Novel agonistic Anti-TNFR2 antibody molecules

Abstract

To provide novel antagonistic antibody molecules that specifically bind to TNFR2 on a target cell and thereby block TNF-α binding to TNFR2 and block TNFR2 signaling.SOLUTION: Described are novel antagonistic antibody molecules that specifically bind to TNFR2 on a target cell and thereby block TNF-α binding to TNFR2 and block TNFR2 signaling, where the antibody molecules also bind to Fc receptors via the Fc region. Also described is the use of such antibody molecules in treatment of cancer or infections caused by intracellular pathogens.SELECTED DRAWING: Figure 5-1

Description

[Technical field]

[0001] The present invention relates to novel antagonistic antibody molecules that specifically bind to tumor necrosis factor receptor 2 (TNFR2) on target cells, thereby blocking the binding of the ligand TNF-α to TNFR2 and also blocking TNFR2 signaling, which also bind to Fc receptors via the Fc region. The present invention also relates to its use in medicine, such as the treatment of cancer or infections caused by intracellular pathogens. [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 described as being highly expressed 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 reg Tregs, 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, 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 WO2014 / 124134 discloses the use of TNFR2 agonists, such as agonist anti-TNFR2 antibodies and / or NF-κB activators, for the in vitro production of Treg-enriched compositions. The compositions are said to be useful for treating immune disorders or infectious diseases in patients. WO2014 / 124134 further discloses TNFR2 antagonist antibodies capable of binding to one or two epitopes of TNFR2. The first of these epitopes included the sequence QTAQMCCSKCSPGQHAKVFC, and the second epitope included one specific amino acid at one specific position in the amino acid sequence of human TNFR. This second epitope may include the sequence RLCAPLRKCRPGF. Such TNFR2 antagonists are said to be useful for producing lymphocyte-enriched and Treg-depleted compositions. In the issued U.S. Patent No. 9,821,010 derived from this PCT application, antagonist antibodies are identified as selectively binding to an epitope within the sequence KQEGCRLCAPLRKCRPGFGV, such as an epitope containing the sequence RLCAPLRKCRPGF. TNFR2 antagonists, and compositions made using the TNFR2 antagonists, are said to be useful in the treatment of proliferative disorders, such as cancer or infectious diseases.

[0008] WO2016 / 187068 discloses antibodies capable of antagonizing tumor necrosis factor receptor superfamily members such as TNFR2. The antibodies are said to be useful for regulating Tregs, such as in immunotherapy for the treatment of proliferative disorders and infectious diseases. In particular, WO2016 / 187068 discloses antagonist TNFR2 antibodies that bind to specific epitopes of human TNFR2, and presents some specific CDR sequences of such antibodies. The data in WO2016 / 187068 are said to indicate that specific binding of the Fab region of antagonist TNFR2 antibodies to TNFR2, rather than non-specific binding of the Fc region of these antibodies, is likely responsible for regulating Treg cell proliferation.

[0009] 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.

[0010] 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.

[0011] WO2017 / 197331 discloses antagonist TNFR2 antibodies comprising a complementarity determining region heavy chain 3 having a specific sequence, and discusses reducing or inhibiting the proliferation of Tregs and / or promoting the proliferation of T effector cells.

[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 crosslink 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 interaction with Fcγ receptors.Specifically, how antibody immune complexes regulate the activation of immune cells is determined by the relative involvement of activating Fcγ receptors and inhibitory Fcγ receptors.Different antibody isotypes bind to activating Fcγ receptors and inhibitory Fcγ receptors with different affinities, resulting in different A:I ratios (activating:inhibitory 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 by 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, which showed 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, which have potent therapeutic activity and 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 (ligand)-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 first group and the antagonist-blocking TNFR2 antibodies that constitute the present invention. This second group of anti-TNFR2 antibodies is not dependent 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 first group of anti-TNFR2 antibodies, namely antagonistic antibody molecules that specifically bind to TNFR2, thereby blocking TNF-α binding to TNFR2 and also blocking TNFR2 signaling. These antibody molecules also have an Fc region that binds to Fc receptors, which is useful for providing FcγR-dependent elimination or functional regulation of TNFR2 positive cells, such as depletion of Tregs or regulation of tumor-associated macrophages.

[0023] Agonistic antibodies belonging to the second group are used in the following examples for comparison with the antagonistic 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] The present invention relates to antagonist antibody molecules that specifically bind to TNFR2 on target cells, thereby blocking the binding of TNF-α to TNFR2 and blocking TNFR2 signaling, which antibody molecules also bind to Fcγ receptors via their Fc region.

[0025] The present invention also relates to specific examples of such novel antagonist-blocking anti-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 one of the above-mentioned nucleotide sequences, or one of the above-mentioned plasmids, or 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 the above antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for use in the treatment of cancer or an infection caused by an intracellular pathogen.

[0032] The present invention also relates to the above antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for use in the treatment of cancer or an infection caused by an intracellular pathogen.

[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 infections caused by intracellular pathogens.

[0034] The present invention also relates to a method for treating cancer or an infection caused by an intracellular pathogen in a patient, comprising administering to the patient 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] The present invention relates to antagonist antibody molecules that specifically bind to TNFR2 on target cells, thereby blocking the binding of TNF-α to TNFR2 and blocking TNFR2 signaling, which antibody molecules also bind to Fcγ receptors via their Fc region.

[0037] The antagonistic antibody molecules disclosed herein block TNF-α binding to TNFR2 and TNFR2 signal transduction. By antagonistic antibody molecules blocking TNF-α binding to TNFR2, it is meant herein that antibody molecules that bind to receptor TNFR2 prevent the binding of ligand TNF-α to the same receptor. This is shown in more detail in Example 3. By antagonistic antibody molecules disclosed herein block TNFR2 signal transduction, it is meant that they block TNFR2-mediated cell activation. 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 NFkappaB (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.

[0038] The 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 the target, not to non-target molecules. "Antibody molecule specifically binds to TNFR2" or "TNFR2-specific antibody molecule" refers to 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 pre-incubating the same cells with a commercially available polyclonal TNFR2 antibody reagent. This shows that non-specific binding can be detected when TNFR2 is masked with a polyclonal reagent. This is shown in Example 2.

[0039] 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.

[0040] Methods for evaluating 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 evaluate the binding of antibody to target and / or the binding of Fc region of antibody to Fc receptor, and the relative strength, or specificity, or inhibition, prevention, or reduction of their interactions. Examples of methods that can be used to evaluate protein binding include, for example, immunoassay, BIAcore, Western blot, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FACS) (for a discussion of antibody specificity, see Fundamental Immunology, 2nd Edition, Raven Press, New York, pp. 332-336 (1989)).

[0041] The target cells expressing TNFR2 to which the blocking antibodies bind according to the present invention include immune cells and / or tumor cells as described above and below. The effect of the binding of the antagonistic antibody molecule according to the present invention to TNFR2 can be a change in the composition of cells in the diseased tissue. This change in composition can occur through a change in the number and / or frequency of TNFR2 expressing cells in the diseased tissue. For example, the effect in cancer can be an increase in the number of intratumoral T cells, an increase in the number of CD8 + Increase in the T cell / Treg ratio (i.e., CD8 + In some embodiments, these effects include a reduction in tissue Treg numbers, CD8 T cell numbers, and / or an increase in myeloid cell numbers associated with anti-tumor as opposed to pro-tumor characteristics. This is shown in Example 5. In some embodiments, these effects include a reduction in tissue Treg numbers, CD8 + This results in an increase in the number of effector T cells and a change in the composition of tissue myeloid cell subsets. The regulation of TNFR2 expressing cells in tissues after in vivo treatment with a surrogate (3-F10) of the antagonistic antibody molecule according to the invention is shown in detail in Example 5. To test a similar effect of the human antagonistic antibody molecule according to the invention, a similar experiment can be performed in mice that are deleted for mouse TNFR2 and transgenic for human TNFR2. Alternatively, and preferably, although it requires considerable time and resources, animals transgenic for human TNFR2 and human FcγR can be generated in a similar manner compared to that previously described for CD40 and hFcγR (Dahan et al. 2016. 'Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcgammaR Engagement', Cancer Cell, 29: 820-31). Such humanized TNFR2 FcγR mice can then be used in turn to test for similar effects of human antagonist antibody molecules according to the invention.

[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 infected with an intracellular pathogen.

[0043] To determine whether an antibody molecule blocks ligand binding to TNFR2, 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. The blocking antibody prevents the ligand TNF-α from binding to the immobilized receptor TNFR2. This is shown and explained in more detail in Example 3 below.

