TNFR2 binding molecules and uses thereof

Abstract

The invention relates to specific TNFR2 binding molecules, epitope peptides of TNFR2 to which the TNFR2 binding molecules bind, and compositions containing the TNFR2 binding molecules. It also relates to nucleic acids encoding the TNFR2 binding molecules and host cells containing the same, as well as methods for preparing the TNFR2 binding molecules. In addition, it relates to therapeutic and diagnostic uses of these TNFR2 binding molecules. In particular, it relates to combined treatments of these TNFR2 binding molecules with other therapies, such as treatment modalities or therapeutic agents.

Description

Technical Field

[0001] The present invention relates to specific TNFR2 binding molecules, epitope peptides of TNFR2 to which the TNFR2 binding molecules bind, and compositions containing the TNFR2 binding molecules. In addition, the present invention relates to nucleic acids encoding the TNFR2 binding molecules and host cells containing the same, as well as methods for preparing the TNFR2 binding molecules. The present invention also relates to therapeutic and diagnostic uses of these TNFR2 binding molecules, in particular, the present invention also relates to combined treatments of these TNFR2 binding molecules with other therapies, such as treatment modalities or therapeutic agents. Background Art

[0002] Tumor necrosis factor receptor 2 (TNFR2, TNFRSF1B) protein belongs to the tumor necrosis factor receptor superfamily and is expressed on the surface of activated regulatory T cells (Regulatory T cells, Tregs), myeloid-derived suppressing cells (Myeloid-derived suppressing cells, MDSCs), CD4 and CD8 positive effector T cells. It is also highly expressed on the surface of various tumor cells, such as Sézary syndrome, mycosis fungoides, etc. (Medler J., Wajant H. (2019). Expert Opin Ther Targets 23, 295-307.). Unlike the widespread expression of TNFR1, TNFR2 is usually expressed more specifically, especially in tumor-infiltrating immune cells, such as regulatory T cells (Tregs), cytotoxic T cells and different myeloid cell subsets (Sheng Y., Li F, Qin Z. (2018). Front Immunol 9, 1170.). TNFR2-positive Treg cells are highly enriched in many tumors, creating a highly suppressive immune microenvironment in the local tumor tissue. At the same time, TNFR2-positive Tregs also show active immunosuppressive activity, becoming the main obstacle affecting the anti-tumor immune response in the tumor microenvironment (Yang Y., Islam MS, Hu Y., Chen X. (2021). Immunotargets Ther 10, 103-122.). Due to the specific high expression of TNFR2 on the surface of Treg, MDSC and many tumor cells in tumors, it is expected to become a promising target for cancer immunotherapy, bringing better efficacy and higher safety.

[0003] Studies have shown that antagonistic antibody drugs targeting TNFR2 can activate anti-tumor immune responses by inhibiting or killing immunosuppressive cells such as Treg and MDSC in the tumor, thereby achieving a therapeutic effect of killing tumors (Sheng Y., Li F., Qin Z. (2018). Front Immunol 9, 1170.).

[0004] Although there are currently antagonistic monoclonal antibody drugs targeting TNFR2 under clinical development (such as BI-1808), there are problems such as insufficient therapeutic effects and high toxicity. As a therapeutic agent, there is still an urgent need to continue to develop small molecule antibodies (such as single domain antibodies) targeting TNFR2 targets. SUMMARY OF THE INVENTION

[0006] The present invention develops a class of TNFR2 binding molecules comprising a single domain antibody (sdAb) portion that specifically recognizes TNFR2, which has one or more of the following properties:

[0007] (1) High affinity binding to human TNFR2, for example, EC binding between the TNFR2 binding molecule and cell surface TNFR2 50 is about 0.01 μg / mL to about 1 μg / mL, for example, about 0.1 μg / mL to about 0.6 μg / mL;

[0008] (2) It does not substantially block the binding of TNFα to TNFR2;

[0009] (3) Inhibition of TNFR2 signaling pathway, for example, in cells expressing TNFR2 such as Treg cells (e.g., Treg cells expressing high CD25), myeloid-derived suppressor cells (MDSCs) and / or TNFR2 + Inhibits TNFR2-mediated signaling in cancer cells;

[0010] (4) It has little effect on the proliferation of Treg cells in PBMCs;

[0011] (5) Inhibit tumor growth in vivo.

[0012] Thus, in a first aspect, the present invention provides a TNFR2 binding molecule comprising at least one single domain antibody (sdAb) portion that specifically binds to TNFR2, wherein the sdAb portion comprises three complementary determining regions from the N-terminus to the C-terminus, namely CDR1, CDR2 and CDR3, wherein:

[0013] (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 3, or a variant in which one or two amino acids in the amino acid sequence of SEQ ID NO: 3 are changed,

[0014] (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 4, or a variant in which one or two amino acids in the amino acid sequence of SEQ ID NO: 4 are changed, and

[0015] (c) CDR3 comprises the amino acid sequence of SEQ ID NO: 5, or a variant in which one or two amino acids in the amino acid sequence of SEQ ID NO: 5 are changed,

[0016] The amino acid change is an addition, deletion or substitution of an amino acid, and the binding molecule comprising the above change at least maintains the ability to bind to TNFR2.

[0017] In some embodiments, the sdAb portion of the TNFR2 binding molecules of the invention comprises

[0018] (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 54:

[0019] GSI-Xaa1-Xaa2-I-Xaa3-Xaa4-MG (SEQ ID NO: 54)

[0020] wherein Xaal is F, W or R, Xaa2 is S or F, Xaa3 is N or L, and Xaa4 is S, D or R;

[0021] (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 55:

[0022] Xaa5-Xaa6-Xaa7-R-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaal3 (SEQ ID NO: 55)

[0023] wherein Xaa5 is A or V, Xaa6 is I, L or H, Xaa7 is G or A, Xaa8 is G, R or T, Xaa9 is G, R, P or S, Xaa10 is G, Q, R, F or V, Xaa11 is S or R, Xaa12 is T or L, and Xaa13 is N or Q; and

[0024] (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 56:

[0025] EISQL-Xaa14-Xaa15-AF-Xaa16-DY(SEQ ID NO: 56)

[0026] Among them, Xaa14 is T, S or G, Xaa15 is W, F or Y, and Xaa16 is R or L.

[0027] In some embodiments, the sdAb portion of the TNFR2 binding molecule of the invention comprises a CDR1, a CDR2, and a CDR3 selected from any one of the following groups:

[0028] (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 3; CDR2 comprises the amino acid sequence of SEQ ID NO: 4; and CDR3 comprises the amino acid sequence of SEQ ID NO: 5;

[0029] (b) CDR1 comprises the amino acid sequence of SEQ ID NO: 10; CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and CDR3 comprises the amino acid sequence of SEQ ID NO: 12;

[0030] (c) CDR1 comprises the amino acid sequence of SEQ ID NO: 14; CDR2 comprises the amino acid sequence of SEQ ID NO: 15; and CDR3 comprises the amino acid sequence of SEQ ID NO: 16;

[0031] (d) CDR1 comprises the amino acid sequence of SEQ ID NO: 18; CDR2 comprises the amino acid sequence of SEQ ID NO: 19; and CDR3 comprises the amino acid sequence of SEQ ID NO: 20;

[0032] (e) CDR1 comprises the amino acid sequence of SEQ ID NO: 22; CDR2 comprises the amino acid sequence of SEQ ID NO: 23; and CDR3 comprises the amino acid sequence of SEQ ID NO: 24;

[0033] (f) CDR1 comprises the amino acid sequence of SEQ ID NO: 26; CDR2 comprises the amino acid sequence of SEQ ID NO: 27; and CDR3 comprises the amino acid sequence of SEQ ID NO: 28;

[0034] (g) CDR1 comprises the amino acid sequence of SEQ ID NO: 30; CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and CDR3 comprises the amino acid sequence of SEQ ID NO: 32;

[0035] (h) CDR1 comprises the amino acid sequence of SEQ ID NO: 34; CDR2 comprises the amino acid sequence of SEQ ID NO: 35; and CDR3 comprises the amino acid sequence of SEQ ID NO: 36;

[0036] (i) CDR1 comprises the amino acid sequence of SEQ ID NO: 38; CDR2 comprises the amino acid sequence of SEQ ID NO: 39; and CDR3 comprises the amino acid sequence of SEQ ID NO: 40;

[0037] (j) CDR1 comprises the amino acid sequence of SEQ ID NO: 42; CDR2 comprises the amino acid sequence of SEQ ID NO: 43; and CDR3 comprises the amino acid sequence of SEQ ID NO: 44;

[0038] (k) CDR1 comprises the amino acid sequence of SEQ ID NO: 46; CDR2 comprises the amino acid sequence of SEQ ID NO: 47; and CDR3 comprises the amino acid sequence of SEQ ID NO: 48;

[0039] (1) CDR1 comprises the amino acid sequence of SEQ ID NO: 50; CDR2 comprises the amino acid sequence of SEQ ID NO: 51; and CDR3 comprises the amino acid sequence of SEQ ID NO: 52.

[0040] In some embodiments, the sdAb portion of the TNFR2 binding molecules of the invention comprises

[0041] (i) any one of the amino acid sequences selected from SEQ ID NO: 6, 7, 8, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53; or

[0042] (ii) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the amino acid sequences selected from SEQ ID NO: 6, 7, 8, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53;

[0043] Preferably, the sdAb portion is a camelid VHH, a partially or fully humanized VHH, a chimeric VHH.

[0044] In some embodiments, the TNFR2 binding molecules of the invention are further linked to an additional protein domain at the N-terminus or C-terminus of the sdAb portion, e.g., to an Fc region of an immunoglobulin, e.g., an Fc region from an IgG, e.g., IgG1, IgG2, IgG3, or IgG4; or, e.g., to a fluorescent protein.

[0045] In some embodiments, the TNFR2 binding molecule of the present invention is a bispecific or multispecific antibody. Preferably, the bispecific antibody molecule specifically binds to the TNFR2 molecule and a second target protein, wherein the second target protein is selected from, for example, tumor antigens (such as tumor-associated antigens and tumor-specific antigens), immunomodulatory receptors and immune checkpoint molecules, such as CTLA-4, TIM-3 or LAG-3.

[0046] In a second aspect, the present invention provides a method for preparing the TNFR2 binding molecule of the present invention, the method comprising culturing a host cell introduced with a nucleic acid encoding the TNFR2 binding molecule of the present invention or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the TNFR2 binding molecule of the present invention, isolating the TNFR2 binding molecule, and optionally the method further comprises recovering the TNFR2 binding molecule from the host cell.

[0047] In a third aspect, the present invention provides a pharmaceutical composition comprising a TNFR2 binding molecule of the present invention, and optionally a pharmaceutically acceptable excipient.

[0048] In some embodiments, the present invention provides a pharmaceutical composition comprising a TNFR2 binding molecule of the present invention, and other therapeutic agents, and optionally pharmaceutical excipients; preferably, the other therapeutic agents are selected from chemotherapeutic agents, other antibodies (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies).

[0049] In some embodiments, the invention provides combination products comprising a TNFR2 binding molecule of the invention and one or more other therapeutic agents, such as chemotherapeutic agents, other antibodies, such as anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0050] In a fourth aspect, the invention provides a method of treating a disease associated with TNFR2 in a subject, comprising administering to the subject a therapeutically effective amount of a TNFR2 binding molecule, pharmaceutical composition, or combination product of the invention.

[0051] In some embodiments, the disease associated with high TNFR2 expression treated by the TNFR2 binding molecules, pharmaceutical compositions, or combination products of the invention is, for example, a cancer that expresses or overexpresses TNFR2.

