A novel agonist anti-TNFR2 antibody molecule

Abstract

Novel antibody molecules are described that specifically bind to TNFR2 on target cells, thereby agonizing TNFR2, but not blocking the binding of the ligand TNF-α to TNFR2. Also described are uses of such antibody molecules in medicine, particularly in the treatment of cancer or chronic inflammatory diseases.

Description

[Technical field]

[0001] The present invention relates to novel agonistic antibodies that specifically bind to tumor necrosis factor receptor 2 (TNFR2) but do not block the binding of the ligand TNF-α to TNFR2. The present invention also relates to their use in medicine, such as in the treatment of cancer or chronic inflammatory diseases. [Background technology]

[0002] Tumor necrosis factor (TNF) receptor 2 (TNFR2, TNFR-2 or TNFRII), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) and CD120b, is a membrane receptor that binds tumor necrosis factor-α (TNF-α or TNFα). It is found on the surface of T cells, monocytes and macrophages and can activate the proliferation of TNFR2 receptor-expressing cells via nuclear factor kappa B (NF-κB). In particular, TNFR2 is a key regulator of cancer, particularly tumor-infiltrating immune cells, such as regulatory T cells (Tregs), CD8 + It is highly upregulated in T cells (e.g., cytotoxic effector T cells) and various myeloid cell subpopulations.

[0003] TNFR2 has been discussed as a promising target for cancer immunotherapy and has been described to be highly expressed, especially in intratumoral Tregs and on the surface of many human tumor cells (Williams GS et al,Oncotarget. 2016;7(42):68278-68291; Vanamee ES et al,Trends in Molecular Medicine, 2017,vol.23,issue 11,1037-1046; Frontiers in Immunology,November 2017|Volume 8|Article 1482,Sci Signal. 2018 Jan 2;11(511)).

[0004] Regulatory T cells (Treg cells, T or T regTregs, sometimes called T cell suppressors (previously known as suppressor T cells or suppressive regulatory T cells), constitute a subpopulation of T cells that are able to suppress other immune cells under normal and pathological immune circumstances. + Other CD4 cells that are not Tregs + T cells present but not Treg CD4 + The cells were FOXP3 negative (FOXP3 - ), whereas Tregs are also FOXP3 positive (FOXP3 + ), Tregs are non-Treg CD4 + It can be isolated from non-Treg CD4 + The cells were CD25 - CD127 + or CD25 + CD127 + Tregs are either CD25 or CD45+. + CD127 陰性 / 低 In that sense, Tregs are non-Treg CD4 + Can be separated from cells.

[0005] TNFR2 has also been discussed in relation to autoimmune diseases (Faustman DL et al, Front Immunol. 2013;4:478, Clin Transl Immunology. 2016 Jan 8;5(1):J Neurosci. 2016 May 4;36(18):5128-43) and inflammatory diseases (Ait-Ali D et al, Endocrinology. 2008 Jun;149(6):2840-52, Sci Rep. 2016 Sep 7;6:32834).

[0006] Different types of anti-TNFR2 antibodies with various properties have also been previously described. For example, Williams et al. (Oncotarget. 2016 Oct 18; 7(42):68278-68291) describe both ligand-blocking and non-ligand-blocking agonist antibodies.

[0007] WO2014 / 124134 is CD4+CD25 高 The present invention discloses the use of TNFR2 agonists, such as agonistic anti-TNFR2 antibodies and / or NF-κB activators, for the in vitro production of Treg-enriched compositions, which are said to be useful for treating immune disorders or infectious diseases in patients.

[0008] WO2017 / 040312 discloses anti-TNFR2 antibodies, particularly agonistic anti-TNFR2 antibodies, that can promote TNFR2 signaling and affect Treg expansion or proliferation. WO2017 / 040312 discloses antibodies that specifically bind to an epitope containing the sequence KCSPG, but not to an epitope containing the sequence KCRPG (thus excluding the antibodies of US9,821,010) or to another TNFR superfamily member. The agonistic antibodies are said to be useful in the treatment of immune diseases. WO2017 / 040312 further presents the complete sequence of human TNFR2.

[0009] WO2017 / 083525 discusses pharmacological compositions comprising anti-TNFR2 antibodies and their use in the treatment of disorders associated with TNF-α and / or TNFR2, such as cancer. WO2017 / 083525 further discusses antibodies comprising a human IgG1 Fc domain with null binding to Fcγ receptors, and the inhibition of Treg proliferation.

[0010] In addition, anti-TNFR2 antibodies capable of acting as TNFR2 agonists have been described in Galloway et al. (Eur. J. Immunol. 22:3045-3048, 1992), Tartaglia et al. (J. Biol. Chem. 268:18542-18548, 1993), Tartaglia et al. (J. Immunol. 151:4637-4641, 1993), Smith et al. (J. Biol. Chem. 269:9898-9905, 1994) and Amrani et al. (Am. J. Respir. Cell. Mol. Biol. 15:55-63, 1996).

[0011] However, none of these documents teach or suggest agonistic TNFR2 antibodies that specifically bind to TNFR2 but do not block the binding of the ligand TNF-α to the same TNFR2.

[0012] Fc receptors are membrane proteins found on the cell surface of immune effector cells such as monocytes, macrophages, dendritic cells, neutrophils, mast cells, basophils, eosinophils, and natural killer cells, and B lymphocytes. The name is derived from their binding specificity to the Fc region of antibodies. Fc receptors are found on cell membranes, also known as plasma membranes or cytoplasmic membranes. Fc receptors can be subdivided into activating FcγRs and inhibitory FcγRs, which are known to coordinately control cell activation by binding aggregated immunoglobulin G Fc and transmit activating or inhibitory signals to cells via intracellular ITAM or ITIM motifs. FcR binding of aggregated immunoglobulins or immune complexes may mediate internalization of the antibody into the cell, leading to antibody-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, or antigen presentation or cross-presentation. FcRs are also known to mediate or enhance cross-linking of cell surface receptors bound to antibodies. Such cross-linking is known to be necessary for the ability of some (Li et al 2011.'Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies', Science,333:1030-4.;White et al.2011.'Interaction with FcgammaRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody', J Immunol,187:1754-63), but not all (Richman et al 2014.'Anti-human CD40 monoclonal antibody therapy is potent without FcR crosslinking', Oncoimmunology,3:e28610) antibodies to activate signaling in target cells and may or may not be necessary to achieve a therapeutic effect.

[0013] A subgroup of Fc receptors are the Fcγ receptors (Fc-gamma receptors, Fc gamma R, FcγR), which are specific for IgG antibodies. There are two types of Fcγ receptors: activating Fcγ receptors (also referred to as activating Fcγ receptors) and inhibitory Fcγ receptors. Activating and inhibitory receptors transmit their signals via immunoreceptor tyrosine-based activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs), respectively. In humans, FcγRIIb (CD32b) is an inhibitory Fcγ receptor, while FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), and FcγRIIIa (CD16a) are activating Fcγ receptors. FcγgRIIIb is a GPI-linked receptor expressed on neutrophils that lacks ITAM motifs but is also considered activating due to its ability to cross-link lipid rafts and bind other receptors. In mice, the activating receptors are FcγRI, FcγRIII and FcγRIV.

[0014] It is known that antibodies can regulate immune cell activity through interactions with Fcγ receptors. Specifically, how antibody immune complexes regulate immune cell activation is determined by the relative binding of activating and inhibitory Fcγ receptors. Different antibody isotypes bind to activating and inhibitory Fcγ receptors with different affinities, resulting in different A:I ratios (Nimmerjahn et al; Science. 2005 Dec 2; 310(5753):1510-2).

[0015] By binding to inhibitory FcγRs, antibodies can inhibit, block, and / or downregulate effector cell functions. By binding to inhibitory FcγRs, antibodies can further stimulate cell activation through aggregation of antibody-targeted signaling receptors on target cells (Li et al.2011.'Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies',Science,333:1030-4;White et al 2011.'Interaction with FcgammaRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody',J Immunol,187:1754-63;White et al 2014.'Fcgamma receptor dependency of agonistic CD40 antibody in lymphoma therapy can be overcome through antibody multimerization',J Immunol,193:1828-35).

[0016] By binding to activating Fcγ receptors, antibodies can activate effector cell function, thereby inducing mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine release, and / or antibody-dependent endocytosis, and in the case of neutrophils, NETosis (i.e., activation and release of NETs (neutrophil extracellular traps)). Antibodies that bind to activating Fcγ receptors can also cause an increase in certain activation markers, such as CD40, MHCII, CD38, CD80, and / or CD86.

[0017] In particular, recent data published by the inventors show a critical and specific dependency of CD8 T cell agonist antibodies and Treg-depleting anti-4-1BB antibodies to bind to activating and inhibitory FcγRs, respectively, for therapeutic efficacy (Buchan et al., 'Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function', Immunity 2018 49(5):958-970). Furthermore, and crucially, co-administration of CD8 T cell agonist antibodies and Treg-depleting anti-4-1BB antibodies optimized for binding to activating and inhibitory FcγRs, respectively, reduced therapeutic activity. These data indicate that it is critically important to develop antibodies with appropriate and coordinated binding of activating and inhibitory FcγRs to maximize the therapeutic activity of antibodies with distinct mechanisms of action. Together, these data indicate that suboptimal binding of activating and inhibitory FcγRs can significantly reduce therapeutic efficacy.

[0018] These data were surprising because they contrasted with findings for antibodies against other TNFSR members, particularly immunostimulatory anti-CD40 antibodies, and demonstrated an absolute requirement for binding of inhibitory, but not activating, FcγRs (Li et al. 2011. 'Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies', Science, 333:1030-4; White et al. 2011. 'Interaction with FcgammaRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody', J Immunol, 187:1754-63). Collectively, these results indicate that FcγR dependency may vary in ways that are not easily predictable between antibodies against different targets of the same receptor superfamily, and even between different types of antibodies against the same target, but may be important to understand and exploit when developing antibodies for therapeutic use. Summary of the Invention

[0019] In the research leading to this and parallel inventions, two major distinct groups of anti-TNFR2 antibodies were identified that have potent therapeutic efficacy as well as different characteristics and mechanisms of action.

[0020] The present inventors first identified the potent therapeutic activity of antagonistic anti-TNFR2 antibodies that block the binding of TNF-α to the TNFR2 receptor. The activity of such antibodies was shown to be dependent on FcγR interactions, particularly binding to activating FcγR, for in vivo therapeutic activity. This group or category of potent anti-TNFR2 therapeutic reagents is characterized by 1) significant blocking and suppression of TNF-α-induced TNFR2 signaling, and 2) activity dependent on FcγR binding, and was found to benefit most strongly from binding to activating FcγR rather than inhibitory FcγR.

[0021] The inventors then identified a separate group of anti-TNFR2 antibodies that have equally potent therapeutic activity in vivo, but whose characteristics are in many ways opposite to those of the antagonist-blocking TNFR2 antibodies that constitute the first group. This second group of anti-TNFR antibodies does not depend on TNF-α blockade or inhibition of TNFR2 signaling for its therapeutic activity, but rather is characterized by a strong activation of TNFR2 signaling. In further contrast to the first group of blocking antibodies, the second group of agonistic antibodies does not show an absolute dependency on antibody:FcγR binding, even if their activity is improved with FcγR:binding antibody variants. In further contrast to the first group of antagonist-blocking antibodies, the second group of agonistic antibodies shows maximum activity in antibody variants with improved binding to inhibitory versus activating FcγRs.

[0022] The present invention relates to a second group of anti-TNFR2 antibodies, namely agonistic antibody molecules that specifically bind to TNFR2 but do not block the binding of the ligand TNF-α to TNFR2. Such antibodies are potential therapeutic reagents and are useful in medicine.

[0023] Antagonistic blocking antibodies belonging to the first group are used in the following examples for comparison with the agonistic non-blocking TNFR2 antibody molecules of the present invention. In the examples, other antibodies having some characteristics similar to those of either or both of the first or second groups are also used for comparison, as further described below.

[0024] Thus, the present invention relates to agonistic antibody molecules that specifically bind to TNFR2 on target cells and do not block TNF-α ligand binding to TNFR2.

[0025] The present invention also relates to specific examples of such novel agonistic TNFR2 antibody molecules.

[0026] The present invention also relates to an isolated nucleotide sequence encoding at least one of the above-mentioned antibody molecules.

[0027] The present invention also relates to a plasmid comprising at least one of the above-mentioned nucleotide sequences.

[0028] The present invention also relates to a virus comprising at least one of the above-mentioned nucleotide sequences or plasmids.

[0029] The present invention also relates to a cell comprising at least one of the above-mentioned nucleotide sequences, or at least one of the above-mentioned plasmids, or at least one of the above-mentioned viruses.

[0030] The present invention also relates to the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for use in medicine.

[0031] The present invention also relates to said antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for use in the treatment of cancer or chronic inflammatory diseases.

[0032] The present invention also relates to the use of said antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for use in the treatment of cancer or chronic inflammatory diseases.

[0033] The present invention also relates to a pharmaceutical composition comprising at least one of the above antibody molecules, nucleotide sequences, plasmids, viruses and / or cells, and optionally a pharma- ceutically acceptable diluent, carrier, vehicle and / or excipient. Such a pharmaceutical composition can be used for the treatment of cancer or chronic inflammatory diseases.

[0034] The present invention also relates to a method for treating cancer or a chronic inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of at least one of the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions.

[0035] The invention also relates to antibody molecules, antibody molecules for use, isolated nucleotide sequences, isolated nucleotide sequences for use, plasmids, plasmids for use, viruses, viruses for use, cells, cells for use, uses, pharmaceutical compositions and methods of treatment as herein described with reference to the accompanying description, examples and / or figures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Thus, the present invention relates to agonistic TNFR2 antibody molecules that specifically bind to TNFR2 but do not block the binding of the ligand TNF-α to the same TNFR2. Preferably, the antibody molecule has intrinsic agonistic activity.

[0037] The agonistic antibody molecules disclosed herein do not block TNF-α binding to TNFR2, nor do they block TNFR2 signaling. It has been clearly shown that TNF-α-mediated signaling through TNFR2 initiates a signaling cascade that ends with the activation of the nuclear transcription factor NFkB (Thommesen et al. “Distinct differences between TNF receptor 1-and TNF receptor 2-mediated activation of NFkappaB”. J Biochem Mol Biol. 2005 May 31; 38(3): 281-9; Yang et al. “Role of TNF-TNF Receptor 2 Signal in Regulatory T Cells and Its Therapeutic Implications”. Front Immunol. 2018 Apr 19; 9: 784). This leads to cell activation and the synthesis of several proinflammatory factors. One of them is IFN-gamma in NK cells (Liu et al. "NF-kappaB signaling in inflammation". Signal Transduct Target Ther. 2017;2.pii:17023; Tato et al. "Opposing roles of NF-kappaB family members in the regulation of NK cell proliferation and production of IFN-gamma". Int Immunol. 2006 Apr;18(4):505-13). In this specification, the terms TNFR2 signaling and TNFR2 activation are used interchangeably. An antibody molecule specifically binds to TNFR2. It is well known that an antibody specifically binds to or interacts with a defined target molecule or antigen, and this means that the antibody preferentially and selectively binds to its target rather than non-target molecules. By "antibody molecule specifically binding to TNFR2" or "TNFR2-specific antibody molecule" is meant an antibody that binds to TNFR2 protein in a dose-dependent manner but does not bind to unrelated proteins.In addition, the same antibody binds to cells that endogenously express TNFR2, and this binding can be blocked by preincubating the same cells with a commercially available polyclonal TNFR2 antibody reagent, demonstrating that nonspecific binding can be detected when TNFR2 is masked with a polyclonal reagent, as shown in Example 2.

[0038] An antibody molecule that specifically binds to TNFR2 (or an anti-TNFR2 antibody molecule) refers to an antibody molecule that specifically binds to at least one epitope within the extracellular domain of TNFR2. Cell surface antigens and epitopes are terms that are readily understood by those skilled in the art of immunology or cell biology.

[0039] Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology. Those skilled in the art will understand that these methods can be used to assess the binding of an antibody to a target and / or the binding of an Fc region of an antibody to an Fc receptor, and the relative strength, or specificity, or inhibition, or prevention, or reduction of their interactions. Examples of methods that can be used to assess protein binding are, for example, immunoassays, BIAcore, Western blots, radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) and flow cytometry (FACS). For a discussion of antibody specificity, see Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 (1989).

[0040] The target cells expressing TNFR2 to which the agonist antibodies bind according to the present invention can be any TNFR2-expressing immune cells, such as CD8 positive cells and myeloid cells.

