RU2829587C2 - Novel molecules of antagonist anti-tnfr2 antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are an antagonist antibody molecule which specifically binds to TNFR2 on a target cell, thereby blocking TNF-α binding with TNFR2 and blocks signal transmission through TNFR2; an isolated nucleotide sequence encoding an antibody molecule; an antibody expression plasmid comprising a nucleotide sequence; a virus for expression and extracellular release of an antibody containing a nucleotide sequence or a plasmid, and a cell as a carrier for antibody delivery, as well as their use in medicine in the treatment of cancer or infection caused by an intracellular pathogen. Also disclosed are a pharmaceutical composition for treating cancer or an infection caused by an intracellular pathogen in patients and methods of treating cancer and an infection caused by an intracellular pathogen in patients.

EFFECT: invention can be used in treating cancer or infections caused by intracellular pathogens.

48 cl, 21 dwg, 11 tbl, 7 ex

Description

Field of technology

The present invention relates to novel antagonist antibody molecules that specifically bind to the tumor necrosis factor receptor type 2 (TNFR2) on target cells, thereby blocking the binding of the TNF-α ligand to TNFR2, and which also block signaling through TNFR2, wherein said antibody molecules also bind to the Fc receptor via their Fc region. The invention also relates to the use of said molecules in medicine, for example for the treatment of cancer or infections caused by an intracellular pathogen.

State of the art

Tumor necrosis factor (TNF) receptor type 2 (TNFR-2, TNFR2, or TNFRII), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) and CD120b, is a membrane receptor that binds tumor necrosis factor-alpha (TNF-α or TNFα). It is found, for example, on the surface of T cells, monocytes, and macrophages, and can activate the proliferation of TNFR2-expressing cells via nuclear factor kappa B (NF-κB). Notably, TNFR2 expression is highly upregulated in cancer and, in particular, on tumor-infiltrating immune cells, such as regulatory T cells (Treg cells), cytotoxic effector CD8 ⁺ T cells, and various myeloid cell subsets.

TNFR2 has been considered as a promising target for cancer immunotherapy, and its high level of expression has been described on the surface of, among others, intratumoral Treg cells and 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).

Regulatory T cells (which may also be called Treg cells, Tregs, or _Tregs , and which were previously known as suppressor T cells or suppressor regulatory T cells) are a subset of T cells that are capable of suppressing other immune cells under normal and pathological immune conditions. Tregs are CD4-positive cells (CD4 ⁺ cells). There are other CD4 ⁺ T cells that are not Treg cells; however, Treg cells can be distinguished from non-Treg CD4 ⁺ cells in that Treg cells are also FOXP3-positive (FOXP3 ⁺ ), whereas non-Treg CD4 ⁺ cells are FOXP3-negative ( ^FOXP3- ). Treg cells can also be distinguished from non-Treg CD4 ⁺ cells in that Treg cells also have a CD25 ⁺ CD127neg ^/low phenotype, while non-Treg CD4 ⁺ cells have either a CD25-CD127 ⁺ or CD25 ⁺ CD127 ⁺ phenotype.

TNFR2 has also been implicated in 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).

Anti-TNFR2 antibodies of different types and with different characteristics have also been described previously. For example, Williams et al (Oncotarget. 2016 Oct 18; 7 (42): 68278-68291) describe both ligand blocking and non-ligand blocking agonist antibodies.

WO 2014/124134 discloses the use of a TNFR2 agonist, such as an agonistic anti-TNFR2 antibody and/or an NF-κB activator, for the in vitro production of a composition enriched in CD4+CD25 ^hi Treg cells. The composition is said to be useful for the treatment of immunological disorders or infectious diseases in patients. WO 2014/124134 further describes TNFR2 antagonist antibodies that can bind one or two epitopes of TNFR2. The first of these epitopes comprised the sequence QTAQMCCSKCSPGQHAKVFC and the second epitope comprised one specific amino acid at one specific amino acid position of human TNFR; the second epitope may comprise the sequence RLCAPLRKCRPGF. Such TNFR2 antagonists are said to be useful for the production of lymphocyte-enriched and Treg-depleted compositions. In U.S. Patent No. 9,821,010, derived from this PCT application, an antagonist antibody was characterized by the ability to selectively bind to an epitope in the sequence KQEGCRLCAPLRKCRPGFGV, for example, an epitope comprising the sequence RLCAPLRKCRPGF. It is claimed that TNFR2 antagonists and compositions prepared using TNFR2 antagonists are useful for the treatment of proliferative disorders, such as cancer, or infectious diseases.

Document WO 2016/187068 describes antibodies capable of antagonizing members of the tumor necrosis factor receptor superfamily, such as TNFR2. The antibodies are claimed to be useful for modulating Treg cells, for example in immunotherapy for the treatment of proliferative disorders and infectious diseases. In particular, the document describes TNFR2 antagonist antibodies that bind to specific epitopes of human TNFR2 and provides a number of specific complementarity determining region (CDR) sequences of such antibodies. It is claimed that the data in document WO 2016/187068 demonstrate that it is likely that the specific binding of the Fab regions of TNFR2 antagonist antibodies to TNFR2, rather than non-specific binding of the Fc regions of such antibodies, is responsible for the modulation of Treg cell growth.

WO 2017/040312 describes anti-TNFR2 antibodies, and in particular agonist anti-TNFR2 antibodies, that are capable of stimulating TNFR2 signaling and influencing the expansion or proliferation of Treg cells. WO 2017/040312 describes antibodies that specifically bind to an epitope containing the KCSPG sequence and not to an epitope containing the KCRPG sequence, thereby excluding the antibodies described above from US 9821010, or, alternatively, do not bind to another member of the TNFR superfamily. The agonist antibodies are claimed to be useful in the treatment of immunological diseases. WO 2017/040312 also describes the complete sequence of human TNFR2.

WO 2017/083525 discusses pharmacological compositions comprising anti-TNFR2 antibodies and their use in the treatment of TNF-α and/or TNFR2-associated disorders, such as cancer. WO 2017/083525 also discusses antibodies comprising a human IgG1 Fc domain that is null for binding to the Fcγ receptor, as well as suppression of Treg expansion.

WO 2017/197331 describes TNFR2 antagonist antibodies comprising the complementarity determining region of the heavy chain 3 having specific sequences and discusses reducing or inhibiting the proliferation of Treg cells and/or stimulating the proliferation of effector T cells.