[0052] In a fifth aspect, the present invention provides a kit for detecting TNFR2 in a sample, wherein the kit comprises a TNFR2 binding molecule of the present invention, and is used to perform the following steps:

[0053] (a) contacting a sample with a TNFR2 binding molecule of the invention; and

[0054] (b) detecting formation of a complex between the TNFR2 binding molecule and TNFR2; optionally, the TNFR2 binding molecule is detectably labeled,

[0055] Thus, it is determined whether or not elevated expression levels of TNFR2 are present in a sample from a subject or individual.

[0056] In a sixth aspect, the present invention provides an epitope peptide of TNFR2 bound by a TNFR2 binding molecule of the present invention, which is located in the groove of the TNFR2 CRD3 domain, for example, it is an epitope peptide of TNFR2 comprising amino acid residues 83, 84, 85, 97, 98, 100, 101, 108, 110, 112, 131, 132, and 133, for example, it is an epitope peptide of TNFR2 shown in SEQ ID NO:9 comprising amino acid residues V83, E84, T85, T97, C98, P100, G101, K108, E110, C112, G131, T132, and E133.

[0057] In a seventh aspect, the present invention provides a TNFR2 binding molecule that binds in a groove of the CRD3 domain of TNFR2, for example, it binds to an epitope of TNFR2 comprising amino acid residues 83, 84, 85, 97, 98, 100, 101, 108, 110, 112, 131, 132, and 133, for example, it binds to an epitope of TNFR2 comprising amino acid residues V83, E84, T85, T97, C98, P100, G101, K108, E110, C112, G131, T132, and E133 as shown in SEQ ID NO:9. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Combined with the following Figure 1 When reading together, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the drawings show the currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0060] Figure 1 The FACS identification results of huTNFR2-HEK293 cell line are shown.

[0061] Figure 2 The FACS identification results of huTNFR2-Jurkat cell line are shown.

[0062] Figure 3 Binding of candidate positive clone lysates to recombinant human TNFR2 is shown.

[0063] Figure 4 The binding activity of anti-TNFR2 VHH-Fc chimeric antibody to huTNFR2-HEK293 cells is shown.

[0064] Figure 5 Species cross-reactivity of anti-TNFR2 VHH-Fc chimeric antibodies is shown.

[0065] Figure 6The blocking activity of the anti-TNFR2 VHH-Fc chimeric antibody on TNFα binding to huTNFR2-HEK293 cells was shown (the chimeric antibody did not substantially block).

[0066] Figure 7 The inhibitory activity of anti-TNFR2 VHH-Fc chimeric antibody on TNFα-induced necrosis of huTNFR2-Jurkat cells was shown.

[0067] Figure 8 The inhibitory effect of anti-TNFR2 VHH-Fc chimeric antibody on tumor growth in humanized mice was shown.

[0068] Fig. 9 The binding activity of anti-TNFR2 humanized antibodies to huTNFR2-HEK293 cells is shown.

[0069] Figures 10A-10D The binding activity of the anti-TNFR2 affinity matured molecules to huTNFR2-HEK293 cells is shown.

[0070] Fig.11 The blocking activity of the anti-TNFR2 affinity matured molecules on TNFα binding to huTNFR2-HEK293 cells is shown (the affinity matured molecules do not substantially block).

[0071] Fig.12 The inhibitory activity of anti-TNFR2 affinity matured molecules on TNFα-induced necrosis of huTNFR2-Jurkat cells was shown.

[0072] Fig.13 The effect of anti-TNFR2 affinity maturation molecules on the proliferation of Treg cells in PBMCs is shown (the affinity maturation molecules do not affect the proliferation of Treg cells in normal PBMCs).

[0073] Fig.14 The inhibitory effect of anti-TNFR2 affinity matured molecules on tumor growth in humanized mice was shown.

[0074] Fig.15A The ADCC effect of the affinity matured molecule 161-hVH5-48 on huTNFR2-HEK293 cells compared to a control antibody is shown.

[0075] Fig. 15B The ADCC effect of the affinity matured molecule 161-hVH5-48 on huTNFR2-Jurkat cells compared to a control antibody is shown.

[0076] Fig.16The results of X-ray diffraction analysis of the crystals of the complex formed by 161-hVH5-48 antibody and TNFR2 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0078] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0079] I. Definitions

[0080] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0081] In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the situation consisting of the elements, integers or steps mentioned. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0082] As used herein, the terms "TNFR2 antibody," "anti-TNFR2 antibody," "antibody that specifically binds to TNFR2," "antibody that specifically targets TNFR2," and "antibody that specifically recognizes TNFR2" are used interchangeably and refer to antagonist TNFR2 antibodies that are capable of specifically binding to TNFR2. In particular, in specific embodiments, it refers to antagonist TNFR2 antibodies that specifically bind to human TNFR2. Antagonist TNFR2 antibodies refer to TNFR2 antibodies that are capable of inhibiting or reducing the activation of TNFR2, attenuating one or more signal transduction pathways mediated by TNFR2, and / or reducing or inhibiting at least one activity mediated by the activation of TNFR2. For example, antagonist TNFR2 antibodies can inhibit or reduce the growth and proliferation of regulatory T cells.

[0083] The term "antibody" is used in the broadest sense herein to refer to a protein comprising an antigen binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, complete antibodies, and antibody fragments. Preferably, the antibody of the present invention is a single domain antibody, a chimeric antibody, or a humanized antibody.

[0084] The term "antibody fragment" refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific antibodies or multispecific antibodies formed from antibody fragments.

[0085] "Complementarity determining region" or "CDR region" or "CDR" is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes and include CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard confformations for the canonical structures of immumoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and the NorthCDR definition based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" covers CDR sequences determined in any of the above ways. CDR can also be determined based on having the same AbM numbering position as a reference CDR sequence (such as any sequence of the CDR exemplified in the present invention). In one embodiment, the CDR of the single-domain antibody of the present invention is positioned according to the AbM numbering scheme. Unless otherwise indicated, in the present invention, when referring to the residue positions in the antibody variable region and CDR (including heavy chain variable region residues), it refers to the numbering position according to the AbM numbering system.

[0086] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, IMGT, AbM and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.

[0087] The term "single domain antibody" generally refers to an antibody in which a single variable domain (e.g., a heavy chain variable domain (VH) or a light chain variable domain (VL), a heavy chain variable domain derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) derived from a fish IgNAR) can confer antigen binding. That is, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include single domain antibodies derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks) (WO2005035572A2). Single domain antibodies derived from camelids are also referred to as VHH in this application, which consist of only one heavy chain variable region, and are antibodies that contain only one chain FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from the C-terminus to the N-terminus, also known as "nanobodies". Single domain antibodies are the smallest units currently known to bind to target antigens.

[0088] "Heavy-chain antibody (hcAb)" refers to an antibody without a light chain, which may include VH-CH2-CH3, or VH-CH1-CH2-CH3, or VHH-CH2-CH3, etc. from the N-terminus to the C-terminus; it may constitute a homodimer, such as a heavy chain dimer antibody without a light chain. The heavy chain antibody may include VH from a standard antibody or VHH from a single domain antibody. In one embodiment, the heavy chain antibody of the present invention includes VHH of a single domain antibody.

[0089] As used herein, the term "multispecific antibody" refers to an antibody having at least two antigen binding sites, each of which binds to different epitopes of the same antigen or to different epitopes of different antigens. Multispecific antibodies are antibodies that have binding specificity to at least two different antigenic epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificity for a first antigen and a second antigen. As used herein, "first antigen binding moiety" and "second antigen binding moiety" refer to amino acid sequences that contain antigen binding sites and can bind to antigenic epitopes, and their definitions fall within the meaning of antibodies or antigen binding fragments.

[0090] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is linked to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug), etc. that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a camel antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced antigenicity in humans as compared to the original camel antibody.

[0091] "Humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs in the humanized antibody correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0092] A "human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequence. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0093] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise indicated, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0094] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co. 91 pages (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0095] As used herein, the term "binding" or "specific binding" means that the binding is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry or other conventional binding assays known in the art.

[0096] The term "immune checkpoint molecule" refers to a class of inhibitory signaling molecules present in the immune system, which avoid tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to self-antigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264). Studies have found that one of the reasons why tumor cells can escape the body's immune system and proliferate uncontrollably is that they utilize the inhibitory signaling pathway of immune checkpoint molecules, thereby inhibiting the activity of T lymphocytes, making it impossible for T lymphocytes to effectively exert their killing effect on tumors (Yao S, Zhu Y and Chen L., Advances intargeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146).

[0097] The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary according to a variety of factors such as disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to stimulate the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are outweighed by the therapeutically beneficial effects. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

[0098] The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0099] The terms "tumor" and "cancer" are used interchangeably herein and encompass both solid tumors and liquid tumors.

[0100] The terms "cancer" and "cancerous" refer to the physiological condition in mammals in which cell growth is unregulated.

[0101] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0102] An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An "isolated nucleic acid encoding a TNFR2 binding molecule" refers to one or more nucleic acid molecules that encode a strand of a TNFR2 binding molecule or a fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

[0103] Calculation of sequence identity between sequences was performed as follows.

[0104] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.

[0105] The comparison of sequences and calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm that has been integrated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6 using the GAP program in the GCG software package (available at http: / / www.gcg.com). A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0106] The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).

[0107] Additionally or alternatively, one can further use the nucleic acid sequences and protein sequences described herein as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

[0108] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection, chemical and physical transfection methods such as electroporation.

[0109] The term "TNFR2-associated disease" refers to any condition caused by, exacerbated by, or otherwise associated with increased expression or activity of TNFR2 (eg, human TNFR2).

[0110] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0111] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), carrier, excipient, stabilizer, or the like, which is administered together with an active substance.

[0112] As used herein, "treat" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the development of the disease or to slow the progression of the disease.

[0113] The term "combination product" refers to a fixed combination or non-fixed combination in a dosage unit form or a kit of parts for combined administration, wherein two or more therapeutic agents can be independently administered simultaneously at the same time or separately at certain time intervals, especially when these time intervals allow the combined therapeutic agents to exhibit cooperation, e.g., synergistic effects. The term "fixed combination" means that the TNFR2 binding molecule of the invention and the combination partner (e.g., other therapeutic agent, such as anti-PD-1 antibody or anti-PD-L1 antibody) are administered to the patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the TNFR2 binding molecule of the invention and the combination partner (e.g., other therapeutic agent, such as anti-PD-1 antibody or anti-PD-L1 antibody) are administered to the patient simultaneously, in parallel or sequentially as separate entities without specific time restrictions, wherein such administration provides therapeutically effective levels of the two therapeutic agents in the patient. The latter also applies to cocktail therapy, e.g., administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.

[0114] The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents to treat cancer as described in the present disclosure. Such administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration or separate administration or sequential administration of each active ingredient in a variety of or in separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In some embodiments, administration also includes using each type of therapeutic agent at approximately the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0115] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are referred to herein as "expression vectors."

[0116] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity screened or selected in the initially transformed cells. Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, and also include cells inside transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0117] "Subject / patient sample" refers to a collection of cells, tissues or body fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are naturally not mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

[0118] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0119] II. TNFR2 Binding Molecules of the Invention

[0120] The TNFR2 binding molecule of the present invention comprises at least one single domain antibody (sdAb) portion that specifically binds to TNFR2, wherein the sdAb portion comprises three complementary determining regions from the N-terminus to the C-terminus, namely CDR1, CDR2 and CDR3, wherein:

[0121] (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 3, or a variant in which one or two amino acids in the amino acid sequence of SEQ ID NO: 3 are changed,

[0122] (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 4, or a variant in which one or two amino acids in the amino acid sequence of SEQ ID NO: 4 are changed, and

[0123] (c) CDR3 comprises the amino acid sequence of SEQ ID NO: 5, or a variant in which one or two amino acids in the amino acid sequence of SEQ ID NO: 5 are changed,

[0124] The amino acid change is an addition, deletion or substitution of an amino acid, and the binding molecule comprising the above change at least maintains the ability to bind to TNFR2.