[0041] The effect of binding of an agonistic antibody molecule according to the invention to TNFR2 may be activation of T cells and / or myeloid cells, and / or infiltration of T cells and / or myeloid cells into the diseased tissue, and / or a change in the composition of T cells and / or myeloid cells in the diseased tissue. A change in the composition of T cells and / or myeloid cells means herein a different absolute or relative number of various cell subpopulations such as Tregs, CD8 positive cells, tumor associated macrophages (TAMs) (including their different subpopulations), myeloid derived suppressor cells (MDSCs) and / or proinflammatory macrophages.

[0042] In this context, diseased tissue means either tumor tissue (i.e., all cells within the tumor microenvironment, including tumor cells, immune cells, endothelial cells and stromal cells) or tissue affected by a chronic inflammatory disease.

[0043] To determine whether an antibody molecule blocks or, rather, does not block in the context of the present invention, an ELISA assay can be used to determine the amount of TNF-α ligand bound to immobilized TNFR2 receptor in the presence of a TNFR2-specific antibody. A non-blocking antibody does not prevent the ligand TNF-α from binding to the immobilized receptor TNFR2. This is shown and explained in more detail in Example 3 below. More specifically, a non-blocking TNFR2 antibody molecule according to the present invention is an antibody molecule that reduces TNF-α binding to TNFR2 by less than 50% compared to TNF-α binding in the presence of an isotype control antibody molecule alone. In some embodiments, this is determined in a high-dose one-point ELISA or a dose-titration ELISA, as shown in Example 3 and Figures 6 and 7.

[0044] Conversely, blocking antagonist antibody molecules are full blockers and can further antagonize TNFR2 signaling. Such antibody molecules are used for comparison in the following examples. Full blockers are defined herein as antibody molecules that reduce TNF-α binding to TNFR2 by more than 98%, i.e. up to 100%, compared to TNF-α binding in the presence of only isotype control antibody molecules. Isotype control antibodies are antibodies raised against proteins or other structures that are not present in any form in the assay under study. Isotype controls ideally have the same framework as the antibody being compared, but at least the same Fc portion. This is known to those skilled in the art. In the examples described herein, the isotype control has the same framework, the same Fc portion, and is specific for fluorescein isothiocyanate (FITC). In some embodiments, full blockers reduce TNF-α binding by more than 99.5%. Other types of blockers are partial blockers and weak blockers. As used herein, a partial blocker is an antibody molecule that reduces TNF-α binding to TNFR2 by 60-98% compared to TNF-α binding in the presence of only an isotype control antibody molecule, and a weak blocker is an antibody molecule that reduces TNF-α binding to TNFR2 by less than 60%, e.g., 50-59.9%, compared to TNF-α binding in the presence of only an isotype control antibody molecule.

[0045] For comparison with the agonistic non-blocking antibody molecules of the present invention, the examples use fully blocking antagonistic antibody molecules, partially blocking antibody molecules and weakly blocking antibody molecules.

[0046] Several properties and characteristics may underlie and (co)determine the biological activity of an antibody. In addition to the ability to block or not block ligand binding to the receptor, such important properties include the ability of the antibody molecule to modulate, i.e., agonize or antagonize, receptor signaling, and the antibody's dependence on FcγR interaction to confer therapeutic activity.

[0047] First, the ability of fully blocking, partially blocking and non-blocking antibodies to modulate TNFR2 signaling was characterized. Two extremes were identified.

[0048] At the first extreme, we have identified antibodies that completely block ligand binding to TNFR2, block TNF-α-induced TNFR2 signaling, and do not themselves induce signaling upon binding to endogenously expressed TNFR2. This group of ligand-blocking antagonist antibodies constitutes a separate invention and is included herein for comparison purposes.

[0049] At the other extreme, antibodies have been identified that do not block ligand binding to TNFR2, but that upon binding to TNFR2 agonize the receptor in cells in which they are endogenously expressed. This second group of antibodies forms the basis of the present invention.

[0050] As used herein, a non-blocking antibody is an antibody molecule that reduces TNF-α binding to TNFR2 by 0-50% (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%, including all integers and all decimal points therebetween) compared to TNF-α binding in the presence of only an isotype control antibody molecule.

[0051] Further antibodies and categories defined by partial blocking agonists, partial blocking non-agonists, and full blocking non-antagonists were identified, indicating the complex biology and great heterogeneity of anti-TNFR2 antibodies and clearly indicating that the antibodies of the present invention form a unique group.

[0052] To determine whether an antibody has agonist or antagonist activity, a natural killer (NK) cell assay can be used as described in Example 4. Briefly, NK cells have been reported to secrete IFN-γ in response to IL-2 and IL-12 stimulation. Soluble TNF-α is endogenously produced and is present at robust but suboptimal concentrations (approximately 100 pg / ml) for TNFR2 signaling. This means that IFN-γ can be increased or decreased by modulation of TNFR2 signaling. As a result, exogenous addition of TNF-α at optimal concentrations for TNFR2 signaling increases IFN-γ concentrations in this assay, as does incubation with an agonist anti-TNFR2 antibody. Conversely, co-incubation with an anti-TNF-α antibody or a ligand-blocking antagonist antibody described herein for comparison reduces IFN-γ release in this assay. Thus, this assay can be used to identify the agonist or antagonist activity, or lack thereof, of anti-TNFR2 antibodies. (TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2.Almishri W.et al.J Innate Immun.2016;8:617-629)

[0053] As a result, the ability of an antibody to agonize, i.e., induce, TNFR2 signaling can be monitored using this experimental setup. The ability of an antibody to induce signaling upon binding to TNFR2 in the same natural killer (NK) cell assay can be assessed by monitoring the increase in IFN-γ release and comparing it to that observed after culture in the presence or absence of exogenous TNF-α added at an optimal concentration for signaling, as described in Example 4. Thus, an agonistic TNFR2 antibody can be defined as an antibody that enhances IFN-γ release by NK cells in this assay. Antibodies with intrinsic agonistic potential enhance IFN-γ release by NK cells in a manner that is not dependent on antibody cross-linking or interaction with Fc gamma receptors, nor on the presence of soluble TNF-α ligand. Thus, intrinsic agonistic activity can be assessed using antibody formats that do not productively bind FcγR, for example, aglycosylated antibodies with a N297A mutation in the Fc domain, or in assay systems / cells that lack FcγR. NK cells are known to those skilled in the art (Binyamin, L., et al (2008). Journal of immunology 180, 6392-6401; Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphoma therapy.). Using this assay, agonistic antibodies are defined as those that result in a >100% (>2-fold) increase in IFN-γ release. Because this assay uses primary cells from PBMC donors, at least four donors should be included and the average value should be calculated from all donors. Cells from each donor included in the calculation of the average should have responded to the positive control (soluble TNF-α) treatment with >100% (>2-fold) increased IFN-γ levels compared to treatment with the isotype control.

[0054] The agonistic antibody molecules described herein have inherent agonistic activity, as explained above.

[0055] In some embodiments of the invention, the antibody preferably enhances IFN-γ release by NK cells by at least 100% in the above assay.

[0056] In some embodiments, agonist activity can be improved by the antibody molecule binding to Fcγ receptor in addition to binding to TNFR2.In some such embodiments, the agonist non-blocking TNFR2 antibody molecule binds to inhibitory Fcγ receptor with higher affinity than activating Fcγ receptor.Having higher affinity to inhibitory Fcγ receptor than activating Fcγ receptor means that the variant binds to inhibitory Fcγ receptor with higher affinity than each individual activating Fcγ receptor, for example, compared to any of FcγRIIA, FcγRIIIA and FcγRI.

[0057] The relatively high homology between the mouse and human FcγR systems explains many of the common aspects of the conserved FcγR-mediated mechanisms between species. However, because mouse and human IgG subclasses differ in their affinities for their cognate FcγRs, when translating FcγR-mediated observations in the mouse system to human IgG-based therapeutics, it is important to select antibodies, antibody subclasses and / or engineered subclass variants that demonstrate appropriate binding to human activating versus inhibitory FcγRs. The affinity and / or avidity of human antibody molecules for individual human FcγRs can be determined using surface plasmon resonance (SPR).

[0058] In some embodiments, binding to Fc receptor occurs through normal interaction between the Fc region of agonist antibody molecule and Fc receptor. In some such embodiments, the antibody molecule is an IgG with an Fc region that binds to Fcγ receptor. In some such embodiments, the anti-TNFRII antibody is of human IgG2 isotype, which has similar intermediate affinity to human inhibitory FcγRIIB and human activating FcγRIIA and FcγRIIIA, but does not bind productively to human activating FcγRI. In some embodiments, the anti-TNFRII antibody is of human IgG1 isotype, which binds to FcγRIIB with higher affinity compared to IgG2, but also binds to activating human activating FcγRIIA, FcγRIIIA with higher affinity, and further binds to activating FcγRI with higher affinity. In other embodiments, the anti-TNFRII antibody has been engineered to enhance binding to FcγRIIB (e.g., a "SELF" mutation) (Chu et al. "Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies." Mol Immunol. 2008 Sep;45(15):3926-33) and / or has been engineered to relatively enhance binding to FcγRIIB compared to activating FcγRs (e.g., a V9 or V11 mutation) (Mimoto et al. "Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa R131 and FcγRIIa H131".Protein Eng Des Sel.2013 Oct;26(10):589-598.}, a human IgG. Such IgG variants engineered to enhance binding to inhibitory FcγRIIB or to specifically enhance binding affinity to inhibitory FcγRIIB but not activating FcγRIIA have been shown to increase the in vivo agonistic and therapeutic activity of the CD40 agonist antibody CP-870,893 in animals with humanized activating and inhibitory FcγRs (Dahan et al.2016.'Therapeutic Activity of Agonistic,Human Anti-CD40 Monoclonal Antibodies Requires Selective FcgammaR Engagement',Cancer Cell,29:820-31).

[0059] In addition to TNFR2, Fc receptors to which agonistic antibody molecules may bind are receptors found on the surface of bone marrow-derived cells such as macrophages, monocytes, MDCS, neutrophils, mast cells, basophils or dendritic cells, or on the surface of lymphocytes such as NK cells, B cells or certain T cells.

[0060] As mentioned above, antibody molecules often bind to Fc receptors via their Fc region. The agonistic antibody molecules disclosed herein have intrinsic agonistic activity and therefore do not need to bind to Fc receptors to agonize TNFR2. This means that in some embodiments of the invention, it is possible to use antibody molecules that do not rely on Fc receptor binding via their Fc region, and indeed it is possible to use antibody molecules that do not have an Fc region. In some such embodiments, the antibody molecule may be a Fab'2 or a PEGylated version thereof. In some embodiments, the antibody molecule may be a bivalent or multivalent antibody molecule, including single chain antibodies, Fab, Fv, scFv, Fab' and / or (Fab')2. In other embodiments, the antibody molecule may comprise a modified Fc region, such as an aglycosylated variant of an IgG1 antibody molecule. Such aglycosylation may be achieved, for example, by amino acid substitution of an asparagine at position 297 (N297X) of the antibody chain. The substitution may be made with glutamine (N297Q), or alanine (N297A), or glycine (N297G), or asparagine (N297D), or by serine (N297S). Other substitutions are described, for example, by Jacobsen FW et al., JBC 2017, 292, 1865-1875 (see, for example, Table 1). Such additional substitutions include L242C, V259C, A287C, R292C, V302C, L306C, V323C, I332C, and / or K334C.

[0061] In some embodiments, the agonist TNFR2 antibody molecule is an IgG1, IgG3 or IgG4 antibody molecule.

[0062] In some embodiments, the agonistic TNFR2 antibody molecule is an IgG antibody molecule that exhibits improved binding to one or several activating Fc receptors and / or has been engineered to improve binding to one or several activating Fcγ receptors and / or has been engineered to improve relative binding to activating Fcγ receptors versus inhibitory Fcγ receptors. In some embodiments, the anti-TNFR2 antibody molecule is an Fc-engineered human IgG1 antibody. Examples of such engineered antibody variants include non-fucosylated antibodies with selectively improved antibody binding to FcγRIIIA, and antibodies engineered by directed, mutational, or other means amino acid substitutions that result in improved binding to one or several activating Fcγ receptors compared to the inhibitory FcγRIIB (Richards et al. 2008. 'Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells', Mol Cancer Ther, 7:2517-27; Lazar et al. 2006. 'Engineered antibody Fc variants with enhanced effector function', Proc Natl Acad Sci USA, 103:4005-10).

[0063] In some embodiments, the human IgG antibody engineered to improve binding to activating Fc gamma receptors may be a human IgG antibody with two mutations S239D and I332E, or three mutations S239D, I332E and A330L, and / or a G236A mutation in its Fc portion. In some embodiments, the human IgG antibody engineered to improve binding to activating Fc gamma receptors may be a non-fucosylated human IgG antibody.

[0064] As explained above, antibody molecules being inherent agonists means that they are agonists both in the absence and presence of TNF-α. In some embodiments, the antibodies are agonists in the absence of TNF-α. In some embodiments, the antibodies are agonists in the presence of TNF-α.

[0065] The target cells expressing TNFR2 to which the agonist antibody binds according to the present invention can be selected from the group consisting of immune cells or cancer cells expressing TNFR2.

[0066] Antibodies are known to those skilled in the art of immunology and molecular biology. Typically, an antibody comprises two heavy chains (H) and two light chains (L). The complete antibody molecule is sometimes referred to herein as a full-size antibody or full-length antibody. The heavy chain of an antibody comprises one variable domain (VH) and three constant domains (CH1, CH2, CH3), and the light chain of an antibody molecule comprises one variable domain (VL) and one constant domain (CL). The variable domains (sometimes referred to as F) are V The variable domains (collectively called the complementarity determining regions) bind to the antibody's target, or antigen. Each variable domain contains three loops, called complementarity determining regions (CDRs), which are involved in target binding. The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant regions of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and in humans, some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4; IgA1 and IgA2.

[0067] Another part of an antibody is the Fc region (also known as the fragment crystallizable domain), which contains two constant domains in each of the antibody's heavy chains. As mentioned above, the Fc region is involved in the interaction between the antibody and the Fc receptor.

[0068] As used herein, the term antibody molecule encompasses full-length or full-size antibodies, as well as functional fragments of full-length antibodies and derivatives of such antibody molecules.

[0069] A functional fragment of a full-sized antibody has the same antigen-binding characteristics as the corresponding full-sized antibody, and contains either the same variable domain (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-sized antibody. A functional fragment does not always contain all six CDRs of the corresponding full-sized antibody. It is understood that a molecule containing three or fewer CDR regions (sometimes only a single CDR or a portion thereof) can retain the antigen-binding activity of the antibody derived from that CDR(s). For example, it has been described in Gao et al., 1994, J.Biol.Chem.,269:32389-93 that the entire VL chain (including all three CDRs) has high affinity for its substrate.

[0070] Molecules containing two CDR regions are described, for example, in Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4:417-430. On page 418 (right column-3 (Our Strategy for Design)), a minibody is described that contains only H1 and H2 CDR hypervariable regions interspersed within framework regions. The minibody is described as being capable of binding to a target. Pessi et al., 1993, Nature, 362:367-9, and Bianchi et al., 1994, J. Mol. Biol., 236:649-59, referenced by Vaughan & Sollazzo, describe H1 and H2 minibodies and their properties in more detail. Qiu et al., 2007, Nature Biotechnology, 25:921-9, show that a molecule consisting of two combined CDRs is capable of binding to an antigen. Quiocho 1993, Nature, 362:293-4 provides an overview of "minibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7 observes that molecules containing two CDRs can retain antigen-binding activity.

[0071] Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272:30937-44, where various hexapeptides derived from CDRs are shown to exhibit antigen-binding activity, and where it is noted that synthetic peptides of complete single CDRs exhibit strong binding activity. Monnet et al., 1999, JBC, 274:3789-96, where various 12-mer peptides and associated framework regions are shown to have antigen-binding activity, and where it is observed that CDR3-like peptides alone are capable of binding to antigens. Heap et al., 2005, J. Gen. Virol., 86:1791-1800, where it is reported that "micro-antibodies" (molecules containing a single CDR) are capable of binding to antigens, and where cyclic peptides from anti-HIV antibodies have been shown to have antigen-binding activity and function. Nicaise et al., 2004, Protein Science, 13:1882-91, show that a single CDR can confer antigen-binding activity and affinity for its lysozyme antigen.

[0072] Thus, antibody molecules having five, four, three or fewer CDRs can retain the antigen-binding properties of the full-length antibody from which they are derived.

[0073] An antibody molecule may be a derivative of a full-length antibody or a fragment of such an antibody. A derivative has the same antigen-binding characteristics as the corresponding full-size antibody, meaning that it binds to the same epitope on the target as the full-size antibody.