Fc receptors are membrane proteins found on the cell surface of immune effector cells, including monocytes, macrophages, dendritic cells, neutrophils, mast cells, basophils, eosinophils, as well as natural killer cells and B lymphocytes. The name derives from their binding specificity to the Fc region of antibodies. Fc receptors are found on the cell membrane, also known as the plasma membrane or cytoplasmic membrane. Fc receptors can be divided into activating FcγRs and inhibitory FcγRs, which are known to coordinate cellular activation by binding the Fc region of aggregated immunoglobulin G and transmitting activating or inhibitory signals to the cell via the intracellular immunoreceptor tyrosine-based activation motif (ITAM) or immunoreceptor tyrosine-based inhibitory motif (ITIM). Binding of aggregated immunoglobulin or immune complexes by FcRs can mediate internalization of the antibody into the cell and can result in antibody-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, or antigen presentation or antigen cross-presentation. FcRs are also known to mediate or enhance cross-linking of antibody-binding receptors on the cell surface. Such cross-linking is known to be required 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 required to achieve therapeutic effects.

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

It is well known that antibodies can modulate immune cell activity through interactions with Fcγ receptors. In particular, how antibody immune complexes modulate immune cell activation is determined by the relative engagement of their activating and inhibitory Fcγ receptors. Different antibody isotypes bind activating and inhibitory Fcγ receptors with different affinities, resulting in different A:I (activation to inhibition ratio) values (Nimmerjahn et al.; Science. 2005 Dec 2; 310(5753):1510-2).

By binding to inhibitory Fcγ receptors, the antibody can inhibit, block and/or reduce the functions of effector cells. By binding to inhibitory FcγRs, antibodies can also stimulate cell activation through aggregation of antibody-targeted signaling receptors on the target cell (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).

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

New data published by the present inventors, among others, demonstrate a critical and differential dependence of therapeutic efficacy on the binding of CD8 T cell agonists and Treg-depleting anti-4-1BB antibodies to activating and inhibitory FcγRs, respectively (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). Moreover, importantly, co-administration of CD8 T cell agonists and Treg-depleting anti-4-1BB antibodies optimized for binding to activating and inhibitory FcγRs, respectively, resulted in attenuated therapeutic activity. These data demonstrate the critical importance of designing antibodies with appropriate and tailored engagement of activating and inhibitory FcγRs to maximize the therapeutic activity of antibodies with different mechanisms of action. At the same time, they demonstrate that suboptimal engagement of activating and inhibitory FcγRs may seriously compromise therapeutic efficacy.

These data were unexpected because they were in contrast to results obtained for antibodies to other members of the TNFSR, particularly for immunostimulatory anti-CD40 antibodies, which show an obligatory requirement for engagement of the inhibitory rather than activating FcγR (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). Taken together, these results demonstrate that FcγR dependence can vary between antibodies to different targets of the same receptor superfamily, and even between different types of antibodies to the same target, in ways that are difficult to predict but that may be critical to understand and exploit in the development of antibodies for therapeutic use.

The essence of the invention

In the course of work leading to this invention, as well as a parallel invention, two major distinct groups of anti-TNFR2 antibodies with potent therapeutic effects and distinct characteristics and mechanisms of action were identified.

The inventors have identified for the first time the potent therapeutic activity of antagonist anti-TNFR2 antibodies that block the binding of TNF-α to the TNFR2 receptor. It has been demonstrated that the activity of such antibodies to exert therapeutic activity in vivo is dependent on the engagement of FcγRs, and in particular on binding to an activating FcγR. This group or category of potent therapeutic anti-TNFR2 reagents has been found to be characterized by 1) potent blockade and inhibition of TNF-α (ligand)-induced TNFR2 signaling, and 2) activity that is dependent on FcγR engagement, with the greatest benefit being conferred by engagement of activating rather than inhibitory FcγRs.

The inventors then identified a separate group of anti-TNFR2 antibodies with similarly potent in vivo therapeutic activity, the characteristics of which, however, are in many respects opposite to those of the blocking-type TNFR2 antagonist antibodies that comprise the first group and the present invention. The anti-TNFR2 antibodies of this second group do not depend on blockade of TNF-α or inhibition of TNFR2 signaling to obtain therapeutic activity, but are instead characterized by strong activation of TNFR2 signaling. In addition, unlike the blocking antibodies of the first group, the agonist antibodies of the second group do not exhibit unconditional dependence on the antibody:FcγR pairs involved, even though their activity increases for variants of the FcγR:antibody pairs involved. In addition, in contrast to the antagonist blocking antibodies of the first group, the agonist antibodies of the second group exhibit the greatest activity in antibody variants with improved binding to inhibitory compared to activating FcγRs.

The present invention relates to a first group of anti-TNFR2 antibodies, i.e., antagonist antibody molecules that, by specifically binding to TNFR2, block the binding of TNF-α to TNFR2 and also block signaling through TNFR2. These antibody molecules also contain an Fc region that binds to an Fc receptor, which is suitable for providing FcγR-dependent elimination or functional modulation of TNFR2-positive cells, including, for example, depletion of Treg cells or modulation of tumor-associated macrophages.

Agonist antibodies belonging to the second group are used in the examples below for comparison with the antagonist TNFR2 blocking antibody molecules of the present invention. In the examples for comparison, other antibodies with some characteristics similar to those of the first or second group, or both, are also used, as explained below.

Accordingly, the present invention relates to antagonist antibody molecules that specifically bind to TNFR2 on a target cell, thereby blocking the binding of TNF-α to TNFR2 and blocking signaling through TNFR2, wherein the antibody molecules also bind to the Fcγ receptor via their Fc region.

The present invention also relates to specific examples of such novel agonist and blocking anti-TNFR2 antibody molecules.

The present invention also relates to isolated nucleotide sequences encoding at least one of the above antibody molecules.

The present invention also relates to plasmids comprising at least one of the above nucleotide sequences.

The present invention also relates to viruses containing at least one of the above nucleotide sequences or plasmids.

The present invention also relates to cells containing at least one of the above nucleotide sequences or at least one of the above plasmids or at least one of the above viruses.

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

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

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

The present invention also relates to pharmaceutical compositions comprising or consisting of at least one of the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and/or cells and, optionally, a pharmaceutically acceptable diluent, carrier or excipient. Such a pharmaceutical composition can be used in the treatment of cancer or infections caused by an intracellular pathogen.

The present invention also relates to methods for treating cancer or infections 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 and/or cells.

