinity for at least one FcR (e.g., a higher apparent Kd or a higher IC) than the parent polypeptide. 50 Compared with the parent polypeptide, its binding capacity is reduced by 40% or more.

[0076] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, the ADCC activity of the target molecule can also be assessed in vivo, for example, as described in the animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0077] Polypeptides comprising Fc region variants exhibit "enhanced ADCC activity" or are capable of more effectively mediating ADCC in the presence of human effector cells, compared to polypeptides comprising wild-type IgG Fc polypeptides or parent polypeptides. When tested in substantially the same quantity as polypeptides comprising wild-type IgG Fc polypeptides (or parent polypeptides), such polypeptides comprising Fc region variants are capable of more effectively mediating ADCC, both in vitro and in vivo. Such variants are generally identified using any in vitro ADCC assay known in the art, such as assays or methods for identifying ADCC activity, such as in animal models. In some embodiments, such variants mediate ADCC 5- to 100-fold more efficiently, such as 25- to 50-fold more efficiently, than wild-type Fc (or parent polypeptide).

[0078] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate structural subclass) that binds to the cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0079] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in some forms.

[0080] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling expression of the latter. For example, a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, can link two protein coding regions in the same reading frame.

[0081] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. If the same base or amino acid monomer subunit is present at the same position in two compared sequences, for example, adenine is present at the same position in two DNA molecules, then the two DNA molecules are homologous at that position. The percentage homology between two sequences refers to the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the homology between the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.

[0082] An "effective amount" of an antibody, combination thereof, or composition thereof disclosed herein is an amount sufficient to achieve a specific purpose. An "effective amount" can be determined empirically and by known methods related to the purpose.

[0083] The term "therapeutically effective amount" refers to an amount of an antibody, combination thereof, or composition thereof disclosed herein that can effectively "treat" an individual's disease or condition. In the case of cancer, a therapeutically effective amount of an antibody, combination thereof, or composition thereof disclosed herein can reduce the number of cancer cells; reduce tumor size and weight; inhibit (i.e., delay to some extent, preferably stop) cancer cells from infiltrating surrounding organs; inhibit (i.e., delay to some extent, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. To the extent that the antibodies, combinations thereof, or compositions disclosed herein can prevent growth and / or kill existing cancer cells, they can be cytostatic and / or cytotoxic. In some embodiments, a therapeutically effective amount is a growth-inhibiting amount. In some embodiments, a therapeutically effective amount is an amount that prolongs a patient's survival. In certain embodiments, a therapeutically effective amount is an amount that improves a patient's progression-free survival.

[0084] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is free of biological activity or other undesirable properties, for example, a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction, or that does not interact in a deleterious manner with any other component contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology and manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0085]

[0066] The embodiments of the present application described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0086] Reference herein to "about" for a value or parameter includes (and describes) variations of the value or parameter itself. For example, a description involving "about X" includes a description of "X".

[0087] As used herein, reference to "not" a value or parameter generally indicates and describes "other than" a value or parameter. For example, "the method cannot be used to treat type X cancer" means that the method is generally used to treat other types of cancer besides type X.

[0088] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Anti-TNFR2 antibodies TNFR2

[0089] Tumor necrosis factor (TNF) receptor 2 (TNFR2) is a signaling molecule located on the surface of a subset of potent regulatory T cells (Tregs) that activates these cells to proliferate through nuclear factor κB (NFκB). TNFR2 is also abundantly expressed on the surface of many human tumors (Vanamee et al.,TNFR2:A Novel Target for Cancer Immunotherapy.Trends Mol Med.2017Nov;23(11):1037-1046). TNFR2 is a cell surface receptor that regulates cell survival and proliferation (Chen, X. et al. (2007) Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+T regulatory cells. J. Immunol. 179, 154-161), and targeting this receptor has become a potential next-generation cancer treatment method (Chen, X. and Oppenheim, JJ (2017) Targeting TNFR2, an immune checkpoint stimulator and oncoprotein, is a promising treatment for cancer. Sci. Signal. 10, eaal2328). Certain human tumor cells can abnormally express TNFR2, and tumor infiltration is mainly composed of highly suppressive TNFR2+ Treg cells (Shimizu, J. et al. (1999) Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J. Immunol. 163, 5211-5218, Ungewickell, A. et al. (2015) Genomic analysis of mycosis fungoides and Sezary syndrome identifies recurrent alterations in TNFR2. Nat. Genet. 47, 1056–1060).

[0090] An exemplary amino acid sequence of the extracellular domain (ECD) of human TNFR2 comprises the amino acid sequence of SEQ ID NO:25. Anti-TNFR2 antibodies

[0091] In some embodiments, the anti-TNFR2 antibodies disclosed herein block the binding of TNFα to TNFR2 and TNFR2 signaling. The anti-TNFR2 antibodies described herein block the binding of TNFα to TNFR2, which means that the antibody molecule binds to the receptor TNFR2, thereby preventing the ligand TNFα from binding to the same receptor. The anti-TNFR2 antibodies described herein block TNFR2 signaling, which means that it blocks TNFR2-mediated cell activation. In some embodiments, the anti-TNFR2 antibodies disclosed herein have a depleting effect on TNFR2-positive cells, which means that when administered to a patient (e.g., a human), such antibody molecules specifically bind to TNFR2 expressed on the surface of TNFR2-positive cells, and such binding results in the depletion of such target cells. As described above, TNFR2 is highly expressed on Tregs in tumors of various cancer patients. In such patients, the antibody molecules of the present invention will preferentially bind to Tregs, thereby resulting in the depletion of Tregs. Tregs have an inhibitory effect on the proliferation, activation, and cytotoxicity of other immune cells, such as CD8-positive (CD8+) cells. Therefore, depletion of Tregs will, at least indirectly, lead to increased proliferation, activation, and migration of CD8+ cells, thereby increasing the number of CD8+ cells within the tumor.

[0092] In one aspect, the present application provides anti-TNFR2 antibodies that specifically bind to human and / or cynomolgus monkey TNFR2. The anti-TNFR2 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy and / or light chain CDRs as described herein. In one aspect, the present application provides isolated antibodies that bind to TNFR2. Contemplated anti-TNFR2 antibodies include, for example, full-length anti-TNFR2 antibodies (e.g., full-length IgG1 or IgG4), anti-TNFR2 single-chain antibodies (scFvs), anti-TNFR2 Fc fusion proteins, multispecific (e.g., bispecific) anti-TNFR2 antibodies, anti-TNFR2 immunoconjugates, and the like. In some embodiments, the anti-TNFR2 antibody is a full-length antibody (e.g., full-length IgG1 or IgG4) or an antigen-binding fragment thereof that specifically binds to TNFR2. In some embodiments, the anti-TNFR2 antibody is a Fab, Fab', F(ab')2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody, or linear antibody.

[0093] In some embodiments, the anti-TNFR2 antibodies described herein specifically bind to a linear epitope of human TNFR2. In some embodiments, the anti-TNFR2 antibodies described herein specifically bind to a non-linear epitope of human TNFR2. In some embodiments, the anti-TNFR2 antibodies described herein specifically bind to an epitope on human TNFR2 comprising Arg99, Lys108, Glu110, Gly111, Arg113, Leu114, and Asp136 of the human TNFR2 sequence set forth in SEQ ID NO:25.

[0094] In some embodiments, the properties of the antibody include, but are not limited to: (i) inhibition of TNF-α binding to TNFR2; (ii) inhibition of TNFR2 signaling pathway; (iii) cross-reactivity with human TNFR2 and cynomolgus monkey TNFR2; (iv) lower non-specific binding to dsDNA, insulin or baculovirus particles; (v) inhibition of Treg cell proliferation; (vi) inhibition of tumor growth or depletion of tumor cells; (vii) reduction of Treg-mediated immunosuppression; (viii) conversion of Treg into effector T cells; (ix) in vivo pharmacokinetic (PK) profile; (x) thermal stability (e.g., high Tm or Tagg); (xi) developability; (xii) reduced toxicity or immunogenicity; or (xiii) easier to manufacture.

[0095] In some embodiments, any of the anti-TNFR2 antibodies described herein comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-TNFR2 antibody comprises an IgG1 heavy chain constant region. In some embodiments, the anti-TNFR2 antibody comprises an IgG2 heavy chain constant region. In some embodiments, the anti-TNFR2 antibody comprises an IgG3 heavy chain constant region. In some embodiments, the anti-TNFR2 antibody comprises an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 21. In some embodiments, the heavy chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-TNFR2 antibody comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-TNFR2 antibody comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-TNFR2 antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain.

[0096] Exemplary antibody sequences are shown in Tables 2-4, wherein CDR numbering is performed according to the EU numbering system Kabat definition. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining the variable regions of antibody light and heavy chains. H and / or V L Antibodies that are based on sequences other than those exemplified in the table below are also within the scope of this application. The anti-TNFR2 antibody sequence of our patent international application number PCT / US2022 / 073523 is incorporated into the present invention. PD-L1 or PD-1 antagonists

[0097] The PD-L1 or PD-1 antagonist can be any drug that inhibits the biological activity of PD-L1 or PD-1. As described herein, "the biological activity of PD-L1 or PD-1" includes the biological activity of PD-L1 or PD-1 or the biological activity of both PD-L1 and PD-1. As described herein, the term "PD-L1 or PD-1 antagonist" refers collectively to PD-L1 or PD-1 antagonists. The biological activity of PD-L1 or PD-1 can be inhibited in any form, for example, by inhibiting the expression of any one or more of PD-L1 or PD-1 mRNA, PD-L1 or PD-1 protein, or by inhibiting the binding of PD-L1 to PD-1. The biological activity can be inhibited to any extent that can achieve a beneficial therapeutic effect. For example, in some embodiments, the biological activity can be completely inhibited (i.e., blocked), while in other embodiments, the biological activity can be partially inhibited (i.e., reduced). As used herein, unless otherwise indicated, the term "PD-L1 or PD-1" refers to PD-L1 or PD-1, respectively, in any form (e.g., mRNA or protein) and from any species (e.g., human or mouse).