[0074] Thus, as used herein, the term "antibody molecule" includes all types of antibody molecules, including monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, human-derived antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFvs), Fab fragments, F(ab')2 fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), antibody heavy chains, antibody light chains, antibody heavy chain homodimers, antibody light chain homodimers, antibody heavy chain heterodimers, antibody light chain heterodimers, antigen-binding functional fragments of such homodimers and heterodimers, as well as functional fragments thereof and derivatives thereof.

[0075] Furthermore, as used herein, the term "antibody molecule", unless otherwise specified, includes all classes of antibody molecules and functional fragments, including IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE.

[0076] In some embodiments, the antibody molecule is a human antibody molecule, a humanized antibody molecule, or an antibody molecule of human origin. In some such embodiments, the antibody molecule is an IgG antibody. It is known that optimal costimulation of agonist receptors of the TNFR superfamily, such as TNFR2, depends on antibody binding to inhibitory FcγRII. In mice, the IgG1 isotype, which preferentially binds to inhibitory Fc gamma receptors (FcγRIIB) and only weakly binds to activating Fc gamma receptors, is known to be optimal for the costimulatory activity of monoclonal antibodies targeting the TNFR superfamily. Although a direct equivalent of the mouse IgG1 isotype in humans has not been described, antibodies can be engineered to show similarly improved binding to inhibitory human Fc gamma receptors over activating human Fc gamma receptors. Such engineered TNFR superfamily-targeting antibodies also have improved in vivo costimulatory activity in transgenic mice engineered to express human activating and inhibitory Fc gamma receptors (Dahan et al, 2016, Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement. Cancer Cell. 29(6):820-31). Thus, in some embodiments, the antibody molecule is of an isotype that binds to inhibitory Fc receptors in an optimal manner. In some embodiments, the antibody molecule is an IgG2 antibody.

[0077] In some embodiments, the agonistic antibody molecule that specifically binds to TNFR2 may be a llama antibody, in particular a llama hcIgG. Like all mammals, camelids produce conventional antibodies with two heavy chains and two light chains linked in a Y-shape by disulfide bonds (IgG1). However, they also produce two unique subclasses of immunoglobulin G, IgG2 and IgG3, also known as heavy chain IgG (hcIgG). These antibodies are composed of only two heavy chains that lack the CH1 region, but contain a V at their N-terminus.H They retain an antigen-binding domain called H. Conventional Igs require the combination of variable regions from both heavy and light chains to allow a high degree of diversity in antigen-antibody interactions. Isolated heavy and light chains still exhibit this ability, but compared to heavy and light chain pairs, they exhibit extremely low affinity. A unique feature of hcIgGs is the ability of their monomeric antigen-binding regions to bind antigens with specificity, affinity, and especially diversity comparable to conventional antibodies, without the need for pairing with another region.

[0078] As outlined above, different types and forms of antibody molecules are encompassed by the present invention and would be known to one skilled in the art of immunology. It is known that antibodies used for therapeutic purposes are often modified with additional moieties that modify the properties of the antibody molecule.

[0079] Thus, the antibody molecules described herein or used as described herein (e.g., monoclonal and / or polyclonal and / or bispecific antibody molecules) include those that comprise a detectable moiety and / or a cytotoxic moiety.

[0080] A "detectable moiety" includes one or more from the group consisting of an enzyme, a radioactive atom, a fluorescent moiety, a chemiluminescent moiety, a bioluminescent moiety. The detectable moiety allows for visualization of the antibody molecule in vitro, and / or in vivo, and / or ex vivo.

[0081] "Cytotoxic moieties" include radioactive moieties and / or enzymes, e.g., enzymes such as caspases and / or toxins, e.g., toxins such as bacterial toxins or venoms, and cytotoxic moieties are capable of inducing cell lysis.

[0082] Further, it is included that the antibody molecule may be in isolated and / or purified form and / or may be PEGylated, which is the method of adding polyethylene glycol polymers to a molecule such as an antibody molecule or derivative to modify its behavior, e.g., to increase its hydrodynamic size and prevent renal clearance, thereby increasing its half-life.

[0083] As described above, the CDRs of an antibody bind to the antibody target. The amino acid assignments for each CDR described herein are as defined by Kabat EA et al. 1991, "Sequences of Proteins of Immunological Interest" Fifth Edition, NIH Publication No. 91-3242, pp xv-xvii.

[0084] As those skilled in the art will recognize, there are other methods for assigning amino acids to each CDR, such as the International ImMunoGeneTics information system (IMGT™) (http: / / www.imgt.org / and Lefranc and Lefranc "The Immunoglobulin Facts Book" published by Academic Press, 2001).

[0085] In some embodiments, the antibody molecule that specifically binds to TNFR2 is a human antibody.

[0086] In some embodiments, an antibody molecule that specifically binds to TNFR2 is a human antibody, ie, an antibody of human origin that has been modified as described herein.

[0087] In some embodiments, an antibody molecule that specifically binds to TNFR2 is a humanized antibody, i.e., an antibody of non-human origin that has been modified to increase similarity to a human antibody. A humanized antibody can be, for example, a mouse antibody or a llama antibody.

[0088] In some embodiments, the antibody molecule that specifically binds to TNFR2 is a human IgG2 antibody molecule.

[0089] In some embodiments, the anti-TNFR2 antibody is also an antibody in the form of a human IgG2 antibody that exhibits improved binding to one or several inhibitory Fc receptors and / or has been engineered for improved binding to one or several inhibitory Fc receptors, and thus in some embodiments the anti-TNFR2 antibody is an Fc-engineered human IgG2 antibody.

[0090] In some embodiments, the anti-TNFR2 antibody is a murine or humanized murine IgG3 antibody.

[0091] In some embodiments, the anti-TNFR2 antibody is a monoclonal antibody.

[0092] In some embodiments, the anti-TNFR2 antibody is a polyclonal antibody.

[0093] In some embodiments, the antibody molecule that specifically binds to TNFR2 is a mouse IgG2a corresponding or a human IgG1 antibody molecule.

[0094] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VH-CDR1 sequences listed in Table 1 below.

[0095] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VH-CDR2 sequences listed in Table 1 below.

[0096] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VH-CDR3 sequences listed in Table 1 below.

[0097] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VL-CDR1 sequences listed in Table 1 below.

[0098] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VL-CDR2 sequences listed in Table 1 below.

[0099] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VL-CDR3 sequences listed in Table 1 below.

[0100] In some embodiments, the anti-TNFR2 antibody molecule comprises: SEQ ID NOs: 1, 2, 3, 4, 5 and 6; SEQ ID NOs: 9, 10, 11, 12, 13 and 14, SEQ ID NOs: 17, 18, 19, 20, 21 and 22, SEQ ID NOs: 25, 26, 27, 28, 29 and 30, SEQ ID NOs: 33, 34, 35, 36, 37 and 38, SEQ ID NOs: 41, 42, 43, 44, 45 and 46, SEQ ID NOs: 49, 50, 51, 52, 53 and 54, SEQ ID NOs: 57, 58, 59, 60, 61 and 62, SEQ ID NOs: 65, 66, 67, 68, 69 and 70, SEQ ID NOs: 73, 74, 75, 76, 77 and 78, SEQ ID NOs: 81, 82, 83, 84, 85 and 86, SEQ ID NOs: 89, 90, 91, 92, 93 and 94, and The antibody molecule is selected from the group consisting of antibody molecules comprising six CDRs selected from the group consisting of SEQ ID NOs: 97, 98, 99, 100, 101 and 102.

[0101] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising six CDRs having SEQ ID NOs: 1, 2, 3, 4, 5 and 6, or an antibody molecule comprising six CDRs having SEQ ID NOs: 9, 10, 11, 12, 13 and 14, or an antibody molecule comprising six CDRs having SEQ ID NOs: 17, 18, 19, 20, 21 and 22, or an antibody molecule comprising six CDRs having SEQ ID NOs: 25, 26, 27, 28, 29 and 30, or an antibody molecule comprising six CDRs having SEQ ID NOs: 33, 34, 35, 36, 37 and 38, or an antibody molecule comprising six CDRs having SEQ ID NOs: 41, 42, 43, 44, 45 and 46.

[0102] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising six CDRs having SEQ ID NOs: 1, 2, 3, 4, 5 and 6.

[0103] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules comprising a VH selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95 and 103.

[0104] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules comprising a VL selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96 and 104.

[0105] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VH having SEQ ID NO: 7, 15, 23, 31, 39 or 47.

[0106] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VH having SEQ ID NO:7.

[0107] In some embodiments, the anti-TNFR2 antibody molecule is preferably an antibody molecule comprising a VL having SEQ ID NO:8, 16, 24, 32, 40 or 48.

[0108] In some embodiments, it is more preferred that the anti-TNFR2 antibody molecule is an antibody molecule comprising a VL having SEQ ID NO:8.

[0109] In some embodiments, the anti-TNFR2 antibody molecule preferably comprises a VH having SEQ ID NO:7 and a VH having SEQ ID NO:8.

[0110] In some embodiments, the anti-TNFR2 antibody molecule comprises a CH having SEQ ID NO:217.

[0111] In some embodiments, the anti-TNFR2 antibody molecule comprises a CL having SEQ ID NO:218.

[0112] In some embodiments, the anti-TNFR2 antibody molecule comprises a VH having SEQ ID NO:7, a VH having SEQ ID NO:8, a CH having SEQ ID NO:217, and a CL having SEQ ID NO:218. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0113] To determine or demonstrate the characteristics of the antibody molecules of the present invention, they were compared with antibody molecules that block TNF-α binding to TNFR2. Such antibodies are shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0114] All sequences in Tables 1 and 2 above are of human origin and are derived from the n-CoDeR® library, as described in detail in Example 1.

[0115] In some embodiments, antibody molecules that specifically bind to TNFR2 described herein may also comprise one or both of the constant regions (CH and / or CL) shown in Table 3 below. [Table 3]

[0116] The first CH (SEQ ID NO:217) and first CL (SEQ ID NO:218) sequences in Table 3 above are of human origin. The second CH (SEQ ID NO:219) and third CH (SEQ ID NO:220) in Table 3 are both of mouse IgG2a origin, with the difference that the third CH sequence (SEQ ID NO:220) contains the N297A mutation. The second CL sequence (SEQ ID NO:221) is of mouse lambda light chain constant region origin. These mouse sequences are used in the surrogate antibody examples.

[0117] In some embodiments, the antibody molecules bind to human TNFR2 (hTNFR2). In some embodiments, it is preferred that the agonist antibody molecules bind strongly to human TNFR2, i.e., they have low EC50 values. This is further demonstrated in Example 2.

[0118] In some embodiments, the antibody molecule advantageously binds to both hTNFR2 and cynomolgus monkey TNFR2 (cmTNFR2 or cynoTNFR2). Cross-reactivity with TNFR2 expressed on cells of cynomolgus monkeys, also called crab-eating macaques or Macaca fascicularis, can be advantageous as it allows animal testing of the antibody molecule without the use of surrogate antibodies, with a particular focus on tolerability.

[0119] In some embodiments, it is necessary to use a surrogate antibody to test the functional activity of an antibody molecule in a relevant in vivo model in mice.To ensure the comparability between the effect of an antibody molecule in humans and the in vivo results of a surrogate antibody in mice, it is essential to select a functionally equivalent surrogate antibody that has the same in vitro properties as the human antibody molecule.In some embodiments, the antibody molecule does not specifically bind to the epitope of TNFR2 that comprises or consists of the sequence KCSPG.

[0120] In some embodiments, the antibody molecule of the invention or used in accordance with the invention is an antibody molecule that can compete with a particular antibody provided herein for binding to TNFR2, e.g., an antibody molecule comprising a VH selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95 and 103, and / or a VL selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96 and 104.

[0121] By "capable of competing" it is meant that the competing antibody is capable of at least partially inhibiting or otherwise interfering with the binding of an antibody molecule as defined herein to its specific target, TNFR2.

[0122] For example, such a competing antibody molecule may be able to inhibit the binding of an antibody molecule described herein to TNRF2 by at least about 10%, e.g., at least about 20%, or at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100%.

[0123] Competitive binding can be determined by methods known to those skilled in the art, such as enzyme linked immunosorbent assay (ELISA).

[0124] ELISA assay can be used to evaluate epitope-modifying or blocking antibody.Additional suitable methods for identifying competitive antibody are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane (see, for example, pages 567-569, 574-576, 583, and 590-612, 1988, CSHL, NY, ISBN0-87969-314-2), which is incorporated herein by reference.

[0125] In some embodiments, it is contemplated to use nucleotide sequences encoding such antibody molecules rather than the antibody molecules themselves. Thus, the present invention encompasses nucleotide sequences encoding the above-described agonist-non-blocking TNFR-2 antibody molecules.

[0126] The agonist non-blocking antibody molecules and nucleotide sequences may be used in medicine, and such antibody molecules and / or nucleotide sequences may be included in pharmaceutical compositions, as discussed further below.

[0127] The above agonistic non-blocking antibody molecules, nucleotide sequences and / or pharmaceutical compositions may be used in the treatment of cancer, as discussed further below.

[0128] The above agonistic non-blocking antibody molecules, nucleotide sequences and / or pharmaceutical compositions may be used in the treatment of chronic inflammatory diseases, as discussed further below.

[0129] The above agonistic non-blocking antibody molecules and / or nucleotide sequences may be used in the manufacture of a pharmaceutical composition for use in the treatment of cancer.

[0130] The above agonist, non-blocking antibody molecules and / or nucleotide sequences may be used in the manufacture of a pharmaceutical composition for use in the treatment of chronic inflammatory diseases.

[0131] The above agonistic non-blocking antibody molecules and / or pharmaceutical compositions may be used in methods of treating cancer in a patient, wherein a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the subject.

[0132] The above agonistic non-blocking antibody molecules and / or pharmaceutical compositions may be used in a method for treating a chronic inflammatory disease in a patient, wherein a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the patient.

[0133] In some embodiments related to the treatment of cancer, the cancer is a solid cancer or leukemia cancer. A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. A solid tumor can be benign (not cancer) or malignant (cancer). A malignant solid tumor is referred to herein as a solid tumor. Various types of solid tumors or cancers are named after the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

[0134] More specific examples of solid cancers are lung cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, kidney cancer, liver cancer, pancreatic cancer, thyroid cancer, brain cancer, central nervous system cancer, melanoma, neuroblastoma, lymphoma, Wilms' tumor, rhabdomyosarcoma, retinoblastoma, and bone cancer.

[0135] More specific examples of leukemic cancers are acute lymphocytic leukemia, chronic myeloproliferative disorders, acute non-lymphocytic leukemia, B-cell acute lymphocytic leukemia, chronic lymphocytic leukemia, T-cell acute lymphocytic leukemia, non-Hodgkin's lymphoma and chronic lymphoproliferative disorders. In some embodiments, the agonistic non-blocking antibody molecule can be used in combination with an antibody molecule that specifically binds to a checkpoint inhibitor. Alternatively, the nucleotide sequence encoding the agonistic non-blocking TNFR2 antibody molecule can be used in combination with an antibody molecule that specifically binds to a checkpoint inhibitor or a costimulatory agonist antibody. Examples of antibodies against checkpoint inhibitors are antibodies that target CTLA4, PD1, PD-L1, VISTA, TIGIT, CD200, CD200R, BTLA, LAG3, TIM3, B7-H3, B7-H4, B7-H7. Examples of costimulatory agonist antibodies are antibodies targeting OX40, 41BB, OX40L, 41BBL, GITR, ICOS, DR3, DR4, DR5, CD40, CD27, RANK, HVEM, LIGHT and B7-H6. Alternatively, the agonist non-blocking TNFR2 antibody molecule can be used in combination with a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor or costimulatory agonist. Alternatively, the agonist non-blocking TNFR2 antibody molecule can be used in combination with a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor or costimulatory agonist. In some such embodiments, the antibody molecule that specifically binds to a checkpoint inhibitor is an anti-PD-1 antibody. PD1 antibodies are primarily targeted to CD8 + It is believed to block PD-L1-mediated inhibitory signals in T cells, allowing for increased T cell-mediated antitumor responses. These treatments may synergize with each other. The same is true for other checkpoint inhibitors and agonistic costimulatory antibodies.

[0136] Additionally, the above agonist non-blocking TNFR2 antibody molecules may be used in combination with chemotherapy (such as, but not limited to, doxorubicin, paraplatin, cyclophosphamide, paclitaxel, gemcitabine, 5-fluorouracil, docetaxel, vincristine, mitoxantrone, mutamycin, epirubicin, and methotrexate), small molecule tyrosine kinase or serine / threonine kinase inhibitors (ibrutinib, imatinib, It can be used in combination with other anti-cancer treatments such as, but not limited to, suntinib, regorafenib, sorafenib, dasatinib, erlotinib, vandetanib, midostaurin, vemurafenib, dabrafenib, palbociclib, ribociclib, trametinib or alectinib), inhibitors targeting growth factor receptors (such as, but not limited to, EGFR / HER1 / ErbB1, EGFR2 / HER2 / ErbB2, EGFR3 / HER3 / ErbB3, VEGFR, PDGFR HGFR, RET, insulin-like growth factor receptor IGFR, FGFR targeting agents), anti-angiogenic drugs (such as, but not limited to, bevacizumab, everolimus, lenalidomide, thalidomide, dibuaflibercept) or irradiation. Usually, all of the above anti-cancer drugs cause cancer cell death, which leads to exposure of neoantigens and inflammation. Exposure of neoantigens and influx of inflammatory cells into tumors may result in synergistic effects of anticancer drugs.