[0044] The blocking antibody molecules according to the present invention are full blockers and are further capable of antagonizing TNFR2 signaling.

[0045] A full blocker is defined herein as an antibody molecule that reduces TNF-α binding to TNFR2 by more than 98%, i.e. up to 100%, compared to TNF-α binding in the presence of only an isotype control antibody molecule. An isotype control antibody is an antibody raised against a protein or other structure that is not present in any form in the assay under study. An isotype control ideally has 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 example described herein, the isotype control has the same framework, the same Fc portion, and is specific for fluorescein isothiocyanate (FITC). In some embodiments, a full blocker reduces TNF-α binding by more than 99.5%.

[0046] Other types of blockers are partial blockers and weak blockers. As used herein, a partial blocker is a blocker that inhibits TNF-α binding to TNFR2 by 60-98% (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94) compared to TNF-α binding in the presence of only an isotype control antibody molecule. , 95, 96, 97 or 98%, and all small numbers in between), and weak blocking agents are antibody molecules that reduce TNF-α binding to TNFR2 by less than 60%, for example 50-59.9% (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 59.9%, and all small numbers in between), compared to TNF-α binding in the presence of only an isotype control antibody molecule.

[0047] Conversely, a non-blocking TNFR2 antibody molecule is one 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, single-point ELISA or a dose-titration ELISA, as shown in Example 3 and Figures 6 and 7.

[0048] Partially blocking, weakly blocking and non-blocking antibodies are used in the examples for comparison with the antagonist blocking antibody molecules of the present invention.

[0049] Several properties and characteristics may underlie and (co)determine the biological activity of an antibody. In addition to the ability to block ligand binding to the receptor, such important properties include the ability of the antibody to modulate, i.e., agonize or antagonize, receptor signaling, and the dependence of the antibody on FcγR interactions to confer therapeutic activity.

[0050] First, the ability of fully blocking, partially blocking and non-blocking antibodies to modulate TNFR2 signaling was characterized. Two extremes were identified.

[0051] At the first extreme, we have identified antibodies that completely block ligand binding to TNFR2, block TNF-α-induced TNFR2 signaling, and bind to endogenously expressed TNFR2 but do not themselves induce signaling. This group of ligand-blocking antagonist antibodies forms the basis of the present invention.

[0052] At the other extreme, we have identified antibodies that do not block ligand binding to TNFR2, but which upon binding to TNFR2 agonize the receptor in cells in which they are endogenously expressed. This second group of antibodies constitutes a separate invention and is included herein for purposes of comparison.

[0053] 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.

[0054] To determine whether an antibody has agonist or antagonist activity, it is possible to use a natural killer (NK) cell assay 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 produced endogenously and is present at robust but suboptimal concentrations (approximately 20-100 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 enhances IFN-γ concentrations in this assay (Figure 8C), 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 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) As a result, in this experimental setup, the antagonist antibody prevents TNF-α-induced signaling in TNFR2-expressing cells and does not stimulate the TNFR2 receptor itself when it binds to the TNFR2 receptor. Specifically, the antagonist antibody in this assay inhibits IFN-γ release rather than increasing it when it binds to the NK cells. As shown in FIG. 8, the full blocking antibody described in the present invention reduced TNFR2 signaling rather than inducing it in this TNF-α-containing NK cell assay, resulting in a reduced amount of IFN-γ released. Thus, as shown in FIG. 8, Example 4, the antibody of the present invention can be classified as a ligand-blocking antagonist anti-TNFR2 antibody.Considering that basal TNF-α levels in the cultures are at least 20 pg / ml, using this assay, antagonist antibodies are defined as those that reduce IFN-γ release by >30% (e.g., 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%). 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.

[0055] Antagonist activity can also be demonstrated using IL-2-mediated activation of memory T cells. Here, activation is measured by the upregulation of T cell activation marker CD25. Using this assay, the addition of non-blocking agonist TNFR2 antibody further upregulates CD25 expression, while blocking antagonist TNFR2 antibody according to the present invention reduces CD25 expression compared to isotype control. This is true for the human antibody of the present invention as well as the mouse surrogate antibody, and is shown in Example 4.

[0056] In addition to binding to TNFR2 and thereby blocking TNF-α binding and signaling, the antibody molecule according to the present invention also binds to Fcγ receptors. The absolute dependence of anti-TNFR2 antibodies belonging to the TNF-α blocking antagonist group of antibodies of the present invention on FcγR interaction for therapeutic effect has been demonstrated in mouse cancer experimental models using antibody variants that either productively bind or do not productively bind FcγR binding. The absolute dependence of such ligand blocking antagonist antibodies for in vivo therapeutic activity and their preferential binding / engagement of activating FcγR over inhibitory FcγR for maximum in vivo therapeutic activity is shown in Example 5. The data demonstrating the FcγR-independent in vivo activity of agonist non-blocking anti-TNFR2 antibodies and their different preferential binding of inhibitory FcγR over activating FcγR for maximum therapeutic activity are included in this example only for comparison and control purposes. Taken together, our data show that several types of anti-TNFR2 antibodies can be generated. Furthermore, our data show that it is not trivial and could not be predicted which antibody variants will be most effective therapeutically, whether they depend on blocking agonist or antagonist (exogenous or endogenous) properties, and whether they are most effective in antibody formats that depend on FcγR-binding or are associated with preferential / strong binding to activating or inhibitory Fc gamma receptors.

[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 have different 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 show proper binding to human activating versus inhibitory FcγRs. The affinity and / or avidity of human antibody molecules for individual human Fcγ receptors can be determined using surface plasmon resonance (SPR). In some embodiments, blocking TNFR2 antibody molecules bind with higher affinity to activating Fcγ receptors than inhibitory Fcγ receptors. Higher affinity to activating Fcγ receptors than inhibitory Fcγ receptors implies variants that bind with higher affinity to activating Fcγ receptors, e.g., FcγRIIA, FcγRIIIA and / or FcγRI, compared to inhibitory Fcγ receptors.

[0058] In some embodiments, the antibody molecule is an IgG that can bind to an Fcγ receptor via the normal interactions between the Fc region of an antibody molecule and an Fcγ receptor.

[0059] In some embodiments, the antagonist blocking TNFR2 antibody molecule is human IgG1.It is well known that human IgG1 binds with high affinity to activating human FcγRI, and with lower and similar affinity to human activating Fcγ receptors FcγRIIA, FcγRIIIA, and to human inhibitory FcγRIIB.This has been demonstrated using surface plasmon resonance (SPR).

[0060] In some embodiments, the antagonist-blocking 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).

[0061] 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.

[0062] The Fcγ receptor to which the Fc region of the antagonist blocking antibody molecule of the present invention binds may be an Fcγ receptor expressing immune effector cells, as described above.

[0063] Binding of TNFR2-specific antibody molecules to TNFR2 surface receptors on target cells and co-binding of FcγR on the same cells or nearby immune effector cells can result in depletion or functional modulation of the TNFR2-positive target cells to which the antibody molecules bind. Cell removal, as used herein, refers to the removal, deletion or elimination of cells via physical clearance of the cells.

[0064] Depletion of cells can be achieved through ADCC, i.e. antibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular cytotoxicity, and / or ADCP, i.e. antibody-dependent cellular phagocytosis. This means that when the antibody molecule described herein is administered to a patient, such as a human, it specifically binds to TNFR2 expressed on the surface of cells, such as Tregs, and this binding results in depletion of cells. In general, high expressing cells are more effectively deleted compared to low expressing cells. As shown in Example 5, Figure 14, Tregs are the most highly expressed cells in the tumor environment.

[0065] ADCC is an immune mechanism by which Fc receptor-bearing effector cells can recognize and kill (i.e., deplete) antibody-coated target cells that express a tumor-derived antigen, in this case TNFR2, on their surface. ADCP is a similar mechanism, but kills (i.e., depletes) target cells by phagocytosis rather than cytotoxicity.

[0066] Furthermore, improved binding of the Fc region of an antibody molecule to Fcγ receptors may also improve depletion of target cells by Fc receptor-dependent killing via ADCC or ADCP, which is particularly relevant for antibody molecules with improved binding to activating Fcγ receptors.

[0067] That an antibody molecule has a depleting effect on TNFR2-positive cells means that, when administered to a patient, such as a human, such an antibody molecule specifically binds to TNFR2 expressed on the surface of TNFR2-positive cells, and this binding results in the depletion of such target cells.

[0068] The cells that are depleted can be several different cells, as explained above in relation to the discussion of what the target cells are. Generally, it is the cells with the highest expression of TNFR2 that are depleted. Other cells that also express TNFR2 but less highly may also be depleted, but only slightly compared to the cells with the highest expression of TNFR2.