[0098] In some embodiments of the present invention, the PD-L1 or PD-1 antagonist is a drug that inhibits PD-L1 or PD-1 signaling, and PD-L1 or PD-1 signaling can be inhibited in any form. For example, the PD-L1 or PD-1 antagonist can inhibit the activation and / or binding of any one or more downstream targets in the PD-L1 or PD-1 signaling pathway (e.g., nuclear factor kappa light chain enhancer of activated B cells (NFκB), mitogen-activated protein kinase (MAPK) 3 / extracellular signal-regulated kinase (ERK) 1, MAPK8 / Jun N-terminal protein kinase (Jun N-Terminal Protein Kinase (JNK)), AKT, and B-catenin). For example, the PD-L1 or PD-1 antagonist may be an agent that binds to PD-L1 or PD-1, thereby reducing or blocking PD-L1 or PD-1 signaling and inhibiting its function. The following examples are for illustration only, and the PD-L1 or PD-1 antagonist that inhibits PD-L1 or PD-1 signaling can be any antibody or antibody fragment, antisense nucleic acid or chemical inhibitor (e.g., small molecule or peptide (or polypeptide) inhibitor) described herein.

[0099] In some embodiments, the PD-L1 or PD-1 antagonist is an agent that inhibits the binding of PD-L1 to PD-1. In this regard, the PD-L1 or PD-1 antagonist may be any agent that binds to PD-L1 or PD-1 protein, thereby reducing or blocking the binding of PD-L1 to PD-1 and inhibiting its function, as well as an agent that competes with PD-L1 or PD-1 protein for natural PD-L1 or PD-1 binding sites. For example, the agent that inhibits the binding of PD-L1 to PD-1 can be any antibody or antibody fragment, antisense nucleic acid or chemical inhibitor (e.g., small molecule or peptide (or polypeptide) inhibitor) described herein.

[0100] In some embodiments of the present application, the PD-L1 or PD-1 antagonist is an antibody that specifically recognizes human PD-L1 or PD-1. In some embodiments, the antagonist is an antibody fragment that specifically binds to human PD-L1 or PD-1. The antibody can be any type of immunoglobulin known in the art. For example, the antibody can be of any isotype, such as IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal. The antibody can be a naturally occurring antibody, for example, an antibody isolated and / or purified from a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody can be a genetically engineered antibody, for example, a humanized antibody or a chimeric antibody, and may contain a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments. The antibody may be in monomeric or multimeric form. In some embodiments, the PD-L1 or PD-1 antibody specifically binds to human PD-L1 or human PD-1. The antibody may be a human antibody, a humanized antibody, or a chimeric antibody. The antibody may also have any degree of binding affinity or functional affinity for PD-L1 or PD-1 or PD-L1 or a functional fragment of PD-1, e.g., a PD-L1 or PD-1 binding portion.

[0101] Anti-PD-L1 or anti-PD-1 antibodies, i.e., antibody fragments, can be prepared using the PD-L1 or PD-1 proteins disclosed herein and conventional procedures. Suitable methods for preparing antibodies are known in the art. For example, standard hybridoma methods are described in, for example, Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). Alternatively, other methods, such as the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74 (2), 361-67 (1984), and Roder et al, Methods Enzymol, 121, 140-67 (1986)), and phage vector expression systems (see Huse et al, Science, 246, 1275-81 (1989)), are known in the art. Further, methods for preparing antibodies in non-human animals are described, for example, in US Patents 5,545,806, 5,569,825 and 5,714,352, US Patent Application No. 2002 / 0197266A1 and US Patent 7,338,929.

[0102] In some embodiments, examples of antibodies that bind to PD-1 and are used in various aspects and embodiments of the invention are described in US Pat. Nos. 8,552,154; 8,354,509; 8,168,757; 8,008,449; 7,521,051; 7,488,802; WO2004072286; WO2004056875; and WO2004004771. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, sintilimab, camrelizumab, toripalimab, tislelizumab, zimberelimab, prolgolimab, dostarlimab, AMG-404, balstilimab, budigalimab, siliglimab, cetrelimab), ezabenlimab, genolimzumab, LZM-009, nofazinlimab, penpulimab, pimivalimab, pucotenlimab, QL-1604, retifanlimab, rulonilimab, sasanlimab, SCT-I10A, serplulimab, SG-001, spartalizumab, SYSA-1802, and TY-101.

[0103] In some embodiments, examples of antibodies that bind to PD-L1 are described in US Pat. Nos. 9,212,224; 8,779,108; 8,552,154; 8,383,796; 8,217,149; US Patent Application Publication Nos. 20110280877; WO2013079174 and WO2013019906. Other exemplary antibodies that bind to PD-L1 (also known as CD274 or B7-H1) and methods of use are disclosed in US Pat. Nos. 8,168,179; 7,943,743; 7,595,048; WO2014055897; WO2013019906 and WO2010077634. Specific anti-human PD-L1 monoclonal antibodies that can be used as PD-1 antagonists in the treatment methods, drugs and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, and MSB0010718C. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of avelumab, durvalumab, atezolizumab, KN035, CK-301, ADG-104, BCD-135, garivulimab, CBT-502, HLX-20, IMC-001, tagitanlimab, LAE-005, LP-002, MSB-2311, adebrelimab, sugemalimab, and socazolimab. Pharmaceutical composition or combination

[0104] Also provided herein are compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising an antibody or combination of antibodies (e.g., a combination of an anti-TNFR2 antibody and an anti-PD-L1 or anti-PD-1 antibody). Nucleic acids encoding anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies, vectors comprising nucleic acids encoding any of the antibodies, or host cells comprising nucleic acids or vectors described herein. In some embodiments, a pharmaceutical composition is provided comprising an antibody or a combination of an anti-TNFR2 antibody and an anti-PD-L1 or anti-PD-1 antibody as described herein, and a pharmaceutically acceptable carrier.

[0105] Suitable anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody formulations can be prepared in the form of lyophilized formulations or liquid formulations by mixing an anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG); exemplary formulations are described in WO98 / 56418, expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration formulation, and the reconstituted formulation can be administered subcutaneously to the subject to be treated herein. Cationic liposomes or liposomes can be used to deliver the anti-TNFR2 antibodies of the present application to cells.

[0106] In addition to anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies, the formulations described herein may also contain one or more other active substances to meet the needs of the specific indication being treated, preferably substances with complementary activities and no negative effects on each other. For example, in addition to anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies, it may be necessary to further include, for example, anti-tumor agents, growth inhibitory agents, cytotoxic agents or chemotherapeutic agents. These molecules are present in combination in amounts effective for the intended purpose. The effective amount of these other substances depends on the amount of anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies in the formulation, the type of disease or condition or treatment, and other factors as described above. These drugs are generally used in the same dosages and routes of administration as described herein, or at 1% to 99% of the currently used dosages.

[0107] The anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody (e.g., full-length anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody) can also be encapsulated in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Sustained-release formulations can be prepared.

[0108] Sustained-release formulations of anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies (e.g., full-length anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies) can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semipermeable matrices containing the antibody (or fragment thereof), which are in the form of shaped articles, for example, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can enable release of molecules for more than 100 days, certain hydrogels can release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate due to exposure to a humid environment at 37°C, which may lead to loss of bioactivity or altered immunogenicity. Rational strategies can be designed to stabilize anti-TNFR2 antibodies, anti-PD-L1, or anti-PD-1 antibodies based on the corresponding mechanism. For example, if the aggregation mechanism is found to be through the formation of intermolecular SS bonds through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in acidic solutions, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.

[0109] In some embodiments, the anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody (e.g., full-length anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody) is formulated in a buffer comprising citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof. In some embodiments, the anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody is formulated in a buffer comprising 100 mM to 150 mM glycine. In some embodiments, the anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody is formulated in a buffer comprising sodium chloride. In some embodiments, the anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody is formulated in a buffer comprising acetate. In some embodiments, the anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody is formulated in a buffer comprising succinate. In some embodiments, the anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody is formulated in a buffer comprising polysorbate 80. In some embodiments, the anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody is formulated in a buffer having a pH of 5.1 to 5.6.

[0110] Preparations for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through sterile filtration membranes.

[0111] In one aspect, the present application provides a pharmaceutical composition comprising an antibody that specifically recognizes human TNFR2 and a PD-L1 or PD-1 antagonist.

[0112] In one aspect, the present application provides a pharmaceutical composition comprising an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

[0113] In another aspect, the present application provides a combination of an antibody that specifically recognizes human TNFR2 and a PD-L1 or PD-1 antagonist.

[0114] On the other hand, the present application provides a combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1. Combination therapy

[0115] As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably, whether in the singular or plural, to refer to cells that have undergone malignant transformation and become pathological to the host. Primary cancer cells can be readily distinguished from non-cancerous cells by well-known techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from primary cancer cells. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to a cancer that typically manifests as a solid tumor, a "clinically detectable" tumor refers to a tumor that is detectable based on the mass of the tumor; for example, by procedures such as computed tomography (CT), magnetic resonance imaging (MRI), X-rays, ultrasound, or palpation during physical examination, and / or due to the expression of one or more cancer-specific antigens in a sample that can be obtained from the patient. The tumor may be a hematopoietic (or blood-derived or hematological or blood-related) cancer, for example, a cancer derived from blood cells or immune cells, which may be referred to as a "liquid tumor." Specific examples of clinical conditions based on hematological neoplasms include leukemias, such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia; plasma cell malignancies, such as multiple myeloma, MGUS, and Waldenstrom's macroglobulinemia; lymphomas, such as non-Hodgkin's lymphoma, Hodgkin's lymphoma, and the like.