The present 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 described herein in light of the accompanying description, examples and/or figures.

Detailed description of the essence of the invention

Accordingly, the present invention relates to antagonist antibody molecules that specifically bind to TNFR2 on a target cell, thereby blocking the binding of TNF-α to TNFR2 and blocking signaling through TNFR2, wherein said antibody molecules also bind to the Fcγ receptor via their Fc region.

The antagonist antibody molecules disclosed herein block both the binding of TNF-α to TNFR2 and signaling through TNFR2. By antagonist antibody molecules block the binding of TNF-α to TNFR2 is meant herein that an antibody molecule that binds to a TNFR2 receptor will, as a result, prevent a TNF-α ligand from binding to the same receptor. This is shown in detail in Example 3. By antagonist antibody molecules disclosed herein block signaling through TNFR2 is meant that they block TNFR2-mediated cell activation. It has been clearly demonstrated that TNF-α-mediated signaling through TNFR2 initiates a signaling cascade that culminates in the activation of the nuclear transcription factor NFκB (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, in turn, leads to cell activation and the synthesis of several proinflammatory factors, one of which is interferon-gamma (IFN-γ) in natural killer (NK) cells (Liu et al. “NF-κB 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 paper, the terms “TNFR2 signaling” and “TNFR2-mediated activation” are used interchangeably.

Antibody molecules bind specifically to TNFR2. It is well known that an antibody specifically binds or interacts with a particular target molecule or antigen, meaning that the antibody preferentially and selectively binds its target over a molecule that is not a target. The terms "antibody molecule that specifically binds TNFR2" or "TNFR2-specific antibody molecule" mean an antibody that binds the TNFR2 protein in a dose-dependent manner but does not bind to an indistinguishable protein. Furthermore, this antibody binds cells that endogenously express TNFR2, and this binding can be blocked by pre-incubating the same cells with a commercially available TNFR2 polyclonal antibody reagent, demonstrating that non-specific binding cannot be detected when TNFR2 is masked by the polyclonal reagent. This is shown in Example 2.

An antibody molecule that specifically binds TNFR2 (or an anti-TNFR2 antibody molecule) means an antibody molecule that specifically binds to at least one epitope in the extracellular domain of TNFR2. "Surface antigen" and "epitope" are terms that can be easily understood by a person skilled in the art of immunology or cell biology.

Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology. Those skilled in the art will appreciate that these methods can be used to assess the binding of an antibody to a target and/or the binding of the Fc region of an antibody to an Fc receptor, and to assess the relative potency or specificity, or inhibition, or prevention, or reduction in the intensity of such interactions. Examples of methods that can be used to assess protein binding include, for example, immunoassays, the Biacore instrumentation suite, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISA), and fluorescence-activated cell sorting (FACS) flow cytometry (for a discussion of antibody specificity, see Fundamental Immunology Second Edition, Raven Press, New York, at pages 332-336 (1989)).

The TNFR2-expressing target cells to which the blocking antibody of the invention binds include immune cells and/or tumor cells as discussed above and below. The effect of binding of the antagonist antibody molecules of the invention to TNFR2 may be to alter the composition of cells in the diseased tissue. Such a change in composition may be due to a change in the number and/or frequency of TNFR2-expressing cells in the diseased tissue. For example, the effect in cancer includes an increase in the number of intratumoral T cells, an increase in the CD8 ⁺ T cell/Treg cell ratio (i.e., the ratio of the number of CD8 ⁺ T cells to the number of Treg cells), and/or an increase in the number of myeloid cells associated with antitumor rather than protumor characteristics. This is shown in Example 5. In some embodiments, such effects result in a decrease in the number of tissue Treg cells, an increase in the number of CD8 ⁺ effector T cells, and a change in the composition of tissue myeloid cell subpopulations. The modulation of TNFR2-expressing cells in tissue after the therapeutic implementation of the invention in vivo using a surrogate (3-F10) of the antagonist antibody molecules of the invention is shown in detail in Example 5. To test similar effects from the human antagonist antibody molecules of the invention, a similar experiment can be carried out in mice from which mouse TNFR2 has been deleted and which have been made transgenic for human TNFR2. In an alternative and preferred embodiment, although time and resource consuming, animals transgenic for human TNFR2 and human FcγR can be generated using a procedure similar 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 TNFR2 and FcγR humanized mice can then be similarly used to test similar effects of the human antagonist antibody molecules of the invention.

In this context, the term “lesion tissue” means either tumor tissue (i.e., all cells in the tumor microenvironment, including tumor cells, immune cells, endothelial cells, and stromal cells) or tissue infected with an intracellular pathogen.

To determine whether an antibody molecule blocks ligand binding to TNFR2, an ELISA assay can be used to measure the amount of TNF-α ligand bound to an immobilized TNFR2 receptor in the presence of TNFR2-specific antibodies. A blocking antibody will prevent TNF-α ligand from binding to the immobilized TNFR2 receptor. This is shown and explained in detail below in Example 3.

The blocking antibody molecule according to the invention is a complete blocker which is further capable of antagonizing signal transduction through TNFR2.

The term "complete blocker" is defined herein as an antibody molecule that reduces the binding of TNF-α to TNFR2 by greater than 98%, i.e., up to 100%, compared to the binding of TNF-α in the presence of an isotype control antibody molecule alone. An isotype control antibody is an antibody to a protein or other structure that is not present in any form in the assay. An isotype control ideally has a similar structure or at least an Fc region similar to the comparison antibodies. This is well known to those skilled in the art. In the examples described herein, the isotype control had a similar structure, a similar Fc region, and was specific for fluorescein isothiocyanate (FITC). In some embodiments, a complete blocker reduces TNF-α binding by greater than 99.5%.

Other types of blockers are partial blockers and weak blockers. In the context of this document, a partial blocker is an antibody molecule that reduces the binding of TNF-α 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%, 95%, 96%, 97%, or 98%, with all decimal places included). between them) compared to TNF-α binding in the presence of an isotype control antibody molecule alone, and a weak blocker is an antibody molecule that reduces TNF-α binding to TNFR2 by less than 60%, such as by 50 to 59.9% (or by 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 59.9%, with all decimal places in between), compared to TNF-α binding in the presence of an isotype control antibody molecule alone.

In contrast, a non-blocking TNFR2 antibody molecule is an antibody molecule that reduces the level of 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 Figs. 6, 7.

Partial blocking, weak blocking and non-blocking antibodies are used in the examples for comparison with the antagonist blocking antibody molecules of the present invention.