[0069] As mentioned above, TNFR2 is highly expressed on Tregs found in tumors of various cancer patients, and in such patients, the antibody molecules of the present invention preferentially bind to and therefore deplete Tregs. Tregs are CD8 positive (CD8 + Treg depletion may, at least indirectly, suppress the proliferation, activation and cytotoxic capacity of other immune cells, such as CD8 + Increased cell proliferation, activation and possibly migration, thus intratumoral CD8 + This leads to an increase in the number of CD8 cells. + T cells are essential for immune-mediated clearance of tumor cells (McKinney et al. Curr Opin Immunol. 2016 Dec;43:74-80; Klebanoff et al. Immunol Rev. 2006 Jun;211:214-24; Alexander-Miller. Immunol Res. 2005;31(1):13-24). したがって、CD8+ Increasing the proliferation, activation and cytotoxic capacity of T cells would be of great benefit to cancer patients and may lead to the eradication of cancer.

[0070] CD8 + Increased proliferation, activation and cytotoxic capacity of T cells is also highly beneficial in the treatment of infections caused by intracellular pathogens. Antigens from intracellular pathogens are usually expressed by CD8 + The MHCI molecule is presented to help activate T cells. This allows CD8 + T cells recognize and lyse infected cells, destroying the pathogen.

[0071] Binding of TNFR2-specific antibody molecules and blockade of TNF-α signaling may also result in the functional modulation of cell phenotype, for example, modulation of tumor-promoting myeloid cells into myeloid cells with tumoricidal properties.

[0072] In some embodiments, TNFR2 positive cells are CD4 positive (CD4 + ) cells, i.e., cells that express CD4.

[0073] In some embodiments, the TNFR2 positive cells are CD4 + and FOXP3 + These cells are Tregs. + Although T cells also express TNFR2, as shown in FIG. 14 and Example 5, Tregs express significantly higher levels of TNFR2 than CD8 positive T cells. This suggests that Tregs are more sensitive to TNFR2 than CD8 positive T cells, which express less TNFR2. + are more susceptible to depletion than cells.

[0074] In some circumstances, TNFR2 is preferentially expressed on immune cells in the tumor microenvironment (tumor infiltrating cells, TILS).

[0075] In some embodiments, Tregs represent the cells with the highest expression of TNFR2 in the tumor microenvironment, and antibody molecules that specifically bind to TNFR2 (or anti-TNFR2 antibody molecules) have a Treg-depleting effect, as described in more detail below, e.g., in Example 5, and in connection with Figures 13 and 15.

[0076] In some embodiments, the TNFR2 positive cells are Tregs in solid tumors. Such Tregs have a very high expression of TNFR2, so administering an antibody molecule that specifically binds to TNFR2 preferentially results in the depletion of such Tregs.

[0077] To determine whether an antibody molecule has a depleting effect on TNFR2 positive cells as referred to herein, an in vivo test in the PBMC-NOG / SCID model can be used. This in vivo test is based on the combined use of PBMC mice and NOG / SCID mice, referred to herein as the PBMC-NOG / SCID model. Both NOG mice and SCID mice are known to those skilled in the art (Ito M et al, (2002) NOD / SCID / γc null The PBMC-NOG model is also known (Cox et al. “Antibody-mediated targeting of the Orai1 calcium channel inhibits T cell function”.PLoS One.2013 Dec 23;8(12):e82944.;and Sondergaard et al. “Human T cells depend on functional calcineurin, tumor necrosis factor-α and CD80 / CD86 for expansion and activation in mice.”Clin Exp Immunol.2013 May;172(2):300-10. The in vivo study in the PBMC-NOG / SCID model consists of nine sequential steps: 1) Isolate, wash, and resuspend human PBMCs (peripheral blood mononuclear cells) in sterile PBS. In some embodiments, PBMCs are at a concentration of 75×10 6 Cells / ml are resuspended in PBS. 2) Inject the NOG mice iv (intravenously) with an appropriate amount of the cell suspension from step 1), for example 200 μl. If you are injecting 200 μl, this will give you 15×10 6 Equivalent to cells / mouse. 3) At an appropriate time after injection, for example 2 weeks, isolate the spleen from the NOG mouse and make it into a single cell suspension. Optionally, take a small sample from the single cell suspension to determine the expression of TNFR2 on human T cells by FACS to confirm the expression of TNFR2. 4) Resuspending the cell suspension of step 3) in sterile PBS. In some embodiments, the cell suspension is at least 50×10 6 The remaining cell suspension is resuspended in sterile PBS at 1000 cells / ml. If the optional TNFR2 expression determination is included in step 3, the remaining cell suspension is resuspended in step 4. 5) Inject the SCID mice ip (intraperitoneally) with an appropriate amount of the suspension from step 4, for example 200 μl. If you are injecting 200 μl, this is 10×10 6 Equivalent to cells / mouse. 6) After injection of step 5), for an appropriate time, e.g., 1 hour, treat the SCID mice with an appropriate amount, e.g., 10 mg / kg, of either the antibody molecule to be tested, a positive control antibody (e.g., an anti-CD25 antibody known to deplete Tregs) or an isotype control monoclonal antibody. 7) Collecting the intraperitoneal fluid of the treated SCID mice at an appropriate time, for example 24 hours, after treatment in step 6). 8) Identifying and quantifying human T cell subsets by FACS using the markers CD45, CD4, CD8, CD25 and / or CD127. Human Tregs are CD4 + CD25 + CD127 低 / - It is well established that they can be distinguished as 9) comparing the results of identification and quantification of T cell subsets from mice treated with the tested antibody molecule with the results of identification and quantification of T cell subsets from mice treated with a positive control antibody and with the results of identification and quantification of T cell subsets from mice treated with an isotype control monoclonal antibody. A lower number of Tregs in the peritoneal fluid of mice treated with the tested antibody molecule compared to the number of Tregs in the peritoneal fluid of mice treated with the isotype control indicates that the antibody molecule has a depleting effect on TNFR2 positive Tregs.

[0078] This assay is presented in more detail in Example 5 below, in conjunction with FIG.

[0079] As mentioned above, other cells, such as cancer cells, may also express TNFR2. In some embodiments, the antibody molecule preferentially binds to TNFR2 expressed on cancer cells, which then directly results in the depletion of the cancer cells.

[0080] 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 region (VH) and three constant regions (CH1, CH2, CH3), and the light chain of an antibody molecule comprises one variable region (VL) and one constant region (CL). The variable regions (sometimes referred to as F) are VThe variable regions (collectively called the complementarity determining regions) bind to the antibody's target, or antigen. Each variable region contains three loops, called complementarity determining regions (CDRs), which are involved in target binding. The constant regions 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272:30937-44, where a series of hexapeptides derived from the CDRs were demonstrated to exhibit antigen-binding activity, and it was noted that synthetic peptides of complete single CDRs exhibited strong binding activity. Monnet et al., 1999, JBC, 274:3789-96, showed that various 12-mer peptides and associated framework regions have antigen-binding activity, and opined that CDR3-like peptides alone are capable of binding to antigens. Heap et al., 2005, J. Gen. Virol., 86:1791-1800, reported that "micro-antibodies" (molecules containing a single CDR) are capable of binding to antigens, and 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.

[0086] 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.

[0087] The antibody molecule may be a derivative of a full-length antibody or a fragment of such an antibody, provided that such derivative or fragment retains Fcγ receptor binding ability. 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.

[0088] 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 antibodies, antibody heavy chains, antibody heavy chain homodimers, antibody heavy chain heterodimers, and antibody light chain heterodimers, as well as functional fragments and derivatives thereof.

[0089] 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.

[0090] 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. In some embodiments, the antibody molecule is of an isotype that binds to an activating Fc receptor in an optimal manner. In some embodiments, the antibody molecule is an IgG1 antibody.

[0091] Those skilled in the art will recognize that mouse IgG2a and human IgG1 share the ability to bind activating Fcγ receptors and activate target cell deletion by activation of immune cells bearing activating Fcγ receptors, for example, by ADCP and ADCC. In some embodiments, the anti-TNFR2 antibody is a mouse or humanized mouse IgG2a antibody.

[0092] In some embodiments, the antibody molecule that specifically binds to TNFR2 is a human IgG2 antibody molecule.

[0093] In some embodiments, the anti-TNFR2 antibody is a murine antibody that is cross-reactive with human TNFR2.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] "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.

[0098] 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.

[0099] 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.

[0100] 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).

[0101] In some embodiments, the antibody molecule that specifically binds to TNFR2 is a human antibody.

[0102] 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.