[0116] These diseases include, but are not limited to, non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, gastroesophageal junction adenocarcinoma, esophageal cancer, esophageal adenocarcinoma, gallbladder cancer, stomach cancer, cervical cancer, gastric adenocarcinoma, head and neck cancer, heart cancer, hepatocellular carcinoma, kidney cancer, liver cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, prostate adenocarcinoma, spleen cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and infectious diseases, including, but not limited to, human Human papillomavirus (HPV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), varicella-zoster virus (VSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Escherichia coli, Chlamydia, Mycobacterium tuberculosis, Streptococcus, Pneumococcus, Pseudomonas, Campylobacter, Salmonella, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans, and Histoplasma capsulatum.

[0117] In some embodiments, the cancer is breast cancer, colon cancer, cervical cancer, kidney cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., NSCLC), ovarian cancer, melanoma, skin cancer (e.g., squamous cell carcinoma or basal cell carcinoma), lymphoma, or leukemia. In certain embodiments, the cancer is melanoma.

[0118] "Combination," "in combination with," or "combined with" are not intended to imply that the therapies or therapeutic agents must be administered simultaneously and / or formulated for delivery together (e.g., in the same composition), although such methods and compositions are within the scope described herein. For example, the anti-TNFR2 antibody and anti-PD-L1 or anti-PD-1 antibody can be administered simultaneously or sequentially with each other (e.g., before or after). The agents in the combination can be administered in any order. Generally, each agent will be administered at a dose and / or schedule determined for that agent. As will be further understood, the therapeutic agents used in combination can be administered together in a single composition or separately in different compositions. Generally, it is expected that the therapeutic agents used in combination can be used at levels no greater than those used individually. In some embodiments, the levels used in combination can be lower than those used individually.

[0119] In some embodiments, to treat cancer or infectious diseases, the antibody that specifically recognizes human TNFR2 can be administered in combination with one or more anti-cancer drugs, such as immune checkpoint inhibitors, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, or anti-tumor compositions.

[0120] In some embodiments, the antibody that specifically recognizes human TNFR2 is administered to a subject together with a second therapeutic drug (such as an immune checkpoint inhibitor (e.g., a PD-1 or PD-L1 signaling inhibitor, an anti-PD-1 antibody, or an anti-PD-L1 antibody)), and stimulating the immune system has a beneficial effect on the disease suffered by the subject, for example, cancer or an infectious disease.

[0121] In one aspect, a method of treating cancer or an infectious disease in an individual in need thereof is provided, comprising administering to the individual a composition comprising an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

[0122] In some embodiments, a pharmaceutical composition comprising an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 is provided for treating cancer or an infectious disease in an individual in need thereof.

[0123] In some embodiments, a pharmaceutical composition comprising an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 is used in the preparation of a medicament for treating cancer or an infectious disease in an individual in need thereof.

[0124] In another aspect, a method of treating cancer or an infectious disease in an individual in need thereof is provided, comprising administering to the individual a combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

[0125] In some embodiments, a combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 is provided for use in treating cancer or an infectious disease in an individual in need thereof.

[0126] In some embodiments, there is provided a use of a combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 in the preparation of a medicament for treating cancer or an infectious disease in an individual in need thereof.

[0127] In another aspect, a method of treating cancer or an infectious disease in an individual in need thereof is provided, comprising administering to the individual an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

[0128] In some embodiments, antibodies that specifically recognize human TNFR2 and antibodies that specifically recognize human PD-L1 or PD-1 are provided for use in treating cancer or infectious diseases in an individual in need thereof.

[0129] In some embodiments, there is also provided use of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 in the preparation of a medicament for treating cancer or infectious diseases in an individual in need thereof. In other embodiments, there is also provided use of an antibody that specifically recognizes human TNFR2 in the preparation of a medicament for treating cancer or infectious diseases in an individual in need thereof in combination with an antibody that specifically recognizes human PD-L1 or PD-1.

[0130] Dosage

[0131] In some embodiments, the antibody that specifically recognizes human TNFR2 and the antibody that specifically recognizes human PD-L1 or PD-1 can be administered simultaneously or sequentially.

[0132] In some embodiments, an antibody that specifically recognizes human TNFR2 is administered after administration of an antibody that specifically recognizes human PD-L1 or PD-1. In some embodiments, an antibody that specifically recognizes human PD-L1 or PD-1 is administered after administration of an antibody that specifically recognizes human TNFR2.

[0133] In some embodiments, the antibody that specifically recognizes human PD-L1 or PD-1 is administered simultaneously with the antibody that specifically recognizes human TNFR2.

[0134] In some embodiments, the antibody that specifically recognizes human PD-L1 or PD-1 is administered during treatment with an antibody that specifically recognizes human TNFR2.

[0135] In some embodiments, the antibody that specifically recognizes human TNFR2 is administered during treatment with an antibody that specifically recognizes human PD-L1 or PD-1.

[0136] In some embodiments, the pharmaceutical composition or combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 as described herein is administered to an individual in need thereof in a therapeutically effective amount. In some embodiments, the pharmaceutical composition or combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 as described herein is administered to an individual in need thereof in a therapeutically effective amount.

[0137] In some embodiments, the antibody specifically recognizing human TNFR2 described herein binds to an epitope of human TNFR2, wherein the epitope comprises 1, 2, 3, 4, 5, 6 or 7 amino acid residues selected from the group consisting of Arg99, Lys108, Glu110, Gly111, Arg113, Leu114 and Asp136 of the human TNFR2 sequence shown in SEQ ID NO: 25.

[0138] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, said variant comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof, said variant comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof, said variant comprising up to about 3 amino acid substitutions; and V L , the V L Comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises substitutions of up to about 3 amino acids; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises substitutions of up to about 3 amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof, wherein the variant comprises substitutions of up to about 3 amino acids.

[0139] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Comprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 7-15 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 16-20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0140] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Contains the amino acid sequence V shown in SEQ ID NO: 7 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 16 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0141] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V HContains the amino acid sequence V shown in SEQ ID NO:8 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 17 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0142] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Contains the amino acid sequence V shown in SEQ ID NO:9 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 18 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0143] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Contains the amino acid sequence V shown in SEQ ID NO:9 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 19 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0144] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Contains the amino acid sequence V shown in SEQ ID NO:9 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 17 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0145] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Comprising the amino acid sequence V shown in SEQ ID NO: 10H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0146] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Comprising the amino acid sequence V shown in SEQ ID NO: 11 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0147] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Contains the amino acid sequence V shown in SEQ ID NO: 12 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0148] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Comprising the amino acid sequence V shown in SEQ ID NO: 13 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0149] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Contains the amino acid sequence V shown in SEQ ID NO: 14 HHC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0150] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H Comprising the amino acid sequence V shown in SEQ ID NO: 15 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising the amino acid sequence V shown in SEQ ID NO: 20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0151] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 7-15 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one of SEQ ID NOs: 7-15; and V L , the V L Comprising the amino acid sequence shown in any one of SEQ ID NOs: 16-20 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NOs: 16-20.

[0152] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L, which comprises the amino acid sequence of SEQ ID NO:16 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:16.

[0153] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 8; and V L , which comprises the amino acid sequence of SEQ ID NO:17 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:17.

[0154] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , which comprises the amino acid sequence of SEQ ID NO:18 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:18.

[0155] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , which comprises the amino acid sequence of SEQ ID NO:19 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:19.

[0156] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , which comprises the amino acid sequence of SEQ ID NO:17 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:17.

[0157] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 10; and V L , which comprises the amino acid sequence of SEQ ID NO:20 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0158] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 11; and V L , which comprises the amino acid sequence of SEQ ID NO:20 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0159] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 12; and V L , which comprises the amino acid sequence of SEQ ID NO:20 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0160] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 13; and V L , which comprises the amino acid sequence of SEQ ID NO:20 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0161] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 14; and V L , which comprises the amino acid sequence of SEQ ID NO:20 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0162] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 15; and V L , which comprises the amino acid sequence of SEQ ID NO:20 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0163] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:37.

[0164] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , which comprises the amino acid sequence of SEQ ID NO:38 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:38.

[0165] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30; and V L , which comprises the amino acid sequence of SEQ ID NO:39 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:39.

[0166] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30; and V L , which comprises the amino acid sequence of SEQ ID NO:40 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:40.

[0167] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30; and V L , which comprises the amino acid sequence of SEQ ID NO:38 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:38.

[0168] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31; and V L , which comprises the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:41.

[0169] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32; and V L , which comprises the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:41.

[0170] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33; and V L , which comprises the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:41.

[0171] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 34; and V L , which comprises the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:41.

[0172] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 35; and V L , which comprises the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:41.

[0173] In some embodiments, the antibody that specifically recognizes human TNFR2 comprises V H , the V H comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 36; and V L , which comprises the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:41.

[0174] In some embodiments, the antibody that specifically recognizes human PD-L1 is selected from the group consisting of avelumab, durvalumab, atezolizumab, KN035, CK-301, ADG-104, BCD-135, garivulimab, CBT-502, HLX-20, IMC-001, tagitanlimab, LAE-005, LP-002, MSB-2311, adebrelimab, sugemalimab, and socazolimab.

[0175] In some embodiments, the antibody that specifically recognizes human PD-1 is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, sintilimab, camrelizumab, toripalimab, tislelizumab, zimberelimab, prolgolimab, dostarlimab, AMG-404, balstilimab, budigalimab, The group consisted of cetrelimab, ezabenlimab, genolimzumab, LZM-009, nofazinlimab, penpulimab, pimivalimab, pucotenlimab, QL-1604, retifanlimab, rulonilimab, sasanlimab, SCT-I10A, serplulimab, SG-001, spartalizumab, SYSA-1802, and TY-101.