[0125] In some embodiments, the TNFR2 binding molecules of the invention bind to mammalian TNFR2, such as human TNFR2.

[0126] In some embodiments, the TNFR2 binding molecules of the invention have one or more of the following properties:

[0127] (1) High affinity binding to human TNFR2, for example, EC binding between the TNFR2 binding molecule and cell surface TNFR2 50 is about 0.01 μg / mL to about 1 μg / mL, for example, about 0.1 μg / mL to about 0.6 μg / mL;

[0128] (2) It does not substantially block the binding of TNFα to TNFR2;

[0129] (3) Inhibition of TNFR2 signaling pathway, for example, in cells expressing TNFR2 such as Treg cells (e.g., Treg cells expressing high CD25), myeloid-derived suppressor cells (MDSCs) and / or TNFR2 + Inhibits TNFR2-mediated signaling in cancer cells;

[0130] (4) It has little effect on the proliferation of Treg cells in PBMCs;

[0131] (5) Inhibit tumor growth in vivo.

[0132] In some embodiments, the TNFR2 binding molecules of the invention suppress the proliferation of Treg cells and / or directly kill Treg cells by binding to TNFR2 on the surface of Treg cells and inactivating them (e.g., thereby reducing the number of Treg cells in a cell population by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% relative to the number of Treg cells in a cell population not exposed to the TNFR2 binding molecules of the invention).

[0133] In some embodiments, the TNFR2 binding molecules of the invention suppress the proliferation of MDSCs and / or directly kill MDSCs by binding to and inactivating TNFR2 on the surface of MDSCs (e.g., thereby reducing the number of MDSCs in a cell population by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% relative to the number of MDSCs in a cell population not exposed to the TNFR2 binding molecules of the invention).

[0134] In some embodiments, the TNFR2 binding molecules of the invention suppress the proliferation of and / or kill cancer cells expressing TNFR2 (e.g., thereby reducing the number of cancer cells expressing TNFR2 in a cell population by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the number of cancer cells expressing TNFR2 in a cell population not exposed to the TNFR2 binding molecules of the invention). Cancer cells of bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sex and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancers, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas.

[0135] In some embodiments, the TNFR2 binding molecules of the invention reduce Treg cells or cancer cells (such as TNFR2 + cancer cells), and / or decrease in Treg cells or cancer cells (e.g., TNFR2 + Cancer cells) secretion of soluble TNFR2.

[0136] In some embodiments, the TNFR2 binding molecules of the present invention cannot block TNFα from binding to TNFR2, but can extremely effectively inhibit cell necrosis induced by the TNFα-TNFR2 signaling pathway. Therefore, the binding epitope of the TNFR2 binding molecules of the present invention is likely to be located in the domain of the TNFR2 transmembrane protein close to the cell membrane end.

[0137] In some embodiments, the sdAb portion of the TNFR2 binding molecule of the invention comprises a CDR1, a CDR2, and a CDR3 selected from any one of the following groups:

[0138] (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 3; CDR2 comprises the amino acid sequence of SEQ ID NO: 4; and CDR3 comprises the amino acid sequence of SEQ ID NO: 5;

[0139] (b) CDR1 comprises the amino acid sequence of SEQ ID NO: 10; CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and CDR3 comprises the amino acid sequence of SEQ ID NO: 12;

[0140] (c) CDR1 comprises the amino acid sequence of SEQ ID NO: 14; CDR2 comprises the amino acid sequence of SEQ ID NO: 15; and CDR3 comprises the amino acid sequence of SEQ ID NO: 16;

[0141] (d) CDR1 comprises the amino acid sequence of SEQ ID NO: 18; CDR2 comprises the amino acid sequence of SEQ ID NO: 19; and CDR3 comprises the amino acid sequence of SEQ ID NO: 20;

[0142] (e) CDR1 comprises the amino acid sequence of SEQ ID NO: 22; CDR2 comprises the amino acid sequence of SEQ ID NO: 23; and CDR3 comprises the amino acid sequence of SEQ ID NO: 24;

[0143] (f) CDR1 comprises the amino acid sequence of SEQ ID NO: 26; CDR2 comprises the amino acid sequence of SEQ ID NO: 27; and CDR3 comprises the amino acid sequence of SEQ ID NO: 28;

[0144] (g) CDR1 comprises the amino acid sequence of SEQ ID NO: 30; CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and CDR3 comprises the amino acid sequence of SEQ ID NO: 32;

[0145] (h) CDR1 comprises the amino acid sequence of SEQ ID NO: 34; CDR2 comprises the amino acid sequence of SEQ ID NO: 35; and CDR3 comprises the amino acid sequence of SEQ ID NO: 36;

[0146] (i) CDR1 comprises the amino acid sequence of SEQ ID NO: 38; CDR2 comprises the amino acid sequence of SEQ ID NO: 39; and CDR3 comprises the amino acid sequence of SEQ ID NO: 40;

[0147] (j) CDR1 comprises the amino acid sequence of SEQ ID NO: 42; CDR2 comprises the amino acid sequence of SEQ ID NO: 43; and CDR3 comprises the amino acid sequence of SEQ ID NO: 44;

[0148] (k) CDR1 comprises the amino acid sequence of SEQ ID NO: 46; CDR2 comprises the amino acid sequence of SEQ ID NO: 47; and CDR3 comprises the amino acid sequence of SEQ ID NO: 48;

[0149] (1) CDR1 comprises the amino acid sequence of SEQ ID NO: 50; CDR2 comprises the amino acid sequence of SEQ ID NO: 51; and CDR3 comprises the amino acid sequence of SEQ ID NO: 52.

[0150] In some embodiments, the TNFR2 binding molecules of the invention comprise at least one single domain antibody (sdAb) portion that specifically binds to TNFR2, wherein the sdAb portion is a VHH. In some embodiments, the VHH comprises or consists of the following sequence:

[0151] (i) any one of the amino acid sequences selected from SEQ ID NO: 6, 7, 8, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53;

[0152] (ii) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the amino acid sequences selected from SEQ ID NO: 6, 7, 8, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53; or

[0153] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 6, 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to any one of the amino acid sequences selected from SEQ ID NO: 6, 7, 8, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, and preferably, the amino acid changes do not occur in the CDR regions.

[0154] In some embodiments, the TNFR2 binding molecule of the present invention comprises at least one single domain antibody (sdAb) portion that specifically binds to TNFR2, wherein the sdAb portion is a partially humanized or fully humanized VHH, a chimeric VHH. Compared with the VHH of camelid animals, the partially humanized or fully humanized VHH, a chimeric VHH of the present invention has a reduced human anti-camelid antibody response to the human body, improves the safety of antibody application, and is an affinity matured VHH.

[0155] In some embodiments, the TNFR2 binding molecules of the invention are linked to the Fc region of an immunoglobulin at the N-terminus or C-terminus of the sdAb portion thereof, optionally via an amino acid linker, e.g., an amino acid linker of between 1 and 20 amino acids in length. In some embodiments, at least 90% of the amino acid linker is glycine and / or serine amino acids. In some embodiments, the Fc region is from IgG, e.g., IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is from human IgG1. In some embodiments, the Fc region is from human IgG2.

[0156] In some embodiments of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0157] In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the VHH. In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to a substitution of an amino acid by another amino acid within the same class (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p.224), e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid.

[0158] In certain embodiments, the TNFR2 binding molecules provided herein are altered to increase or decrease the extent of their glycosylation. The addition or deletion of glycosylation sites to the TNFR2 binding molecules can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the TNFR2 binding molecule comprises an Fc region, the carbohydrate attached to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, such as removing fucose moieties to improve antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosylation modifications can be performed to modulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the TNFR2 binding molecules provided herein to generate Fc region variants to enhance, for example, the effectiveness of the TNFR2 binding molecules of the present invention in treating cancer.

[0159] In some embodiments, the TNFR2 binding molecules of the present invention are in the form of bispecific or multispecific antibody molecules, which specifically bind to the TNFR2 molecule and the second target protein. In one embodiment, the second target protein can be any antigen of interest, for example, selected from tumor antigens (e.g., tumor-associated antigens and tumor-specific antigens), immunomodulatory receptors, and immune checkpoint molecules. As used herein, "tumor-associated antigens" refer to antigens that are highly expressed in tumor cells and also exist in healthy cells but at a lower expression level. As used herein, "tumor-specific antigens" refer to antigens that are specifically expressed in tumor cells and hardly expressed in healthy cells. Non-limiting examples of tumor antigens may include CD19, CD20, EGFR, GPC3, HER-2, and FOLR1. Non-limiting examples of immune checkpoint molecules may include CTLA-4, LAG-3, and TIM-3. Immunomodulatory receptors may include, for example, immune activating receptors (e.g., CD27, CD137, CD40, GITR, and OX40) and immunosuppressive receptors (e.g., BTLA, CTLA4, and LAG-3). The multispecific antibody molecule can be, for example, a trispecific antibody molecule comprising a first binding specificity for TNFR2 and second and third binding specificities for one or more of the following molecules: EGFR, GPC3, 4-1BB, OX40, or LAG-3.

[0160] III. Nucleic acids of the present invention and host cells containing the same

[0161] In one aspect, the invention provides nucleic acids encoding any of the above TNFR2 binding molecules or fragments thereof or any of their chains. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. In some embodiments, the vector is a viral vector, such as an adenoviral vector, a retroviral vector, a poxvirus vector, an adeno-associated virus vector, a baculovirus vector, a herpes simplex virus vector, or a vaccinia virus vector.

[0162] In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0163] In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes but is not limited to a virus, a plasmid, a cosmid, a lambda phage or a yeast artificial chromosome (YAC). In one embodiment, the vector is a pcDNA34-TOPO vector.

[0164] Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultivated in a culture medium and screened for suitable activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the specific expression vector and mammalian host cell used. Variation or optimization.

[0165] In addition, one or more markers of the host cell that allows to select transfected can be introduced to select the cell that has stably incorporated DNA into its chromosome. The marker can for example provide prototrophy, biocidal resistance (for example, antibiotic) or heavy metal (such as copper) resistance etc. to the auxotrophic host. The selectable marker gene can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be needed so that the best synthetic mRNA can be obtained. These elements can include splicing signals, as well as transcription promoters, enhancers and termination signals.

[0166] In one embodiment, a host cell comprising a polynucleotide of the present invention is provided. In some embodiments, a host cell comprising an expression vector of the present invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli. The host cell can also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells, etc. Vertebrate cells can also be used as hosts. For example, a mammalian cell line modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), Chinese hamster ovary cells (CHO cells), CHO-S cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2 / 0, etc. For a review of mammalian host cell lines suitable for producing proteins, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a HEK293 cell.

[0167] IV. Production and Purification of TNFR2 Binding Molecules of the Invention

[0168] In one embodiment, the invention provides a method for preparing a TNFR2 binding molecule, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the TNFR2 binding molecule or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the TNFR2 binding molecule, and optionally isolating the TNFR2 binding molecule. In a certain embodiment, the method further comprises recovering the TNFR2 binding molecule from the host cell (or host cell culture medium).

[0169] To recombinantly produce the TNFR2 binding molecules of the invention, nucleic acids encoding the TNFR2 binding molecules of the invention are first isolated and inserted into vectors for further cloning and / or expression in host cells. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to nucleic acids encoding the TNFR2 binding molecules of the invention.