[0103] 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.

[0104] In some embodiments, the anti-TNFR2 antibody is a monoclonal antibody.

[0105] In some embodiments, the anti-TNFR2 antibody is a polyclonal antibody.

[0106] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VH-CDR1 sequences listed in Table 1 below.

[0107] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VH-CDR2 sequences listed in Table 1 below.

[0108] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VH-CDR3 sequences listed in Table 1 below.

[0109] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VL-CDR1 sequences listed in Table 1 below.

[0110] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VL-CDR2 sequences listed in Table 1 below.

[0111] In some embodiments, an antibody molecule that specifically binds to TNFR2 comprises one of the VL-CDR3 sequences listed in Table 1 below.

[0112] 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, 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.

[0113] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule that comprises six CDRs having SEQ ID NOs: 1, 2, 3, 4, 5 and 6.

[0114] 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 and 23.

[0115] 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 and 24.

[0116] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VH having SEQ ID NO:7.

[0117] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule that comprises a VL having SEQ ID NO:8.

[0118] In some embodiments, the anti-TNFR2 antibody molecule comprises a VH having SEQ ID NO:7 and a VH having SEQ ID NO:8.

[0119] In some embodiments, the anti-TNFR2 antibody molecule comprises a CH having SEQ ID NO:217.

[0120] In some embodiments, the anti-TNFR2 antibody molecule comprises a CL having SEQ ID NO:218.

[0121] 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] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0122] To determine or demonstrate the characteristics of the antibody molecules of the present invention, they were compared to antibody molecules that do not block TNF-α ligand binding to TNFR2. Such antibodies are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0123] All sequences in Tables 1, 2 and 3 above are of human origin and are derived from the n-CoDeR® library, as described in detail in Example 1.

[0124] 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 4 below. [Table 4]

[0125] The first CH (SEQ ID NO:217) and first CL (SEQ ID NO:218) sequences in Table 4 above are of human origin. The second CH (SEQ ID NO:219) and third CH (SEQ ID NO:220) in Table 4 are both derived from mouse IgG2a, with the difference being that the third CH sequence (SEQ ID NO:220) contains the N297A mutation. The second CL sequence (SEQ ID NO:221) is derived from the mouse lambda light chain constant region. These mouse sequences are used in the surrogate antibody examples.

[0126] In some embodiments, the antibody molecule binds to human TNFR2 (hTNFR2).

[0127] 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.

[0128] In some embodiments, testing the functional activity of an antibody molecule in a relevant in vivo model in mice requires the use of a surrogate antibody. To ensure comparability between the effect of the antibody molecule in humans and the in vivo results of the 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.

[0129] In some embodiments, the antibody molecule does not specifically bind to an epitope of TNFR2 comprising or consisting of the sequence KCSPG.

[0130] In some embodiments, an 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 that can compete with an antibody molecule comprising a VH selected from the group consisting of SEQ ID NOs: 7, 15 and 23, and / or a VL selected from the group consisting of SEQ ID NOs: 8, 16 and 24.

[0131] 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.

[0132] For example, such a competing antibody molecule may be capable of inhibiting binding of an antagonist blocking antibody molecule described herein 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%, about 100% and / or may be capable of inhibiting the binding ability of an antibody described herein to prevent or reduce binding of TNFR2 to a specific target ligand TNF-α by at least about 10%, e.g., at least about 20%, 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%.

[0133] Competitive binding can be determined by methods known to those skilled in the art, such as enzyme linked immunosorbent assay (ELISA).

[0134] 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.

[0135] 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 antagonist-blocking TNFR-2 antibody molecules.

[0136] The antagonist 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.

[0137] The antagonist blocking antibody molecules, nucleotide sequences and / or pharmaceutical compositions may be used in the treatment of cancer, as discussed further below.

[0138] The antagonist blocking antibody molecules, nucleotide sequences and / or pharmaceutical compositions may be used in the treatment of infections caused by intracellular pathogens, as discussed further below.

[0139] The antagonist blocking antibody molecules and / or nucleotide sequences may be used in the manufacture of a pharmaceutical composition for use in the treatment of cancer.

[0140] The antagonist blocking antibody molecules and / or nucleotide sequences described above may be used in the preparation of a pharmaceutical composition for use in the treatment of infections caused by intracellular pathogens.

[0141] The antagonist blocking antibody molecules and / or pharmaceutical compositions may be used in a method of treating cancer in a patient, wherein a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the patient.

[0142] The antagonist blocking antibody molecules and / or pharmaceutical compositions described above may be used in a method for treating an infection caused by an intracellular pathogen in a patient, in which a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the patient.

[0143] In some embodiments related to the treatment of cancer, the cancer is a solid or leukemic 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 cancerous) or malignant (cancerous). Malignant solid tumors are referred to herein as solid cancers. Various types of solid tumors are named after the type of cells that form them. Examples of solid tumors or cancers are sarcomas, carcinomas, and lymphomas.

[0144] More specific examples of solid cancers are lung 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, Wilms' tumor, rhabdomyosarcoma, retinoblastoma, head and neck cancer, gastric cancer, lymphoma, and bone cancer.

[0145] 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.

[0146] In some embodiments relating to the treatment of infections caused by intracellular pathogens such as viruses or bacteria, specific examples of intracellular pathogens are Legionella pneumophila, R. rickettsia, Mycobacterium tuberculosis, Listeria monocytogenes, Salmonella spp, invasive Escherichia coli, Neisseria spp, Brucella spp, Shigella spp, influenza viruses, herpes viruses, hepatitis viruses, coxsackie viruses, Epstein-Barr virus or rhinoviruses.

[0147] In some embodiments of cancer treatment, the antagonist 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 antagonist 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. Antibodies against checkpoint inhibitors include 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 that target OX40, 41BB, OX40L, 41BBL, GITR, ICOS, DR3, DR4, DR5, CD40, CD27, RANK, HVEM, LIGHT and B7-H6. Alternatively, the antagonist-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 a costimulatory agonist. Alternatively, the antagonist-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 a costimulatory agonist. In some such embodiments, the antibody molecule that specifically binds to a checkpoint inhibitor is an anti-PD-1 antibody. PD-1 (or PD1) antibodies are primarily targeted to CD8 + It is believed to block PD-L1-mediated inhibitory signals in T cells, allowing increased T cell-mediated antitumor responses. Treg-depleting antagonist TNFR2 antibodies work by a different mechanism to increase antitumor responses. Thus, these treatments may synergize with each other. The same is true for other checkpoint inhibitors and agonistic co-stimulatory antibodies.

[0148] Additionally, the antagonist 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, , 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, etc.), antiangiogenic drugs (such as but not limited to bevacizumab, everolimus, lenalidomide, thalidomide, dibuaflibercept, etc.) or irradiation. Usually, all of the above anticancer drugs cause cancer cell death, which leads to neoantigen exposure and inflammation. When neoantigens are exposed and inflammatory cells influx into the tumor occurs, a synergistic effect of anticancer drugs can occur and antagonist ligand-blocking TNFR2 antibodies can be added to deplete Tregs, thereby further strengthening the immune system.

[0149] 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.

[0150] Thus, it is included that the antagonist 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.

[0151] The pharmaceutical compositions described herein may comprise, or in some embodiments consist of, antibody molecules, nucleotide sequences, plasmids, viruses or cells.

[0152] 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.

[0153] In some embodiments, the pharmaceutical composition may comprise a nucleotide sequence encoding a portion or a complete antibody molecule described herein integrated into a cellular or viral genome or viriome. And the pharmaceutical composition may comprise a cell or virus as a delivery vehicle for the antibody of the invention (or a delivery vehicle for the nucleotide sequence encoding the antibody of the 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.

[0154] In some embodiments, the present invention 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.

[0155] 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.

[0156] As an example, the nucleotide sequence encoding antibody 001-H10 may be as shown in Table 5. [Table 5]

[0157] Some oncolytic viruses have the ability to accept DNA inserts 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 et al. 2014. 'Oncolytic Poxviruses', Annu Rev Virol, 1:119-41; Bommareddy.et al 2018. 'Integrating oncolytic viruses in combination cancer immunotherapy', Nat Rev Immunol, 18:498-513). Full-length IgG antibodies are successfully integrated 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 et al. 2016. 'Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition', Oncoimmunology, 5: e1220467). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted upon cell infection (Marino et al. 2017. 'Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes', PLoS One, 12: e0177810).

[0158] 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.

[0159] The invention also includes other therapeutics or drug "forms" such as antibody drug conjugates, fusion proteins, and pharmaceutical compositions comprising such therapeutics.

[0160] 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.