[0176] In some embodiments, the combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 is superior to the antibody that specifically recognizes human TNFR2 of the same molar mass or the antibody that specifically recognizes human PD-L1 or PD-1 of the same molar mass, respectively. In some embodiments, the anti-tumor effect of the combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1 is improved by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, respectively, compared to the antibody that specifically recognizes human TNFR2 of the same molar mass or the antibody that specifically recognizes human PD-L1 or PD-1 of the same molar mass. Binding affinity

[0177] Binding affinity can be expressed as Kd, Koff, Kon or Ka. As described herein, the term "Koff" refers to the rate constant at which the antigen binding domain dissociates from the antigen binding domain / antibody complex, as determined by a kinetic selection apparatus. The term "Kon" used herein refers to the association rate constant at which the antibody binds to the antigen to form the antigen binding domain / antibody complex. The term dissociation constant "Kd" used herein refers to the dissociation constant for a specific antibody-antigen interaction, describing the antigen concentration required for the antigen to occupy half of all antigen binding domains and reach equilibrium in a solution of antibody molecules, which is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In cases where the antigen binding domain is attached to the cell wall, for example in a yeast expression system, the corresponding dissociation rate constant is expressed as EC50, which is a good approximation of Kd. The affinity binding constant Ka is the reciprocal of the dissociation constant Kd.

[0178] The equilibrium dissociation constant (Kd) can be used as an indicator of the affinity of the antigen-binding domain for the antigen. For example, a simple analysis can be performed using antibodies labeled with various markers and a Biacore instrument (manufactured by Amersham Biosciences) by the Scatchard method, and the interaction between biomolecules can be analyzed by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values ​​obtained using these methods are expressed in units of M (mol). An antibody that specifically binds to a target may have, for example, a Kd of ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 Kd value of M.

[0179] The binding specificity of an antibody can be determined experimentally by methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning. Nucleic Acids

[0180] Nucleic acid molecules encoding anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies are also contemplated. In some embodiments, a nucleic acid (or a set of nucleic acids) encoding a full-length anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody is provided, including any of the full-length anti-TNFR2 or anti-PD-L1 or anti-PD-1 antibodies described herein. In some embodiments, the nucleic acid (or a set of nucleic acids) encoding the antibodies described herein may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His-tag, an HA tag).

[0181] Also contemplated herein are isolated host cells comprising an anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody, isolated nucleic acids encoding an anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody polypeptide component, or vectors comprising nucleic acids encoding an anti-TNFR2 antibody, anti-PD-L1 or anti-PD-1 antibody polypeptide component as described herein.

[0182] The present application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibodies of the present application under at least moderately stringent hybridization conditions.

[0183] The present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.

[0184] In brief, a natural or synthetic nucleic acid encoding an antibody is inserted into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, such as a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), to express the antibody. The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate the expression of the target nucleic acid sequence.

[0185] The nucleic acids described herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. For example, see US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the present application also provides gene therapy vectors.

[0186] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0187] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin that works in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0188] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.

[0189] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30–110 bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are interchanged or moved relative to one another. In the thymidine kinase (Tk) promoter, activity begins to decline only when the spacing between promoter elements increases to 50 bp.

[0190] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, this application should not be limited to using only constitutive promoters. Inducible promoters are also considered in this application. The use of an inducible promoter provides a molecular switch that can activate expression of an operably linked polynucleotide sequence when such expression is desired, and deactivate expression when such expression is not desired. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0191] In some embodiments, expression of the antibody is inducible. In some embodiments, the nucleic acid sequence encoding the antibody is operably linked to an inducible promoter, including any of the inducible promoters described herein. Inducible promoter

[0192] The use of an inducible promoter provides a molecular switch that turns on the expression of a polynucleotide sequence operably linked thereto when expression is desired, and turns off expression when expression is not desired. Exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, DM et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al (1993) Biochemistry 32:10607-10613; Datta, R. et al (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters suitable for use in in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system used to express antibodies is the Tet system. In some embodiments, the inducible promoter system used to express antibodies is the E. coli lac repression system.

[0193] An exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or its active analogs, the inducer releases TetR from TetO, thereby causing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0194] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, so that the sequence of a given nucleic acid has a large number of variants without any change in the amino acid sequence it encodes. However, many organisms differ in codon usage, also known as "codon preference" (i.e., the preference for using a specific codon for a given amino acid). Codon preference is generally associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).

[0195] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, the tetracycline-regulated transcription activator protein (tTA), composed of a fusion of TetR and the strong transcription activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of the tetracycline-responsive promoter element (TRE). The TRE element consists of a TetO sequence fused in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and the target gene cannot be expressed.

[0196] In contrast, in the Tet-On system, transcription is activated in the presence of either Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transactivation domain. However, a four-amino acid change in the DNA-binding region of TetR alters rtTA's binding properties, allowing it to only recognize the tetO sequence within the target transgene's TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can activate transcription of its TRE-regulated target gene only in the presence of Dox.

[0197] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49: 603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising the lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducing agent, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0198] In order to evaluate the expression of a polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments. Either the selectable marker gene or the reporter gene can be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0199] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and encodes a polypeptide whose expression is expressed as some easily detectable properties, such as enzymatic activity. After DNA is introduced into the recipient cell, the expression of the reporter gene is detected at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tel et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared by known techniques or obtained commercially. Typically, a construct with a minimal 5' flanking region that can show the highest expression level of a reporter gene is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0200] In some embodiments, nucleic acid encoding any of the antibodies described herein is provided. In some embodiments, the nucleic acid includes one or more nucleic acid sequences encoding antibody heavy and light chains. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).

[0201] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0202] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistic methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0203] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0204] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0205] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to import nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, the nucleic acid can be combined with lipids. Nucleic acids combined with lipids can be wrapped into the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule combined with the liposome and the oligonucleotide, embedded in the liposome, form a complex with the liposome, be dispersed in a solution containing lipids, mix with lipids, combine with lipids, be suspended in lipids, be contained in micelles or mix with micelles, or otherwise combine with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist with a bilayer structure, with micelles or with a "collapsed" structure. They can also simply be dispersed in solution and may form aggregates of uneven size or shape. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0206] Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present application, a variety of experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such experiments include, for example, "molecular biology" experiments well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or identification by the assays described herein, all fall within the scope of the present application. Antibody preparation

[0207] In some embodiments, the antibody (e.g., an antibody that specifically recognizes TNFR2, PD-L1, or PD-1) is a monoclonal antibody. In some embodiments, the antibody is derived from a monoclonal antibody. In some embodiments, the antibody comprises a V from a monoclonal antibody. H and V L , or variants thereof. In some embodiments, the antibody further comprises a C from a monoclonal antibody H 1 and C L region, or variants thereof. Monoclonal antibodies can be prepared using methods known in the art, such as hybridoma cell methods, yeast display, phage display methods, or recombinant DNA methods. In addition, exemplary yeast display and phage display methods are described herein and in the following examples.

[0208] In the hybridoma method, hamsters, mice or other suitable host animals are usually immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the target protein. Typically, if human cells are needed, peripheral blood lymphocytes ("PBLs") are used, and if non-human mammalian derived cells are needed, spleen cells or lymph node cells are used. Lymphocytes are fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0209] In some embodiments, the immortalized cell line is effectively fused, ensures high-level stable expression of the antibody by the selected antibody-producing cells, and is sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for use in preparing human monoclonal antibodies.

[0210] The presence of monoclonal antibodies against the polypeptide in the culture medium of the hybridoma cells can then be determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding experiments, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by Scatchard analysis as described in, for example, Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0211] After identifying the desired hybridoma cells, the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium. Alternatively, hybridoma cells can be grown in mammalian ascites.

[0212] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0213] In some embodiments, according to any of the antibodies described herein, the antibody comprises a sequence selected from a clone of an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening combinatorial libraries of antibody fragments having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. It is further described in Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0214] In some phage display methods, V H and V LAll components of the gene are randomly recombined in a phage library, and then phages that can bind to the antigen are screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments in the form of scFv fragments or Fab fragments. Library phages of immune origin provide high-affinity antibodies against immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies against a variety of non-self antigens and self-antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0215] The antibody is prepared by screening the library for antigen-binding portions that can specifically bind to the target antigen (such as TNFR2, PD-L1 or PD-1) through phage display. The library can be a human scFv phage display library with at least 1×10 9 (For example, at least 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11) kinds of unique human antibody fragments with different diversity. In some embodiments, the library is a human natural library constructed by DNA extracted from PMBCs and spleens of healthy subjects, comprising all human heavy and light chain subfamilies. In some embodiments, the library is a human natural library constructed by DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semisynthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, RM et al., Nat. Biotechnol. 23 (3): 344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semisynthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) amino acids. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0216] Phage clones with high affinity for target antigens (such as TNFR2, PD-L1 or PD-1) can be screened by iterative binding of phage to target antigens, which are bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage and elution of the specifically bound phage. Subsequently, the bound phage clones are eluted and used to infect suitable host cells, such as E. coli XL1-Blue, for expression and purification. Multiple rounds of panning (e.g., 2, 3, 4, 5, 6 or more rounds), such as solution panning, cell panning or a combination of the two, can be performed to enrich for phage clones that specifically bind to the target antigen. The specific binding of the enriched phage clones to the target antigen can be detected by any method known in the art, including, for example, ELISA and FACS.

[0217] Monoclonal antibodies and bispecific antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described in this application can be easily isolated and sequenced by conventional methods (e.g., by oligonucleotide probes that specifically bind to the genes encoding the light and heavy chains of the murine antibody). Hybridoma cells as described above or antigen-specific phage clones of this application can be used as a source of such DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a simian COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain monoclonal antibodies synthesized in the recombinant host cell. The DNA can also be modified, for example, by replacing homologous non-human sequences with coding sequences for human heavy and light chain constant structures and / or framework regions (US Patent No. 4,816,567; Morrison et al., supra), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can replace the constant region of the antibody in the present application, or can replace one antigen binding site in the variable domain of the antibody in the present application to form a chimeric bivalent antibody. In some embodiments, other variable domains directed against different epitopes or antigens can be included to produce chimeric bispecific antibodies.

[0218] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is typically truncated at any position in the Fc region to prevent cross-linking of the heavy chains. Alternatively, the relevant cysteine ​​residues are replaced with other amino acid residues or deleted to prevent cross-linking.