Several properties and features may underlie and (together) determine the biological activity of antibodies. In addition to the ability to block ligand binding to a receptor, such important properties include the ability of an antibody to modulate receptor signaling, i.e., to agonize or antagonize receptor signaling, and the dependence of the antibody on FcγR interactions to exert therapeutic activity.

Initially, the ability of blocking, partially blocking, and non-blocking antibodies to modulate TNFR2 signaling was characterized. Two extreme cases were identified.

In the first case, antibodies were identified that completely blocked ligand binding to TNFR2, blocked TNF-α-induced signaling through TNFR2, and that did not themselves induce signaling when bound to a cell endogenously expressing TNFR2. This group of ligand-blocking antagonist antibodies forms the basis of the present invention.

In another case, antibodies were identified that did not block ligand binding to TNFR2, but which, when bound to cells endogenously expressing TNFR2, agonized the receptor. This second group of antibodies constitutes a separate invention and is included herein for comparison.

Antibodies and variants thereof, referred to as partially blocking agonist, partially blocking non-agonist, and fully blocking non-antagonist, were further identified, demonstrating the complex biology and significant heterogeneity of anti-TNFR2 antibodies and clearly showing that the antibodies of the present invention form a unique group.

To determine whether an antibody has agonist or antagonist activity, a natural killer (NK) cell assay can be used, as described in Example 4. Briefly, NK cells are shown to respond to IL-2 and IL-12 stimuli by secreting IFN-γ. Soluble TNF-α is produced endogenously and is present at consistent but suboptimal concentrations (~20-100 pg/mL) for TNFR2 signaling, indicating that IFN-γ can be either increased or decreased by modulating TNFR2 signaling. Therefore, exogenous addition of TNF-α at an optimal concentration for TNFR2 signaling increases IFN-γ concentrations in this assay, as does incubation with an agonist anti-TNFR2 antibody (Fig. 8C). In contrast, co-incubation with anti-TNF-α antibody or the antagonist ligand blocking antibodies described herein reduces IFN-γ release in this assay. As a result, this assay can be used to identify the agonist, antagonist, 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) Therefore, in this experimental setup, the antagonist antibody prevents TNF-α-induced signaling in TNFR2-expressing cells without itself stimulating the TNFR2 receptor upon binding to it. In particular, the antagonist antibodies in this assay do not increase the release of IFN-γ upon binding to the aforementioned NK cells, but instead inhibit the release of IFN-γ. As shown in Fig. 8, the fully blocking antibodies described in this invention do not induce signaling through TNFR2, but instead reduce signaling through TNFR2 in this TNF-α-containing NK cell assay, resulting in a decrease in the amount of IFN-γ released. As shown in Fig. 8 in Example 4, the antibodies of this invention can be characterized as ligand blocking antagonist anti-TNFR2 antibodies. Using this assay, an antagonist antibody is defined as an antibody that results in a greater than 30% (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) reduction in IFN-γ release, provided that the basal TNF-α level in the culture is at least 20 pg/mL. Because this assay uses primary cells from peripheral blood mononuclear cell (PBMC) donors, at least 4 donors must be included and average values must be calculated for all donors. Cells from each donor to be included in the calculation of the mean must respond to positive control (soluble TNF-α) treatment with a greater than 100% (more than 2-fold) increase in IFN-γ levels compared to isotype control treatment.

Antagonist activity can also be demonstrated using IL-2-mediated activation of memory T cells. Activation in this case is measured by upregulation of the T cell activation marker CD25. Using this assay, addition of non-blocking agonist TNFR2 antibodies increases the expression level of CD25, whereas blocking antagonist TNFR2 antibodies according to the invention result in lower expression of CD25 compared to the isotype control. This is true for the human antibodies of the invention as well as for surrogate murine antibodies and is shown in Example 4.

In addition to binding to TNFR2 and, through it, blocking TNF-α binding and signaling, the antibody molecules of the invention also bind to Fcγ receptors. The unconditional dependence of therapeutic efficacy on interactions between FcγR and anti-TNFR2 antibodies belonging to the TNF-α-blocking antagonist group of antibodies of the invention has been demonstrated in experimental mouse cancer models using antibody variants that productively or non-productively engage FcγR binding. The unconditional dependence of maximal in vivo therapeutic activity for such ligand-blocking antagonist antibodies and their preferential binding/engagement of activating, rather than inhibitory, FcγRs is shown in Example 5. Data demonstrating the in vivo FcγR-independent activity of non-blocking anti-TNFR2 agonist antibodies and their distinctive preferential engagement of inhibitory, rather than activating, FcγRs to obtain maximal therapeutic activity are included in this example for comparison and contrast only. Taken together, the data demonstrate that several types of anti-TNFR2 antibodies can be generated. Furthermore, the data demonstrate that it is non-trivial and impossible to predict which antibody variants will be most therapeutically effective, whether they will be based on blocking, agonist or antagonist (extrinsic or intrinsic) properties, and whether they will be dependent on FcγR engagement, or whether they will be most effective in antibody formats associated with preferential/strong binding to activating or inhibitory Fc-gamma receptors.

The relatively high homology between the mouse and human FcγR systems explains many common aspects of the conserved FcγR-mediated mechanisms between the species. However, mouse and human IgG subclasses differ in affinity for their cognate FcγRs, which becomes important when translating FcγR-mediated observations in the mouse system to human IgG-based therapeutics to select an antibody, antibody subclass, and/or engineered subclass variant that exhibits more favorable binding to activating versus inhibitory human FcγRs. The affinity and/or avidity of human antibody molecules for individual human FcγR receptors can be determined using surface plasmon resonance (SPR). In some embodiments, a TNFR2 blocking antibody molecule binds with higher affinity to activating Fcγ receptors than to inhibitory Fcγ receptors. The expression “with higher affinity for activating Fcγ receptors than for inhibitory Fcγ receptors” refers to variants that bind with higher affinity to activating Fcγ receptors, such as FcγRIIA, FcγRIIIA and/or FcγRI, compared to inhibitory Fcγ receptors.

In some embodiments, the antibody molecule is an IgG that can bind to Fcγ receptors through the normal interaction between the Fc region of the antibody molecule and the Fcγ receptor.

In some embodiments, the TNFR2 blocking antagonist 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 activating human Fcγ receptors FcγRIIA, FcγRIIIA, and inhibitory human FcγRIIB. This has been demonstrated, for example, using surface plasmon resonance (SPR).