[0161] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, and / or granules and / or tablets of the kind previously described.

[0162] 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.

[0163] 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.

[0164] 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).

[0165] 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, sugar, 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 generate pharma-ceutically acceptable salt forms of the agents according to the present 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.

[0166] 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.

[0167] The term "patient" (or subject), as used herein, refers to an animal, including a human, that has been diagnosed with a particular disease.

[0168] In some embodiments, a patient (or subject) is an animal, including a human, that has been diagnosed with cancer and / or exhibits symptoms of cancer.

[0169] In some embodiments, a patient (or subject) is an animal, including a human, that has been diagnosed with and / or exhibits symptoms of an infection caused by a pathogen.

[0170] 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.

[0171] 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.

[0172] "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.

[0173] 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.

[0174] Cancer treatments are often administered as a course of treatment, i.e., the therapeutic agents are 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.

[0175] "Currently undergoing treatment" includes when a patient is currently undergoing a course of treatment and / or is currently receiving a therapeutic agent and / or is currently receiving a course of therapeutic agent.

[0176] In some embodiments, the cancer treated according to the present invention is a solid tumor.

[0177] 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.

[0178] 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.

[0179] "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.

[0180] 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.

[0181] "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.

[0182] 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).

[0183] "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.

[0184] 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]

[0185] In the following examples, reference is made to the following figures:

[0186] [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 the mean fluorescence intensity (MFI) after subtraction of isotype control background (Figure 2A (full and partial blockers), Figure 2B (partial blockers), Figures C and D (non-blockers), Figure 2E (mouse full blockers (3-F10) and non-blockers (5-A05)). Human TNFR2 antibodies bind to in vitro activated CD4s 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 all antibodies could be blocked by the polyclonal TNFR2 antibody. [Figure 4]Figure 1 shows the 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] We demonstrate that all TNFr2-specific n-CoDeR® antibodies described herein 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. Activated cells were blocked with 40 μg / ml MR2-1 antibody (Figure 5A, black bar) or left for 30 min with PBS (Figure 5A, grey bar), 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 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 minutes. Cells were then analyzed by FACS. The MR2-1 antibody did not interfere with the binding of the TNFR2-specific n-CoDeR® antibody, and the n-CoDeR® antibody did not affect the binding of MR2-1 to activated cells, indicating that all n-CoDeR® antibodies bind to other domains of the TNFR2 protein than the MR2-1 antibody. [Figure 6]Figure 6 shows the ligand blocking activity of anti-human TNFR2 antibodies. A blocking ELISA was performed with n-CoDeR® mAb specific for hTNFR2 to evaluate 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% (shown 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 as non-blocking mAbs. [Figure 7] Figure 7 shows the ligand blocking activity of anti-mouse TNFR2 antibodies. A blocking ELISA was performed with n-CoDeR® mAb specific for mTNFR2 to evaluate 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% (shown by the dotted line) were then administered to further investigate the possibility of ligand blocking. 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. On this basis, antibodies 3-F10 and 5-A05 were selected to represent full blocking and non-blocking antibodies, respectively. [Figure 8]Classification of TNFR2-specific n-CoDeR® antibodies according to their ability to agonize / antagonize TNFR2 signaling and to block TNF-α binding to TNFR2. The ability of TNFR2-specific n-CoDeR® antibodies to enhance or reduce IFN-γ production was monitored using NK cells stimulated with IL-2 and IL-12 and plotted as a function of the antibody's ability to block TNF-α ligand binding to TNFR2 as described above. 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 (IFN-γ value of isotype control=1 in the figure) and is shown in FIG. 8A. Human antibodies that showed EC50 values ​​higher than 25 nM against in vitro activated CD4+ T cells were not included in the analysis. FIG. 8B: Human NK cells also produce TNF-α in these cultures (data show the average TNF-α levels of two donors). Cell culture supernatants were collected and the amount of IFN-γ produced was analyzed using MSD. Results are normalized to the isotype control. IFN-γ results are the average of three donors in two independent experiments. The results reveal two extreme groups characterized by 1) antibodies with full blocking and antagonist properties and 2) antibodies with agonistic non-blocking properties, respectively. Agonistic non-blocking antibodies are agonistic and enhance IFN-γ production from cytokine-stimulated NK cells, whereas blocking antibodies are antagonistic and suppress IFN-γ release. Figure 8C shows that IFN-γ release is dependent on TNF-α, as neutralization of soluble TNF-α reduces IFN-γ and addition of exogenous TNF-α increases IFN-γ. Figure 8D shows that addition of a blocking anti-TNF-α antibody neutralizes soluble TNF-α in a dose-dependent manner. At a dose of 1 μg / ml, no soluble TNF-α is detectable in the supernatant. [Figure 9]We show that TNFR2-specific n-CoDeR® antibodies, which are non-blocking agonists but not blocking antagonists, increase the percentage of CD25+ cells in the memory CD4+ T cell population. 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)) population 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]A shows that ligand-blocking antagonist antibodies have the most pronounced antitumor effects as mIgG2a, an isotype that preferentially binds to activating Fc receptors. 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 weekly with 10mg / kg antibody as indicated. Tumors were measured twice weekly 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 antagonist ligand-blocking antibody in FcγR-deficient Ig format (middle figure) and an agonist nonligand-blocking antibody (lower figure). The middle panel shows the same antibody in mouse IgG2a format that binds primarily to activating FcγRs, and the right panel shows the antibody in mouse IgG1 format that binds primarily to inhibitory FcγRIIb. For Figure 10B, surviving mice were followed for 70 days. As can be seen, blocking antagonist antibodies are most effective as tumor therapy in the IgG2a format, which binds primarily to activating FcγRs, and ineffective in formats deficient in FcγR binding. On the other hand, non-blocking agonist antibodies are most effective as tumor therapy in the IgG1 format, which binds preferentially to inhibitory FcγRs. In addition, agonist antibodies have an intrinsic, FcγR-independent antitumor effect, as seen using the N297A format. ***=p<0.001 (compared to isotype control calculated by log-rank Mantel-Cox test) [Figure 11]We show that ligand-blocking antagonist antibodies are effective as antitumor therapy in combination with anti-PD1. 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. The figure shows tumor growth curves for individual mice. Figure 11A: isotype control, Figure 11B: PD-1 targeting antibody, Figure 11C: 3-F10 antibody (surrogate antibody, ligand blocker, antagonist), Figure 11D: 3-F10 in combination with PD1. Tumors were measured twice weekly until they reached 15mm in diameter, after which mice were sacrificed. Figure 11E shows survival curves for the four different treatment groups. **=p<0.01, ***=p<0.001 compared to isotype control calculated by log-rank Mantel-Cox test. [Figure 12] We show that blocking antagonist antibodies are effective in combination with anti-PD-L1 as anti-tumor therapy. C57 / BL6 mice were injected subcutaneously with 1x106 MC38 cells. With an average tumor size of 5x5mm, mice were treated with isotype control antibody or 3F10 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. Figures show mean tumor growth + / - SEM, n=10 / group. *=<0.05, ***=p<0.001 (calculated using one-way ANOVA). [Figure 13] : C57 / BL6 mice were injected subcutaneously with 1x106 B16.F10 cells. Three days later, mice were treated intraperitoneally twice a week with 10 mg / kg of antibody as indicated. Tumors were measured twice a week until they reached a diameter of 15 mm, after which mice were sacrificed. Figure 13A: Isotype control, Figure 13B: 3-F10 antibody (surrogate antibody, ligand blocker, antagonist). Figure 13C 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 14]We show that the ligand-blocking antagonist surrogate antibody 3F10 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. Fig. 14A: Ligand-blocking antagonist surrogate antibody 3F10 causes depletion of Tregs, and Fig. 14B: Influx or proliferation of CD8+ T cells. This shifts the CD8+ / Treg T cell ratio as shown in Fig. 14C. Fig. 14D shows not only T cell counts, but also myeloid cell counts. Here, the number of tumor-associated macrophages (TAMs, defined as CD11b+F4 / 80+MHCII+, but both negative for Ly6G and Ly6C) is very significantly reduced. The non-ligand blocking agonist surrogate antibody 5A05 also modulates TAM numbers, but still significantly differently than the ligand blocking antibody 3F10. [Figure 15] We show that T cells from human tumors express similar levels of TNFR2 as T cells recovered from PBMC-reconstituted NOG mice. Briefly, NOG mice were injected intravenously with 15–20 × 106 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 human T cells grown and activated in vivo in NOG mice and those derived from human tumors. [Figure 16]We show that the ligand blocking antagonist antibody 1-H10 depletes Tregs in vivo in an FcγR-dependent manner. NOG mice were injected intravenously with 15-20×106 PBMC cells. 10-12 days later, spleens were removed from the mice, single cell suspensions were prepared, and injected intraperitoneally into SCID mice (10-15×106 cells / mouse). One hour later, mice were treated intraperitoneally with 10 mg / kg antibody, and 24 hours after antibody injection, intraperitoneal fluid was collected from the mice and cells in the fluid were analyzed using FACS. Figure 16A shows the average percentage of stained Tregs (defined as CD45+CD3+CD4+CD25+CD127 low / negative) from the human CD45+ population, demonstrating that Tregs are significantly depleted by blocking antibody 1-H10. Figure 16B shows the average percentage of CD8+ T cells among human CD45+, indicating that 1-H10 significantly increases the CD8+ T cell population. Figure 16C shows that the ratio of CD8+ T cells to Tregs is significantly increased by 1-H10. Data in Figures 16A-C are shown as the average of four different experiments, with each dot representing one mouse. Yervoy and commercial anti-CD25 antibodies were used as positive controls. All data were normalized to isotype controls, so in Figures 16A and B the isotype control is set to 100% and in Figure 16C it is set to 1%. Figure 16D shows another experiment in which the dependency of FcγR binding on Treg depletion was evaluated using the FcγR binding-deficient 1-H10 IgG1N297Q antibody (designated as 1-H10NQ in the figure). As can be seen, depletion is most effective with the wild type IgG1 format (1-H10) compared to the Fc deleted format (1-H10NQ). [Figure 17]It shows that antagonist ligand non-blocking TNFR2 antibodies do not induce cytokine release in vitro. IFN-γ release induced by various TNFR2-specific antibodies was measured in three different in vitro systems. As positive controls, anti-CD3 antibody = OKT3, 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 17A shows the results from a high-density cell culture. PBMCs were cultured at 1 x 107 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 17B shows a solid-phase in vitro culture performed by coating the wells of a 96-well plate with antibody before adding PBMCs. Again, both alemtuzumab and OKT3 induced significant IFN-γ release along with several TNFR2-specific antibodies. However, the full blocking antibody 1-H10 did not induce cytokine release above the isotype control antibody. Figure 17C shows whole blood stimulation with antibodies. Here, alemtuzumab induced significant IFN-γ release, whereas none of the TNFR2-specific antibodies induced it. [Figure 18] We show that antagonist ligand non-blocking TNFR2 antibodies do not induce cytokine release in vivo. NOG mice were intravenously injected with 25 x 106 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 h after injection (Figure 18A). 5 h after injection, the experiment was terminated and blood was analyzed for the content of IFN-γ (Figure 18B) or TNF-α (Figure 18C). ****=p<0.0001 and **=p<0.01 (calculated by one-way ANOVA). [Figure 19]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 19A and B), 3 and partially 4 completely abolish the interaction between the antibody and TNFR2 (Figures 19C and D). Similarly, the absence of domains 1+3 completely prevents binding of all antibodies (except 1F06) (Figure 19E), 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 19F). Dark grey indicates positive control and white indicates negative control antibody. [Figure 20] 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 21] Binding to wild-type human and mouse TNFR2 is shown (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