[0219] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0220] The antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused with an immunoglobulin constant region. Preferably, they are fused with an immunoglobulin heavy chain constant region, which includes at least a portion of a hinge, CH2, and CH3 region. In certain embodiments, the first heavy chain constant region (CH1) comprising the necessary sites for light chain binding is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion, if desired, may also include the DNA encoding the immunoglobulin light chain, is inserted into an independent expression vector, and is co-transfected into a suitable host organism. In certain embodiments, the antibody variable domains targeting different epitopes or different antigens can be fused with immunoglobulin constant domain sequences to produce chimeric bispecific antibodies. Human and humanized antibodies

[0221] The antibody can comprise a humanized antibody portion or a human antibody portion. The humanized form of a non-human (such as mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab', F(ab')2, scFv or other antigen-binding subsequences of an antibody), which generally includes a minimum sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins, immunoglobulin chains or its fragment (receptor antibody), wherein the residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues with desired specificity, affinity and performance, such as the CDR of a mouse, rat or rabbit. In certain embodiments, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies can also include amino acid residues that are neither part of the receptor antibody nor in the CDR or framework region sequences introduced. Generally, humanized antibodies include at least one, generally two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the framework regions are human immunoglobulin consensus sequences.

[0222] Typically, a humanized antibody or humanized antibody portion contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are generally referred to as "imported" residues, typically from the "imported" variable domain. According to some embodiments, humanization can be performed essentially according to the following method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, this "humanized" antibody portion (US Patent No. 4,816,567) is essentially less than a complete human antibody, in which the variable domains have been replaced by corresponding sequences from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0223] Human antibodies are an alternative to humanization. For example, it is now possible to prepare transgenic animals (e.g., mice) that can produce a complete human antibody library after immunization without producing endogenous immunoglobulins. For example, it has been reported that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits the production of endogenous antibodies. Transferring the human germline immunoglobulin gene array into such germline mutant mice can produce human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, human antibody production can be found to be very similar to that in humans in all aspects, including gene rearrangement, assembly, and antibody libraries. This method is described in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0224] Human antibodies or human antibody portions can also be produced by in vitro activated B cells (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147 (1):86-95 (1991). Anti-TNFR2, anti-PD-L1, or anti-PD-1 antibody variants

[0225] In some embodiments, the amino acid sequences of variants of the antibodies provided herein (e.g., antibodies that specifically recognize TNFR2, PD-L1, or PD-1) are also contemplated. For example, it may be necessary to improve the binding affinity and / or other biological activities of the antibody. The amino acid sequence of the antibody variant can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. The final construction can be completed by any combination of deletions, insertions, and substitutions of amino acid residues to give it the desired characteristics. For example, antigen binding.

[0226] In some embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody to screen for products of desired activity, for example, improved biological activity, maintained / improved antigen binding ability, and reduced immunogenicity. In some embodiments, the amino acid substitutions described herein are limited to the "exemplary substitutions" shown in Table A of this application. In some embodiments, amino acid substitutions are limited to the "preferred substitutions" shown in Table A of this application.

[0227] Conservative substitutions are shown in Table A below.

[0228] Amino acids are divided into different categories based on common side chain properties: a. Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that affect chain direction: Gly, Pro; f. Aromatic: Trp, Tyr, Phe.

[0229] Non-conservative substitutions entail substituting a member of one of these classes for a member of another class.

[0230] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display. In short, one or more CDR residues are mutated, the variant antibody portion is displayed on phage, and variants with specific biological activity (e.g., biological activity or binding affinity based on an RBC lysis inhibition assay) are screened. Changes (e.g., substitutions) can be made in the HVRs region, for example, to obtain improved biological activity or antibody affinity based on an RBC lysis inhibition assay. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at specific deterministic residues (SDRs), and the resulting variant V H and V L Methods for constructing and reselecting affinity maturation from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0231] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes HVR-mediated methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.

[0232] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be produced in HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments, the variant V provided above H and V L Sequences, each HVR is either unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0233] A useful method for identifying amino acid residues or regions in antibodies that can be targeted for mutation is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are replaced with neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the amino acid position to demonstrate that the position has functional sensitivity to the initial substitution. Alternatively or additionally, the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine whether they have the desired properties.

[0234] Insertions into the amino acid sequence include fusions at the amino and / or carboxyl termini ranging in length from one residue to polypeptides containing 100 or more residues, and also include insertions of one or more amino acid residues within the sequence. Examples of terminal insertions include antigen-binding portions having a methionyl residue at the N-terminus. Other insertional variants of the antigen-binding portion include fusions to the N-terminus or C-terminus of the antigen-binding portion to an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antigen-binding portion. Fc region variants

[0235] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody described herein (e.g., an anti-TNFR2, anti-PD-L1, or anti-PD-1 antibody), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC potency, typically associated with receptors that bind to Fc (FcRs). In some embodiments, the Fc region variant has reduced ADCC potency. There are many examples of how changes or mutations in the Fc sequence affect its potency, for example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9 (2): 6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0236] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., infected cells) when antigens on the surface of the target cell membrane are bound by specific antibodies (e.g., anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies). Typically, the ADCC effect involves NK cells activated by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the surface of target cells. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of the antibody leads to activation of the NK cell, release of cytolytic granules, and subsequent apoptosis of the target cell.

[0237] In some embodiments, the present application also provides antibodies (e.g., anti-TNFR2, anti-PD-L1 or anti-PD-1 antibodies) comprising an Fc region with some but not all effector functions, such that the antibody has an extended half-life in vivo, but a specific effector function (e.g., CDC or ADCC) is unnecessary or harmful, and such antibodies become ideal candidates for the present application. The reduction / elimination of CDC and / or ADCC activity is confirmed by performing cytotoxicity assays in vitro and / or in vivo. For example, an Fc receptor (FcR) binding assay is used to confirm that the antibody lacks FcγR binding ability (and therefore may lack ADCC activity) but still retains FcRn binding ability. Among the main cells that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assessment of ADCC activity of a molecule of interest are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see, e.g., ACTI TM Flow cytometry nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and CytoTox 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.) can be used. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be tested in vivo, for example, in animal models as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and M. J. Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0238] Antibodies with reduced effector function comprising one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region ( US Pat. No. 6,737,056 ). These Fc variants include Fc variants with substitutions at two or more residues at positions 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine ( US Pat. No. 7,332,581 ).

[0239] Such antibody variants with increased or decreased binding to FcRs have been described (see, eg, US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0240] In some embodiments, alterations in the Fc region result in altered (ie, enhanced or diminished) regulatory effects, as described in Moore et al., MAbs. 2(2): 181-189 (2010).

[0241] In some embodiments, anti-TNFR2 antibodies, anti-PD-L1 or anti-PD-1 antibodies are provided, which comprise Fc region variants having one or more amino acid substitutions that extend half-life and / or enhance binding to Fc receptors (FcRn). Antibodies with extended half-life and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise one or more amino acid substitutions in the Fc region that enhance binding of the Fc region to FcRn. These Fc variants comprise one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 in the Fc region, such as a substitution at residue 434 in the Fc region ( U.S. Pat. No. 7,371,826 ).

[0242] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821 and WO 94 / 29351 for additional examples of Fc region variants.

[0243] The present application contemplates anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies (e.g., full-length anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies) comprising any one of the Fc variants described herein, or a combination thereof. Glycosylation variants

[0244] In some embodiments, the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies provided herein are altered to increase or decrease the extent of glycosylation of the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies. Adding or deleting glycosylation sites on an antibody can be conveniently accomplished by altering the amino acid sequence of the antibody or its polypeptide portion to add or remove one or more glycosylation sites.

[0245] When anti-TNFR2, anti-PD-L1 or anti-PD-1 antibodies contain an Fc region, the sugars attached thereto can be changed. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically connected to the CH2 domain Asn297 of the Fc region via an N-link, see, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as trehalose connected to the GlcNAc of the "stem" portion of the biantennary oligosaccharide structure. In some embodiments, the antibodies of the present application may be oligosaccharide-modified to produce antibody variants with certain improved properties.

[0246] The N-glycans attached to the CH2 domain of the Fc region are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140: 777-83). Typically, a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for binding to FcγRs; however, non-fucosylated N-glycans enhance the binding ability of Fc to FcγRIIIa. Enhanced binding to FcγRIIIa results in an enhanced ADCC effect, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0247] In some embodiments, when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental. In some embodiments, the Fc fragment or CH2 domain is non-glycosylated. In some embodiments, glycosylation is prevented by mutating the N-glycosylation site in the CH2 domain.

[0248] In some embodiments, anti-TNFR2, anti-PD-L1 or anti-PD-1 antibody variants are provided, which comprise an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may enhance ADCC function. Specifically, antibodies provided herein have reduced fucose relative to the same antibody produced by wild-type CHO cells. That is, they are characterized in that they have a smaller amount of fucose than antibodies produced by natural CHO cells (e.g., CHO cells that produce natural glycosylated forms, CHO cells containing natural FUT8 genes). In some embodiments, the N-linked glycans of the anti-TNFR2, anti-PD-L1 or anti-PD-1 antibody have less than 50%, 40%, 30%, 20%, 10% or 5% fucose. For example, the fucose content of the anti-TNFR2, anti-PD-L1 or anti-PD-1 antibody may be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. In some embodiments, the N-linked glycans of the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies do not contain fucose, i.e., the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies are completely fucose-free, have no fucose, or are defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chains attached to Asn297 relative to the total amount of all sugar structures (e.g., complex, hybrid, or mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at position 297 of the Fc region (Fc region residues are numbered according to the EU numbering system). However, due to minor sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have enhanced ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.), US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. al. Biotech. Bioeng. 87:614 (2004). Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO 2003 / 085107).