In some embodiments, the antagonist TNFR2 blocking antibody molecule is an IgG antibody molecule that exhibits improved binding to one or more activating Fc receptors and/or is engineered to have improved binding to one or more activating Fcγ receptors and/or is engineered to have improved relative binding to activating receptors compared to inhibitory Fcγ receptors. In some embodiments, the anti-TNFR2 antibody is an Fc-engineered human IgG1 antibody. Examples of such engineered antibody variants include afucosylated antibodies with selectively improved antibody binding to FcγRIIIA and antibodies engineered by targeted, mutational, or other amino acid substitution resulting in improved binding to one or more activating Fcγ receptors compared to 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 U S A, 103: 4005-10)

In some embodiments, a human IgG antibody engineered to improve binding to activating Fc gamma receptors may be a human IgG antibody that has two mutations S239D and I332E or three mutations S239D, I332E and A330L and/or G236A mutations in its Fc region. In some embodiments, a human IgG antibody engineered to improve binding to activating Fc gamma receptors may be an afucosylated human IgG antibody.

Said Fcγ receptor to which said Fc region of said antagonist blocking antibody molecules of the present invention binds may be an Fcγ receptor expressed by an immune effector cell as described above.

Binding of a TNFR2-specific antibody molecule to TNFR2 surface receptors on a target cell and simultaneous engagement of FcγR by the same cell or an immune effector cell in close proximity may result in depletion or functional modulation of TNFR2-positive target cells to which the antibody molecule binds. As used herein, the term "cell depletion" means depletion, deletion, or elimination of the cell by physical removal of the cell.

The cell depletion can be achieved by 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 such binding results in cell depletion. In general, cells with high expression are eliminated more efficiently compared to cells with low expression. As shown in Example 5, Fig. 14, Tregs are the highest expressing cells in a tumor setting.

ADCC is an immune mechanism by which Fc receptor-bearing effector cells can recognize and kill, i.e. deplete, antibody-coated target cells expressing tumor-derived antigens on their surface, i.e., in this case, TNFR2. ADCP is a similar mechanism, although it results in the killing, i.e. depletion, of target cells by phagocytosis rather than cytotoxicity.

In addition, improved binding of the Fc region of antibody molecules to the Fcγ receptor may also enhance the depletion of target cells through Fc receptor-dependent cell death via ADCC or ADCP. This is particularly relevant for antibody molecules with improved binding to activating Fcγ receptors.

That antibody molecules have 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 such binding leads to depletion of these target cells.

The exhausted cells can be several different cells, as explained above when considering the characteristics of the target cell. Typically, the cells that are exhausted are those with the highest TNFR2 expression. Other cells that also express TNFR2, but not as strongly, can also be exhausted, but to a lesser extent than the cells that have the highest TNFR2 expression.

As mentioned above, TNFR2 is highly expressed on Treg cells found in tumors of various cancer patients, and in such patients, the antibody molecule of the invention will preferentially bind to Treg cells and as a result lead to Treg cell depletion. Treg cells have an inhibitory effect on the proliferation, activation and cytotoxic capacity of other immune cells, such as CD8-positive (CD8 ⁺ ) cells, and therefore Treg cell depletion will at least indirectly lead to increased proliferation, activation and possibly migration of CD8 ⁺ cells, and thus to an increase in the number of intratumoral CD8 ⁺ cells. CD8 ⁺ T cells are required 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). Therefore, increasing the proliferation, activation, and cytotoxic capacity of CD8 ⁺ T cells would provide significant benefit to the cancer patient and may lead to disease eradication.

Increasing the proliferation, activation, and cytotoxic capacity of CD8 ⁺ T cells will also be of significant benefit in treating infection caused by an intracellular pathogen. Intracellular pathogen antigens are typically present on MHC I molecules, which play an important role in activating CD8 ⁺ T cells. As a result, CD8 ⁺ T cells recognize infected cells and lyse them, thereby destroying the pathogen.

Binding of TNFR2-specific antibody molecule and blocking TNF-α signaling can also lead to functional modulation of cellular phenotypes, such as modulation of a pro-tumor myeloid cell into a myeloid cell with anti-tumor properties.

In some embodiments of the invention, the TNFR2-positive cells, i.e., the target cells, are CD4-positive (CD4 ⁺ ) cells, i.e., cells that express CD4.

In some embodiments, the TNFR2-positive cells are both CD4 ⁺ and FOXP3 ⁺ , i.e., they express both CD4 and FOXP3. These cells are Treg cells. CD8 ⁺ T cells also express TNFR2, but Treg cells express significantly higher levels of TNFR2 than CD8-positive T cells, as shown in Fig. 14 in Example 5. This makes Treg cells more susceptible to exhaustion compared to low-expressing CD8 ⁺ cells.

In some situations, TNFR2 is preferentially expressed on immune cells in the tumor microenvironment (tumor infiltrating cells (TILS)).

In some embodiments, the Treg cells will be the cells in the tumor microenvironment that have the highest expression of TNFR2, such that antibody molecules that specifically bind to TNFR2 (or anti-TNFR2 antibody molecules) have a Treg cell depleting effect. This is discussed in detail below, for example, in Example 5 and for Figs. 13 and 15.

In some embodiments, the TNFR2-positive cells will be Treg cells in a solid tumor. Such Treg cells will have very high expression of TNFR2, and therefore administration of antibody molecules that specifically bind to TNFR2 will preferably result in depletion of such Treg cells.

To determine whether an antibody molecule is an antibody molecule that has an enhanced depletion effect on TNFR2-positive cells as described 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, which is referred to herein as the PBMC-NOG/SCID model. Both NOG and SCID mice are known to those skilled in the art (Ito M et al, (2002) NOD/SCID/γc ^null mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100(9):3175-3182; Bosma GC et al; Nature. 1983 Feb 10; 301(5900):527-30; A severe combined immunodeficiency mutation in the mouse), and 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 Søndergaard 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 test in the PBMC-NOG/SCID model includes the following nine sequential steps:

1) Human PBMCs (peripheral blood mononuclear cells) are isolated, washed, and resuspended in sterile phosphate-buffered saline (PBS). In some embodiments, PBMCs are resuspended in PBS at a concentration of 75 x 10 ⁶ cells/mL.

2) NOG mice are injected intravenously with an appropriate amount, e.g. 200 µl, of the cell suspension from step 1. When 200 µl is injected, this corresponds to 15×10 ⁶ cells/mouse.