[0170] The TNFR2 binding molecules of the invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and such factors, and these will be apparent to those skilled in the art. The purity of the TNFR2 binding molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0171] V. Activity Assays for TNFR2 Binding Molecules of the Invention

[0172] The TNFR2 binding molecules provided herein can be identified, screened or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art. In one aspect, the TNFR2 binding molecules of the invention are tested for their antigen binding activity, for example, by known methods such as FACS, ELISA or Western blot. Binding to TNFR2 can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, FACS is used to determine the binding of the TNFR2 binding molecules of the invention to cell surface TNFR2 (e.g., human TNFR2).

[0173] Cells for use in any of the above in vitro assays include cell lines that naturally express TNFR2 or are engineered to express TNFR2. The cell lines engineered to express TNFR2 are cell lines that do not normally express TNFR2 but express TNFR2 after transfection of DNA encoding TNFR2 into the cells.

[0174] VI. Pharmaceutical compositions and pharmaceutical preparations

[0175] In some embodiments, the present invention provides a composition comprising any TNFR2 binding molecule described herein, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises a TNFR2 binding molecule of the present invention, and a combination of one or more other therapeutic agents (e.g., a chemotherapeutic agent, a cytotoxic agent, other antibodies, small molecule drugs or immunomodulators, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).

[0176] In some embodiments, the composition is used to treat a tumor. In some embodiments, the tumor is cancer.

[0177] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising TNFR2 binding molecules and / or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding TNFR2 binding molecules. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0178] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents and absorption delaying agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Haudbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Seskey and SCOwen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of a wetting or emulsifying agent, or a pH buffering agent. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0179] Pharmaceutical formulations comprising the TNFR2 binding molecules described herein can be prepared by mixing the TNFR2 binding molecules of the invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0180] The pharmaceutical composition or formulation of the present invention may also contain more than one active ingredient, which is required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anti-cancer active ingredients, such as chemotherapeutic agents, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, etc. The active ingredients are suitably combined in an amount effective for the intended use.

[0181] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the TNFR2 binding molecules of the invention, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0182] VII. Combination Products or Kits

[0183] In some embodiments, the present invention also provides a combination product comprising a TNFR2 binding molecule or antigen binding fragment thereof of the present invention and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, small molecule drugs or immunomodulators, etc.). In some embodiments, other antibodies are, for example, anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0184] In some embodiments, the combination product is used to treat a tumor. In some embodiments, the tumor is cancer or the like.

[0185] In some embodiments, two or more components of the combination product may be co-administered to a subject sequentially, separately or simultaneously.

[0186] In some embodiments, the invention also provides kits comprising the TNFR2 binding molecules, pharmaceutical compositions or combination products of the invention, and optionally a package insert directing administration.

[0187] In some embodiments, the present invention also provides a pharmaceutical product comprising the TNFR2 binding molecules, pharmaceutical compositions, combination products of the present invention, optionally further comprising a package insert for directing administration.

[0188] VIII. Uses of TNFR2 Binding Molecules of the Invention

[0189] In one aspect, the invention relates to a method of treating a disease associated with TNFR2 in a subject, the method comprising administering to the subject a therapeutically effective amount of a TNFR2 binding molecule disclosed herein, or a pharmaceutical composition or combination product comprising the same.

[0190] In some embodiments, the invention relates to a method of treating a cancer that expresses or overexpresses TNFR2 in a subject, the method comprising administering to the subject a therapeutically effective amount of a TNFR2 binding molecule disclosed herein, or a pharmaceutical composition or combination product comprising the same. In some embodiments, the cancer that expresses or overexpresses TNFR2 is, for example, bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sex and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas.

[0191] The subject can be a mammal, e.g., a primate, preferably, a higher primate, e.g., a human (e.g., a patient suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., a tumor as described herein) or at risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and / or radiation therapy.

[0192] In some embodiments, the cancers described herein include, but are not limited to, solid tumors, blood cancers, soft tissue tumors, and metastatic lesions.

[0193] In some embodiments, the treatment methods described herein further comprise administering to the subject or individual a TNFR2 binding molecule or pharmaceutical composition or combination product disclosed herein in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents.

[0194] In some embodiments, the treatment modality includes surgery (e.g., tumor resection); radiotherapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiotherapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed to a preselected target or organ) or focused irradiation), etc. Focused irradiation can be selected from stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiotherapy. Focused irradiation can have a radiation source selected from particle beams (protons), cobalt-60 (photons), and linear accelerators (X-rays), for example, as described in WO2012177624A1.

[0195] Radiation therapy can be administered by one or a combination of several methods including, but not limited to, external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, systemic radiation therapy, radiotherapy, and permanent or transient interstitial brachytherapy.

[0196] In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, other antibodies.

[0197] Exemplary other antibodies include, but are not limited to, inhibitors of immune checkpoint molecules (e.g., anti-PD-1, anti-PD-L1, anti-TIM-3, anti-CEACAM, or anti-LAG-3); antibodies that stimulate immune cells (e.g., agonistic GITR antibodies or CD137 antibodies), etc. Preferably, other antibodies are selected from anti-PD-1 antibodies and / or anti-PD-L1 antibodies. More preferably, the anti-PD-1 antibody is Nivolumab from Bristol-Myers Squibb (BMS), Pembrolizumab from Merck; the anti-PD-L1 antibody is atezolizumab developed by Roche, avelumab developed by Merck KGaA in Germany and Pfizer in the United States, and durvalumab developed by AstraZeneca.

[0198] The combination therapy of the present invention encompasses combined administration (wherein two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration. In the case of separate administration, administration of the TNFR2 binding molecules of the present invention, etc., can be implemented before, simultaneously with, and / or after administration of the other therapy.

[0199] In one embodiment, administration of the TNFR2 binding molecule and administration of the other therapy (eg, treatment modality or therapeutic agent) occur within about one month, or within about one, two or three weeks, or within about 1, 2, 3, 4, 5 or 6 days of each other.

[0200] The TNFR2 binding molecules of the present invention (and pharmaceutical compositions comprising the same) can be administered by any suitable method, including parenteral administration, intrapulmonary administration and intranasal administration, and, if required for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term, the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various medication schedules are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, push administration, and pulse infusion.

[0201] For the prevention or treatment of disease, the appropriate dosage of the TNFR2 binding molecules of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of TNFR2 binding molecule, the severity and course of the disease, whether the TNFR2 binding molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the TNFR2 binding molecule, and the discretion of the attending physician. The TNFR2 binding molecules are suitably administered to the patient as a single treatment or over a series of treatments. The dosage and treatment regimen of the TNFR2 binding molecule can be determined by a skilled artisan.

[0202] It will be appreciated that any of the above-described prevention or treatments can be performed using the compositions or combination products of the present invention in place of the TNFR2 binding molecule.

[0203] IX. Methods and compositions for diagnosis and detection

[0204] In certain embodiments, any TNFR2 binding molecule provided herein can be used to detect the presence of TNFR2 in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays. In certain embodiments, the biological sample is a sample of blood, serum, or other body fluid of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

[0205] In one embodiment, a TNFR2 binding molecule for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of TNFR2 in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of TNFR2 protein in a biological sample. In certain embodiments, TNFR2 is human TNFR2. In certain embodiments, the method comprises contacting a biological sample with a TNFR2 binding molecule as described herein under conditions that allow the TNFR2 binding molecule to bind to TNFR2, and detecting whether a complex is formed between the TNFR2 binding molecule and TNFR2. The formation of a complex indicates the presence of TNFR2. The method can be an in vitro or in vivo method. In one embodiment, the TNFR2 binding molecule is used to select a subject suitable for treatment with the TNFR2 binding molecule, for example, wherein TNFR2 is a biomarker for selecting the subject.

[0206] In one embodiment, the TNFR2 binding molecules of the invention can be used to diagnose cancer or tumors, e.g., to evaluate (e.g., monitor) the treatment or progression of a disease described herein (e.g., a hyperproliferative or cancerous disease), its diagnosis and / or staging in a subject. In certain embodiments, labeled TNFR2 binding molecules are provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferase, for example, firefly luciferase and bacterial luciferase (U.S. Patent No. US4737456A), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, and the like.

[0207] In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with a TNFR2 binding molecule. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin-fixed, paraffin-embedded (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

[0208] In some embodiments, TNFR2 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0209] In some embodiments, a method of treating a tumor is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample comprising cancer cells) for the presence of TNFR2, thereby determining a TNFR2 value, comparing the TNFR2 value to a control value (e.g., the value of TNFR2 in a sample from a healthy individual), and if the TNFR2 value is greater than the control value, administering to the subject a therapeutically effective amount of a TNFR2 binding molecule (e.g., a TNFR2 binding molecule described herein), optionally in combination with one or more other therapies, thereby treating the tumor.

[0210] It is understood that the various embodiments described in each section of the present invention, such as diseases, therapeutic agents, treatment methods and administration, are also applicable to the embodiments of other sections of the present invention, or can be combined with the embodiments of other sections. The embodiments described in each section of the present invention that are applicable to the properties, uses and methods of TNFR2 binding molecules are also applicable to compositions, conjugates, combination products and kits containing TNFR2 binding molecules. Example

[0211] The following examples are intended to illustrate the present invention only and therefore should not be construed as limiting the present invention in any way.

[0212] Example 1. Preparation of TNFR2 ligands, anti-TNFR2 positive control antibodies, and cell lines overexpressing TNFR2

[0213] 1.1 Preparation of TNFR2 ligands

[0214] The ligand of TNFR2 used in the embodiment is TNFα. The human TNFα extracellular region (as shown in SEQ ID NO: 1) was synthesized according to the sequence provided by the GeneCards database, and the C-terminus of the gene sequence encoding the human TNFα extracellular region shown in SEQ ID NO: 1 was connected to the human IgG1 Fc segment (as shown in SEQ ID NO: 2), and then constructed into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen). The obtained expression vector was expressed using the ExpiCHO transient expression system (Gibco, A29133), and the obtained supernatant was purified by Protein A / G affinity purification method after being filtered at 0.22 μm, and then eluted with 100 mM glycine (pH 3.0) to obtain a TNFα-Fc fusion protein that passed the quality inspection.

[0215] 1.2 Preparation of anti-TNFR2 positive control antibody

[0216] The anti-TNFR2 positive control antibody used in the example is hSBT-002e (hereinafter also referred to as "SBT002e"), which was synthesized according to the sequence disclosed in the international application WO2017083525A1, and a plasmid containing the SBT002e light chain gene and a plasmid containing the SBT002e heavy chain gene were constructed by molecular cloning methods. The ExpiCHO transient transfection system was used to express SBT002e, and the resulting supernatant was purified by Protein A / G affinity purification method after 0.22 μm filtration, and then eluted with 100 mM glycine (pH 3.0) to obtain the positive control antibody SBT002e.

[0217] 1.3 Preparation of cell lines overexpressing human TNFR2

[0218] HEK293 cell line overexpressing human TNFR2 (hereinafter referred to as huTNFR2-HEK293 cell line) and Jurkat cell line overexpressing human TNFR2 (hereinafter referred to as huTNFR2-Jurkat cell line) were constructed.

[0219] A DNA fragment encoding the full-length human TNFR2 protein (amino acid sequence as shown in SEQ ID NO: 9) was synthesized by gene synthesis technology (General Biosystems (Anhui) Co., Ltd.), and cloned into the expression vector pLVX-puro (Clontech, Cat#632164). The plasmid was introduced into E. coli DH5α by transformation, and the correct plasmid clone was obtained by sequencing after picking a single E. coli clone, and the plasmid was extracted and sequenced again for confirmation.