[0137] For example, it will be known to those skilled in the art of medicine that drugs can be modified with different additives to alter the rate at which they are absorbed by the body, and can be modified in different forms, for example to allow for particular routes of administration to the body.

[0138] Thus, it is included that the agonist non-blocking antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described herein can be combined with pharma- ceutically acceptable excipients, carriers, diluents, vehicles and / or adjuvants to form a pharmaceutical composition. In this context, the term pharmaceutical composition can be used interchangeably with the terms pharmaceutical preparation, pharmaceutical formulation, therapeutic composition, therapeutic preparation, therapeutic formulation and therapeutic entity.

[0139] The pharmaceutical compositions described herein may comprise, or in some embodiments consist of, antibody molecules, nucleotide sequences, plasmids, viruses or cells.

[0140] The pharmaceutical compositions described herein may in some embodiments consist of or comprise a plasmid comprising a nucleotide sequence encoding the above-described antibody molecule or comprising the above-described nucleotide sequence.

[0141] In some embodiments, the pharmaceutical composition may comprise a nucleotide sequence encoding a part or a complete antibody molecule described herein, integrated into a cell or viral genome or viriome. And the pharmaceutical composition may comprise a cell or virus as a delivery vehicle for the antibody of the present invention (or a delivery vehicle for a nucleotide sequence encoding the antibody of the present invention). For example, in some embodiments, the virus may be in the form of a therapeutic oncolytic virus comprising a nucleotide sequence encoding at least one of the antibody molecules described herein. In some embodiments, such an oncolytic virus comprises a nucleotide sequence encoding a full-length human IgG antibody. In some embodiments, such an oncolytic virus comprises a nucleotide sequence encoding an scFv, Fab or F(ab')2 antibody molecule.

[0142] As described in the appended claims, the present invention in some embodiments relates to a virus comprising the nucleotide sequence of the present invention or the plasmid of the present invention.Preferably, the virus is an oncolytic virus, such as a therapeutic oncolytic virus.Oncolytic viruses are known to those skilled in the art of medicine and virology.

[0143] In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding an amino acid sequence having at least 80% identity to a sequence shown in Table 1 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 85% identity to a sequence shown in Table 1 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 90% identity to a sequence shown in Table 1 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 95% identity to a sequence shown in Table 1 above.

[0144] In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:15 and SEQ ID NO:16. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:23 and SEQ ID NO:24. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:39 and SEQ ID NO:40. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:47 and SEQ ID NO:48. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:55 and SEQ ID NO:56. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:63 and SEQ ID NO:64. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:71 and SEQ ID NO:72. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:79 and SEQ ID NO:80. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:87 and SEQ ID NO:88. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO: 95 and SEQ ID NO: 96. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO: 103 and SEQ ID NO: 104.

[0145] In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding an amino acid sequence having at least 80% identity to a sequence shown in Table 1 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding an amino acid sequence having at least 85% identity to a sequence shown in Table 1 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding an amino acid sequence having at least 90% identity to a sequence shown in Table 1 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding an amino acid sequence having at least 95% identity to a sequence shown in Table 1 above.

[0146] As an example, the nucleotide sequence encoding antibody 001-F02 may be as shown in Table 4. [Table 4-1] [Table 4-2]

[0147] Some oncolytic viruses have the ability to accept DNA insertions large enough to accommodate the integration of full-length human antibody sequences. Attenuated vaccinia virus and herpes simplex virus are examples of therapeutic oncolytic viruses whose genomes are large enough to allow the integration of full-length IgG antibody sequences (Chan,WM et al 2014 Annu Rev Virol 1(1):119-141;Bommareddy,PK,et al.2018 Nat Rev Immunol 18(8):498-513). Full-length IgG antibodies are successfully incorporated into oncolytic vaccinia viruses, resulting in the expression and extracellular release (production) of full-length IgG antibodies upon infection of virus-susceptible host cells, e.g., cancer cells. (Kleinpeter,P.,et al.2016,Oncoimmunology 5(10):e1220467). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted upon cell infection (Marino, N., et al. 2017 J Clin Invest 123(6):2447-2463).

[0148] The present invention also encompasses pharmaceutical compositions comprising a virus, such as an oncolytic virus as described above, and a pharma- ceutically acceptable diluent, vehicle and / or adjuvant.

[0149] The pharmaceutical composition may, in some embodiments, be in the form of a CAR-T cell having a partial or complete antibody sequence described herein as part of its chimeric antigen T cell receptor encoding sequence.

[0150] The present invention also encompasses pharmaceutical compositions comprising the above-described CAR-T cells and a pharma- ceutically acceptable diluent, vehicle and / or adjuvant.

[0151] The invention also includes other therapeutics or drug "forms" such as antibody drug conjugates, fusion proteins, and pharmaceutical compositions comprising such therapeutics.

[0152] The antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein may be suitable for parenteral administration, including aqueous and / or non-aqueous sterile injection solutions which may contain antioxidants, and / or buffers, and / or bacteriostats, and / or solutes that render the formulation isotonic with the blood of the intended recipient; and / or aqueous and / or non-aqueous sterile suspensions which may contain suspending agents and / or thickening agents. The antibody molecules, nucleotide sequences, plasmids, cells and / or pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water for injections, immediately prior to use.

[0153] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, and / or granules and / or tablets of the kind previously described.

[0154] For parenteral administration to human patients, the daily dosage level of the anti-TNFR2 antibody molecule will usually be 1 mg / kg to 20 mg / kg of patient weight, or in some cases up to 100 mg / kg, administered in single or divided doses. Lower doses may be used under special circumstances, for example in combination with chronic administration. In any event, the physician will determine the actual dosage that will be most suitable for an individual patient, which will vary with the age, weight, and response of the particular patient. The dosages described above are exemplary of the average case. Of course, there may be individual cases in which higher or lower dosage ranges are merited, and these are within the scope of the present invention.

[0155] Typically, a pharmaceutical composition (or medicament) described herein comprising an antibody molecule will contain the anti-TNFR2 antibody molecule at a concentration of about 2 mg / ml to 150 mg / ml or about 2 mg / ml to 200 mg / ml.

[0156] Generally, in humans, oral or parenteral administration of the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein is the preferred route and is the most convenient. For veterinary use, the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein are administered as an appropriately acceptable pharmaceutical formulation in accordance with normal veterinary practice, and the veterinarian will determine the dosing regimen and route of administration that will be most appropriate for a particular animal. Thus, the present invention provides pharmaceutical formulations comprising an amount of the antibody molecules, nucleotide sequences, plasmids, viruses, and / or cells of the present invention effective to treat various conditions (discussed above and further below). Preferably, the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein are adapted for delivery by a route selected from the group including intravenous (IV or iv), intramuscular (IM or im), subcutaneous (SC or sc).

[0157] The present invention also includes the antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein that contain pharma- ceutically acceptable acid or base addition salts of the target binding molecules or moieties of the present invention. The acids used to prepare pharma- ceutically acceptable acid addition salts of the aforementioned base compounds useful in the present invention are, inter alia, those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts. Pharmaceutically acceptable base addition salts may also be used to produce pharma-ceutically acceptable salt forms of the agents according to the invention. Chemical bases that may be used as reagents to prepare pharma-ceutically acceptable base salts of the agents that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, those derived from such pharmacologically acceptable cations, such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts, such as N-methylglucamine-(meglumine), and lower alkanolammonium, as well as other pharma-ceutically acceptable organic amine base salts. The antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technique may be used. One of skill in the art will appreciate that lyophilization and reconstitution may lead to varying degrees of loss of antibody activity (e.g., in conventional immunoglobulins, IgM antibodies tend to have greater loss of activity than IgG antibodies) and that usage levels may need to be adjusted upward to compensate.In one embodiment, a lyophilized (freeze-dried) polypeptide binding moiety loses less than about 20%, or less than about 25%, or less than about 30%, or less than about 35%, or less than about 40%, or less than about 45%, or less than about 50% of its activity (before lyophilization) when rehydrated.

[0158] The anti-TNFR2 antibody molecules, nucleotide sequences and pharmaceutical compositions described herein can be used for treating cancer in a subject or patient. As used herein, the terms subject and patient are used interchangeably.

[0159] The term "patient" (or subject), as used herein, refers to an animal, including a human, that has been diagnosed with cancer and / or exhibits symptoms of a particular disease.

[0160] In some embodiments, the patient (or subject) is an animal, including a human, that has been diagnosed with cancer.

[0161] In some embodiments, the patient (or subject) is an animal, including a human, diagnosed with and / or exhibiting symptoms of a chronic inflammatory disease. As used herein, chronic inflammatory disease includes autoimmune diseases. As mentioned above, several different immune cells may express TNFR2, and the relative levels of expression may vary depending on the disease and situation. For example, it is known that regulatory T cells can express high levels of TNFR2, and that they can be expanded by TNFR2 agonists. Regulatory T cells constitute a subpopulation of T cells that can suppress other immune cells in normal and pathological immune settings, and are thought to help prevent autoimmune attacks on self-tissues. Thus, stimulating the activity of regulatory T cells may be of great importance in the treatment of autoimmune disorders (Sharabi et al. "Regulatory T cells in the treatment of disease". Nat Rev Drug Discov. 2018 Oct 12). Examples of chronic inflammatory diseases other than autoimmune disorders are osteoarthritis and celiac disease. Examples of autoimmune disorders are rheumatoid arthritis (RA), multiple sclerosis (MS), type I diabetes, systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease (IBD) or myasthenia gravis (MG).

[0162] In some embodiments, the patient (or subject) is a patient with high TNFR2 expression in diseased tissue. In this context, high expression means a higher level of TNFR2 expression compared to corresponding healthy tissue. Usually, the healthy tissue used for such comparison is a reference tissue (or standard reference) collected from the healthy tissue of one or several healthy individuals. The expression level can be measured by standard techniques such as immunohistochemistry (IHC), fluorescence activated cell sorting (FACS) or mRNA expression measurement.

[0163] The patient may be a mammal or a non-mammal. Preferably, the mammalian patient is a human, horse, cow, sheep, pig, camel, dog or cat. Most preferably, the mammalian patient is a human.

[0164] "Exhibiting symptoms of cancer" includes when a subject exhibits cancer symptoms and / or cancer diagnostic markers and / or when the cancer symptoms and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0165] It will be readily apparent to one skilled in the art of medicine what cancer symptoms and cancer diagnostic markers are, and how to measure and / or assess and / or quantify whether there is a reduction or increase in the severity of a cancer symptom or whether there is a reduction or increase in a cancer diagnostic marker, and how the cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0166] Cancer treatments are often administered as a course of treatment, i.e., the therapeutic agent is administered over a period of time. The length of time for the course of treatment depends on several factors, including the type of therapeutic agent being administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the patient, among other reasons.

[0167] "Currently undergoing treatment" includes when a patient is currently undergoing a course of treatment and / or receiving a therapeutic agent and / or receiving a series of therapeutic agents.

[0168] In some embodiments, the cancer treated according to the present invention is a solid tumor.

[0169] Any of the above mentioned cancers are known and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Thus, the symptoms, cancer diagnostic markers and therapeutic agents used to treat the above types of cancers will be known to those skilled in the art of medicine.

[0170] The clinical definition of the diagnosis, prognosis and progression of most cancers is by a certain classification, known as staging. These staging systems work by collating many different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and / or prognosis and / or progression of cancer. Those skilled in the art of oncology will know how to use staging systems to evaluate the diagnosis and / or prognosis and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms should be used for this purpose.

[0171] "Cancer staging" includes the Rai staging system, including stage 0, stage I, stage II, stage III, and stage IV, and / or the Binet staging system, including stage A, stage B, and stage C, and / or the Ann Arbour staging system, including stage I, stage II, stage III, and stage IV.

[0172] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur reproducibly in a particular cancer, which means that the examination of these changes in morphology (also known as histological examination) can be used to diagnose or prognose cancer. Techniques for visualizing and preparing samples for visualizing to examine cell morphology are known in the art, such as, for example, optical microscopy or confocal microscopy.

[0173] "Histological examination" includes the presence of small mature lymphocytes, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders, the presence of small mature lymphocytes with dense nuclei lacking discernible nucleoli, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders and dense nuclei lacking discernible nucleoli, and / or the presence of atypical cells, and / or cleaved cells, and / or prolymphocytes.

[0174] It is known that cancer is the result of mutations in the DNA of cells, which can lead to cell death avoidance or uncontrollable proliferation.Therefore, the examination of these mutations (also known as cytogenetic examination) can be a useful tool for evaluating the diagnosis and / or prognosis of cancer.An example of this is the deletion of chromosomal position 13q14.1, which is characteristic of chronic lymphocytic leukemia.Techniques for examining mutations in cells are known in the art, such as, for example, fluorescent in situ hybridization (FISH).

[0175] "Cytogenetic testing" includes testing of DNA, specifically chromosomes, in cells. Cytogenetic testing can be used to identify DNA alterations that may be associated with the presence of refractory and / or recurrent cancer. These include deletion of the long arm of chromosome 13, and / or deletion at chromosomal location 13q14.1, and / or trisomy of chromosome 12, and / or deletion of the long arm of chromosome 12, and / or deletion of the long arm of chromosome 11, and / or deletion of 11q, and / or deletion of the long arm of chromosome 6, and / or deletion of 6q, and / or deletion of the short arm of chromosome 17, and / or deletion of 17p, and / or t(11:14) translocation, and and / or (q13:q32) translocations, and / or antigen gene receptor rearrangements, and / or BCL2 rearrangements, and / or BCL6 rearrangements, and / or t(14:18) translocations, and / or t(11:14) translocations, and / or (q13:q32) translocations, and / or (3:v) translocations, and / or (8:14) translocations, and / or (8:v) translocations, and / or t(11:14) and (q13:q32) translocations.