3) At a suitable time, such as 2 weeks after injection, the spleens of NOG mice are isolated and converted into a single cell suspension. Optionally, to confirm TNFR2 expression, a small sample from the single cell suspension is taken to determine TNFR2 expression on human T cells using FACS.

4) The cell suspension from step 3 is resuspended in sterile PBS. In some embodiments, the cell suspension is resuspended in sterile PBS at a concentration of 50 x 10 ⁶ cells/mL. If the optional TNFR2 expression determination is included in step 3, the remainder of the cell suspension is then resuspended in step 4.

5) SCID mice are injected i.p. (intraperitoneally) with an appropriate amount, e.g. 200 µl, of the suspension from step 4. When 200 µl is injected, this corresponds to 10 x 10 ⁶ cells/mouse.

6) At a suitable time, such as 1 hour after the injection in step 5, SCID mice are administered an appropriate amount, such as 10 mg/kg, of either the antibody molecule being tested, a positive control antibody (such as an anti-CD25 antibody known to deplete Treg cells), or an isotype control monoclonal antibody.

7) Intraperitoneal fluid from treated SCID mice is collected at a suitable time, such as 24 hours after treatment in step 6.

8) Using the FACS method, human T cell subpopulations are identified and quantified using the following markers: CD45, CD4, CD8, CD25 and/or CD127. It has been well established that human Treg cells in the PBMC population are characterized by the CD4+CD25+CD127 ^low/- phenotype.

9) The results of identification and quantification of T cell subpopulations in mice receiving the test antibody molecule are compared with the results of identification and quantification of T cell subpopulations in mice receiving the positive control antibody and with the results of identification and quantification of T cell subpopulations in mice receiving the isotype control monoclonal antibody. The lower number of Treg cells in the intraperitoneal fluid of mice receiving the test antibody molecule compared to the number of Treg cells in the intraperitoneal fluid of mice receiving the isotype control demonstrates that the antibody molecule has a depleting effect on TNFR2-positive Treg cells.

This analysis is demonstrated in detail below in Example 5 using Fig. 15.

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

Antibodies are well known to those skilled in the art of immunology and molecular biology. Typically, an antibody is composed of two heavy (H) chains and two light (L) chains. This complete antibody molecule is sometimes referred to herein as a full-length antibody or full-length antibody. The heavy chain of an antibody contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), and the light chain of an antibody molecule contains one variable domain (VL) and one constant domain (CL). These variable domains (sometimes collectively referred to as F _V regions) bind to the antibody target, or antigen. Each variable domain consists of three loops, called complementarity determining regions (CDRs), that are responsible for binding to the target. These constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions. Antibodies or immunoglobulins can be classified into different classes depending on the amino acid sequence of the constant region of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and in humans, some of these immunoglobulins are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2.

Another part of the antibody is the Fc region (also known as the crystallizable fragment domain), which consists of two constant domains from each of the antibody's heavy chains. As previously mentioned above, the Fc region is responsible for the interaction between the antibody and the Fc receptor.

As used herein, the term "antibody molecule" refers to full-length or complete antibodies, as well as functional fragments of full-length antibodies and derivatives of such antibody molecules.

Functional fragments of a full-length antibody have antigen-binding properties similar to the corresponding full-length antibody and contain either similar variable domains (i.e., VH and VL sequences) and/or similar CDR sequences to the corresponding full-length antibody. A functional fragment does not always contain all six CDRs of the corresponding full-length antibody. Molecules containing three or fewer CDR regions (in some cases, even only one CDR region or part of it) are considered to be able to retain the antigen-binding activity of the antibody from which the CDRs are derived. For example, Gao et al., 1994, J. Biol. Chem., 269: 32389-93, report that the entire VL chain (including all three CDR regions) has high affinity for its substrate.

Molecules containing two CDR regions are described, for example, in the paper "Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4: 417-430" on page 418 (right column, section "3 Our Strategy for Design") a minibody is described that includes only the hypervariable H1 and H2 CDR regions interspersed within the framework regions. It was shown that such a minibody is able to bind to the target. Vaughan and Sollazzo refer to the paper "Pessi et al., 1993, Nature, 362: 367-9, and Bianchi et al., 1994, J. Mol. Biol., 236: 649-59" and describe in detail the minibodies from H1 and H2 and their properties. In the work «Qiu et al., 2007, Nature Biotechnology, 25:921-9» it is shown that a molecule consisting of two linked CDR regions is capable of binding antigen. In the work «Quiocho 1993, Nature, 362: 293-4» a brief description of the «minibody» technology is given. In the work «Ladner 2007, Nature Biotechnology, 25:875-7» it is noted that molecules containing two CDR regions are capable of retaining antigen-binding activity.

Single CDR antibody molecules are described, for example, in Laune et al., 1997, JBC, 272: 30937–44, which showed that a number of hexapeptides derived from the CDR region exhibit antigen-binding activity and noted that synthetic peptides of the complete single CDR region exhibit strong binding activity. In Monnet et al., 1999, JBC, 274: 3789–96, a number of 12-mer peptides and their associated framework regions exhibit antigen-binding activity and noted that only one CDR3-like peptide is capable of binding antigen. In Heap et al., 2005, J. Gen. Virol., 86: 1791–1800" reported that a "microantibody" (a molecule containing a single CDR region) was able to bind antigen and showed that a cyclic peptide of an anti-HIV antibody had antigen-binding activity and function. "Nicaise et al., 2004, Protein Science, 13: 1882–91" showed that a single CDR region could confer antigen-binding activity and affinity for its lysozyme antigen.

It follows that antibody molecules containing five, four, three or fewer CDR regions are capable of retaining the antigen-binding properties of the full-length antibodies of which they are derived.

Such an antibody molecule may also be a derivative of a full-length antibody or a fragment of such an antibody, provided that such a derivative or fragment retains the ability to bind to the Fcγ receptor. If a derivative is used, it must have the same antigen-binding characteristics as the corresponding full-length antibody, in the sense that it binds to the same epitope on the target as the full-length antibody.

Therefore, in the context of this document, the term "antibody molecule" refers to all types of antibody molecules and functional fragments thereof, as well as derivatives thereof, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, antibodies of human origin, humanized antibodies, chimeric antibodies, single chain antibodies, antibody heavy chains, antibody heavy chain homodimers, antibody heavy chain heterodimers, and antibody light chain heterodimers.