[0220] HEK293 cells were cultured using Gibco's DMEM medium (Cat. No. 11995-665). CRL-1573 TM ), Jurkat cells were cultured using Gibco's RPMI1640 medium (Cat. No. 11875093) TIB-152). One day before electroporation, cells were passaged to 5×10 5 The next day, the electroporation kit (Cat. No.: MPK10096) and electroporator (Invitrogen, Neon TMTransfection System, MP922947) to introduce the constructed plasmid into the cells. The electroporated cells were transferred to DMEM culture medium containing 10% FBS and placed in a 37°C cell culture incubator for 48 hours. Then 1500-4000 cells / well were plated into a 96-well plate, and puromycin (Gibco, A1113803) was added at a final concentration of 2μg / mL, and cultured in a 37°C carbon dioxide incubator. After 10 days, DMEM culture medium containing 2μg / mL puromycin was added. The cell clones grown in the 96-well plate were picked up and transferred to a 24-well culture plate for continued expansion and culture. After that, the control antibody SBT002e was used by FACS to identify the cell line that had been stably transformed with human TNFR2. The identification results of the huTNFR2-HEK293 cell line are shown as follows. Figure 1 As shown, the results of huTNFR2-Jurkat cell line identification are as follows Figure 2 shown.

[0221] Example 2. Animal immunization and serum immune titer detection

[0222] 2.1 Animal immunization

[0223] Recombinant human TNFR2 (SinoBiological, 10417-H03H) was used as an antigen to immunize two alpacas (Nanchang Dajia Technology). Each alpaca was immunized with 500 μg of antigen each time, once every two weeks, for a total of 4 immunizations.

[0224] 2.2 Serum immune titer detection

[0225] After the alpaca immunization is completed, the alpaca serum is taken for immune titer detection. The immune titer determination is to determine the binding ability of the immune serum to recombinant human TNFR2 by the ELISA method, and the immune effect is determined based on the antibody titer of the antigen.

[0226] The specific method is as follows: On the day before the immunopotency assay, the recombinant human TNFR2 was diluted with PBS to a final concentration of 2 μg / mL to obtain a diluent. 30 μL of the diluent was added to the ELISA plate and coated at 4°C overnight. On the day of the immunopotency assay, the coated plate was rinsed three times with PBST, then blocked with PBST containing 5% skimmed milk powder at room temperature for 2 hours, and then rinsed three times with PBST. On another 96-well dilution plate, the unimmunized negative serum and the post-immunization serum were diluted with PBS, the first well was diluted 2000 times, and then the subsequent 7 wells were diluted by 3-fold gradient. The diluted serum was added to the first ELISA plate coated with recombinant human TNFR2 and incubated at room temperature for 1 hour. After washing the plate three times with PBST, anti-IgG (H+L)-HRP (Milliipore) was added at 1:10000 and incubated at room temperature for 1 hour. After incubation, the plate was washed six times with PBST, TMB (SurModics, TMBS-1000-01) was added for color development, and 2M HCl was added to terminate the reaction according to the color development results. The OD value was read at OD450 wavelength using a microplate reader (Molecular Devices, SpecterMax190).

[0227] The results showed that the serum titer after 4 immunizations reached 256,000, which can be used for the next step of constructing an alpaca peripheral blood immune antibody library.

[0228] Example 3. Construction and preliminary screening of alpaca immune library

[0229] 3.1 Library construction

[0230] After the animal immunization, 50 mL of fresh blood from the alpaca was taken, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque density gradient separation solution (GE, 17144003S), and RNA was extracted from the separated PBMC cells, and the extracted RNA was reverse transcribed into cDNA by a reverse transcription kit (TaKaRa, 6210A). Based on the situation of the VHH antibody germline gene (germline), degenerate primers were designed to obtain a DNA fragment encoding VHH-CH2 by PCR amplification and recovery of the PCR product by agarose gel electrophoresis. Then all VHH genes were amplified using the recovered DNA fragment product as a template, and finally the target antibody gene fragment was inserted into the phage display vector by double enzyme digestion and connection. The ligation product was recovered using a recovery kit (Omega, D6492-02), and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser), and coated on a 2-YT solid plate containing ampicillin resistance to construct an anti-human TNFR2 single domain antibody library.

[0231] The library capacity was determined to be 1.8×10 9 The helper phage M13KO7 (NEB) was used to package the anti-human TNFR2 single domain antibody library to obtain the phage library corresponding to the anti-human TNFR2 single domain antibody library.

[0232] 3.2 Phage library magnetic bead screening

[0233] The biotin-labeled TNFR2 protein and the ovalbumin-coupled magnetic beads (Thermo fisher, 11205D) were incubated together to bind the TNFR2 protein to the magnetic beads. The magnetic beads bound to the TNFR2 antigen and the phage library with nanoantibody display prepared in 3.1 above were incubated at room temperature for 2 hours. After washing 6-8 times with PBST, the non-specifically adsorbed phages were removed, and trypsin (Gibco) was added and gently mixed for 20 minutes to elute the nanoantibody display phage that specifically binds to the human TNFR2 protein. The eluted phages were then infected with SS320 bacteria (Lucigen, MC1061F) in the logarithmic phase, and the SS320 bacteria infected with the phages were coated on 50μg / mL carbenicillin resistance plates, cultured overnight at 37°C, and the bacteria were collected the next day. Phages were prepared using SS320 bacteria for the next round of screening.

[0234] 3.3 Monoclonal screening

[0235] The positive phage libraries in the first and second rounds of products obtained by magnetic bead screening were selected for monoclonal screening. The specific method is as follows: the recombinant human TNFR2 was coated on a 96-well ELISA plate one day before the monoclonal screening, and the phage supernatant was prepared in a 96-well plate the next day. The positive clones for human recombinant TNFR2 (SinoBiological, 10417-H03H) were screened by phage ELISA, and then all positive clones were picked for sequencing analysis. The bacterial solution of the positive monoclonal clone was inoculated into 50mL 2-YT medium at 1:100, cultured on a constant temperature shaker at 37°C for 14h, centrifuged at 10000g for 5min at room temperature, and resuspended with 1mL Tris-HCl buffer containing benzonase at pH 9.0, lysed on ice for 30min, centrifuged at 4°C for 10min, and the supernatant was collected to obtain the positive clone lysate.

[0236] The prepared positive clone lysate was subjected to ELISA affinity detection. The specific method is as follows: 2 μg / mL recombinant human TNFR2 was coated on a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed 3 times with PBST and blocked with 5% skim milk for 2 hours. Subsequently, the plate was washed 3 times with PBST, and the gradient dilution of the positive clone lysate was added and incubated for 1 hour. Subsequently, the plate was washed 3 times with PBST, and Rabbit Anti-Camelid-VHH-HRP (Genescript, A01861-200) diluted 1:8000 was added and incubated for 1 hour. Subsequently, the plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The values ​​under OD450 were read by an enzyme reader (Molecular Devices, SpecterMax 190) and four-parameter fitting was used.

[0237] The results are as follows Figure 3 As shown, the binding of the lysate of the selected positive clone NB92-161 to recombinant human TNFR2 exhibited a dose-dependent binding effect. The amino acid sequence of the VHH of the positive clone NB92-161 is shown in SEQ ID NO: 6, and the amino acid sequences of the CDR1, CDR2 and CDR3 of the positive clone NB92-161 defined by AbM are shown in SEQ ID NO: 3-5, respectively.

[0238] Example 4. Production and expression of VHH-Fc chimeric antibodies

[0239] The VHH obtained by screening in Example 3 was fused with the human IgG1 Fc segment (as shown in SEQ ID NO: 2), wherein the C-terminus of the VHH gene sequence was connected to the N-terminus of the human IgG1 Fc segment gene sequence to construct the expression vector pcDNA3.4-TOPO (Invitrogen) of the VHH-Fc chimeric antibody. The expression was performed using the ExpiCHO transient expression system, and the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 minutes. The obtained supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100mM sodium acetate (pH3.0), followed by neutralization with 1M Tris-HCl, and finally the obtained protein was replaced into PBS buffer using an ultrafiltration concentration tube (Millipore, UFC901096) to obtain a VHH-Fc chimeric antibody that passed the quality inspection.

[0240] Example 5. Evaluation of affinity activity of VHH-Fc chimeric antibodies

[0241] The affinity activity of the obtained VHH-Fc chimeric antibody was evaluated. The binding activity of the VHH-Fc chimeric antibody to the TNFR2 protein on the cell was detected by FACS method. The specific method is as follows: the cultured huTNFR2-HEK293 cells were collected, centrifuged at 300g to remove the supernatant, and the cells were resuspended in the prepared FACS buffer (PBS containing 1% BSA), counted and the cell suspension density was adjusted to 2×10 6 cells / mL; huTNFR2-HEK293 cells were added to a 96-well round-bottom plate at 100 μL per well, and the supernatant was removed by centrifugation at 300 g; gradient dilutions of chimeric antibody NB92-161 (the antibodies were named according to the clone numbers) and control antibody SBT002e were added to the corresponding wells of the 96-well round-bottom plate, the cells were resuspended and incubated at 4°C for 30 min; the incubated cell mixture was washed 3 times and PE-labeled anti-human IgG Fc flow cytometry antibody (Abcam, ab98596) was added; the cells were resuspended and incubated at 4°C for 30 min; the incubated cell mixture was washed 3 times and the cells were resuspended and detected by flow cytometer (Beckman, CytoFLEXAOO-1-1102).

[0242] The results of flow cytometry were as follows Figure 4 As shown, the binding activity of chimeric antibody NB92-161 on huTNFR2-HEK293 cells was lower than that of control antibody SBT002e, where the binding EC 50 The binding EC of SBT002e was 0.5127 μg / mL. 50The cellular affinity of the chimeric antibody NB92-161 for TNFR2 was much lower than that of the control antibody SBT002e.

[0243] Example 6. Verification of species cross-reactivity of VHH-Fc chimeric antibodies

[0244] The obtained VHH-Fc chimeric antibody was verified for species cross-reactivity. The specific method is as follows: 2 μg / mL recombinant cynomolgus monkey TNFR2 (SinoBiological, 90102-C08H) and recombinant mouse TNFR2 (SinoBiological, 50128-M08H) were coated on a 96-well ELISA plate and incubated overnight at 4°C; the next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours; then, the plate was washed three times with PBST and a gradient dilution of the chimeric antibody NB92 was added. -161 and control antibody SBT002e were incubated for 1 hour; after the incubation, the plate was washed 3 times with PBST, and Goat-Anti-Human-IgG-Fc-HRP (abcam, ab97225) diluted 1:4000 was added and incubated for 1 hour; then, the plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and color was developed in the dark for 5-10 minutes, and 2M HCl was added to terminate the reaction according to the color development. The values ​​under OD450 were read by an ELISA reader (Molecular Devices, SpecterMax 190) and four-parameter fitting was used.

[0245] The results are as follows Figure 5 As shown in the figure, the chimeric antibody NB92-161 has good cross-binding activity with recombinant cynomolgus monkey TNFR2. In addition, the chimeric antibody NB92-161 has no cross-binding with recombinant mouse TNFR2, so this result is not shown in the figure.