[0176] Cancer patients are known to exhibit certain physical symptoms, which are often the result of cancer taking a toll on the body. These symptoms often recur with the same cancer, and can be diagnostic and / or prognostic and / or progression features of the disease. Those skilled in the art of medicine will understand which physical symptoms are associated with which cancers, and how the evaluation of these body systems can correlate with the diagnosis and / or prognostic and / or progression of the disease. "Physical symptoms" include hepatomegaly and / or splenomegaly. [Brief description of the drawings]

[0177] In the following examples, reference is made to the following figures:

[0178] [Figure 1] The antibodies of the invention bind to TNFR2. Figure 1A-D: ELISA shows that human antibodies bind to human TNFR2 protein in a dose-dependent manner, generating different EC50 values. Figure 1E: Mouse antibodies 3-F10 and 5-A05 bind to mTNFR2 with similar affinity. [Diagram 2]Figure 2 shows the binding of TNFR2-specific n-CoDeR® antibodies to in vitro activated CD4+ T cells. Human blood-derived CD4+ T cells (Figure 2A-D) and mouse splenic CD4+ T cells (Figure 2E) were activated with IL-2 and CD3 / CD28 Dynabeads®. The affinity of TNFR2-specific n-CoDeR® antibodies for activated cells was analyzed by FACS at concentrations ranging from 0.002 to 267 nM (human) and 0.00003 to 133 nM (mouse). Curves show MFI after subtraction of isotype control background (Figure 2A (full and partial blockers), Figure 2B (partial blockers), Figure 2C and D (non-blockers), Figure 2E (mouse surrogate full blockers (3-F10) and non-blockers (5-A05)). Human TNFR2 antibodies bind to in vitro activated CD4+ T cells with different affinities (EC50 values ​​range from 0.59 to 53 nM), whereas mouse TNFR2 antibodies bind with similar affinities (EC50 values ​​range from 0.072 to 0.11 nM). [Diagram 3] This shows that the TNFR2n-CoDeR® antibody specifically binds to TNFR2. Human blood-derived CD4+ T cells (Figure 3A) and mouse splenic CD4+ T cells (Figure 3B) were activated for 3 days with recombinant IL-2 and CD3 / CD28 activation beads. In vitro activated cells were either blocked with polyclonal TNFR2 antibody (gray line) or left in PBS (black line) for 30 min, and then stained for 15 min with different suboptimal concentrations of TNFR2n-CoDeR® antibodies or isotype control (dashed line). Cells were then washed and incubated with APC conjugated secondary antibody for 30 min before analysis by flow cytometry. This shows that the TNFR2n-CoDeR® antibody (human and mouse) is specific for TNFR2, since the binding of all antibodies could be blocked by the polyclonal TNFR2 antibody. [Figure 4]Figure 1 shows cross-reactivity of human TNFR2-specific n-CoDeR® antibodies to cynomolgus monkeys. CD4+ T cells were isolated from cynomolgus monkey blood and stimulated with PMA and ionomycin. Two days later, cells were labeled with 0.1, 1, or 10 μg / ml of TNFR2-specific n-CoDeR® antibodies or isotype control, followed by incubation with APC-conjugated secondary α-human antibodies. Cells were analyzed by flow cytometry. The figure shows the percentage of TNFR2+ T cells for each antibody relative to the isotype control. Results are the mean and SD from 2-3 individual experiments. Most TNFR2 antibodies show cross-reactive binding to cynomolgus monkey cells. [Diagram 5]Activated cells were blocked with 40 μg / ml MR2-1 antibody (FIG. 5A, black bar) or left with PBS (FIG. 5A, grey bar) for 30 min, after which TNFR2-specific n-CoDeR® antibody / polyclonal TNFR2 (pTNFR2) was added and cells were incubated for 15 min. The percentage of bound TNFR2 n-CoDeR® antibody was analyzed by FACS after incubation with APC-conjugated secondary antibody. In FIG. 5B, activated CD4+ T cells were blocked with 40 μg / ml TNFR2-specific n-CoDeR® antibody / pTNFR2 (black bar) or left with PBS (grey bar) and then incubated with PE-conjugated MR2-1 antibody for 15 min. Cells were then analyzed by FACS. The MR2-1 antibody does not interfere with the binding of the TNFR2-specific n-CoDeR® antibody, and the n-CoDeR® antibody does not affect the binding of MR2-1 to activated cells, indicating that all of the n-CoDeR® antibodies bind to other domains of the TNFR2 protein other than the MR2-1 antibody. This indicates that all of the TNFr2-specific n-CoDeR® antibodies bind to other epitopes on the TNFR2 protein other than the TNFR2 clone MR2-1. CD4+ T cells from human blood were stimulated with rhIL-2 and CD3 / CD28 activation beads for 2-3 days. The activated cells were blocked with 40 μg / ml MR2-1 antibody (Figure 5A, black bars) or left for 30 min using PBS (Figure 5A, grey bars), after which the TNFR2-specific n-CoDeR® antibody / polyclonal TNFR2 (pTNFR2) was added and the cells were incubated for 15 min. The percentage of bound TNFR2 n-CoDeR® antibody was analyzed by FACS after incubation with APC-conjugated secondary antibody. In Figure 5B, activated CD4+ T cells were blocked with 40 μg / ml TNFR2-specific n-CoDeR® antibody / pTNFR2 (black bars) or left with PBS (gray bars) and then incubated with PE-conjugated MR2-1 antibody for 15 min. Cells were then analyzed by FACS.The MR2-1 antibody does not interfere with the binding of the TNFR2-specific n-CoDeR® antibody, and the n-CoDeR® antibody does not affect the binding of MR2-1 to activated cells, indicating that all of the n-CoDeR® antibodies bind to other domains of the TNFR2 protein other than the MR2-1 antibody. [Figure 6] Data for ligand blocking antibodies are shown. A blocking ELISA was performed with n-CoDeR® mAb specific for hTNFRII to assess the ligand blocking properties. Figure 6A) All antibodies were incubated at 10 μg / ml. All antibodies that reduced the signal achieved with the isotype control by more than 50% (indicated by the dotted line) were then administered to further investigate the ligand blocking potential. Figure 6B) shows a full blocking mAb, Figures 6C) and D) show partial blocking mAbs, and Figure 6E) shows a weak blocking mAb. All other mAbs are considered non-blocking mAbs. [Figure 7] Data for ligand blocking antibodies are shown. A blocking ELISA was performed with n-CoDeR® mAb specific for mTNFRII to assess the ligand blocking properties. Figure 7A) All antibodies were incubated at 10 μg / ml. All antibodies that reduced the signal achieved with the isotype control by more than 50% (indicated by the dotted line) were then administered to further investigate the ligand blocking potential. Figure 7B) shows a full blocking mAb, Figures 7C) and D) show partial blocking mAbs, and Figure 7E) shows a weak blocking mAb. All other mAbs are considered as non-blocking mAbs. [Figure 8]We show that agonistic non-blocking TNFR2-specific n-CoDeR® antibodies enhance IFN-γ production in NK cells stimulated with IL-2 and IL-12, whereas antagonistic blocking TNFR2-specific n-CoDeR® antibodies do not. Figure 8A: Human blood-derived NK cells were stimulated with 20 ng / ml rhIL-2 and 20 ng / ml rhIL-12, and 10 μg / ml TNFR2-specific n-CoDeR® antibodies, isotype control or 100 ng / ml rhTNF-α were added for 24 hours. The amount of IFN-γ in the culture supernatant was measured using MSD. The amount of IFN-γ was normalized to the isotype control and is shown in Figure 8A. Human antibodies that showed EC50 values ​​higher than 25 nM against in vitro activated CD4+ T cells were not included in the analysis. Human NK cells also produce TNF-α in these cultures (see Figure 8D below). IFN-γ results are averages of three donors in two independent experiments. Results show that non-blocking TNFR2 antibodies are antagonistic, enhancing IFN-γ production from cytokine-stimulated NK cells, while blocking antibodies are fairly agonistic, decreasing IFN-γ production by NK cells. In addition, Figures 8B-D show that agonistic antibodies have intrinsic agonistic activity, even in the absence of measurable TNF-α ligand. As shown in Figure 8B, the addition of blocking TNF-α antibodies reduces the amount of IFN-γ released, but maintains the ratio to the isotype control (Figure 8C), even in the complete absence of measurable TNF-α in the supernatant (Figure 8D). Figure 8D shows the average TNF-α levels in two donors in the presence or absence of TNF-α neutralizing antibodies. [Figure 9]It is shown that agonistic non-blocking TNFR2-specific n-CoDeR® antibodies activate memory CD4+ T cell populations (as indicated by an increase in the percentage of CD25+ cells), whereas antagonistic blocking TNFR2-specific n-CoDeR® antibodies do not. Human blood-derived CD4+ T cells (FIG. 9A) and mouse splenic CD4+ T cells (FIG. 9B) were activated with recombinant IL-2 and TNFR2-specific n-CoDeR® antibodies, isotype control or recombinant TNF-α. After 3 days of culture, cells were stained with CD25 and CD45RO (human) / CD44 and CD62L (mouse) and analyzed by flow cytometry. The results show the percentage of memory CD25-expressing cells (CD45RO+ cells (human) / CD44+CD62L- (mouse)) populations relative to the percentage of CD25+ cells recovered in cultures with isotype control. Results are means and SEM from seven donors (Figure 9A, human) and three mice (two independent experiments) (Figure 9B). In both human and mouse cultures, non-blocking TNFR2 antibodies induced the percentage of CD25+ memory cells, whereas blocking antibodies had no such effect on the memory population. For both human cultures (Figure 9A) and mice (Figure 9B), the addition of exogenous TNF-α increases the CD25+ memory T cell population. *=p<0.05 (calculated by one-way ANOVA). [Figure 10]Figure 10 shows that anti-TNFR2 mAb modulates the suppressive function of myeloid-derived suppressor cells (MDSC). For Figure 10A and B, MDSC were pre-incubated with human anti-TNFR2 mAb for 30 min. Then, without washing the cells, titrated numbers of MDSC were incubated with CFSE-labeled CD3+ T cells in the presence of CD3 / CD28 Dynabeads®. Figure 10A: The percentage of activated CD25+ T cells was determined by FACS after 3 days. To normalize between the different assays, the background of the isotype control was subtracted from all data points. The figure shows a summary of 6 different donors in 3 independent experiments with a ratio of MDSC to T cells of 1:4. Figure 10B: The amount of secreted cytokines in the supernatant was evaluated by MSD and the ratio of IFN-γ and IL-10 release was calculated in relation to the isotype control. Data were pooled from the analysis of culture supernatants obtained from experiments with 5 different donors. For Fig. 10C, CD11b+ myeloid cells from mouse CT26 tumors were isolated and pre-incubated with mouse anti-TNFR2 mAbs for 30 min. Titrated numbers of myeloid cells were co-cultured with CFSE-labeled CD3+ T cells purified from naive Balb / C spleens. Cells were stimulated with CD3 / CD28 Dynabeads® for 3 days and the percentage (%) of proliferating CFSE-low cells was analyzed by FACS. (n=4 independent experiments). Only agonistic / non-blocking anti-TNFR2 mAbs were shown to reverse the suppressive function of MDSCs in T cell / MDSC co-culture assays. For Fig. 10D, MDSCs were pre-incubated with three different isotypes (including Fc-deleted forms) of two different human anti-TNFR2 mAbs (one blocking=1H10) and one non-blocking (=1F02) for 30 min. Substitution at amino acid position 297 resulted in loss of glycosylation and reduced binding to FcγR (herein referred to as N297Q). Titrated numbers of MDSCs were then incubated with CFSE-labeled CD3+ T cells in the presence of CD3 / CD28 Dynabeads® without washing the cells, and the percentage of activated CD25+ T cells was measured by FACS after 3 days. To normalize between different assays, the background of the isotype control was subtracted from all data points.This figure shows that the agonist activity of the non-blocking 1-F02 antibody is independent of antibody isotype and FcγR binding.This figure shows a summary of 4 different donors in 2 independent experiments with a 1:4 ratio of MDSC to T cells. [Figure 11] For Figure 11A, Balb / c mice were injected subcutaneously with 1x106 CT26 cells. Eight days later, with an average tumor size of 3x3mm, mice were treated intraperitoneally twice a week with 10mg / kg antibody as indicated. Tumors were measured twice a week until they reached 15mm in diameter, after which mice were sacrificed. The top figure shows tumor growth in mice treated with isotype control, while the bottom two figures show, in the left panel, an antagonistic ligand blocking antibody in FcγR-deficient Ig format (middle figure) and an agonistic non-ligand blocking antibody (bottom figure). The middle panel shows the same antibody in mouse IgG2a format that binds primarily to activating FcγRs, while the right panel shows the antibody in mouse IgG1 format that binds primarily to inhibitory FcγRIIb. For Figure 11B, surviving mice were followed for 70 days. As can be seen, the non-blocking agonistic antibody is most effective as a tumor treatment in the IgG1 format that binds primarily to inhibitory FcγRs. In addition, agonist antibodies have FcγR-independent antitumor effects, as seen using the N297A format. Blocking antagonist antibodies, on the other hand, are most effective as tumor treatments in IgG2a formats that bind primarily to activating FcγRs, and are ineffective in formats that are deficient in FcγR binding. ***=p<0.001 compared to isotype control calculated by log-rank Mantel-Cox test. [Figure 12]C57 / BL6 mice were injected subcutaneously with 1x106 MC38 cells. At an average tumor size of 3x3mm, mice were treated intraperitoneally twice weekly with 10mg / kg antibody as indicated in the figure. The figure shows the tumor growth curves of individual mice. Figure 12A: isotype control, Figure 12B: PD-1 targeting antibody, Figure 12C: 5A05 antibody (surrogate antibody, ligand non-blocking agent, agonist), Figure 12D: 5A05 in combination with PD1. Tumors were measured twice weekly until they reached a diameter of 15mm, after which mice were sacrificed. Figure 12E shows the survival curves of the four different treatment groups. *=p<0.05, ***=p<0.001 compared to isotype control calculated by log-rank Mantel-Cox test. [Figure 13] This shows that ligand-blocking agonist antibodies are effective in combination with anti-PD-L1 as an anti-tumor treatment. C57 / BL6 mice were injected subcutaneously with 1x106 MC38 cells. At an average tumor size of 5x5mm, mice were treated with isotype control antibody or 5A05 twice (days 1 and 4), with anti-PD-L1 for 4 consecutive days followed by a fifth injection 2 days later (for a total of 5 injections on days 1, 2, 3, 4, and 7), or a combination of both. All antibodies were administered intraperitoneally at 10mg / kg. Figure 13 shows the mean tumor growth + / - SEM, n=10 / group. *=<0.05, ***=p<0.001 (calculated using one-way ANOVA). [Figure 14] C57 / BL6 mice were injected subcutaneously with 1x106B16 cells. Three days later, mice were treated intraperitoneally with 10mg / kg of antibody twice a week. Tumors were measured twice a week until they reached 15mm in diameter, after which mice were sacrificed. Figure 14A: Isotype control, Figure 14B: 5A05 antibody (surrogate antibody, ligand non-blocker, agonist). Figure 14C shows the survival curves of the two different treatment groups. *=p<0.05 compared to isotype control calculated by log-rank Mantel-Cox test. [Figure 15]The ligand-blocking agonist surrogate antibody 5A05 alters the immune cell composition of tumors. Mice were inoculated with CT26 tumor cells as described, and when tumors reached a size of approximately 7x7mm, antibodies were injected as indicated. After three injections, mice were sacrificed 8 days after treatment initiation, and tumors were harvested. Tumor single cell suspensions were analyzed for immune cell content by FACS. A) The ligand-blocking agonist surrogate antibody 5A05 causes depletion of Tregs and B) an influx or proliferation of CD8+ T cells. This shifts the Treg / CD8+ T cell ratio as shown in C). Figure D) shows not only T cells, but also myeloid cells. Here, the number of tumor-associated macrophages (TAMs, defined as CD11b+, but negative for both Ly6G and Ly6C) is very significantly reduced. The ligand-blocking antagonist surrogate antibody 3F10 also modulates TAM numbers, but still very differently than 5A05. [Figure 16] NOG mice were injected intravenously with 15–20x106 PBMC cells. After 10–12 days, spleens were removed from the mice, single cell suspensions were prepared, and TNFR2 expression was assessed by FACS. Previously, TNFR2 expression was assessed in T cells recovered from blood and tumor samples from three or nine cancer patients, respectively. As shown in the figure, TNFR2 expression in Tregs and CD8+ T cells is highly similar between in vivo grown and activated human T cells in NOG mice and T cells derived from human tumors. [Figure 17]NOG mice were injected intravenously with 15-20x106 PBMC cells. After 10-12 days, spleens were removed from the mice, single cell suspensions were prepared, and cells were analyzed using FACS. Figure 17A shows the mean percentage of stained Tregs, defined as CD45+CD3+CD4+CD25+CD127 low / negative, out of the total number of cells in the spleen. Figure 17B shows the mean percentage of T cells (CD3+) out of the total number of cells in the spleen. As shown in Figure 17, the non-ligand blocking agonist antibody 1F02 increases the number of Tregs (which express the highest levels of TNFR2) as well as the total number of T cells in this model. In comparison, the ligand blocking antagonist antibody 1H10 does not have this effect. [Figure 18] The IFN-γ release induced by various TNFR2-specific antibodies was measured in three different in vitro systems. As positive controls, anti-CD3 antibody-OKT3 or muromonab-CD3, anti-CD52 antibody-alemtuzumab, and anti-CD28 antibody were used. Isotype controls were used as negative controls. Each dot represents PBMC from one human donor. Figure 18A shows the results from a high-density cell culture. PBMCs were cultured at 1x107 cells / ml. After 48 hours, 10 μg / ml of antibody was added and incubated for 24 hours. As can be seen, both alemtuzumab and OKT3 induced significant IFN-γ release, whereas none of the TNFR2-specific antibodies induced it. Figure 18B shows a solid-phase in vitro culture performed by coating the wells of a 96-well plate with the antibody before adding the PBMCs. Again, both alemtuzumab and OKT3 induced significant IFN-γ release, especially in one of the donors, along with several TNFR2-specific antibodies. Figure 18C shows whole blood stimulation with antibodies, where alemtuzumab induced significant IFN-γ release, but none of the TNFR2-specific antibodies did. [Figure 19]NOG mice were intravenously injected with 25x106 PBMC cells. 14 days later, when the mouse blood was shown to consist of approximately 40% human T cells, the mice were treated with 10 μg of antibody. Body temperature was measured 1 hour after injection (Figure 19A). 5 hours after injection, the experiment was terminated and blood was analyzed for IFN-γ (Figure 19B) or TNF-α (Figure 19C) content. ****=p<0.0001 and **=p<0.01 (calculated by one-way ANOVA). [Figure 20] Binding to TNFR2 mutants lacking individual domains is shown. Antibody binding to TNFR2 mutants expressed in HEK cells was tested with a flow cytometry approach. While the absence of domains 1 and 2 does not significantly affect binding (Figures 20A and B), 3 and partially 4 completely abolish the interaction between the antibody and TNFR2 (Figures 20C and D). Similarly, the absence of domains 1+3 completely prevents binding of all antibodies (except 1F06) (Figure 20E), and the absence of domains 2+4 completely abolishes binding to agonist antibodies (1F02, 1F06, 4E08) and also greatly reduces binding to antagonists (1H10, 4H02, 5B08) (Figure 20F). Dark grey indicates positive control and white indicates negative control antibody. [Figure 21] Shown is a comparison of the amino acid sequences of human (H-D3) and mouse (M-D3) domain 3 of TNFR2. Similar amino acids are shown in white and differences are shown in grey. The five sequences below represent the five different constructs on which the antibodies will be tested. The exchanges from human to mouse sequences are underlined, while non-underlined sequences are fully human. Domains 1, 2 and 4 are human and contain no substitutions or mutations. [Figure 22]Binding to wild-type human and mouse TNFR2 is shown in the left panel. Mutated hTNFR2 constructs (m1, m2, m3 and m4) were used to narrow down the binding site for various anti-hTNFR2 antibodies. Flow cytometry analysis revealed that mutations in aa119-132 do not affect antibody binding, whereas mutations in aa151-160 completely abolish binding of all antibodies. Mutations in 134-144 only disrupt binding to blocking antagonist antibodies and do not significantly affect agonist antibodies. Dark grey bars indicate positive control antibodies and white indicates negative control antibodies. Dashed lines are the levels of negative control antibodies. EXAMPLES

[0179] Certain non-limiting examples are now described which illustrate certain aspects of the present invention.