[0249] Anti-TNFR2, anti-PD-L1 or anti-PD-1 antibody variants further involve bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93 (5): 851-861 (2006). Anti-TNFR2, anti-PD-L1 or anti-PD-1 antibody variants are also provided, which have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0250] In some embodiments, the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibody variants can comprise an Fc region that can bind to FcγRIII. In some embodiments, the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibody variants comprising an Fc region have ADCC activity in the presence of human effector cells (e.g., T cells), or have enhanced ADCC activity in the presence of human effector cells compared to an otherwise identical antibody having a human wild-type IgG1 Fc region. Cysteine ​​engineered variants

[0251] In some embodiments, it is desirable to prepare cysteine-engineered anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies in which one or more amino acid residues are substituted with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites of these antibodies. By replacing those residues with cysteine, reactive sulfhydryl groups are located at accessible sites of the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibodies, which can be used to conjugate these antibodies to other moieties, such as drug moieties or linker-drug moieties, to prepare anti-TNFR2, anti-PD-L1, or anti-PD-1 antibody immunoconjugates as further described herein. Cysteine-engineered antibodies can be prepared, for example, as described in US Pat. No. 7,521,541. derivative

[0252] In some embodiments, the anti-TNFR2, anti-PD-L1 or anti-PD-1 antibodies provided herein may be further modified to include other non-protein moieties known in the art and readily available. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, whether it is desired to improve the properties or function of the anti-TNFR2, anti-PD-L1, or anti-PD-1 antibody, whether the antibody derivative is intended for treatment of a particular condition, etc. Products and kits

[0253] In some embodiments of the present application, an article of manufacture is provided, comprising a substance that can be used to treat cancer or an infectious disease. The article of manufacture may include a container and a label or package insert on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be made of a variety of materials, such as glass or plastic. Typically, the container contains a composition that is effective for treating the disease or condition described herein and has a sterile port (for example, the container can be an intravenous infusion bag or a vial with a cap pierceable by a hypodermic injection needle). At least one active substance in the composition is the anti-TNFR2 antibody, anti-PD-L1, or anti-PD-1 antibody described herein. The label or package insert indicates the specific condition that the composition can be used to treat. The label or package insert further includes instructions for administering the combination of the anti-TNFR2 antibody and the anti-PD-L1 or anti-PD-1 antibody to a patient. Articles of manufacture and kits including combination therapies are contemplated herein.

[0254] Package insert refers to instructions typically included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, contraindications and / or warnings related to the use of these therapeutic products. In some embodiments, the package insert indicates that the composition can be used to treat cancer or infectious diseases. In some embodiments, the package insert indicates that the composition can be used to treat a cancer or infectious disease selected from non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, gastroesophageal junction adenocarcinoma, esophageal cancer, esophageal adenocarcinoma, gallbladder cancer, gastric cancer, cervical cancer, gastric adenocarcinoma, head and neck cancer, heart cancer, hepatocellular carcinoma, kidney cancer, liver cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, prostate adenocarcinoma, spleen cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and infectious diseases, including but not limited to Diseases or conditions in the group consisting of human papillomavirus (HPV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), varicella-zoster virus (VSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Escherichia coli, Chlamydia, Mycobacterium tuberculosis, Streptococcus, Pneumococcus, Pseudomonas, Campylobacter, Salmonella, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans and Histoplasma capsulatum.

[0255] In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. Other materials desirable from a commercial and user perspective may also be included, including other buffers, diluents, filters, needles, and syringes.

[0256] Also provided are kits that can be used for various purposes, such as for the treatment of cancer or infectious diseases, optionally in combination with products. The kit of the present application includes one or more containers comprising a composition (or single dose form and / or product) of an anti-TNFR2 antibody and an anti-PD-L1 or anti-PD-1 antibody, and in some embodiments, further comprising another drug (e.g., a drug described herein) and / or instructions for use consistent with any of the methods described herein. The kit may further include a description of selecting an individual suitable for treatment. The instructions for use included in the kit in the present application are typically written instructions on a label or package insert (e.g., a paper sheet contained in the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0257] For example, in some embodiments, the kit includes a composition comprising an anti-TNFR2 antibody and an anti-PD-L1 or anti-PD-1 antibody. In some embodiments, the kit includes: a) any composition described herein, and b) at least one other drug in an effective amount that can enhance the effect of the composition (e.g., therapeutic effect, detection effect). In some embodiments, the kit includes: a) any composition described herein, and b) instructions for administering the composition to an individual for treating cancer or an infectious disease. In some embodiments, the kit includes: a) any composition described herein, and b) at least one other drug in an effective amount that can enhance the effect of the composition (e.g., therapeutic effect, detection effect), and c) instructions for administering the composition and the other drug to an individual for treating cancer or an infectious disease. The anti-TNFR2 antibody and anti-PD-L1 or anti-PD-1 antibody composition and the other drug can be present in separate containers or in the same container. For example, the kit can include one specific composition or two or more compositions, wherein one composition includes an anti-TNFR2 antibody and an anti-PD-L1 or anti-PD-1 antibody and the other composition includes another drug.

[0258] In some embodiments, the kit comprises a nucleic acid (or a set of nucleic acids) encoding an anti-TNFR2 antibody. In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an anti-TNFR2 antibody, and b) a host cell that expresses the nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an anti-TNFR2 antibody, and b) instructions for: i) expressing the anti-TNFR2 antibody in a host cell, ii) preparing a composition comprising the anti-TNFR2 antibody, and iii) administering the composition comprising the anti-TNFR2 antibody to a subject to treat cancer or an infectious disease. In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an anti-TNFR2 antibody, b) a host cell that expresses the nucleic acid (or a set of nucleic acids), and c) instructions for: i) expressing the anti-TNFR2 antibody in a host cell, ii) preparing a composition comprising the anti-TNFR2 antibody, and iii) administering the composition comprising the anti-TNFR2 antibody to a subject to treat cancer or an infectious disease.

[0259] In some embodiments, the kit comprises one (or a set of) nucleic acids encoding anti-PD-L1 or anti-PD-1 antibodies. In some embodiments, the kit comprises: a) one (or a set of) nucleic acids encoding anti-PD-L1 or anti-PD-1 antibodies, and b) a host cell expressing one (or a set of) nucleic acids. In some embodiments, the kit comprises: a) one (or a set of) nucleic acids encoding anti-PD-L1 or anti-PD-1 antibodies, and b) instructions for use, for: i) expressing anti-PD-L1 or anti-PD-1 antibodies in host cells, ii) preparing a composition comprising anti-PD-L1 or anti-PD-1 antibodies, and iii) administering a composition comprising anti-PD-L1 or anti-PD-1 antibodies to an individual to treat cancer or an infectious disease. In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an anti-PD-L1 or anti-PD-1 antibody, b) a host cell that expresses the nucleic acid (or a set of nucleic acids), and c) instructions for: i) expressing the anti-PD-L1 or anti-PD-1 antibody in the host cell, ii) preparing a composition comprising the anti-PD-L1 or anti-PD-1 antibody, and iii) administering the composition comprising the anti-PD-L1 or anti-PD-1 antibody to an individual to treat cancer or an infectious disease.

[0260] The kits described herein are packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kits may optionally provide other components, such as buffers and instructional information. Therefore, the application also provides articles, which include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0261] Instructions for use of a composition comprising an anti-TNFR2 antibody and an anti-PD-L1 or anti-PD-1 antibody typically include information such as dosage, dosing cycle, and route of administration. The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. For example, a kit is provided that contains a sufficient dose of a composition as described herein to provide long-term effective treatment for an individual, such as one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. The kit may also contain multiple unit doses of the pharmaceutical composition and instructions for use, and is packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy or compounding pharmacy.

[0262] Those skilled in the art will recognize that several embodiments are possible within the scope and purpose of this application. The application will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the application, but should not be construed as limiting its scope in any way. DETAILED DESCRIPTION

[0263] The following exemplary embodiments illustrate various features and embodiments of the present invention. These embodiments are intended to be illustrative and not limiting. Those skilled in the art will readily appreciate that the specific examples are merely illustrative of the present invention, which is more fully described in the claims that follow. Each embodiment and feature described herein should be understood to be interchangeable and combinable with each embodiment included in this application. Example 1: Characterization of anti-TNFR2 antibodies

[0264] This example illustrates methods for preparing anti-TNFR2 antibodies, as well as methods for screening and selecting antibodies for further characterization. The contents of International Application No. PCT / US2022 / 073523 are incorporated herein by reference.

[0265] Immunization and Fusion: Balb / c and NZB mice were immunized with recombinant human TNFR2 ECD (SEQ ID NO: 25) fused to His- or mouse IgG2a Fc produced in Expi293 or CHO cells, with RIBI (Sigma Aldrich, cat# S6322-1VL), Titermax (Sigma Aldrich, cat# T2684-1ML), or / and Freund's adjuvant (Freund's adjuvant, incomplete) (Sigma Aldrich, cat# F5506-10x-10mL) as adjuvants. Three days after the last immunization, spleens and lymph nodes were harvested and processed according to the manufacturer's recommendations. Mouse B cells were isolated using the EasySep Mouse B Cell Isolation Kit (StemCell, cat# 19854A) and fused with myeloma SP2 / 0-Ag14 cells (ATCC, CRL1581) using PEG. According to the standard operating instructions, the fused cells were seeded in six-well plates containing semi-solid cloning cell HY cloning medium D (StemCell, cat# 03804). Monoclonal hybridoma clones were picked into 96 wells / plate using a ClonePix 2 instrument (Molecular Devices) and cultured in HT medium.

[0266] Hybridoma Screening: After 10-14 days of culture, the supernatant was collected and coated with the extracellular domain of human or cynomolgus macaque TNFR2 (SEQ ID NO: 27) with a His or human Fc tag on a 96-well ELISA plate for primary screening. Parent hybridomas identified in the primary screening were expanded. Hybridoma supernatants identified in the primary screening were further tested for their ability to block biochemical binding between human TNFR2 and human TNFα. Selected hybridomas were preferentially subcloned and further characterized.

[0267] Purification of hybridoma antibodies: Positive clone 51B5 was amplified and the antibody was purified using Protein A resin. Positive clone 51B5 was sequenced and amplified, and its sequence is shown in Table 2-3.