[0246] Example 7. Evaluation of the blocking activity of VHH-Fc chimeric antibodies against TNFα binding to TNFR2

[0247] The obtained VHH-Fc chimeric antibody was evaluated for its ligand blocking activity. The FACS method was used to detect whether the VHH-Fc chimeric antibody blocked the binding of TNFα to TNFR2. The specific method was as follows: the cultured huTNFR2-HEK293 cells were collected, centrifuged at 300g to remove the supernatant, and the cells were resuspended in the prepared FACS buffer, counted, and the cell suspension density was adjusted to 2×10 6cells / mL; huTNFR2-HEK293 cells were added to a 96-well round-bottom plate at 100 μL per well, and the supernatant was removed by centrifugation at 300 g; chimeric antibody NB92-161 and control antibody SBT002e were added to the corresponding wells of the 96-well round-bottom plate in a gradient dilution, and the cells were resuspended and incubated at 4°C for 30 min; the incubated cell mixture was washed 3 times and a dilution of biotin-labeled TNFα-Fc fusion protein (prepared in Example 1.1 of the present application) (0 .1μg / mL) 100μL, resuspend the cells and incubate the cells at 4°C for 30min; wash the incubated cell mixture three times, then add PE-labeled streptavidin (eBioscience, 12-4317-87), and incubate at 4°C for 30min; wash the incubated cell mixture three times, then add FACS buffer to the wells, 200μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0248] The results showed that the anti-TNFR2 VHH-Fc chimeric antibody did not substantially block the binding activity of TNFα to TNFR2 on huTNFR2-HEK293 cells. Figure 6 From this result, it can be seen that the chimeric antibody NB92-161 does not basically block the binding of TNFα to TNFR2, while the control antibody SBT002e can completely block the binding of TNFα to TNFR2, and its IC 50 It is 0.0917μg / mL.

[0249] Example 8. Evaluation of the inhibitory activity of VHH-Fc chimeric antibodies on cell necrosis induced by TNFα-TNFR2 signaling pathway

[0250] In this example, a TNFα-induced cell necrosis experiment was used to evaluate whether the candidate antibodies of the present invention have inhibitory activity on the TNFα-TNFR2 signaling pathway. The specific method is as follows: the cultured huTNFR2-Jurkat cells were collected, centrifuged at 300 g to remove the supernatant, the huTNFR2-Jurkat cells were resuspended in culture medium, counted and the cell suspension density was adjusted to 2×10 5cells / mL; huTNFR2-Jurkat cells were added to a 96-well round-bottom plate at 50 μL per well; gradient dilutions of chimeric antibody NB92-161 and control antibody SBT002e were added to the corresponding wells of the 96-well round-bottom plate, 25 μL per well, and incubated at 37°C for 2 hours; after the incubation, a dilution of TNFα-Fc fusion protein (prepared in Example 1.1 of the present application) (5 ng / mL) was added to the corresponding wells, 25 μL per well, and incubated at 37°C for 24 hours; after the incubation, Cell-Titer Glo (Promega, G7572) was added, 50 μL per well, incubated for 10 minutes, and then placed in a microplate reader (MD, SpectraMax i3x) to detect the fluorescence value.

[0251] The results are as follows Figure 7 As shown in Figure 2, although the affinity of the chimeric antibody NB92-161 for binding to TNFR2 is much lower than that of the control antibody SBT002e, it can significantly inhibit TNFα-induced huTNFR2-Jurkat cell necrosis, and the inhibitory activity is much better than that of the control antibody SBT002e. 50 The ED of the control antibody SBT002e was 0.03390 μg / mL. 50 The concentration of TNFR2 in the chimeric antibody was 0.4993 μg / mL.

[0252] Example 9. Validation of the efficacy of VHH-Fc chimeric antibody in TNFR2 humanized mouse tumor model

[0253] In order to confirm the activity of candidate anti-TNFR2 VHH-Fc chimeric antibodies in inhibiting tumor growth in vivo, a tumor-bearing model based on TNFR2 humanized mice MC38 was established. The specific method is as follows: TNFR2 humanized C57BL / 6 mice with similar body weight around 8 weeks of age (Shanghai Model Organisms Technology Co., Ltd.) were selected and divided into PBS control group, chimeric antibody NB92-161 group and positive control antibody SBT002e group, a total of 3 groups, 5 mice in each group. Mouse colon cancer cell line MC38 (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences) was cultured in vitro, and 1.5×10 6 MC38 cells were injected subcutaneously into mice, which was recorded as day 0. On the seventh day, each group of mice was injected with 7.5 mg / kg of chimeric antibody NB92-161, 15 mg / kg of positive control antibody or PBS, and then the drug was administered twice a week for 6 consecutive times. The weight of the mice and the size of the tumor were recorded weekly from the 7th day until the tumor in the PBS control group grew to 1500 mm 3 Tumor size was measured by digital calipers and calculated using the formula (L×W2 ) / 2, where L is the longest and W is the shortest of the tumor diameters (mm). Relative tumor volume is equal to the tumor volume at a given time point divided by the tumor volume before treatment began.

[0254] The results are as follows Figure 8 As shown, the chimeric antibody NB92-161 exhibited strong tumor growth inhibitory activity, and there was no significant difference in relative tumor volume compared with the positive control antibody SBT002e.

[0255] Example 10. Humanized antibody modification

[0256] In order to reduce the immunogenicity of antibody molecules in vivo, the anti-TNFR2 VHH-Fc chimeric antibody NB92-161 was humanized. The antibody sequence was compared with the human antibody germline gene database to find 1-3 germline genes with high homology to each VHH sequence. Taking into account the drugability of germline genes, appropriate germline gene templates were selected for comparison, and the number of non-human sequence sites in the VHH framework region was analyzed. Homology modeling was performed on VHH, and the homology modeling was referenced to the nanoantibody model in the PDB database (http: / / www.rcsb.org / ). Combining the simulated structural model of VHH and the non-human site situation, combined back mutation design was performed (while avoiding the introduction of potential post-translational modification sites), and sequences with different degrees of humanization were designed. The VHH amino acid sequences of the humanized antibodies NB92-161-hVH5 and NB92-161-hVH4 after the anti-TNFR2 VHH-Fc chimeric antibody modification are shown in SEQ ID NO: 7 and SEQ ID NO: 8, and the degree of humanization is 95.87% and 94.21%, respectively.

[0257] Example 11. Humanized Antibody Binding to huTNFR2-HEK293 Cells

[0258] In order to detect the binding activity of humanized antibodies to human TNFR2 antigen, anti-TNFR2 VHH-Fc chimeric antibodies and their corresponding humanized antibodies were detected by FACS. The specific method is similar to that of Example 5.

[0259] The results are as follows Fig. 9 As shown. Fig. 9 As can be seen, the ability of humanized antibodies NB92-161-hVH5 and NB92-161-hVH4 to bind to human TNFR2 is comparable to that of their anti-TNFR2 VHH-Fc chimeric antibody NB92-161 (parent molecule).

[0260] Example 12. Affinity maturation of humanized antibodies

[0261] In this embodiment, affinity maturation modification was performed on humanized antibody NB92-161-hVH5 to improve affinity and biological activity. Affinity maturation modification is based on M13 phage display technology, using codon-based primers (during primer synthesis, a single codon consists of NNK) to introduce mutations in the CDR region, and a total of 4 phage display libraries were constructed: Library 1 is a single-point combination mutation of CDR1+CDR2+CDR3; Library 2 is a double-point combination mutation of CDR1+CDR2; Library 3 is a double-point combination mutation of CDR1+CDR3, and Library 4 is a double-point combination mutation of CDR2+CDR3. The library capacity is shown in Table 1.

[0262] Using humanized antibody NB92-161-hVH5 as a template, a single CDR region mutation fragment was obtained by PCR, and then the VHH full-length fragment was obtained by overlapping PCR. The point mutation antibody was connected to the phage display vector by double enzyme digestion (Hind III and Not I) and double sticky end ligation. Finally, the VHH sequence with the mutation site was transferred into Escherichia coli SS320 by electroporation.

[0263] After the 4 constructed libraries were packaged into phages, solid phase panning was performed. The phages displaying the full-length fragment of VHH were bound to the antigen coated on the immunotube, and antibodies with high affinity were selected by reducing the pressure of the coated antigen. After panning, elution, and infection of Escherichia coli SS320 for the next cycle of panning, after 2-3 cycles of panning, monoclones were selected for affinity ELISA detection. According to affinity and sequence analysis, 11 candidate anti-TNFR2 affinity mature molecules were selected for sample preparation. The preparation method is detailed in Example 4. The variable region amino acid sequence information of the candidate anti-TNFR2 affinity mature molecules is shown in Table 2.

[0264] Table 1 Library design and library capacity

[0265]

[0266]

[0267] Table 2 Variable region amino acid sequences of candidate anti-TNFR2 affinity mature molecules (SEQ ID NO:

[0268] Molecule name HCDR1 HCDR2 HCDR3 VHH 161-hVH5-1 10 11 12 13 161-hVH5-3 14 15 16 17 161-hVH5-8 18 19 20 21 161-hVH5-10 22 23 24 25 161-hVH5-19 26 27 28 29 161-hVH5-22 30 31 32 33 161-hVH5-24 34 35 36 37 161-hVH5-36 38 39 40 41 161-hVH5-37 42 43 44 45 161-hVH5-48 46 47 48 49 161-hVH5-49 50 51 52 53

[0269] Example 13. Evaluation of affinity activity of affinity matured molecules

[0270] In order to detect the binding activity of affinity matured molecules to human TNFR2 antigen, this example uses FACS method to detect candidate affinity matured molecules. For specific methods, see Example 5.

[0271] Test results such as Figures 10A-10D As shown. Figures 10A-10C As can be seen from the table, the 10 candidate affinity mature molecules presented have comparable or superior affinity activities to the parent molecule NB92-161-hVH5. Fig. 10D As shown in Table 3, the affinity activity of the affinity matured molecule 161-hVH5-48 is close to that of the control antibody SBT002e, and the affinity activity is increased by about 5 times compared with the parent molecule NB92-161-hVH5. 50 The values ​​are shown in Table 3.

[0272] Table 3 Affinity activity of affinity matured molecules

[0273] Molecule name <![CDATA[EC 50 (μg / mL)]]> NB92-161-hVH5 0.3359 161-hVH5-48 0.0664 SBT002e 0.0698

[0274] Example 14. Evaluation of the blocking activity of affinity matured molecules on TNFα binding to TNFR2

[0275] In order to detect whether the affinity matured molecule has the activity of blocking TNFα from binding to TNFR2, this example conducted a ligand blocking activity evaluation, and the specific method is as described in Example 7.

[0276] The results are as follows Fig.11 As shown, the affinity matured molecule 161-hVH5-48, like the parent molecule NB92-161-hVH5, does not block the binding of TNFα to TNFR2.

[0277] Example 15. Evaluation of the inhibitory activity of affinity mature molecules on cell necrosis induced by TNFα-TNFR2 signaling pathway

[0278] In order to detect whether the affinity matured molecule still has strong inhibitory activity on the TNFR2 signaling pathway induced by TNFα, this example evaluated the inhibitory activity through a TNFα-induced cell necrosis experiment. The specific method is as described in Example 8.

[0279] Test results such as Fig.12 As shown in Figure 2, the affinity matured molecule 161-hVH5-48 still has excellent inhibitory activity, which is much higher than the control antibody SBT002e. The ED 50 The ED of the control antibody SBT002e was 0.008109 μg / mL. 50 It is 0.5137μg / mL.