[0187] Certain non-limiting examples will now be described which illustrate certain aspects of the present invention.

[0188] In many examples, particularly in vivo examples, antibody 3-F10 is used. This is a murine antibody that is a surrogate antibody for the human antibody disclosed herein. This antibody was selected based on its ability to bind to murine TNFR2, its blocking of murine TNF-α ligand binding to TNFR2, and its antagonist activity in murine T cell activation assays as described in Example 4. In some examples, the 3-F10 antibody was tested and compared with various antibody formats associated with strong and preferential binding to activating Fc gamma receptors over inhibitory Fc gamma receptors (mIgG2a), strong and preferential binding to murine inhibitory FcγR (mIgG1), or lack of binding to murine FcγR (mIgG2aN297A).

[0189] In some examples, antibody 5-A05 is used. This is a mouse surrogate antibody for the human anti-TNFR2 non-blocking agonist antibody included herein for reference and comparison reasons. 5-A05 was selected based on its ability to bind mouse TNFR2, lack of blocking effect on mouse TNF-α ligand binding to TNFR2, and based on its antagonist activity in mouse T cell activation assay as described in Example 4. In some examples, 5-A05 antibody was tested and compared with various antibody formats related to strong and preferential binding to activating Fc gamma receptors over inhibitory Fc gamma receptors (mIgG2a), strong and preferential binding to mouse inhibitory FcγR over activating FcγR (mIgG1), or lack of binding to mouse Fcγ (N297A).

[0190] In some examples and figures, slightly different names for antibody clones are used, for example, clone 001-H10 may be abbreviated to 1-H10 or 1H10, and 005-B08 may be abbreviated to 5-B08 or 5B08.

[0191] 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 or mouse TNFR2.

[0192] 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.

[0193] 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).

[0194] 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.

[0195] 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.

[0196] The data presented in Table 6 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. 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 6 and FIG. 1E). [Table 6]

[0197] Example 2 - Antibody specificity (See also Figures 2-5 and their descriptions above.) CD4 + Isolation of T cells PBMCs from human buffy coats and whole blood from cynomolgus monkeys (M. fascicularis) 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).

[0198] 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) for 2-3 days at 37°C. 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 2A-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) for 2–3 days at 37 °C. 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 2E. EC50 values ​​of the titration curves were calculated in Microsoft Excel and are shown in Table 7. 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)).

[0199] 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 3, 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.

[0200] 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.

[0201] 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) or PBS (gray bars in Fig. 5). After 30 min of incubation, cells were immediately stained with TNFR2-specific n-CoDeR® antibody or polyclonal TNFR2 (pTNFR2) for 15 min. After incubation with APC-conjugated secondary anti-human IgG reagent (Jackson), cells were analyzed by flow cytometry (FACSVerse, BD). In Fig. 5B, activated CD4 + T cells were blocked with 40 μg / ml of TNFR2-specific n-CoDeR® antibody or 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 percentage of MR2-1+ cells was the same for unblocked and n-CoDeR® blocked cells (Figure 5B), and n-CoDeR® antibody binding was the same with or without MR2-1 blocking (Figure 5A). These data indicate that n-CoDeR® antibody binds to other epitopes of the TNFR2 protein other than the MR2-1 antibody.

[0202] 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 to upregulate TNFR2. 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.

[0203] In summary, the data in Example 2 show that human antibodies bind specifically to TNFR2 endogenously expressed on human immune cells. Moreover, 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 7]

[0204] 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 hTNFR2 (Sinobiologics Catalog No. 10414-H08H) or mTNFR2 (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% Tween20), 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.

[0205] The data are presented below in Tables 8 and 9, and in Figures 6 and 7, respectively. [Table 8] [Table 9]

[0206] 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.

[0207] 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.

[0208] Example 4 - In vitro functionality of antibodies (See also Figures 8-9 and their descriptions above.) Ability of TNFR2 antibodies to modulate cytokine-stimulated NK cell IFN-γ production 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).

[0209] 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.

[0210] 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 1 μg / ml completely neutralized soluble TNF-α, and this dose also reduced IFN-γ release.

[0211] 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 antagonist effects on NK cells and reduced IFN-γ production (Figure 8A).

[0212] This study was deemed not representative to perform with mouse surrogate antibodies due to the lack of endogenously produced TNF-α in mouse cultures and the expression of inhibitory FcγRs on mouse NK cells, whereas their human counterparts express only activating FcγRs. Instead, the agonist or antagonist properties of the mouse surrogate antibodies were addressed using a memory T cell activation assay (induction of CD25) as described below.

[0213] CD25 expressing memory CD4 by TNFR2 antibody + T cell induction 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.

[0214] In brief, the Miltenyi CD4 + Isolate human CD4 T cells from PBMCs by MACS using a "T cell isolation kit" + T cells were isolated. CD4s 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).

[0215] 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).

[0216] The percentage of CD25 expressing cells was enhanced in memory cell cultures stimulated with non-blocking TNFR2 in both humans and mice, however, stimulation with blocking antibodies did not increase, but rather reduced, CD25 expression in these cultures.

[0217] In summary, the data in Example 4 demonstrate that the ligand blocking antibodies are antagonists, as measured in vitro by several methods: inhibition of NK cell-mediated IFN-γ release, and CD4+ as measured by CD25 expression. +Activation of Memory Cells Antagonist ligand blocking antibodies are presented in accordance with the present invention, and agonist ligand non-blocking antibodies are included for comparison.