[0268] Preparation of recombinant 51B5 chimeric antibody in IgG1 format: Recombinant 51B5 chimeric antibody constructs were prepared using methods known in the art, comprising mouse heavy and light chain variable regions and human constant regions. Exemplary human heavy and light chain constant region sequences are shown in Table 4. The 51B5 chimeric antibody was tested for binding affinity and activity in blocking TNFα binding to TNFR2 according to the following assays.

[0269] Preparation of humanized antibodies from hybridoma clone 51B5: The light chain variable region (VL) and heavy chain variable region (VH) sequences of the mouse antibody from hybridoma 51B5 were aligned with human germline antibody sequences. Human germline kappa light and heavy chains served as human frameworks.

[0270] The complementarity determining regions (CDRs) of the light and heavy chains of the mouse TNFR2 antibody 51B5 were transplanted into the closest human framework identified to prepare humanized antibody clones. In this process, the antibody 51B5 was humanized by transplanting the CDRs from the mouse antibody V region onto the human germline antibody V region framework. The CDRs transplanted from the donor to the recipient sequence were defined by Kabat (Kabat et al., 1987). In order to restore the activity of the antibody, many framework residues from the mouse V region were also retained in the humanized sequence, and these V regions were found to be V H -V L Framework residues that are part of the interaction interface, or serve as “Vernier” regions, can adjust the CDR structure and fine-tune it for antigen binding (Foote et al., 1992).

[0271] The sequences of the humanized antibodies are summarized in Tables 2-3. Example 2: Characterization and activity evaluation of anti-TNFR2 antibodies Epitope resolution by alanine scanning assay

[0272] The TNFR2 antibody 51B5 has been identified to bind to CRD3 of the human TNFR2 receptor. In this assay, the epitope to which the humanized anti-TNFR2 antibodies SB1901-19, SB1901-72, or SB1901-76 bind to human TNFR2 was resolved by alanine scanning. Alanine scanning mutagenesis was performed within the huTNFR2 CRD3 region of the chimeric protein musTNFR2-huCRD3(ECD) (SEQ ID NO:26). A His-tag was added to the C-terminus of the chimeric protein mutant to facilitate purification and detection. Plates were coated with the humanized TNFR2 antibody SB1901-19, SB1901-72, or SB1901-76 at 4°C overnight. After washing, the alanine mutants of the chimeric protein were added and incubated at room temperature for 2 hours with shaking. The plates were washed again. Then, an anti-His antibody conjugated to AP was added to the plates and incubated at room temperature for 1 hour. The plates were washed and developed with pNPP substrate for 30 minutes and read at 450 nm.

[0273] Alanine scanning results indicate that the epitopes of humanized anti-TNFR2 antibodies SB1901-19, SB1901-72, or SB1901-76 are identical (data not shown). Figures 1A-1B show the results of alanine scanning of an exemplary antibody SB1901-76. Amino acids R99, K108, E110, G111, R113, L114, and D136 in the CRD3 region of human TNFR2 (SEQ ID NO: 25) are essential for binding of the antibody to human TNFR2. The results indicate that the conformational epitope of the SB1901-19, SB1901-72, or SB1901-76 antibody comprises or consists of amino acid residues R99, K108, E110, G111, R113, L114, and D136 of SEQ ID NO: 25.

[0274] Binding affinity

[0275] Binding affinities (monovalent Kd) of anti-TNFR2 antibodies were determined using biolayer interferometry on an Octet RED96 instrument (ForteBio) at 30°C and 1200 rpm. Kinetic analysis was performed using an anti-human IgG Fc capture (AHC) biosensor (ForteBio) in kinetic buffer (PBS, 0.1% Tween-20, and 1% bovine serum albumin) with the following steps: (a) antibody (2 μg / mL) for 300 seconds, (b) baseline for 120 seconds, (c) binding to His-tag-huTNFR2 (2.5, 0.5, and 0 μg / mL) for 420 seconds, and (d) dissociation for 1200 seconds. After Savitzky-Golay filtration, data were fitted and analyzed using a 1:1 binding model using Octet Data Analysis Software 8.0 (ForteBio). The equilibrium dissociation constant (Kd) was calculated as the ratio of Koff / Kon. Examples of binding affinities for humanized antibodies are shown in Table 5. Table 5: Binding affinity of humanized antibodies to TNFR2 antigen Antibody Kd(nM) Kon(1 / Ms) Koff(1 / s) SB1901-72 1.11 4.12E+05 4.56E-04 SB1901-80 2.52 3.73E+05 9.37E-04

[0276] Binding to cells expressing huTNFR2

[0277] To test the binding of anti-TNFR2 antibodies to cells expressing huTNFR2, we performed FACS analysis using Expi293 cells stably expressing huTNFR2.

[0278] The coding sequence of huTNFR2 (Uniprot, P20333) was cloned into a lentiviral vector and the vector was transformed into a lentiviral vector according to the viral packaging kit (Lenti-X TM The virus was packaged according to the instructions of the ELISA Packaging Single Shots (Cat# 631275, Takada). The recombinant virus was transfected into Expi293 cells and selected with puromycin. The cell line stably expressing huTNFR2 was incubated with anti-TNFR2 antibody in PBS containing 0.5% BSA, 1 mM EDTA and 0.1% sodium azide (FACS buffer) at 4°C for 30 minutes. The cells were washed and incubated with 10 nM phycoerythrin (PE)-conjugated anti-human Fc antibody (Biolegend, cat# 409304) at 4°C for 20 minutes. The cells were washed and separated using an Attune (ThermoFisher Scientific) flow cytometer. The data were analyzed using FlowJo software. Antibody binding was represented by mean fluorescence intensity (MFI).

[0279] like Figure 2AAs shown, the chimeric anti-TNFR2 antibody 51B5 and humanized antibodies SB1901-72, SB1901-74, SB1901-76, SB1901-78, SB1901-80, and SB1901-82 effectively bound to Expi293-TNFR2 cells in a dose-dependent manner.

[0280] Binds to huTNFR2-Expi293 cells and blocks TNFα binding

[0281] Expi293 cells stably expressing huTNFR2 were incubated with anti-TNFR2 antibodies for 30 minutes at 4°C. The cells were washed and incubated with human TNFα (SinoBiogical, cat#10602-HNAE) conjugated with 10 nM Alexa Fluor 647 (ThermoFisher Scientific, cat#A20186) for 20 minutes at 4°C. The cells were washed and collected using a flow cytometer. Data were analyzed using FlowJo software. TNFα binding is expressed as MFI.

[0282] like Figure 2B As shown, the chimeric anti-TNFR2 antibody 51B5 and humanized antibodies SB1901-72, SB1901-74, SB1901-76, SB1901-78, SB1901-80 and SB1901-82 can effectively inhibit the binding of soluble TNFα to Expi293 cells expressing TNFR2, and the inhibition is dose-dependent.

[0283] In vitro human primary Treg cell proliferation assay

[0284] The functional activity of anti-TNFR2 antibodies on primary human Treg cells was examined. PBMCs were incubated in round-bottom plates with 200 U / ml IL-2 and 20 ng / ml TNFα in complete culture medium in the presence or absence of anti-TNFR2 antibodies at 37°C for 72 hours. Cells were stained with anti-CD3 and anti-CD4 antibodies in FACS buffer for 30 minutes at 4°C. Cells were washed and fixed / permeabilized with fixation / permeabilization buffer for 30 minutes at 4°C. Cells were then washed with 1× permeabilization buffer and stained with anti-human Foxp3 antibody in 1× permeabilization buffer for 30 minutes at 4°C. Cells were washed, fixed with 2% PFA, and isolated and analyzed using an Attune flow cytometer. The percentage of Foxp3+ cells among CD4+ cells was analyzed using FlowJo software.

[0285] like Figure 3As shown, the chimeric anti-TNFR2 antibody 51B5 and exemplary humanized TNFR2 antibodies SB1901-19, SB1901-25, SB1901-26, and SB1901-27 were able to inhibit Treg cell proliferation in vitro. Example 3: In vivo anti-tumor effect study to evaluate the activity of the combination of anti-TNFR2 antibody and anti-PDL1 antibody

[0286] This example demonstrates the functional activity of humanized anti-TNFR2 antibodies administered alone or in combination with anti-PD-L1 or anti-PD-1 antibodies in in vivo tumor model studies.

[0287] Subcutaneous tumor model: MC38 was used in this example.

[0288] Animals and husbandry: Forty female C57BL / 6-Tnfrsf1b tm1(TNF-RSF1B) / Bcgen mice (6-9 weeks old). The animals were fed with a breeding diet of "SPF rats and mice growth" and had free access to water. For easy identification, the animals were ear-tagged and the left back area was shaved in preparation for cell transplantation. The animals were housed in polycarbonate cages (cage size 320×200×135 mm). The ambient temperature was controlled at 20°C to 26°C and the humidity was controlled at 40-70%. Animal care and use complied with the standard operating procedures of JOINN LABORATORIES (Suzhou) Inc., the Guide for the Care and Use of Laboratory Animals (8th edition, Institute of Laboratory Animal Resources, Council on Life Sciences, National Research Council; National Academy Press; Washington, DC, 2010), and the Animal Welfare Act (Public Act 99-198) passed by the United States Department of Agriculture.

[0289] Cell preparation and transplantation: Mouse colon cancer cell line MC38 purchased from the Institute of Basic Medical Sciences was cultured and expanded in RPMI medium containing 2 mM L-glutamine, 10% fetal bovine serum (FBS) and 1% 100× penicillin / streptomycin (PS). The growth environment was maintained at 37°C and 5% CO2 in an incubator. After expansion, the cells (passage 3) were trypsinized using 0.25% trypsin EDTA solution. The cells were then washed and counted. The cell viability before transplantation was 92%-94%. The cells were suspended in Dulbecco's phosphate-buffered saline (DPBS) at a concentration of 1×10 7 The transplantation site of the test animal was disinfected with an alcohol prep pad, and 0.2 mL was transplanted subcutaneously using a 25-gauge needle and a 1-ml syringe.