Additionally, as used herein, the term "antibody molecule" encompasses all classes of antibody molecules and functional fragments, including: IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE, unless otherwise specified.

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. Therefore, in some embodiments, the antibody molecule has an isotype that optimally engages activating Fc receptors. In some embodiments, the antibody molecule is an IgG1 antibody.

One skilled in the art will appreciate that murine IgG2a and human IgG1 engage activating Fcγ receptors and are both capable of activating target cell deletion by activating immune cells bearing activating Fcγ receptors, such as through ADCP and ADCC mechanisms. In some embodiments, the anti-TNFR2 antibody is a murine or humanized murine IgG2a antibody.

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

In some embodiments, the anti-TNFR2 antibody is a murine antibody that is cross-specific to human TNFR2.

As emphasized above, the present invention relates to various types and forms of antibody molecules known to a person skilled in the art of immunology. It is known that antibodies used for therapeutic purposes are often modified by additional components that change the properties of the antibody molecule.

Therefore, it is assumed that an antibody molecule according to this description or an antibody molecule used according to this description (e.g., a monoclonal antibody molecule and/or a polyclonal antibody molecule and/or a bispecific antibody molecule) contains a detectable moiety and/or a cytotoxic moiety.

The term "detectable fragment" means one or more of the group consisting of: an enzyme; a radioactive atom; a fluorescent fragment; a chemiluminescent fragment; a bioluminescent fragment. The detectable fragment of the molecule allows the antibody molecule to be visualized in vitro and/or in vivo and/or ex vivo.

The term "cytotoxic fragment" means a radioactive fragment and/or an enzyme, wherein, for example, said enzyme is a caspase; and/or a toxin, wherein, for example, said toxin is a bacterial toxin or poison; wherein said cytotoxic fragment is capable of inducing cellular lysis.

It is also contemplated that the antibody molecule may be in an isolated form and/or purified form, and/or may be PEGylated. PEGylation is a method by which polyethylene glycol polymers are added to a molecule, such as an antibody molecule or a derivative thereof, in order to alter its characteristics, such as to prolong its half-life by increasing its hydrodynamic size, preventing renal clearance.

As discussed above, the CDR regions of an antibody bind to the antibody target. The amino acid assignments to each CDR region described herein are as defined in "Kabat EA et al. 1991, In "Sequences of Proteins of Immunological Interest" Fifth Edition, NIH Publication No. 91-3242, pp xv-xvii."

As may be known to one skilled in the art, there are other ways of assigning amino acids to each CDR region. For example, the International Immunogenetics Information System (IMGT(R), ImMunoGeneTics) (http://www.imgt.org/ and Lefranc and Lefranc "The Immunoglobulin FactsBook" published by Academic Press, 2001).

In some embodiments, the antibody molecule that specifically binds TNFR2 is a human antibody.

In some embodiments of the invention, an antibody molecule that specifically binds TNFR2 is an antibody of human origin, i.e., an originally human antibody that has been modified as described herein.

In some embodiments, an antibody molecule that specifically binds TNFR2 is a humanized antibody, i.e., an originally non-human antibody that has been modified to increase its similarity to a human antibody. Humanized antibodies can be, for example, mouse antibodies or llama antibodies.

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

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

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

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

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

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

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

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

In some embodiments, an anti-TNFR2 antibody molecule is an antibody molecule comprising 6 CDRs comprising the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6; or an antibody molecule comprising 6 CDRs comprising SEQ ID NOs: 9, 10, 11, 12, 13, and 14; or an antibody molecule comprising 6 CDRs comprising SEQ ID NOs: 17, 18, 19, 20, 21, and 22.

In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising 6 CDRs comprising the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

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.

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.

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

In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VL of SEQ ID NO: 8.

In some embodiments, an anti-TNFR2 antibody molecule comprises a VH comprising SEQ ID NO: 7 and a VH comprising SEQ ID NO: 8.

In some embodiments, the anti-TNFR2 antibody molecule comprises a CH comprising SEQ ID NO: 217.

In some embodiments, the anti-TNFR2 antibody molecule comprises a CL comprising SEQ ID NO: 218.

In some embodiments, an anti-TNFR2 antibody molecule comprises a VH comprising SEQ ID NO: 7, a VH comprising SEQ ID NO: 8, a CH comprising SEQ ID NO: 217, and a CL comprising SEQ ID NO: 218.

Table 1. Individual sequences of antagonist TNFR2 blocking antibody molecules according to the invention (in the VH and VL sequences, the CDR sequences are highlighted in bold)

Table 2. Selected sequences of TNFR2-blocking antibody molecules that are not antagonists and that are used in this document for comparison (in the VH and VL sequences, the CDR sequences are shown in bold)

To determine or demonstrate the properties of the antibody molecules of the present invention, they were compared with antibody molecules that do not block the binding of TNF-α ligand to TNFR2. Such antibodies are presented in Table 3.

Table 3. Selected sequences of TNFR2 non-blocking antibody molecules mentioned in this document as reference antibodies (in VH and VL sequences, CDR sequences are highlighted in bold)

All sequences given above in Tables 1, 2 and 3 are of human origin and come from the n-CoDeR® library, as explained in detail in Example 1.

In some embodiments, the antibody molecules described herein that specifically bind TNFR2 may also comprise one or both of the constant regions (CH and/or CL) listed below in Table 4.

Table 4

Plot Subsequence SEQ ID NO: CH ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 217 CL QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 218 CH AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMS GKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 219 CH AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSG KEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 220 CL QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS 221

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

In some embodiments, the antibody molecule does not bind human TNFR2 (hTNFR2).

In some embodiments, it is preferred that the antibody molecule binds to both hTNFR2 and cynomolgus macaque TNFR2 (cmTNFR2 or cynoTNFR2). Cross-reactivity with TNFR2 expressed on cynomolgus macaque cells, also called crab-eating macaque or Macaca fascicularis, may provide the advantage of being able to test the antibody molecule in animals without the need to use a surrogate antibody, with particular attention to tolerability.

In some embodiments of the invention, it is necessary to use a surrogate antibody to test the functional activity of the antibody molecule in appropriate in vivo mouse models. To achieve comparability between the effect of the antibody molecule in humans and the in vivo results for the surrogate antibody in mice, it is important to select a functionally equivalent surrogate antibody that has in vitro characteristics similar to the human antibody molecule.

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

In some embodiments, an antibody molecule of the invention or used in accordance with the invention is an antibody molecule that is capable of competing in binding to TNFR2 with the specific antibodies provided herein, such as capable of competing with antibody molecules 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.