[0218] Example 5 - Surrogate ligand blocking, antagonistic anti-mouse TNFR2 mAb has in vivo anti-tumor effects (See also Figures 10-16 and their descriptions above.) Therapeutic efficacy in various tumor models To evaluate the in vivo antitumor efficacy of a ligand-blocking antagonist anti-TNFR2 mAb, a murine surrogate called 3F10 was investigated in vivo in different tumor models using different isotype formats, either alone or in combination with anti-PD-1 as described below.

[0219] 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.F10 cells (ATCC) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells were semi-confluent, they were detached with trypsin and 10 × 10 6 The mice were resuspended in sterile PBS at 1 × 10 cells / ml. 6 100 μl of cell suspension equivalent to cells / mouse were injected subcutaneously. 3-8 days after injection depending on the model, mice were treated intraperitoneally with 10 mg / kg of antibody as indicated (isotype control, 3-F10 or 5-A05) twice a week. Tumors were measured twice a week until they reached 15 mm in diameter, after which mice were sacrificed.

[0220] The ligand-blocking antagonist anti-mouse TNFR2 mAb 3-F10 exhibited therapeutic antitumor effects in three different tumor models (Figures 10-13), including therapeutic effects in the more treatment-sensitive CT26 (Figure 10) and tumor growth inhibitory effects in the more treatment-resistant MC38 and B16 (Figures 11-13).

[0221] The antitumor effect of ligand-blocking antagonist anti-mouse TNFR2 mAbs is Fc:FcγR dependent. To assess the importance of FcFcγR interactions for the in vivo antitumor efficacy of a ligand-blocking antagonist anti-TNFR2 murine surrogate mAb, different Fc formats of this antibody were investigated in vivo in the CT26 tumor model as described below.

[0222] Mice were bred and maintained as described above. CT26 cells (ATCC) were grown and injected as described above. When tumors reached 3×3 mm, mice were treated intraperitoneally twice weekly with 10 mg / kg of antibody (isotype control, 3-F10 IgG1, 3-F10 IgG2a, or 3-F10-N297A (Fc-deficient). Tumors were measured twice weekly until they reached 15 mm in diameter, after which mice were sacrificed.

[0223] Fc-deleted 3-F10-N297A showed the lowest or no therapeutic activity compared to the isotype control, indicating that Fc binding is crucial for the therapeutic efficacy of this ligand-blocking antagonist anti-mouse TNFR2 mAb (Figures 10A and B). Both IgG1 and IgG2a formats show significant therapeutic efficacy. However, the IgG2a format, which preferentially binds to activating Fcγ receptors, shows superior therapeutic efficacy indicative of Treg depletion / phagocytosis as one important mechanism of action of this ligand-blocking antagonist anti-mouse TNFR2 mAb (Figures 10A-B). This is in contrast to the non-blocking agonist surrogate antibody 5A05, which shows some activity in the Fc-deleted format and the highest activity in the mouse IgG1 format, which is known to preferentially bind inhibitory FcγRs. The ligand-blocking antagonist antibody (3F10) is according to the invention, and the ligand-blocking agonist antibody (5A05) is included for reference.

[0224] Combination effect with anti-PD-1 mAb To evaluate the in vivo antitumor efficacy of the combination of ligand-blocking antagonist anti-TNFR2 mAb, anti-PD-1 with a murine surrogate (3-F10), the therapeutic combination was investigated in vivo in the MC38 tumor model as described below.

[0225] 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 (isotype control, anti-mouse PD-1, 3-F10, or a combination of anti-mouse PD-1 and 3-F10) as shown in Figures 11A-E. Tumors were measured twice weekly until they reached 15 mm in diameter, after which mice were sacrificed.

[0226] Both anti-mouse PD-1 and the ligand-blocking antagonist anti-mouse TNFR2 mAb 3-F10 indeed show tumor growth suppressive therapeutic effects in the MC38 model (Figures 11A-E). Combining anti-PD1 with the antagonist anti-mouse TNFR2 mAb 3-F10 cures tumors in treatment-resistant MC38 models (Figures 11D-E).

[0227] Combination effect with anti-PD-L1 mAb To evaluate the in vivo anti-tumor efficacy of the combination ligand-blocking antagonist anti-TNFR2 mAb, a murine surrogate (3F10) was further combined with anti-PD-L1 for treatment in the MC38 tumor model, as described below.

[0228] 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.

[0229] Both anti-mouse PD-L1 and the ligand-blocking antagonist anti-mouse TNFR2 mAb 3-F10 demonstrate a therapeutic effect in inhibiting tumor growth in the MC38 model (Figure 12). Combining anti-PD-L1 with the antagonist anti-mouse TNFR2 mAb 3-F10 further enhances the anti-tumor effect (Figure 12).

[0230] 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 7 × 7 mm, mice were treated intraperitoneally with 10 mg / kg of antibody as indicated. Mice were treated on days 1, 4, and 7, and sacrificed on day 8. Tumors were dissected, mechanically divided into small pieces, and digested with a mixture of collagenase, Liberase 100 μg / ml, and DNase 100 μg / ml for 2 × 5 min at 37 °C with vortexing in between. After filtration through a 70 μm filter, the cell suspension was washed with PBS containing 10% FBS (400 g for 10 min). 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 + The results are shown in Figure 14.

[0231] As seen in Figure 14, treatment with ligand-blocking / antagonist TNFR2 antibodies depletes tumor Tregs. + There is also a weak trend towards increased T cell influx. At the same time, this significantly improves the ratio of CD8+ T cells to Tregs (Figure 14C). In addition, the antagonist antibody modulates the myeloid compartment by reducing the number of tumor-associated macrophages (Figure 14D).

[0232] PBMC-NOG / SCID model To confirm the in vivo findings regarding the depletion activity of the ligand-blocking antagonist anti-mouse TNFR2 surrogate mAbs, the depletion capacity of the ligand-blocking antagonist anti-human TNFR2 mAb 1-H10 in the PBMC-NOG / SCID model was analyzed in vivo as described below.

[0233] Mice were bred and maintained in a local facility in accordance with Home Office guidelines. Eight-week-old female SCID and NOG mice were supplied by Taconic (Bomholt, Denmark) and maintained in a local animal facility. For the PBMC-NOG / SCID (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and washed, after which the cells were cultured at 75 × 10 6 The cells were resuspended in sterile PBS at 15 × 10 cells / ml. 6200 μl of cell suspension, equivalent to 100 cells / mouse, was injected intravenously. Two weeks after injection, the spleen was isolated and reduced to a single cell suspension. A small sample was then taken to measure TNFR2 expression in human T cells by FACS (FIG. 15). This FACS showed that TNFR2 expression in Treg and CD8+ T cells was very similar between human T cells grown and activated in vivo in NOG mice and T cells derived from human tumors. The majority of cells were 50×10 6 The cells were resuspended in sterile PBS at 10 × 10 cells / ml. 6 200 μl of the suspension, corresponding to cells / mouse, were injected intraperitoneally. After 1 h, mice were treated with 10 mg / kg of either Yervoy, anti-CD25, 1-H10, 1-H10-N297Q (Fc-deleted version of 1-H10) or isotype control mAb. Mouse peritoneal fluid was collected after 24 h. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD3, CD4, CD8, CD25, CD127 (all from BD Biosciences).

[0234] The Treg depletion activity of 1-H10 was superior to Yervoy and 1-H10N297Q (Figure 16), confirming that the ligand-blocking antagonist anti-TNFR2 mAb 1-H10 depleted Tregs and that Fc interactions were involved in this depletion (Figure 16D).

[0235] In summary, Example 5 shows that: 1. Antagonist ligand-blocking antibodies may exert potent antitumor effects across several tumor models. 2. This effect can be enhanced by combining it with an anti-PD1 antibody. 3. This effect is dependent on the binding of activating FcγR. 4. Treatment with antagonistic ligand-blocking surrogate antibodies significantly alters the T cell composition of tumors, resulting in CD8 + Increases T cell / Treg ratio and reduces tumor-associated macrophages. 5. In human tumors, the cells that most highly express TNFR2 are Tregs. 6. In a human xenograft model in which tumor TNFR2 expression is mimicked in T cells, human Tregs are deleted and CD8 + T cell levels increase. 7. This Treg deletion is most pronounced when the antibody is able to bind to activating FcγRs.

[0236] Example 6 - Antagonist ligand-blocking antibodies do not induce large amounts of proinflammatory cytokines (See also Figures 17-18 and their descriptions above.) 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 antagonistic ligand-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:

[0237] 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 Cells were cultured at 1 × 10 cells / ml. 2 ml of cell culture was plated in a 12-well plate. After 48 hours, 10 μg / ml of antibody was added to 1 × 10 cells in a 96-well flat-bottom plate. 5 The cells were added to the pre-incubated PBMCs and incubated for 24 hours.