[0290] Measurements and Antibody Treatment: Tumors were allowed to grow, and mice were then randomly assigned to different study groups. Mice were assigned to ensure that the average weight of all groups was within 10% of the total average tumor burden of the study population. Human MOPC21 IgG1 isotype antibody (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of animmunoglobulin mRNA using specific priming and the dideoxynucleotide methodof RNA sequencing. Nucleic Acids Res. 9(18): 4485-4494), SB1901-72, and anti-PD-L1 antibody (Atezolizumab, Genentech) were prepared in-house. Anti-PD-1 antibody (Clone No. RMP1-14) was purchased from BioXcell. For each antibody, mice were treated with intraperitoneal injections twice a week for 3 weeks, and tumor volume was monitored (n = 10 mice / group). The long and short axes of the tumor were measured with a vernier caliper and recorded to calculate the tumor volume. A tumor growth curve was drawn based on the tumor volume to compare the differences between the groups. Tumor volume was calculated according to the following formula: V = 1 / 2 × long axis × short axis 2 .

[0291] Side effect assessment: All animals were observed for clinical signs or toxicity at least once a day. Animals were weighed weekly. Animals were euthanized if weight loss exceeded 20% or other clinical signs requiring euthanasia appeared. Animals were euthanized when the tumor volume of an individual animal reached or exceeded 2500 mm. 3 When , the animals were euthanized.

[0292] Results: The changes in tumor volume and the average tumor volume of each group were measured.

[0293] Combination therapy of anti-TNFR2 antibodies and anti-PD-L1 antibodies

[0294] The results of the combined treatment of antibody SB1902-72 and anti-PD-L1 antibody in the MC38 tumor model are shown in Table 6. Human TNFR2 transgenic mice bearing MC38 tumors were treated with SB1901-72 (10 mg / kg), anti-PD-L1 antibody (5 mg / kg), a combination of the two, or isotype antibody (10 mg / kg) by intraperitoneal injection twice a week for a total of 6 injections. Tumor size was monitored twice a week. Table 6: Overview of in vivo subcutaneous tumor study protocols

[0295] Tumor growth curves of mice in the single treatment group or combined treatment group are shown in Figure 2 Figure 4A The average tumor growth curves of each group are shown in Figure 4B As shown. The results showed that on day 22, the tumor volume of all treatment groups was significantly lower than that of the isotype antibody treatment group (P<0.001), indicating that SB1901-72 blocking TNFR2 can effectively inhibit tumor growth. In addition, the combination of anti-PD-L1 antibody and SB1901-72 showed a trend of being more effective than antibody treatment alone. At the same time point, there was no significant difference in body weight between the groups. The animals did not show any abnormalities in general clinical observation.

[0296] Combination therapy of anti-TNFR2 antibody and anti-PD-1 antibody

[0297] The results of the combined treatment of SB1902-72 and anti-PD-1 antibodies in the MC38 tumor model are shown in Table 7. Human TNFR2 transgenic mice bearing MC38 tumors were treated with SB1901-72, anti-PD-L1 antibodies, a combination of the two, or isotype-specific antibodies at 3 mg / kg via intraperitoneal injection twice weekly for a total of six injections. Tumor size was monitored twice weekly. Table 7: Overview of in vivo subcutaneous tumor study protocols

[0298] Tumor growth curves of mice in the single treatment group or combined treatment group are shown in Figure 2 Figure 4C The average tumor growth curves of each group are shown in Figure 4D The results showed that tumor growth was significantly inhibited in all treatment groups compared to the isotype antibody treatment group (P<0.001). Tumor growth curves for each group showed that 4 / 10 mice in the anti-PD-1 antibody group had their tumors completely eliminated, while 8 / 10 mice in the combination treatment group had their tumors completely eliminated. Combination therapy with anti-PD-1 antibodies and SB1901-72 was more effective than single antibody treatment.

[0299] Taken together, these results suggest that combination therapy of anti-TNFR2 antibody (SB1901-72) with anti-PD-1 or anti-PD-L1 antibodies enhances the anti-tumor effect compared with single antibody therapy.

Claims

1. A method for treating cancer or infectious diseases in an individual in need thereof, comprising administering to the individual an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

2. An antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1, for use in treating cancer or infectious diseases in individuals in need thereof.

3. An antibody that specifically recognizes human TNFR2, for use in combination with an antibody that specifically recognizes human PD-L1 or PD-1 to treat cancer or infectious diseases in an individual in need thereof.

4. The method according to claim 1, or the antibody for use according to claim 2 or 3, wherein the antibody that specifically recognizes human TNFR2 and the antibody that specifically recognizes human PD-L1 or PD-1 are administered to an individual in need thereof simultaneously or sequentially.

5. A pharmaceutical composition comprising an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

6. A combination of an antibody that specifically recognizes human TNFR2 and an antibody that specifically recognizes human PD-L1 or PD-1.

7. The pharmaceutical composition according to claim 5 or the combination according to claim 6, for use in treating cancer or infectious diseases in a subject in need thereof.

8. A method of treating cancer or an infectious disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 5 or the combination of claim 6.

9. The combination for use according to claim 7 or the method according to claim 8, wherein the antibody that specifically recognizes human TNFR2 and the antibody that specifically recognizes human PD-L1 or PD-1 are administered to an individual in need thereof simultaneously or sequentially.

10. The method according to any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition according to claim 5, or the combination according to claim 6, wherein the anti-PD-1 antibody is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, sintilimab, camrelizumab, toripalimab, tislelizumab, zimberelimab, prolgolimab, dostarlimab, b), AMG-404, balstilimab, budigalimab, cetrelimab, ezabenlimab, genolimzumab, LZM-009, nofazinlimab, penpulimab, pimivalimab, pucotenlimab, QL-1604, retifanlimab, rulonilimab, sasanlimab, SCT-I10A, serplulimab, SG-001, spartalizumab, SYSA-1802, and TY-101.

11. The method according to any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition according to claim 5, or the combination according to claim 6, wherein the anti-PD-L1 antibody is selected from the group consisting of avelumab, durvalumab, atezolizumab, KN035, CK-301, ADG-104, BCD-135, garivulimab, CBT-502, HLX-20, IMC-001, tagitanlimab, LAE-005, LP-002, MSB-2311, adebrelimab, sugemalimab, and socazolimab.

12. The method of any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition of claim 5, or the combination of claim 6, wherein the anti-TNFR2 antibody comprises: a heavy chain variable domain (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising DDYID (SEQ ID NO: 1); HC-CDR2 comprising EIYPGSGNTYYNEKFKG (SEQ ID NO: 2); and HC-CDR3 comprising SQVYGKIAMDH (SEQ ID NO: 3); and the light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising RASESVDNSGNSFMH (SEQ ID NO: 4); a LC-CDR2 comprising RASNLES (SEQ ID NO: 5); and a LC-CDR3 comprising QQSKEDPYT (SEQ ID NO: 6).

13. The method of any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition of claim 5, or the combination of claim 6, wherein the anti-TNFR2 antibody comprises: V H , which comprises V as shown in any one of the amino acid sequences of SEQ ID NOs: 7-15 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in any one of the amino acid sequences of SEQ ID NOs: 16-20 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

14. The method of any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition of claim 5, or the combination of claim 6, wherein the anti-TNFR2 antibody comprises: V H , comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7-15 or a variant thereof, wherein the variant has at least about 80% sequence identity with any one of SEQ ID NOs: 7-15; and V L , which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 16-20 or a variant thereof, wherein the variant has at least about 80% sequence identity with any one of the amino acid sequences in SEQ ID NOs: 16-20.

15. The method of any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition of claim 5, or the combination of claim 6, wherein the anti-TNFR2 antibody comprises: (i)V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 16; (ii)V H , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 17; (iii)V H , comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 18; (iv)V H , comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 19; (v)V H , comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 17; (vi)V H , comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 10; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 20; (vii)V H , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 20; (viii)V H , comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 12; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 20; (ix)V H , comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 13; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 20; (x)V H , comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 14; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 20; or (xi)V H , comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 15; and V L , comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

20.

16. The method according to any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition of claim 5, or the combination of claim 6, wherein: (i) the anti-TNFR2 antibody further comprises an Fc fragment; or (ii) the anti-TNFR2 antibody is a full-length IgG antibody, optionally, a full-length IgG1, IgG2, IgG3 or IgG4 antibody; or (iii) the anti-TNFR2 antibody is chimeric, human or humanized; or (iv) The anti-TNFR2 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd and diabody.

17. The method of any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, the pharmaceutical composition of claim 5, or the combination of claim 6, wherein the anti-TNFR2 antibody comprises: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 28; and a light chain comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 37; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and a light chain comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 38; (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a light chain comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 39; (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a light chain comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 40; (v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a light chain comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 38; (vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 31; and a light chain comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41; (vii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 32; and a light chain comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41; (viii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41; (ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 34; and a light chain comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41; (x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 35; and a light chain comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 41; or (xi) a heavy chain comprising the amino acid sequence of SEQ ID NO:36 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:36; and a light chain comprising the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

41.

18. The method according to any one of claims 1, 4, and 8-9, the antibody for use according to any one of claims 2-4, the pharmaceutical composition for use according to claim 7, the combination for use according to claim 7 or 9, wherein the cancer or infectious disease is selected from the group consisting of lung cancer, skin cancer, lymphoma, leukemia, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, gastroesophageal junction adenocarcinoma, esophageal cancer, esophageal adenocarcinoma, gallbladder cancer, gastric cancer, cervical cancer, gastric adenocarcinoma, head and neck cancer, heart cancer, hepatocellular carcinoma, kidney cancer, liver cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer , prostate adenocarcinoma, spleen cancer, small cell or non-small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and infectious diseases, including but not limited to, human papillomavirus (HPV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), varicella zoster virus (VSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Escherichia coli, Chlamydia, Mycobacterium tuberculosis, Streptococcus, Pneumococcus, Pseudomonas, Campylobacter, Salmonella, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans and capsular Histoplasma capsulatum.

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