[0180] In many examples, particularly in vivo examples, antibody 5-A05 is used. This is a murine antibody that is a surrogate for the human antibodies disclosed herein. It was selected as a surrogate antibody based on its similar properties to the ligand non-blocking agonist antibodies with good EC50 values.

[0181] In some examples and figures, slightly different names for antibody clones are used, for example, clone 001-F02 may be abbreviated to 1-F02 or 1F02, and 005-B08 may be abbreviated to 5-B08 or 5B08.

[0182] Example 1 - Generation of TNFR2-specific antibodies (See also Figure 1 and its description above.) Isolation of scFv antibody fragments The n-CoDeR® scFv library (BioInvent; Soderlind E, et al Nat Biotechnol. 2000; 18(8):852-6) was used to isolate scFv antibody fragments that recognize human TNFR2.

[0183] The phage library was used in three successive rounds of panning against recombinant human or mouse proteins (Sino Biological). After phage incubation, cells were washed to remove unbound phages. Bound phages were eluted with trypsin and amplified in E. coli. The resulting phage stocks were converted to scFv format. E. coli was transformed with the scFv-carrying plasmid to express individual scFv clones.

[0184] Identification of unique TNFR2-binding scFv The converted scFvs from the third round of panning were assayed for binding to transfected 293FT cells expressing human or mouse TNFR2 or an unrelated protein using a homogeneous FMAT assay (Applied Biosystems, Carlsbad, CA, USA).

[0185] Briefly, transfected cells were added to clear-bottom plates along with scFv-containing supernatants from expression plates (diluted 1:7), mouse anti-His Tag antibody (0.4 μg / ml, R&D Systems), and APC-conjugated goat anti-mouse antibody (0.2 μg / ml, catalog number 115-136-146, Jackson Immunoresearch). FMAT plates were incubated at room temperature for 9 hours before being read. Bacterial clones that bound to cells transfected with TNFR2 but not to cells transfected with a non-related protein were classified as actives and cherry-picked into 96-well plates.

[0186] IgG binding to TNFR2 in ELISA 96-well plates (Lumitrac 600 LIA plates, Greiner) were coated with 1 pmol / well of recombinant human or mouse TNFR2-Fc protein (Sino Biological) overnight at 4°C. After washing, titrated doses of anti-TNFR2 mAb ranging from 20 μg / ml to 0.1 ng / ml (133 nM to 1 pM) were allowed to bind for 1 h. Plates were then washed again and bound antibodies were detected with anti-human F(ab)-HRP secondary antibody (Jackson ImmunoResearch) diluted to 50 ng / ml. Plates were analyzed using a Tecan Ultra Microplate reader with Super Signal ELISA Pico (Thermo Scientific) as substrate.

[0187] The data presented in Table 5 and Figures 1A-D show that all human anti-TNFR2 antibodies bind to human TNFR2 protein, with EC50 values ​​ranging from 0.082 nM for 1-C08 to 4.4 nM for 1-A09.

[0188] In addition, mouse antibody surrogate clones 3-F10 and 5-A05 also bind to the mTNFR2 protein. These two clones bind with very similar affinities (Table 5 and FIG. 1E). [Table 5]

[0189] Example 2 - Antibody specificity (See also Figures 2-5 and their descriptions above.) CD4 + Isolation of T cells PBMCs from human buffy coats and cynomolgus monkey (M. fascicularis) whole blood were isolated using Ficoll-Paque PLUS (GE Healthcare) gradients. + T cells, CD4 +T cells were isolated from PBMCs by magnetic cell sorting using a T cell isolation kit (human) or CD4 MicroBeads, non-human primate (cynomolgus monkey) (both from Miltenyi). + T cells were identified using Miltenyi's CD4 + T cells were isolated from the spleen using a T cell isolation kit (mouse).

[0190] Titration of TNFR2-specific n-CoDeR® antibodies The ability and affinity of the TNFR2n-CoDeR® antibody to bind to TNFR2 expressed on cells was measured using in vitro activated CD4 + T cells were obtained using human CD4 + T cells were stimulated with 50ng / ml rhIL-2 (R&D systems) and Dynabeads® T-Activator CD3 / CD28 for T cell proliferation and activation (Gibco) at 37 degrees for 2-3 days. In vitro activated cells were labeled with increasing amounts of n-CoDeR® antibodies specific for TNFR2 or isotype control ranging from 0.002-267nM. Cells were then incubated with APC-conjugated a-human IgG secondary ab (Jackson) and then analyzed by flow cytometry (FACSVerse, BD). The resulting titration curves are shown in Figure 3A-D. Mouse CD4 +T cells were stimulated with 135 U / ml rmIL-2 (R&D systems) and Dynabeads® T-Activator CD3 / CD28 for T cell proliferation and activation (Gibco) at 37 degrees for 2-3 days. In vitro activated cells were labeled with increasing amounts of n-CoDeR® antibodies specific for TNFR2 or isotype control ranging from 0.00003 to 133 nM. Cells were then incubated with APC-conjugated a-mouse IgG secondary ab (Jackson) and then analyzed by flow cytometry (FACSVerse, BD). Titration curves are shown in Figure 3E. EC50 values ​​of the titration curves were calculated in Microsoft Excel and are shown in Table 6. For human antibodies, EC50 values ​​varied from 0.6 nM (4-H02) to 52.7 nM (1-C03). The murine antibodies bound to in vitro activated cells with similar affinities (0.072 nM (3-F10) and 0.11 nM (5-A05)).

[0191] Specificity of TNFR2n-CoDeR® Antibody The specificity of the TNFR2 antibody for TNFR2 was obtained by FACS blocking experiments using a commercially available polyclonal TNFR2 antibody (R&D Systems). +T cells (mouse and human) were stimulated for 2-3 days with 50ng / ml rhIL-2 (R&D systems) (human) / 135U / ml rmIL-2 (R&D systems) (mouse) and Dynabeads® T-Activator CD3 / CD28 for T cell proliferation and activation (Gibco), blocked with 40μg / ml polyclonal TNFR2 antibody (R&D systems) for 30 min, and immediately incubated with TNFR2 n-CoDeR® antibody or isotype control for 15 min. The concentrations of n-CoDeR® antibodies used were based on the titration curves of the individual TNFR2 n-CoDeR® antibodies, choosing suboptimal concentrations of each antibody. Cells were then washed and incubated with APC-conjugated secondary antibodies (Jackson) for 30 min. Cells were analyzed by flow cytometry (FACSVerse, BD). As shown in Figure 4, all binding of TNFR2-specific n-CoDeR® antibodies (both human and mouse) could be blocked by polyclonal TNFR2 antibodies. These results indicate that TNFR2 n-CoDeR® antibodies inhibit the binding of in vitro activated CD4 + It has been confirmed to specifically bind to TNFR2 on T cells.

[0192] Epitope mapping of TNFR2-specific n-CoDeR® antibodies to TNFR2 antibody clone MR2-1 TNFR2 antibody clone MR2-1 (Invitrogen) binds to a specific domain of the TNFR2 protein. TNFR2-specific n-CoDeR® antibodies bound to the same domain as MR2-1 when tested by FACS blocking experiments.

[0193] Human CD4 +T cells were stimulated with 50ng / ml rhIL-2 (R&D systems) and Dynabeads® T-Activator CD3 / CD28 (Gibco) for 2-3 days to allow for T cell proliferation and activation. Activated cells were blocked with 40μg / ml MR2-1 (black bars in Fig. 5A), TNFR2-specific n-CoDeR® antibody / polyclonal TNFR2 (R&D systems) (black bars in Fig. 6B) or PBS (gray bars in Fig. 5). After 30 minutes of incubation, cells were immediately stained with TNFR2-specific n-CoDeR® antibody / pTNFR2 (Fig. 5A) or MR2-1 (Fig. 5B) for 15 minutes. Cells in Fig. 5A were also incubated with APC-conjugated secondary a-human IgG reagent (Jackson). Cells were analyzed by flow cytometry (FACSVerse, BD). MR2-1 + The percentage of cells was the same for unblocked and n-CoDeR blocked cells (Figure 5B), and the binding of n-CoDeR antibodies was the same with or without MR2-1 blockade (Figure 5A). These n-CoDeR antibodies likely bind to other epitopes of the TNFR2 protein other than the MR2-1 antibody.

[0194] Binding of TNFR2n-CoDeR® Antibody to Cynomolgus Monkeys To verify the cross-reactivity of TNFR2 antibodies to cynomolgus monkeys, we used cynomolgus monkey CD4 + T cells were stimulated with 50ng / ml PMA (Sigma) and 100ng / ml ionomycin (Sigma) for 2 days. Cells were incubated with TNFR2-specific n-CoDeR® antibodies at three different concentrations (0.1, 1, and 10 μg / ml) and then incubated with APC-conjugated secondary a-human IgG reagent (Jackson). Cells were analyzed by flow cytometry (FACSVerse, BD) and showed that most of the human TNFR2-specific n-CoDeR® antibodies were able to bind to cynomolgus monkey TNFR2. The results of the individual antibodies are shown in Figure 4.

[0195] In summary, the data in Example 2 show that the human antibodies bind specifically to TNFR2 endogenously expressed on human immune cells. Furthermore, the data show that this binding can be blocked by adding a polyclonal commercial antibody against TNFR2. This shows a very high specificity for TNFR2. The same is true for surrogate clones 3F10 and 5A05 on mouse cells expressing mouse TNFR2. Also, the binding of the human clones is not affected by the MR2-1 antibody, which shows a different epitope specificity compared to MR2-1. [Table 6]

[0196] Example 3 - Testing of ligand blocking properties (See also Figures 6-7 and their descriptions above.) ELISA method 96-well plates were coated with 2.5 pmol / well hTNFRII (Sinobiologics Catalog No. 10414-H08H) or mTNFRII (Sinobiologics Catalog No. 50128 M08H) in ELISA coating buffer (0.1 M sodium carbonate, pH 9.5) and incubated overnight at 4°C. After washing with ELISA wash buffer (PBS with 0.05% Tween 20), plates were incubated with 10 μg / ml (single-dose ELISA) or 33 nM n-CoDeR® mAb for 1 h at room temperature with gentle agitation, followed by 1:2 dilution in blocking buffer containing 0.45% fish gelatin (titration ELISA). Recombinant hTNF-α-bio (R&D Catalog No. BT210) or mTNF-α (Gibco Catalog No. PMC3014) was then added at final concentrations of 5 nM and 2 nM, respectively, and incubated for an additional 15 min. Plates were then washed. For human ELISA, streptavidin-HRP (Jackson Cat. No. 016-030-084) diluted 1:2000 in blocking buffer was added and incubated again for 1 hour at room temperature, followed by washing first with ELISA buffer and then with Tris buffer (pH 9.8). Substrate (Super Signal ELISA Pico from Thermo Scientific (Cat. No. 37069)) was then diluted according to the manufacturer's instructions, added to the wells, incubated for 10 minutes in the dark, and read on a Tecan Ultra. For mouse ELISA, rabbit anti-mTNF-α (Sinobiologicals Cat. No. 50349-RP02) diluted to 1 μg / ml was added and allowed to incubate for 1 hour at room temperature. After washing, anti-rabbit-HRP diluted 1:10000 in blocking buffer was added and again incubated for 1 hour at room temperature. Substrate addition and reading were performed as above.

[0197] The anti-human and anti-mouse antibody data are shown below in Tables 7 and 8, and in Figures 6 and 7, respectively. [Table 7] [Table 8]

[0198] Definition of Blockage Complete blockers are defined as those that reduce TNF-α binding by >98% Partial blockers are defined as those that reduce TNF-α binding by 60-98%. Weak blockers are defined as those that reduce TNF-α binding by less than 60%. Non-blocking antibodies are defined as those that do not exceed 50% blocking in a high dose one-point ELISA, as shown in Figures 6A and 7A.

[0199] The data presented in this example demonstrate that a range of antibodies have been generated, from those that completely inhibit the binding of the ligand TNF-α to those that do not inhibit ligand blockade at all, and this is true for both human and mouse surrogate antibodies.

[0200] Example 4 - In vitro functionality of antibodies (See also Figures 8-10 and their descriptions above.) TNF-α non-blocking TNFR2 antibodies, in contrast to TNF-α blocking TNFR2 antibodies, enhance IFN-γ production from cytokine-stimulated NK cells. The agonist / antagonist properties of TNFR2-specific antibodies were assessed using an NK cell assay described by Almishri et al. (TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2. Almishri W. et al. J Innate Immun. 2016;8:617-629).

[0201] Briefly, human NK cells were isolated from human PBMCs by MACS using the "NK Isolation Kit" (Miltenyi). 100 μl of NK cells (1 × 10 ) were incubated in U-bottom plates (Corning® 96-well TC-treated microplates, Sigma-Aldrich) with 10 μg / ml of TNFR2-specific antibody, 10 μg / ml of isotype control or 100 ng / ml of TNF-α (R&D systems). 6 Cells / ml) were cultured with 20ng / ml rhIL-2 (R&D systems) and 20ng / ml rhIL-12 (R&D systems). Supernatants were harvested after 24 hours and the amount of IFN-γ produced was assessed by MSD. As a control, an anti-TNF-α antibody (catalog no. AF-210-NA, R&D systems) that neutralizes TNF-α was included. As seen in Figure 8D, a dose of 1μg / ml completely neutralized soluble TNF-α and this dose also reduced IFN-γ release.

[0202] Human non-blocking TNFR2 antibodies clearly enhanced IFN-γ production in IL-2- and IL-12-stimulated NK cells (2- to 3-fold higher IFN-γ than isotype control), whereas antagonist antibodies (shown here as full blockers) exhibited antagonistic effects on NK cell IFN-γ production ( Fig. 8A ).

[0203] This testing was deemed unrepresentative to perform with mouse surrogate antibodies due to the lack of endogenously produced TNF-α in mouse cultures, the expression of inhibitory FcγRs on mouse NK cells, and the expression of only activating FcγRs in their human counterparts. Instead, we used memory T cell activation assays (induction of CD25), and myeloid-derived suppressor cell (MDSC) suppression assays (both described below) to address the agonist or antagonist properties of the mouse surrogate antibodies.

[0204] Non-ligand blocking TNFR2 antibodies inhibit memory CD4 + Induces CD25 expression on T cells, but not ligand-blocking TNFR2 antibodies. To further understand the agonist / antagonist properties of TNFR2 antibodies, we investigated the CD25-expressing memory CD4 + Their ability to enhance the proportion of T cells was assessed.

[0205] In brief, the Miltenyi CD4 + Isolate human CD4 T cells from PBMCs by MACS using a "T cell isolation kit" + T cells were isolated. CD4 + T cells were cultured with 10 ng / ml rhIL-2 (R&D Systems) and 10 μg / ml TNFR2-specific antibody or the indicated amount of rhTNF-α (R&D Systems). After 3 days, memory cells (CD45RO + The expression of CD25 on IgG4-associated leukocytes (IL-1, IL-2, and IL-3) was analyzed by FACS (Figure 9A).

[0206] Similarly, mouse CD4 + T cells are called CD4 + Memory cells (CD44 + CD62L - Expression of CD25 on CD4+ / CD5+ cells was analyzed by FACS after 3 days (Figure 9B).