[0238] 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, 1 × 10 5 PBMCs were added per well and incubated for 48 hours.

[0239] Cytokine release was also measured after 200 μl of whole blood was stimulated with 5 μg / ml of antibody for 48 hours.

[0240] 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).

[0241] In summary, the blocking antagonist antibodies did not induce significant cytokine release in any of the in vitro settings. The positive control antibodies alemtuzumab and OKT3 induced cytokines, most notably IFN-γ of all, but the 1H10 antibody did not induce IFN-γ above the isotype control antibody, as shown in Figure 17. No other cytokines were elevated by 1H10 (data not shown).

[0242] PBMC-NOG Tolerance Model To investigate the tolerability of the ligand-blocking antagonist anti-human TNFR2 mAb 1-H10, in vivo cytokine release was analyzed in the PBMC-NOG model as described below.

[0243] 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 125 × 10 6 The cells were resuspended in sterile PBS at 25 × 10 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. The blood consisted of about 40% human T cells and the mice were considered humanized. Mice were then treated with 10 μg of either Yervoy, anti-CD3 (OKT-3), 1-H10 or isotype control mAb. Body temperature was measured before and 1 hour after antibody injection, Figure 18A. As can be seen in Figure 18A, 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-H10 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 18B 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 1H10-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.

[0244] In summary, Example 6 shows that TNFR2 ligand blocking antibodies, exemplified herein by the antibody designated 1-H10, do not induce substantial levels of cytokine release as measured by several previously published methods, which indicates an acceptable safety profile in this regard, as cytokine release is a limiting factor in the clinical development of some immunomodulatory antibodies.

[0245] 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 10. 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 11. The latter is possible because neither antibody cross-reacts with mouse TNFR. In both cases, constructs were purchased from GeneArt (ThermoFisher). Constructs were cloned into an expression vector containing a CMV promoter and an OriP origin of plasmid replication and were transiently expressed in suspension-adapted HEK293-EBNA cells. [Table 10] [Table 11]

[0246] 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.

[0247] 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 19). 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 19E and F).

[0248] 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 20 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.

[0249] 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.

[0250] Our monoclonal antibodies showed strong binding to human TNFR2 but no or little binding to mouse TNFR2 (left panel in Figure 21). 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 at least partial epitopes within their sequence. Testing a slightly larger construct, hTNFR2m4, which has mutations at aa 130-144, showed similar binding to construct hTNFR2m2.

[0251] 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.

[0252] 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 antagonist antibody molecule that specifically binds to TNFR2 on a target cell, thereby blocking the binding of TNF-α to TNFR2 and blocking TNFR2 signaling, wherein the antibody molecule also binds to an Fcγ receptor via its Fc region.

2. The antibody molecule of claim 1 , wherein the antibody binds with higher affinity to activating Fcγ receptors than to inhibitory Fcγ receptors.

3. The antibody molecule of claim 1 or 2, wherein the binding of the antibody molecule to TNFR2 results in a change in the number and / or frequency of TNFR2 expressing cells in diseased tissue.

4. The antibody molecule of any one of claims 1 to 3, 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.

5. The antibody molecule of any one of claims 1 to 4, wherein the antibody molecule is selected from the group consisting of a full size antibody, a chimeric antibody, a single chain antibody, and an antigen-binding fragment thereof that retains the ability to bind to an Fc receptor via the Fc region.

6. The antibody molecule of any one of claims 1 to 5, which binds to human TNFR2 (hTNFR2) and / or to cynomolgus monkey TNFR2 (cmTNFR2).

7. The antibody molecule according to any one of claims 1 to 6, wherein the antibody molecule is selected from the group consisting of a human IgG antibody molecule, a humanized IgG antibody molecule, and an IgG antibody molecule of human origin.

8. The antibody molecule of claim 7, wherein the antibody molecule is a human IgG1 antibody.

9. The antibody molecule of claim 7 or 8, wherein the antibody molecule is engineered to improve binding to an activating Fc gamma receptor.

10. The antibody molecule of any one of claims 1 to 9, wherein the antibody molecule is a monoclonal antibody.

11. The antibody molecule of any one of claims 1 to 10, wherein the antibody molecule does not specifically bind to an epitope comprising or consisting of the sequence KCSPG.

12. the antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3; If present, VH-CDR1 is selected from the group consisting of SEQ ID NOs: 1, 9 and 17; If present, VH-CDR2 is selected from the group consisting of SEQ ID NOs: 2, 10 and 18; If present, the VH-CDR3 is selected from the group consisting of SEQ ID NOs: 3, 11 and 19; If present, VL-CDR1 is selected from the group consisting of SEQ ID NOs: 4, 12 and 20; If present, VL-CDR2 is selected from the group consisting of SEQ ID NOs: 5, 13 and 21; The antibody molecule of any one of claims 1 to 11, wherein the VL-CDR3, if present, is selected from the group consisting of SEQ ID NOs: 6, 14 and 22.

13. The antibody molecule comprises the following CDRs: (i) SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, or (ii) SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, or (iii) comprising a variable heavy chain (VH) comprising SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; and / or said antibody molecule comprises the following CDRs: (i) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or (ii) SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or (iii) the antibody molecule of any one of claims 1 to 12, comprising a variable light chain (VL) comprising SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:

22.

14. The antibody molecule of any one of claims 1 to 13, wherein the antibody molecule comprises a variable heavy chain (VH) amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15 and 23, and / or the antibody molecule comprises a variable light chain (VL) amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16 and 24.

15. The antibody molecule according to any one of claims 1 to 11, which is an antibody molecule capable of competing for binding to TNFR2 with the antibody molecule according to any one of claims 12 to 14.

16. An isolated nucleotide sequence encoding an antibody molecule according to any one of claims 1 to 14.

17. A plasmid comprising the nucleotide sequence of claim 16.

18. 18. A virus comprising the nucleotide sequence 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 nucleotide sequence according to claim 16, a plasmid according to claim 16, or a virus according to claim 18 or 19.

21. An antibody molecule according to any one of claims 1 to 15, a nucleotide sequence 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 nucleotide sequence 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 an infection caused by an intracellular pathogen.

23. 23. The antibody molecule, nucleotide sequence, 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 The antibody molecule of any one of claims 1 to 15, the nucleotide sequence of claim 16, the plasmid of claim 17, the virus of claim 19 and / or the cell of claim 20 for use in combination with 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.

25. Use of an antibody molecule according to any one of claims 1 to 15, a nucleotide sequence 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 an infection caused by an intracellular pathogen.

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

27. the pharmaceutical composition is for use in treating the 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 27. The use of claim 25 or 26, administered in combination with 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.

28. A pharmaceutical composition comprising or consisting of an antibody molecule according to any one of claims 1 to 15, a nucleotide sequence 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, and optionally a pharma- ceutically acceptable diluent, carrier, vehicle and / or excipient.

29. 29. A pharmaceutical composition according to claim 28 for use in the treatment of cancer or an infection caused by an intracellular pathogen.

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

31. A method for treating cancer or an infection caused by an intracellular pathogen in a patient, comprising administering to said patient a therapeutically effective amount of an antibody molecule according to any one of claims 1 to 15, a nucleotide sequence according to claim 16, a plasmid according to claim 20, a virus according to claim 21 or 22, a cell according to claim 23, or a pharmaceutical composition according to claim 29.

32. 32. The method of claim 31, wherein the patient has high TNFR2 expression in diseased tissue.

33. A therapeutically effective amount 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.

33. The method of claim 31 or 32, wherein said patient is also administered

34. The antibody molecule for use according to claim 24, the nucleotide sequence 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, the pharmaceutical composition according to claim 30, or the method according to claim 33, wherein the checkpoint inhibitor is PD-1.

35. The antibody molecule for use according to claim 24, the nucleotide sequence 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, the pharmaceutical composition according to claim 30, or the method according to claim 33, wherein the checkpoint inhibitor is PD-L1.

36. The antibody molecule for use according to claim 22, 23, 24, 34 or 35, the nucleotide sequence for use according to claim 22, 23, 24, 34 or 35, the plasmid for use according to claim 25, 26, 34 or 35, the virus for use according to claim 25, 26, 34 or 35, the cell for use according to claim 22, 23, 24, 34 or 35, the use according to claim 25, 26, 27, 34 or 35, the pharmaceutical composition of claim 29, 30, 34 or 35, or the method according to claim 31, 32, 33, 34 or 35, wherein the cancer is a solid cancer.

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