[0280] Example 16. Study on the effect of affinity maturation molecules on the proliferation of Treg cells in PBMC

[0281] In order to detect whether affinity maturation molecules affect the proliferation of Treg cells in PBMC, this example evaluated the Treg cell proliferation experiment induced by TNFα. The specific method is as follows: PBMC cells are firstly isolated from fresh blood, and then CD4 + T cell isolation kit (Miltenyi, 130-096-533) was used to further isolate CD4 + T cells; collect CD4 + T cells were centrifuged at 300 g and the supernatant was removed. The cells were resuspended in complete medium, counted, and the cell suspension density was adjusted to 2 × 10 6 cells / mL; CD4 + T cells were added to 96-well round-bottom plates at 100 μL per well, and affinity matured molecule 161-hVH5-48 and control antibody SBT002e prepared with culture medium containing 400 U / mL IL2 (Novoprotein, CP09) and 40 ng / mL TNFα (Sino Biological, 10602-H01H) were added to the corresponding wells of the 96-well round-bottom plate, 100 μL per well, and incubated at 37°C for 72 hours; after the incubated cell mixture was washed three times, PE-labeled anti-human CD4 flow cytometry antibody (BioLegend, 357404) and FITC-labeled anti-human CD25 flow cytometry antibody (BioLegend, 356106) were added, the cells were resuspended and incubated at 4°C for 30 minutes; after the incubated cell mixture was washed three times, 4% paraformaldehyde solution was added and fixed at room temperature for 30 minutes; after washing three times with 1X Perm solution, Alexa Fluor The cells were incubated at room temperature for 1 h with 647-labeled anti-human Foxp3 flow cytometry antibody (BioLegend, 320114). After incubation, the cell mixture was washed three times and then the cells were resuspended and detected by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0282] The results of flow cytometry were as follows Fig.13 As shown: The affinity matured molecule 161-hVH5-48 does not affect the proliferation of Treg cells in PBMC; while the positive antibody SBT002e can significantly inhibit the proliferation of Treg cells, which may cause unnecessary blood toxicity.

[0283] Example 17 Evaluation of the anti-tumor activity of affinity matured molecules in humanized mice

[0284] In order to detect the tumor-suppressing ability of affinity matured molecules in mice, this example was evaluated using a TNFR2 humanized mouse model. The specific method is as follows: MC-38 cells (mouse colon cancer cells, Cobioer Biosciences, CBP60825) in the logarithmic growth phase were taken and each mouse was cultured at 1×10 6 Subcutaneous inoculation was performed, and TNFR2 humanized mice (Biocytogen, 110032, female, 5-6 weeks old) were selected. 3 The mice were randomly divided into groups with 6-8 mice in each group and administered by intraperitoneal injection twice a week for 3 weeks.

[0285] The results are as follows Fig.14 As shown in Table 4, at high and medium doses, the tumor inhibition effects of the affinity matured molecule 161-hVH5-48 and the control antibody SBT002e were comparable, but the number of mice with complete tumor regression in the antibody 161-hVH5-48 group was greater than that in the control antibody group, especially at medium doses, the number of mice with complete tumor regression in the 161-hVH5-48 group was far greater than that in the control antibody group.

[0286] Table 4 Number of tumors completely regressed

[0287]

[0288]

[0289] Example 18 ADCC Effect of Affinity Matured Molecules

[0290] In order to detect the ADCC effect of affinity matured molecules, this example was evaluated by an in vitro ADCC model, and the specific method is as follows:

[0291] Adjust the density of huTNFR2-HEK293 cell suspension to 2 × 10 per ml. 5 50 μL of the cell suspension was added to each well of a 96-well round-bottom plate. Then, 50 μL of gradient dilutions of affinity matured molecule 161-hVH5-48, control antibody SIM-0235-001, and control antibody BI-1808 were added to each well and incubated at 37°C for 20 min. The density of the PBMC (Allcells, NF0074) suspension revived one day in advance was adjusted to 5×10 per ml. 5 Cells were added to the pre-incubated 96-well round-bottom plate at 50 μL per well and incubated at 37°C for 4 h. After incubation, 60 μL of LDH detection reagent (Shanghai Biotech Co., Ltd., C0017) was added to each well and incubated at room temperature for 1 h. After incubation, the signal value was read at OD490 using an ELISA reader and the killing rate was calculated.

[0292] Test results such as Fig.15A As shown, the ED50 of the affinity matured molecule 161-hVH5-48 is 0.0032 μg / mL, and the maximum killing rate can reach 36%; the ED50 of the positive antibody SIM-0235-001 (Innovent, WO2021023098A1) is 0.0196 μg / mL, and the maximum killing rate can reach 23%; the ED50 of the positive antibody BI-1808 (BioInvent, WO2020089474A1) is 0.0095 μg / mL, and the maximum killing rate can reach 28%; the ADCC effect of the affinity matured molecule 161-hVH5-48 is much better than that of the control antibody.

[0293] Based on the same method, this example also detected the ADCC effect of the antibody on huTNFR2-Jurkat cells. The test results are as follows: Fig. 15B As shown, the ADCC effect of the affinity matured molecule 161-hVH5-48 is much better than that of the control antibody.

[0294] Example 19 Crystal structure analysis of detailed epitopes

[0295] In order to determine the epitope of 161-hVH5-48 binding to TNFR2, this example prepared a complex crystal of TNFR2 and 161-hVH5-48, and analyzed the binding epitope by X-ray diffraction.

[0296] Specifically, for the expression of antigenic proteins, TNFR2 (33-205aa) protein was expressed by prokaryotic Escherichia coli, and the inclusion body protein expressed by Escherichia coli was purified by dilution and renaturation, and then verified by molecular sieve Superdex75. For the expression of antibodies, the full-length 161-hVH5-48 nanoantibody was expressed by the CHO eukaryotic expression system, and then papain was used to remove the effect of Fc on protein crystallization, and then purified by molecular sieve. After the preparation of antigens and antibodies, the renatured antigen protein TNFR2 (33-205aa) was incubated with the antibody 161-hVH5-48 after Fc cleavage at 4°C overnight, and then the complex was prepared by molecular sieve Superdex75. Subsequently, protein crystal screening was used to obtain crystals grown under specific conditions, and then the quality of the crystals was improved from the aspects of precipitant, salt concentration, pH and protein concentration through subsequent crystal growth optimization. Finally, the diffraction pattern of the protein crystals was obtained through X-ray crystallography diffraction, and phase analysis and model building were performed using software such as HKL3000, CCP4, Coot and Phenix. Based on the structural analysis of the antigen-antibody complex, the key amino acid sites were determined using software PDBePISA and Chimera.

[0297] The results are as follows Fig.16 As shown, the 161-hVH5-48 antibody mainly binds to the groove of the CRD3 domain of the antigen TNFR2, and the key antigen epitopes for binding include 13 amino acids such as V83, E84, T85, T97, C98, P100, G101, K108, E110, C112, G131, T132, and E133. The key antibody interaction amino acid sites include 14 amino acids such as R29, F30, N32, R53, E99, S101, Q102, L103, G104, Y105, A106, F107, R108, and D109. Specific antigen-antibody interactions include salt bonds (E110-R29), hydrogen bonds (E84-R53, T97-G104, C98-Y105, C98-F107, K108-Y105, G131-R108, T132-R108, E133-S101, E133-Q102, E133-L103, E133-G104, E133-Y105, E133-A106) and hydrophobic interactions. By comparing with the known TNF-TNFR2 crystal structure (PDB: 3ALQ), it was found that the TNF trimer mainly binds to its receptor TNFR2 through CRD2 and CRD3, which overlaps with the CRD3 region bound by the 161-hVH5-48 antibody. It is believed that the binding of the 161-hVH5-48 antibody may hinder the normal binding of the TNF trimer to the TNFR2 receptor, indicating that the 161-hVH5-48 antibody may function as a non-classical blocking antibody.

[0298] Exemplary sequences

[0299] SEQ ID NO: 1 TNFα extracellular region

[0300] VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0301] SEQ ID NO:2 hIgG1 Fc

[0302] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKpREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0303] SEQ ID NO:3 NB92-161 CDR1

[0304] GSIFSINDMG

[0305] SEQ ID NO:4 NB92-161 CDR2

[0306] AIGRGGGSTN

[0307] SEQ ID NO:5 NB92-161 CDR3

[0308] EISQLTWAFRDY

[0309] SEQ ID NO:6 NB92-161 VHH

[0310] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKQRELVAAIGRGGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHAEISQLTWAFRDYWGQGTQVTVSS

[0311] SEQ ID NO:7 NB92-161-hVH5

[0312] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAAIGRGGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLTWAFRDYWGQGTLVTVSS

[0313] SEQ ID NO:8 NB92-161-hVH4

[0314] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKQRELVAAIGRGGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLTWAFRDYWGQGTLVTVSS

[0315] SEQ ID NO: 9 TNFR2 full-length protein

[0316] MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS

[0317] SEQ ID NO: 10 161-hVH5-1 CDR1

[0318] GSIFSINDMG

[0319] SEQ ID NO: 11 161-hVH5-1 CDR2

[0320] VHGRGGGSTN

[0321] SEQ ID NO: 12 161-hVH5-1 CDR3

[0322] EISQLTWAFRDY

[0323] SEQ ID NO: 13 161-hVH5-1 VHH

[0324] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAVHGRGGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLTWAFRDYWGQGTLVTVSS

[0325] SEQ ID NO:14 161-hVH5-3 CDR1

[0326] GSIFSINDMG

[0327] SEQ ID NO:15 161-hVH5-3 CDR2

[0328] AIGRGRRSTN

[0329] SEQ ID NO:16 161-hVH5-3 CDR3

[0330] EISQLSFAFRDY

[0331] SEQ ID NO:17 161-hVH5-3 VHH

[0332] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAAIGRGRRSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLSFAFRDYWGQGTLVTVSS

[0333] SEQ ID NO:18 161-hVH5-8 CDR1

[0334] GSIFSINDMG

[0335] SEQ ID NO:19 161-hVH5-8 CDR2

[0336] AIGRGGQRTN

[0337] SEQ ID NO:20 161-hVH5-8 CDR3

[0338] EISQLSFAFRDY

[0339] SEQ ID NO:21 161-hVH5-8 VHH

[0340] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAAIGRGGQRTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLSFAFRDYWGQGTLVTVSS

[0341] SEQ ID NO:22 161-hVH5-10 CDR1

[0342] GSIFSILRMG

[0343] SEQ ID NO:23 161-hVH5-10 CDR2

[0344] AIGRTRGSTN

[0345] SEQ ID NO:24 161-hVH5-10 CDR3

[0346] EISQLTWAFRDY

[0347] SEQ ID NO:25 161-hVH5-10 VHH

[0348] EVQLVESGGGLVQPGGSLRLSCAASGSIFSILRMGWYRQAPGKGLELVAAIGRTRGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLTWAFRDYWGQGTLVTVSS

[0349] SEQ ID NO:26 161-hVH5-19 CDR1

[0350] GSIWSINDMG

[0351] SEQ ID NO:27 161-hVH5-19 CDR2

[0352] AIGRGGGSTN

[0353] SEQ ID NO:28 161-hVH5-19 CDR3

[0354] EISQLTWAFLDY

[0355] SEQ ID NO:29 161-hVH5-19 VHH

[0356] EVQLVESGGGLVQPGGSLRLSCAASGSIWSINDMGWYRQAPGKGLELVAAIGRGGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLTWAFLDYWGQGTLVTVSS

[0357] SEQ ID NO:30 161-hVH5-22 CDR1

[0358] GSIFSINDMG

[0359] SEQ ID NO:31 161-hVH5-22 CDR2

[0360] AIGRRPGSTN

[0361] SEQ ID NO:32 161-hVH5-22 CDR3

[0362] EISQLSFAFRDY

[0363] SEQ ID NO:33 161-hVH5-22 VHH

[0364] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAAIGRRPGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLSFAFRDYWGQGTLVTVSS

[0365] SEQ ID NO:34 161-hVH5-24 CDR1

[0366] GSIFSILSMG

[0367] SEQ ID NO:35 161-hVH5-24 CDR2

[0368] AIGRGGGSLQ

[0369] SEQ ID NO:36 161-hVH5-24 CDR3

[0370] EISQLTWAFRDY

[0371] SEQ ID NO:37 161-hVH5-24 VHH

[0372] EVQLVESGGGLVQPGGSLRLSCAASGSIFSILSMGWYRQAPGKGLELVAAIGRGGGSLQYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLTWAFRDYWGQGTLVTVSS

[0373] SEQ ID NO:38 161-hVH5-36 CDR1

[0374] GSIFSINDMG

[0375] SEQ ID NO:39 161-hVH5-36 CDR2

[0376] AIGRGSVSTN

[0377] SEQ ID NO:40 161-hVH5-36 CDR3

[0378] EISQLTYAFRDY

[0379] SEQ ID NO:41 161-hVH5-36 VHH

[0380] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAAIGRGSVSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLTYAFRDYWGQGTLVTVSS

[0381] SEQ ID NO:42 161-hVH5-37 CDR1

[0382] GSIFSINDMG

[0383] SEQ ID NO:43 161-hVH5-37 CDR2

[0384] AIGRGGFSTN

[0385] SEQ ID NO:44 161-hVH5-37 CDR3

[0386] EISQLSFAFRDY

[0387] SEQ ID NO:45 161-hVH5-37 VHH

[0388] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAAIGRGGFSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLSFAFRDYWGQGTLVTVSS

[0389] SEQ ID NO:46 161-hVH5-48 CDR1

[0390] GSIRFINDMG

[0391] SEQ ID NO:47 161-hVH5-48 CDR2

[0392] AIGRGGGSTN

[0393] SEQ ID NO:48 161-hVH5-48 CDR3

[0394] EISQLGYAFRDY

[0395] SEQ ID NO:49 161-hVH5-48 VHH

[0396] EVQLVESGGGLVQPGGSLRLSCAASGSIRFINDMGWYRQAPGKGLELVAAIGRGGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLGYAFRDYWGQGTLVTVSS

[0397] SEQ ID NO:50 161-hVH5-49 CDR1

[0398] GSIFSINDMG

[0399] SEQ ID NO:51 161-hVH5-49 CDR2

[0400] ALARGGGSTN

[0401] SEQ ID NO:52 161-hVH5-49 CDR3

[0402] EISQLSFAFRDY

[0403] SEQ ID NO:53 161-hVH5-49 VHH

[0404] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINDMGWYRQAPGKGLELVAALARGGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCHAEISQLSFAFRDYWGQGTLVTVSS

[0405] SEQ ID NO: 54 CDR1

[0406] GSI-Xaa1-Xaa2-I-Xaa3-Xaa4-MG

[0407] Among them, Xaal is F, W or R, Xaa2 is S or F, Xaa3 is N or L, and Xaa4 is S, D or R.

[0408] SEQ ID NO: 55 CDR2

[0409] Xaa5-Xaa6-Xaa7-R-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13

[0410] Among them, Xaa5 is A or V, Xaa6 is I, L or H, Xaa7 is G or A, Xaa8 is G, R or T, Xaa9 is G, R, P or S, Xaa10 is G, Q, R, F or V, Xaa11 is S or R, Xaa12 is T or L, and Xaa13 is N or Q.

[0411] SEQ ID NO: 56 CDR3

[0412] EISQL-Xaa14-Xaa15-AF-Xaa16-DY

[0413] Among them, Xaal4 is T, S or G, Xaa15 is W, F or Y, and Xaa16 is R or L.

Claims

1. A TNFR2 binding molecule comprising at least one single domain antibody (sdAb) portion that specifically binds to TNFR2, wherein the sdAb portion comprises three complementary determining regions from the N-terminus to the C-terminus, namely CDR1, CDR2 and CDR3, in: (a) the amino acid sequence of CDR1 is shown in SEQ ID NO:3; the amino acid sequence of CDR2 is shown in SEQ ID NO:4; and the amino acid sequence of CDR3 is shown in SEQ ID NO:5; or (b) the amino acid sequence of CDR1 is shown in SEQ ID NO: 10; the amino acid sequence of CDR2 is shown in SEQ ID NO: 11; and the amino acid sequence of CDR3 is shown in SEQ ID NO: 12; or (c) the amino acid sequence of CDR1 is shown in SEQ ID NO: 14; the amino acid sequence of CDR2 is shown in SEQ ID NO: 15; and the amino acid sequence of CDR3 is shown in SEQ ID NO: 16; or (d) the amino acid sequence of CDR1 is shown in SEQ ID NO: 18; the amino acid sequence of CDR2 is shown in SEQ ID NO: 19; and the amino acid sequence of CDR3 is shown in SEQ ID NO: 20; or (e) the amino acid sequence of CDR1 is shown in SEQ ID NO:22; the amino acid sequence of CDR2 is shown in SEQ ID NO:23; and the amino acid sequence of CDR3 is shown in SEQ ID NO:24; or (f) the amino acid sequence of CDR1 is shown in SEQ ID NO:26; the amino acid sequence of CDR2 is shown in SEQ ID NO:27; and the amino acid sequence of CDR3 is shown in SEQ ID NO:28; or (g) the amino acid sequence of CDR1 is shown in SEQ ID NO:30; the amino acid sequence of CDR2 is shown in SEQ ID NO:31; and the amino acid sequence of CDR3 is shown in SEQ ID NO:32; or (h) the amino acid sequence of CDR1 is shown in SEQ ID NO:34; the amino acid sequence of CDR2 is shown in SEQ ID NO:35; and the amino acid sequence of CDR3 is shown in SEQ ID NO:36; or (i) the amino acid sequence of CDR1 is shown in SEQ ID NO:38; the amino acid sequence of CDR2 is shown in SEQ ID NO:39; and the amino acid sequence of CDR3 is shown in SEQ ID NO:40; or (j) the amino acid sequence of CDR1 is shown in SEQ ID NO:42; the amino acid sequence of CDR2 is shown in SEQ ID NO:43; and the amino acid sequence of CDR3 is shown in SEQ ID NO:44; or (k) the amino acid sequence of CDR1 is shown in SEQ ID NO:46; the amino acid sequence of CDR2 is shown in SEQ ID NO:47; and the amino acid sequence of CDR3 is shown in SEQ ID NO:48; or (l) The amino acid sequence of CDR1 is shown in SEQ ID NO:50; the amino acid sequence of CDR2 is shown in SEQ ID NO:51; and the amino acid sequence of CDR3 is shown in SEQ ID NO:

52.

2. The TNFR2 binding molecule of claim 1, wherein the sdAb portion is an amino acid sequence selected from any one of SEQ ID NOs: 6, 7, 8, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53.

3. The TNFR2 binding molecule of claim 1, wherein the sdAb portion is a camelid VHH, a partially or fully humanized VHH, or a chimeric VHH.

4. The TNFR2 binding molecule of claim 1, wherein the sdAb portion is linked to an Fc region of an immunoglobulin at the N-terminus or the C-terminus; or, is linked to a fluorescent protein.

5. The TNFR2 binding molecule of claim 4, wherein the sdAb portion is linked at the N-terminus or C-terminus to an Fc region from IgG1, IgG2, IgG3 or IgG4.

6. The TNFR2 binding molecule according to any one of claims 1 to 5, which has one or more of the following properties: (1) High affinity binding to human TNFR2, wherein the EC binding between the TNFR2 binding molecule and the cell surface TNFR2 50 0.01 μg / mL to 1 μg / mL; (2) does not block the binding of TNFα to TNFR2; (3) inhibiting the TNFR2 signaling pathway; (4) does not affect the proliferation of Treg cells in PBMCs; (5) Inhibit tumor growth in vivo.

7. The TNFR2 binding molecule of claim 6, having one or more of the following properties: (1) High affinity binding to human TNFR2, wherein the EC binding between the TNFR2 binding molecule and the cell surface TNFR2 50 0.1 μg / mL to 0.6 μg / mL; (2) does not block the binding of TNFα to TNFR2; (3) In Treg cells, myeloid-derived suppressor cells and / or TNFR2 expressing + Inhibits TNFR2-mediated signaling in cancer cells; (4) does not affect the proliferation of Treg cells in PBMCs; (5) Inhibit tumor growth in vivo. The TNFR2 binding molecule according to claim 7 , wherein the Treg cells are Treg cells expressing CD25 high.

9. The TNFR2 binding molecule of any one of claims 1 to 5, which is a bispecific or multispecific antibody.

10. The TNFR2 binding molecule of claim 9, wherein the bispecific antibody molecule specifically binds to a TNFR2 molecule and a second target protein.

11. The TNFR2 binding molecule of claim 10, wherein the second target protein is selected from a tumor antigen, an immunomodulatory receptor, and an immune checkpoint molecule.

12. The TNFR2 binding molecule of claim 11, wherein the tumor antigen is a tumor-associated antigen or a tumor-specific antigen; and the immunomodulatory receptor and immune checkpoint molecule are CTLA-4, TIM-3 or LAG-3.

13. The TNFR2 binding molecule of any one of claims 1 to 5, which binds in a groove of the CRD3 domain of TNFR2.

14. The TNFR2 binding molecule of claim 13, which binds to an epitope of TNFR2 comprising amino acid residues 83, 84, 85, 97, 98, 100, 101, 108, 110, 112, 131, 132, 133.

15. The TNFR2 binding molecule of claim 14, which binds to an epitope of TNFR2 as set forth in SEQ ID NO:9 comprising amino acid residues V83, E84, T85, T97, C98, P100, G101, K108, E110, C112, G131, T132, and E133.

16. An isolated nucleic acid encoding the TNFR2 binding molecule of any one of claims 1 to 15.

17. A vector comprising the nucleic acid of claim 16. The vector according to claim 17 , which is an expression vector. The vector according to claim 18 , which is a pcDNA3.4-TOPO vector.

20. A host cell comprising the nucleic acid of claim 16 or the vector of any one of claims 17 to 19, wherein the host cell is prokaryotic or eukaryotic.

21. The host cell according to claim 20, wherein the host cell is selected from the group consisting of Escherichia coli cells, yeast cells, and mammalian cells.

22. The host cell according to claim 21, wherein the host cell is a HEK293 cell or a CHO cell.

23. A method of making the TNFR2 binding molecule of any one of claims 1 to 15, the method comprising culturing the host cell of any one of claims 20 to 22 under conditions suitable for expression of a nucleic acid encoding the TNFR2 binding molecule of any one of claims 1 to 15.

24. The method of claim 23, comprising isolating the TNFR2 binding molecule.

25. The method of claim 23, further comprising recovering the TNFR2 binding molecule from the host cell.

26. A pharmaceutical composition comprising the TNFR2 binding molecule of any one of claims 1 to 15, and a pharmaceutically acceptable adjuvant.

27. A combination product comprising the TNFR2 binding molecule of any one of claims 1 to 15, and an additional therapeutic agent, wherein the additional therapeutic agent is selected from chemotherapeutic agents, other antibodies.

28. The combination product according to claim 27, wherein the other antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

29. Use of the TNFR2 binding molecule of any one of claims 1 to 15, the pharmaceutical composition of claim 26, or the combination product of claim 27 or 28 for the preparation of a medicament for treating a disease associated with TNFR2 in a subject, wherein the disease associated with TNFR2 is a cancer that expresses or overexpresses TNFR2.

30. The use of claim 29, wherein the cancer expressing or overexpressing TNFR2 is selected from the group consisting of bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal pelvis cancer, spinal axis tumors, gliomas, meningiomas and pituitary adenomas.

31. A kit for detecting TNFR2 in a sample, the kit comprising the TNFR2 binding molecule of any one of claims 1 to 15, for carrying out the following steps: (a) contacting a sample with a TNFR2 binding molecule according to any one of claims 1 to 15; and (b) detecting the formation of a complex between the TNFR2 binding molecule and TNFR2.

32. The kit for detecting TNFR2 in a sample according to claim 31, wherein the TNFR2 binding molecule is detectably labeled.

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