[0207] The percentage of CD25 expressing cells was enhanced in memory cell cultures stimulated with non-blocking TNFR2, again demonstrating agonist activity in both humans and mice, however, stimulation with blocking antibodies did not increase CD25 expression in these cultures.

[0208] Non-ligand-blocking, but not ligand-blocking, anti-TNFR2 mAb reverses the suppressive function of myeloid-derived suppressor cells (MDSCs) in a T cell / MDSC coculture assay The effect of human anti-TNFR2 mAbs on the suppressive function of MDSCs was assessed in a T cell / MDSC co-culture assay. Briefly, MACS-isolated human CD14 + MDSCs were generated by culturing monocytes isolated from cancer patients with 50% ascites and 50% R-10 for 3 days. MDSCs were then washed and pre-incubated with 10 μg / ml anti-TNFR2 mAb for 30 min. Without washing the cells, a titer of MDSCs was incubated with MACS-isolated CFSE-labeled CD3 in the presence of CD3 / CD28 Dynabeads®. + After 72 hours, activated CD25 + The percentage of T cells was assessed by FACS. IFN-γ and IL-10 secretion was measured by MSD according to the manufacturer's instructions.

[0209] In contrast to the blocking anti-TNFR2 mAb (dark grey bars), the non-blocking anti-TNFR2 mAb (light grey bars) inhibited the activation of CD25 + It was shown that blocking TNFR2 antibodies increased the percentage of Th1 / Th2 cytokines in the TNFR2-dependent inflammatory response (Figure 10A). By measuring the amount of secreted interferon gamma (IFN-γ) and interleukin-10 in the supernatant, the ratio of the two cytokines was used to estimate the Th1 / Th2 balance. As shown in Figure 10B, the ratio of IFN-γ to IL-10 was significantly elevated by the non-blocking anti-TNFR2 antibodies compared to the blocking antagonist antibodies, indicating that the non-blocking antibodies induce a shift toward the Th1 pathway.

[0210] As for human antibodies, the effect of mouse anti-TNFR2 antibodies has been tested in a similar inhibitory assay. Here, CD11b + Myeloid cells were directly MACS isolated from CT26 mouse tumors and pre-incubated with anti-TNFR2 mAb for 30 min. CD3 + Responder T cells were purified from naive Balb / C spleens. Myelosuppressor cells and CFSE-labeled T cells were co-cultured at different ratios for 3 days. 低The percentage of T cells was determined by FACS. Similar to the results of the human assay, the percentage of proliferating responder cells is significantly lower after incubation with blocking antibodies than with non-blocking antibodies (Figure 10C).

[0211] In MDCS suppressed cultures, myeloid cells express high levels of various FcγRs. To test whether the observed agonist and antagonist effects are Fc-dependent, the ligand non-blocking agonist antibody 1F02 was tested in several formats. hIgG1 bound well to activating FcγRs, hIgG2 also bound well to inhibitory FcγRIIB, and binding to all FcγRs was significantly reduced in Fc-deficient formats. The results in Figure 10D show that the agonist activity of the non-blocking 1-F02 antibody is independent of antibody isotype and FcγR binding.

[0212] In summary, the data in Example 4 demonstrate that the non-ligand blocking antibodies are agonists as measured in vitro by several methods: CD4 as measured by NK cell-mediated IFN-γ release, CD25 expression, and T cell proliferation. + activation of memory cells, and CD25 expression in MDCS co-culture assays. Furthermore, the data show that this is an intrinsic property that is independent of the presence of ligand and independent of antibody isotype. We also show that the surrogate murine antibody 5A05 has similar agonistic characteristics. T-cell or NK-cell stimulating antibodies could be used in the treatment of cancer, inducing an endogenous immune response that ultimately destroys malignant cells.

[0213] Agonist ligand non-blocking antibodies are presented in accordance with the present invention, and antagonist ligand blocking antibodies are included for comparison.

[0214] Example 5 - Surrogate ligand non-blocking, agonist anti-mouse TNFR2 mAb has in vivo anti-tumor effects (See also Figures 11-17 and their descriptions above.) Therapeutic efficacy in various tumor models To evaluate the in vivo antitumor efficacy of a nonligand-blocking agonist anti-TNFR2 mAb, a murine surrogate called 5-A05 was investigated in vivo in different tumor models using different isotype formats, either alone or in combination with anti-PD-1 as described below.

[0215] Mice were bred and maintained in a local facility in accordance with Home Office guidelines. Female BalbC and C57 / BL6 mice, 6–8 weeks old, were supplied by Taconic (Bomholt, Denmark) and maintained in a local animal facility. CT26, MC38 and B16 cells (ATCC) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells were semi-confluent, they were detached with trypsin and resuspended in sterile PBS at 10 × 106 cells / ml. Mice were injected subcutaneously with 100 μl of cell suspension, corresponding to 1 × 106 cells / mouse. 3–8 days after injection depending on the model, mice were treated intraperitoneally with 10 mg / kg of antibody as indicated in the figure (isotype control, 3-F10 or 5-A05), twice weekly. Tumors were measured twice weekly until they reached 15 mm in diameter, after which mice were sacrificed.

[0216] The non-ligand blocking agonist anti-mouse TNFR2 mAb 5-A05 exhibited therapeutic anti-tumor effects in three different tumor models (Figures 11-14), including therapeutic effects in the more treatment-sensitive CT26 (Figure 11) and tumor growth inhibitory effects in the more treatment-resistant MC38 and B16 (Figures 12-14).

[0217] The antitumor effect of a nonligand-blocking agonist anti-mouse TNFR2 mAb does not require FcγR binding but is enhanced by such binding To assess the importance of Fc-FcγR interactions on the in vivo anti-tumor efficacy of the non-ligand blocking agonist anti-TNFR2 murine surrogate mAb 5A05, different Fc formats of this antibody were investigated in vivo in the CT26 tumor model as described below.

[0218] Mice were bred and maintained as described above. CT26 cells (ATCC) were grown and injected as described above. When tumors reached 3x3mm, mice were treated twice weekly with 10mg / kg of antibody intraperitoneally (isotype control, 5A05 IgG1, 5A05 IgG2a or 5-A05-N297A (Fc-deficient). Tumors were measured twice weekly until they reached 15mm in diameter, after which mice were sacrificed.

[0219] Fc-deleted 5-A05-N297A shows clear therapeutic activity compared to the isotype control, indicating that Fc binding is not completely essential for the therapeutic efficacy of this ligand-nonblocking agonist anti-mouse TNFR2 mAb (Figure 11A and B). In addition, both IgG1 and IgG2a formats show enhanced therapeutic efficacy. However, the IgG1 format, which preferentially binds to inhibiting Fcγ receptors, shows superior therapeutic efficacy indicative of agonism, one of the key mechanisms of action of this anti-mouse TNFR2 mAb (Figure 11A-B). This is in contrast to the ligand-blocking antagonist surrogate antibody 3-F10, which shows no activity in the Fc-deleted format and the highest activity in the mouse IgG2a format, which is known to preferentially bind to activating FcγRs.

[0220] The ligand-blocking agonist antibody (5-A05) is according to the invention, and the ligand-blocking antagonist antibody (3-F10) is included for reference.

[0221] Combination effect with anti-PD-1 mAb To evaluate the in vivo antitumor efficacy of the combination of a non-ligand blocking agonist anti-TNFR2 mAb, anti-PD-1 with a murine surrogate (5-A05), the therapeutic combination was investigated in vivo in the MC38 tumor model as described below.

[0222] Mice were bred and maintained as described above. MC38 cells (ATCC) were grown and injected as described above. Eight days after injection, mice were treated intraperitoneally twice weekly with 10 mg / kg of antibody as shown in Figures 12A-E (isotype control, anti-mouse PD-1, 5-A05, or a combination of anti-mouse PD-1 and 5-A05). Tumors were measured twice weekly until they reached 15 mm in diameter, after which mice were sacrificed.

[0223] Both anti-mouse PD-1 and the non-ligand blocking agonist anti-mouse TNFR2 mAb 5-A05 demonstrate a tumor growth inhibitory therapeutic effect in the MC38 model (Figures S12A-E). Combining anti-PD1 with 5-A05 cures tumors in treatment-resistant MC38 models (Figures S12D-E).

[0224] Combination effect with anti-PD-L1 mAb To evaluate the in vivo anti-tumor efficacy of the combination of agonistic anti-TNFR2 mAbs, the mouse surrogate (5A05) was further combined with anti-PD-L1 for treatment in the MC38 tumor model, as described below.

[0225] Mice were bred and maintained as described above. MC38 cells (obtained from Dr. M. Cragg, University of Southampton) were grown and injected as described above. Six days after injection, mice were treated with isotype control antibody or 3F10 twice (days 1 and 4), with anti-PD-L1 (clone 10F.9G2, Bioxcell) for four consecutive days with a fifth injection two days later (for a total of five injections on days 1, 2, 3, 4, and 7), or a combination of both. All antibodies were administered intraperitoneally at 10 mg / kg. Tumors were measured with calipers twice a week until they reached a volume of 2000 mm3, after which mice were sacrificed.

[0226] Both anti-mouse PD-L1 and agonist anti-mouse TNFR2 mAb 5A05 indeed show therapeutic efficacy in suppressing tumor growth in the MC38 model (Figure 13). Combining anti-PD-L1 with antagonist anti-mouse TNFR2 mAb 5A05 further enhances the anti-tumor effect (Figure 13).

[0227] In vivo immune cell regulation To investigate the effect of immune cells on tumors in vivo, BalbC mice were inoculated with CT26 cells as described above. After tumors reached approximately 7x7mm, mice were treated with 10mg / kg of antibody intraperitoneally, as shown in Figure 15. Mice were treated on days 1, 4, and 7, and sacrificed on day 8. Tumors were dissected, mechanically broken into small pieces, and digested with a mixture of collagenase 100μg / ml Liberase and 100μg / ml DNase for 2x5 minutes at 37°C with vortexing in between. After filtration through a 70μm filter, the cell suspension was washed with PBS containing 10% FBS (400g for 10 minutes). Cells were then resuspended in MACS buffer and stained with a panel of antibodies staining CD45, CD3, CD8, CD4, and CD25 or MHCII, F4 / 80, Ly6C, CD11b, and Ly6G. Prior to staining, cells were blocked for non-specific binding using 100 μg / ml IVIG (purified intravenous immunoglobulin). Cells were analyzed using FACS Verse. Mouse Tregs were cloned using CD45 + CD3 + CD4 + CD25 + and TAMs were quantified as CD11b + Ly6G - Ly6C - F4 / 80 + MHCII + was quantified as:

[0228] As seen in FIG. 15, treatment with an agonistic TNFR2 antibody inhibited CD8 + This leads to increased T cell influx, with a weak tendency towards Treg depletion. At the same time, this + The ratio of T cells to Tregs is significantly improved (Figure 15C). In addition, the agonistic antibody modulates the myeloid compartment by reducing the number of tumor-associated macrophages.

[0229] PBMC-NOG / SCID model To confirm the in vitro findings of agonistic / T cell proliferation activity of the non-ligand blocking agonist anti-TNFR2 mAb 1-F02, we analyzed the ability of this mAb to induce T cell proliferation in the PBMC-NOG model in vivo as described below.

[0230] Mice were bred and maintained in a local facility in accordance with Home Office guidelines. Eight-week-old female NOG mice were supplied by Taconic (Bomholt, Denmark) and maintained in a local animal facility. For the PBMC-NOG (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and washed, after which the cells were cultured at 75x10 6 The cells were resuspended in sterile PBS at 15x10 cells / ml. 6 200 μl of cell suspension, equivalent to 100 cells / mouse, was injected intravenously. Two weeks after injection, mice were treated twice (2 days apart) with 10 mg / kg isotype control, Yervoy, anti-CD25, Campath, 1-F02 or 1-H10 (ligand blocking antagonist anti-TNFR2 mAb). Two days after the last injection, mouse spleens were harvested. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD3, CD4, CD8, CD25, CD127 (all from BD Biosciences). In a separate experiment, spleens from untreated human PBMC were sacrificed to measure the expression of TNFR2 on human T cells by FACS (Figure 16). The FACS data showed that TNFR2 expression on Treg and CD8+ T cells was very similar between human T cells grown and activated in vivo in NOG mice and human tumor-derived T cells.

[0231] 1-F02, but not the antagonist 1-H10, induced T cell proliferation in line with what was seen in vitro and in vivo with a non-ligand blocking agonist anti-mouse surrogate TNFR2 mAb (FIG. 17).

[0232] In summary, Example 5 shows that: 1. Agonist ligand non-blocking antibodies may exert potent anti-tumor effects across several tumor models. 2. This effect can be enhanced by combining it with an anti-PD1 antibody. 3. Agonistic nonligand blocking antibodies do not exhibit absolute FcγR dependency for their antitumor effects, but their effects are enhanced primarily by binding of inhibitory FcγRs. 4. Agonistic nonligand blocking antibodies increase the influx of CD8+ T cells and reduce the number of Tregs in the tumor. In addition, they reduce the number of TAMs in the tumor, thus altering both the T cell and myeloid cell composition of the tumor. 5. In human tumors, T cells express TNFR2. 6. In a human xenograft model where tumor TNFR2 expression is mimicked in T cells, the agonist ligand non-blocking antibody 1-F02 increases the numbers of T cells.

[0233] Example 6 - Agonist ligand non-blocking antibodies do not induce large amounts of proinflammatory cytokines (see also Figures 18-19 and the above descriptions of these figures). The release of large amounts of proinflammatory cytokines is one of the possible side effects of immunomodulatory antibodies used to treat patients. Here, therefore, two different methods were used to measure the cytokine release induced by agonistic ligand non-blocking antibodies. The first is based on antibody stimulation in in vitro cultures, and the second is based on xenotransplantation of human immune cells into immunodeficient mice. In vitro, it has been shown that the culture setting has a significant impact on the release of cytokines (Vessillier et al., J Immunol Methods. 2015 Sep;424:43-52). To account for the methodological differences, three different in vitro culture settings were used, following a recent publication:

[0234] For high density cell culture (HDC) cytokine release assays (CRAs), PBMCs were cultured at 1 × 10 in serum-free CTL-test medium (Cell Technology Limited) supplemented with 2 mM glutamine, 1 mM pyruvate, and 100 IU / ml penicillin and streptomycin. 7 2 ml of cell culture was plated in a 12-well plate. After 48 hours, 10 μg / ml of antibody was added to 1x10 cells in a 96-well flat-bottom plate. 5 The cells were added to the pre-incubated PBMCs and incubated for 24 hours.

[0235] PBMC solid-phase (SP) CRA was performed by coating wells of a 96-well plate with 1 μg / ml of antibody for 1 h. After washing the plate with PBS, 1x10 5 PBMCs were added per well and incubated for 48 hours.

[0236] Cytokine release was also measured after 200 μl of whole blood was stimulated with 5 μg / ml of antibody for 48 hours.

[0237] At the end of the incubation period, plates were centrifuged and culture supernatants were harvested and stored at −20° C. Concentrations of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-8 and TNF-α were measured using custom-made MSD plates according to the manufacturer's instructions (Meso Scale Discovery, USA).

[0238] In summary, the ligand non-blocking agonist antibodies only induced significant cytokine release above IFN-γ in any of the in vitro settings (data not shown). The positive control antibodies alemtuzumab and OKT3 induced cytokines, most notably IFN-γ. As seen in Figure 18, the non-blocking agonist antibodies did not induce IFN-γ in two of the three in vitro settings. Additionally, antibody 1-F02 did not induce IFN-γ in any of the in vitro settings.

[0239] PBMC-NOG Tolerance Model To investigate the tolerability of the non-ligand blocking agonist anti-human TNFR2 mAb 1-F02, in vivo cytokine release was analyzed in the PBMC-NOG model as described below.

[0240] Mice were bred and maintained in a local facility in accordance with Home Office guidelines. Eight-week-old female NOG mice were supplied by Taconic (Bomholt, Denmark) and maintained in a local animal facility. For the PBMC-NOG (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and washed, after which the cells were cultured at 125x10 6 The cells were resuspended in sterile PBS at 25x10 cells / ml. 6 200 μl of cell suspension, corresponding to cells / mouse, were injected intravenously. Two weeks after injection, blood samples were taken to analyze the level of "humanization", meaning the amount of human cells in the blood of NOG mice. Mice were considered humanized if the blood consisted of about 40% human T cells. Mice were then treated with 10 μg of either Yervoy, anti-CD3 (OKT-3), 1-F02 or isotype control mAb. Body temperature was measured before and 1 hour after antibody injection (Figure 19A). As can be seen in Figure 19A, the positive control antibody OKT3 induced a dramatic decrease in body temperature, as previously published and in accordance with the toxicity seen in clinical practice with this antibody. In contrast, 1-F02 had no effect on body temperature at all. Five hours after injection of the antibodies, the experiment was terminated and blood was taken for analysis of cytokine release (MSD). The cytokines measured were human IFN-γ, TNF-α, IL-6 and IL1β. Of these, IFN-γ and TNF-α were reliably quantified at high levels. As seen in Figure 19B and C, the positive control antibody OKT3 induced both significant IFN-γ and TNF-α release (in line with the toxicity seen in clinical practice with this antibody), whereas 1-F02-treated mice had no significant IFN-γ release. However, there was a trend toward increased TNF-α release, although not as significant or dramatic as with OKT3.

[0241] In summary, Example 6 shows that TNFR2 ligand non-blocking agonist antibodies, exemplified herein by the antibody designated 1-F02, do not induce substantial levels of cytokine release as measured by several previously published methods, which is a limiting factor for some immunomodulatory antibodies, indicating an acceptable safety profile in this regard.

[0242] Example 7 - Epitopes of TNFR2-targeting antibodies generated Knockout of domain constructs In the first set of experiments, we used DNA constructs encoding various mutants of TNFR2 lacking one or more of the four extracellular domains listed in Table 9. In the second set of experiments, we used DNA constructs encoding mutants of TNFR2 in which different parts of domain 3 were replaced with the corresponding mouse parts, as listed in Table 10. The latter is possible because none of the antibodies cross-react with mouse TNFR. In both cases, constructs were purchased from GeneArt (ThermoFisher). Constructs were cloned into expression vectors containing a CMV promoter and an OriP origin of plasmid replication and were transiently expressed in suspension-adapted HEK293-EBNA cells. [Table 9] [Table 10]

[0243] Flow cytometry-based binding analysis HEK-293-E cells were transfected with the respective cDNA plasmids of TNFR2 mutants using Lipofectamin2000. 48 hours after transfection, cells were harvested and stained with the indicated antibodies for 30 minutes. After two washes with PBS, surface-bound antibodies were stained with a secondary anti-IgG conjugated to APC. Cells were washed and stained for live / dead before flow cytometry analysis on a BD-Verse flow cytometer.

[0244] Flow cytometry-based binding experiments on transfected HEK293 cells clearly showed that domains 1 and 2 did not affect (domain 1) or only slightly affected (domain 2) the binding of the antibodies to these cells. As a positive control, a polyclonal anti-human TNFR2 antibody was used. The positive control antibody showed high binding to all constructs tested, whereas the negative antibody showed no binding (Figure 20). All antibodies tested showed a complete lack of binding to TNFR2 lacking domain 3. Similarly, most antibodies were unable to bind to TNFR2 when domain 4 was missing. All antagonist antibodies (1H10, 4H02 and 5B08) showed a dramatic reduction in binding to TNFR2Δ4 by more than 50% compared to binding to TNFR2Δ1 and TNFR2Δ2. Similarly, removal of the two domains from TNFR2 clearly demonstrated that the absence of domains 3 or 4 largely abolished binding of all antibodies tested to TNFR2, except for the agonist antibody 1F06, and the absence of domain 4 abolished binding of the agonist antibodies and greatly reduced binding of the antagonist antibodies (Figures 20E and F).

[0245] Binding to mouse-human chimeric TNFR2 To further narrow down the binding site and define the epitope, a portion of human TNFR2 domain 3 was replaced with the corresponding mouse sequence. Since all antibodies show little cross-reactivity to mouse TNFR2, the loss of binding to a particular construct allows the purification of the binding epitope. Figure 21 shows various mouse-human chimeric TNFR2 constructs. Four different substitutions were made, exchanging either 14 (m1), 12 (m2), 10 (m3) or 16 (m4) amino acids from the human sequence with the corresponding mouse sequence. The other three domains (1, 2, 4) contain only human sequences.

[0246] These constructs (TNFR2 domains 1-4 with 3 mutations) were then transfected into HEK293 cells and antibody binding was tested using a flow cytometry approach. As positive controls, polyclonal antibodies against mouse TNFR2 and human TNFR2 were used. As expected, due to sequence similarity, both polyclonal control antibodies showed significant cross-reactivity, recognizing both human and mouse TNFR2. Clearly, matching the antibodies to the intended targets gave the best signals.

[0247] Our monoclonal antibodies showed strong binding to human TNFR2 but no or little binding to mouse TNFR2 (left panel in Figure 22). Similar binding with little reduction was observed for all clones to the hTNFR2m1 construct with mutations at aa 119-132, indicating that none of the antibodies bind to an epitope within that region. However, mutations at aa 134-144 (hTNFR2m2 construct) completely abolished binding of half of the antibodies tested, corresponding to antagonist antibodies 1-H10, 4-H02, 5-B08, indicating that the antibodies bind at least partially within this region. 1-G10 is a partial blocker and is also strongly affected by this substitution. Of note, agonist antibodies (1-F02, 1-F06 and 4-E08) retained binding using construct 2, strongly suggesting a different epitope compared to the antagonist antibodies. Interestingly, all antibodies lost binding to the hTNFR2m3 construct, which has mutations at aa 151-160, indicating that all antibodies, both agonist and antagonist, have partial epitopes within their sequence. Testing a slightly larger construct, hTNFR2m4, which has mutations at aa 130-144, showed similar binding to construct hTNFR2m2.

[0248] Conclusions about epitope binding Grouping the antibodies by their functional role, the agonist antibodies (1-F02, 1-F06, and 4-E08) appear to bind to the very distal C-terminal part of domain 3 encompassing aa 151-160 and likely extend into a larger part of domain 4, whereas the epitopes of the antagonists (1-H10, 5-B08, and 4-H02) are shifted towards the center of domain 3 encompassing aa 134-160 and likely cover a smaller part of domain 4. However, their epitopes nevertheless appear to overlap to some extent.

[0249] None of the antibodies bind to the N-terminal portion of domain 3, aa 119 to 134. The binding site for domain 4 is likely for all antibodies but has not been fully identified.

Claims

1. An agonistic antibody molecule that specifically binds to TNFR2 on a target cell, comprising: agonistic antibody molecule, wherein the antibody molecule does not block TNF-α ligand binding to TNFR2 and has intrinsic agonistic activity, (i) a variable heavy chain (VH) comprising SEQ ID NO:1 as CDR1, SEQ ID NO:2 as CDR2, and SEQ ID NO:3 as CDR3, and a variable light chain (VL) comprising SEQ ID NO:4 as CDR1, SEQ ID NO:5 as CDR2, and SEQ ID NO:6 as CDR3; or (ii) a variable heavy chain (VH) comprising SEQ ID NO: 97 as CDR1, SEQ ID NO: 98 as CDR2, and SEQ ID NO: 99 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 100 as CDR1, SEQ ID NO: 101 as CDR2, and SEQ ID NO: 102 as CDR3; or (iii) a variable heavy chain (VH) comprising SEQ ID NO:9 as CDR1, SEQ ID NO:10 as CDR2, and SEQ ID NO:11 as CDR3, and a variable light chain (VL) comprising SEQ ID NO:12 as CDR1, SEQ ID NO:13 as CDR2, and SEQ ID NO:14 as CDR3; or (iv) a variable heavy chain (VH) comprising SEQ ID NO: 17 as CDR1, SEQ ID NO: 18 as CDR2, and SEQ ID NO: 19 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 20 as CDR1, SEQ ID NO: 21 as CDR2, and SEQ ID NO: 22 as CDR3; or (v) a variable heavy chain (VH) comprising SEQ ID NO:25 as CDR1, SEQ ID NO:26 as CDR2, and SEQ ID NO:27 as CDR3, and a variable light chain (VL) comprising SEQ ID NO:28 as CDR1, SEQ ID NO:29 as CDR2, and SEQ ID NO:30 as CDR3; or (vi) a variable heavy chain (VH) comprising SEQ ID NO: 33 as CDR1, SEQ ID NO: 34 as CDR2, and SEQ ID NO: 35 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 36 as CDR1, SEQ ID NO: 37 as CDR2, and SEQ ID NO: 38 as CDR3; or (vii) a variable heavy chain (VH) comprising SEQ ID NO: 41 as CDR1, SEQ ID NO: 42 as CDR2, and SEQ ID NO: 43 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 44 as CDR1, SEQ ID NO: 45 as CDR2, and SEQ ID NO: 46 as CDR3; or (viii) a variable heavy chain (VH) comprising SEQ ID NO: 49 as CDR1, SEQ ID NO: 50 as CDR2, and SEQ ID NO: 51 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 52 as CDR1, SEQ ID NO: 53 as CDR2, and SEQ ID NO: 54 as CDR3; or (ix) a variable heavy chain (VH) comprising SEQ ID NO:57 as CDR1, SEQ ID NO:58 as CDR2, and SEQ ID NO:59 as CDR3, and a variable light chain (VL) comprising SEQ ID NO:60 as CDR1, SEQ ID NO:61 as CDR2, and SEQ ID NO:62 as CDR3; or (x) a variable heavy chain (VH) comprising SEQ ID NO:65 as CDR1, SEQ ID NO:66 as CDR2, and SEQ ID NO:67 as CDR3, and a variable light chain (VL) comprising SEQ ID NO:68 as CDR1, SEQ ID NO:69 as CDR2, and SEQ ID NO:70 as CDR3; or (xi) a variable heavy chain (VH) comprising SEQ ID NO: 73 as CDR1, SEQ ID NO: 74 as CDR2, and SEQ ID NO: 75 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 76 as CDR1, SEQ ID NO: 77 as CDR2, and SEQ ID NO: 78 as CDR3; or (xii) a variable heavy chain (VH) comprising SEQ ID NO: 81 as CDR1, SEQ ID NO: 82 as CDR2, and SEQ ID NO: 83 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 84 as CDR1, SEQ ID NO: 85 as CDR2, and SEQ ID NO: 86 as CDR3, or (xiii) A variable heavy chain (VH) comprising SEQ ID NO: 89 as CDR1, SEQ ID NO: 90 as CDR2, and SEQ ID NO: 91 as CDR3, and a variable light chain (VL) comprising SEQ ID NO: 92 as CDR1, SEQ ID NO: 93 as CDR2, and SEQ ID NO: 94 as CDR3: The agonist antibody molecule comprising:

2. An antibody molecule as described in claim 1, comprising a variable heavy chain (VH) having SEQ ID NO:1 as CDR1, SEQ ID NO:2 as CDR2, and SEQ ID NO:3 as CDR3, and a variable light chain (VL) having SEQ ID NO:4 as CDR1, SEQ ID NO:5 as CDR2, and SEQ ID NO:6 as CDR3.

3. The antibody molecule may be a full size antibody, a chimeric antibody, a bivalent or multivalent antibody molecule including a single chain antibody, a Fab, an Fv, an scFv, an Fab' and / or an (Fab') ２ and antigen-binding fragments thereof.

4. The antibody molecule of any one of claims 1 to 3, wherein the antibody molecule also binds to an Fcγ receptor.

5. The antibody molecule of any one of claims 1 to 4, wherein the antibody molecule binds with higher affinity to inhibitory Fcγ receptors than to activating Fcγ receptors.

6. The antibody molecule of any one of claims 1 to 5, wherein the binding of said antibody molecule to TNFR2 results in a change in the number and / or frequency of TNFR2 expressing cells in diseased tissue.

7. The antibody molecule of any one of claims 1 to 6, wherein the binding of said antibody molecule to TNFR2 results in infiltration of T cells and / or myeloid cells into diseased tissue and / or a change in the composition of T cells and / or myeloid cells in diseased tissue.

8. The antibody molecule of any one of claims 1 to 7, which binds to human TNFR2 (hTNFR2) and / or to cynomolgus monkey TNFR2 (cmTNFR2).

9. An antibody molecule described in any one of claims 1 to 8, wherein the antibody molecule is an IgG antibody molecule.

10. The antibody molecule described in claim 9, wherein the antibody molecule is an IgG2 antibody molecule.

11. An antibody molecule described in any one of claims 1 to 10, wherein the antibody molecule is a humanized antibody molecule.

12. The antibody molecule of any one of claims 1 to 11, wherein the antibody molecule is a monoclonal antibody.

13. The antibody molecule of any one of claims 1 to 12, wherein the antibody molecule does not specifically bind to an epitope comprising or consisting of the sequence KCSPG.

14. The antibody molecule (i) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 7, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 8; or (ii) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 103, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 104; or (iii) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 15, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 16; or (iv) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 23, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 24; or (v) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 31, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 32; or (vi) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 39, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 40; or (vii) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 47, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 48; or (viii) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 55, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 56; or (ix) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 63, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 64; or (x) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 71, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 72; or (xi) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 79, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 80; or (xii) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 87, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 88; or (xiii) a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 95, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO: 96: The antibody molecule of any one of claims 1 to 13, comprising

15. An antibody molecule described in any one of claims 1 to 14, comprising a variable heavy chain (VH) amino acid sequence represented by SEQ ID NO: 7, and a variable light chain (VL) amino acid sequence represented by SEQ ID NO:

8.

16. An isolated polynucleotide molecule encoding an antibody molecule according to any one of claims 1 to 15.

17. A plasmid comprising the polynucleotide molecule of claim 16.

18. 18. A virus comprising the polynucleotide molecule of claim 16 or the plasmid of claim 17.

19. The virus of claim 18, further comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor.

20. 20. A cell comprising a polynucleotide molecule according to claim 16, or a plasmid according to claim 17, or a virus according to claim 18 or 19.

21. An antibody molecule according to any one of claims 1 to 15, a polynucleotide molecule according to claim 16, a plasmid according to claim 17, a virus according to claim 18 or 19 and / or a cell according to claim 20 for use in medicine.

22. An antibody molecule according to any one of claims 1 to 15, a polynucleotide molecule according to claim 16, a plasmid according to claim 17, a virus according to claim 18 or 19 and / or a cell according to claim 20 for use in the treatment of cancer or a chronic inflammatory disease.

23. 23. The antibody molecule, polynucleotide molecule, plasmid, virus and / or cell for use according to claim 22, wherein the patient to be treated is a patient with high TNFR2 expression in diseased tissue.

24. In the treatment of cancer, an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; a plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and / or A cell comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. An antibody molecule according to any one of claims 1 to 15, a polynucleotide molecule according to claim 16, a plasmid according to claim 17, a virus according to claim 18 and / or a cell according to claim 20 for use in combination with

25. Use of an antibody molecule according to any one of claims 1 to 15, a polynucleotide molecule according to claim 16, a plasmid according to claim 17, a virus according to claim 18 and / or a cell according to claim 20 for the manufacture of a pharmaceutical composition for use in the treatment of cancer or a chronic inflammatory disease.

26. 26. The use according to claim 25, wherein the pharmaceutical composition is for use in the treatment of cancer or chronic inflammatory disease in patients with high TNFR2 expression in diseased tissue.

27. The pharmaceutical composition comprises: an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; a plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and / or A cell comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor.

27. Use for the manufacture of a pharmaceutical composition for use in the treatment of cancer according to claim 25 or 26, administered in combination with

28. A pharmaceutical composition comprising or consisting of an antibody molecule according to any one of claims 1 to 15, a polynucleotide molecule according to claim 16, a plasmid according to claim 17, a virus according to claim 18 and / or a cell according to claim 20, and optionally a pharma- ceutically acceptable diluent, carrier, vehicle and / or excipient.

29. 29. The pharmaceutical composition of claim 28 for use in the treatment of cancer or chronic inflammatory disease.

30. In the treatment of cancer, an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; A plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and / or A cell comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor.

30. The pharmaceutical composition of claim 29, for use in combination with a pharmaceutical composition comprising:

31. 25. The antibody molecule for use according to claim 24, the polynucleotide molecule for use according to claim 24, the plasmid for use according to claim 24, the virus for use according to claim 19, the virus for use according to claim 24, the cell for use according to claim 24, the use according to claim 27, or the pharmaceutical composition according to claim 30, wherein the checkpoint inhibitor is PD-1.

32. 25. The antibody molecule for use according to claim 24, the polynucleotide molecule for use according to claim 24, the plasmid for use according to claim 24, the virus for use according to claim 19, the virus for use according to claim 24, the cell for use according to claim 24, the use according to claim 27, or the pharmaceutical composition according to claim 30, wherein the checkpoint inhibitor is PD-L1.

33. 32. The antibody molecule for use according to claim 22, 23, 24, 31 or 32, the polynucleotide molecule for use according to claim 22, 23, 24, 31 or 32, the plasmid for use according to claim 22, 23, 24, 31 or 32, the virus for use according to claim 22, 23, 24, 31 or 32, the cell for use according to claim 22, 23, 24, 31 or 32, the use according to claim 25, 26, 27, 31 or 32, or the pharmaceutical composition according to claim 29, 30, 31 or 32, wherein the cancer is a solid cancer.

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