The term "capable of competing" means that the competing antibody is capable of inhibiting or otherwise interfering, at least in part, with the binding of an antibody molecule as described herein to a particular TNFR2 target.

For example, such a competing antibody molecule may be capable of inhibiting the binding of an antagonist blocking antibody molecule as described herein by at least about 10%; for example, 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 inhibit the ability of an antibody described herein to interfere with TNFR2 binding or reduce binding to a specific target of a TNF-α ligand by at least about 10%, such as 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%.

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

ELISA assays can be used to evaluate epitope-modifying or blocking antibodies. Additional methods suitable for detecting competing antibody are described in Antibodies: A Laboratory Manual, Harlow & Lane, 1988, CSHL, NY, ISBN 0-87969-314-2, the contents of which are incorporated herein by reference (see, for example, pp. 567–569, 574–576, 583, and 590–612).

In some embodiments of the invention, it is of interest to use not the antibody molecule itself, but a nucleotide sequence encoding such an antibody molecule. As a result, the present invention encompasses nucleotide sequences encoding the above-mentioned antagonist TNFR-2 blocking antibody molecules.

The above-described antagonist blocking antibody molecules and nucleotide sequences can be used in medicine, and then such an antibody molecule and/or nucleotide sequence can be included in a pharmaceutical composition, as discussed below.

The above-described antagonist blocking antibody molecules, nucleotide sequences and/or pharmaceutical compositions can be used in the treatment of cancer, as discussed below.

The above-described antagonist blocking antibody molecules, nucleotide sequences and/or pharmaceutical compositions can be used in the treatment of an infection caused by an intracellular pathogen, as discussed below.

The above-described antagonist blocking antibody molecules and/or nucleotide sequences can be used in the preparation of a pharmaceutical composition for the treatment of cancer.

The above-described antagonist blocking antibody molecules and/or nucleotide sequences can be used in the preparation of a pharmaceutical composition for the treatment of an infection caused by an intracellular pathogen.

The above-described antagonist blocking antibody molecules and/or pharmaceutical compositions can be used in a method of treating cancer in a patient, in which a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the subject.

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

In some embodiments of the invention relating to the treatment of cancer, the cancer is a solid cancer or leukemic cancer. A solid tumor is an abnormal mass of tissue that typically does not contain cysts or fluid areas. Solid tumors can be benign (not cancer) or malignant (cancer). Malignant solid tumors are referred to herein as solid cancer. The various types of solid tumors are named according to the types of cells that form them. Examples of solid tumors or cancers include sarcomas, carcinomas, and lymphomas.

More specific examples of solid cancers include 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, stomach cancer, lymphoma, and bone cancer.

More specific examples of leukemic cancers include acute lymphocytic leukemia, chronic myeloproliferative disorder, acute nonlymphocytic leukemia, B-cell acute lymphocytic leukemia, chronic lymphocytic leukemia, T-cell acute lymphocytic leukemia, and non-Hodgkin lymphoproliferative disorders.

In some embodiments of the invention, relating to the treatment of an infection caused by an intracellular pathogen, such as a virus or bacteria. Specific examples of intracellular pathogens are Legionella pneumophila, R. rickettsia, Mycobacterium tuberculosis, Listeria monocyotogenes, Salmonella spp, invasive Escherichia coli, Neisseria spp, Brucella spp, Shigella spp, influenza virus, herpes virus, hepatitis virus, coxsackievirus, Epstein-Barr virus or rhinovirus.

In some embodiments of the invention relating to the treatment of cancer, the antagonist blocking antibody molecules described above can be used in combination with an antibody molecule that specifically binds to a checkpoint inhibitor. In an alternative embodiment of the invention, the nucleotide sequences encoding an antagonist TNFR2 blocking antibody molecule discussed above can be used in combination with an antibody molecule that specifically binds to a checkpoint inhibitor or together with a stimulatory agonist antibody. Antibodies to checkpoint inhibitors include antibodies targeting CTLA4, PD1, PD-L1, VISTA, TIGIT, CD200, CD200R, BTLA, LAG3, TIM3, B7-H3, B7-H4, B7-H7. Examples of co-stimulatory agonist antibodies include antibodies targeting OX40, 41BB, OX40L, 41BBL, GITR, ICOS, DR3, DR4, DR5, CD40, CD27, RANK, HVEM, LIGHT, and B7-H6. In an alternative embodiment, the antagonist TNFR2 blocking antibody molecules described above may be used in combination with a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor or in combination with a stimulatory agonist. In an alternative embodiment, the nucleotide sequences encoding a TNFR2 blocking antibody molecule described above may be used in combination with a nucleotide sequence that encodes an antibody molecule that specifically binds to a checkpoint inhibitor or in combination with a stimulatory agonist. In some such embodiments, the antibody molecule that specifically binds to the checkpoint inhibitor is an anti-PD-1 antibody. Anti-PD1 (or PD1) antibodies are believed to block the inhibitory signal mediated by PD-L1, primarily in CD8 ⁺ T cells; thereby allowing for an enhancement of the T cell-mediated anti-tumor response. Antagonist TNFR2 antibodies that deplete Treg cells would work through a separate mechanism to enhance the anti-tumor response. Therefore, such therapies may interact with each other. A similar approach is true for other checkpoint inhibitors and agonist co-stimulatory antibodies.

In addition, the antagonist TNFR2 blocking antibody molecules described above can be used in combination with other anti-cancer drugs, such as chemotherapy drugs (e.g., 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 (e.g., but not limited to, ibrutinib, imatinib, suntinib, regorafenib, sorafenib, dasatinib, erlotinib, vandetanib, midostaurin, vemurafenib, dabrafenib, palbociclib, ribociclib, trametinib, or alectinib), inhibitors targeting growth factor receptors (e.g. but not limited to drugs targeting EGFR/HER1/ErbB1, EGFR2/HER2/ErbB2, EGFR3/HER3/ErbB3, VEGFR, PDGFR HGFR, RET, insulin-like growth factor IGFR, FGFR), antiangiogenic agents (e.g. but not limited to bevacizumab, everolimus, lenalidomide, thalidomide, ziv-aflibercept) or radiation. Generally, all of the above-mentioned anticancer drugs induce cancer cell death, which leads to neoantigen exposure and inflammation. During neoantigen exposure and inflammatory cell influx into the tumor, synergistic effects of the anticancer drug and the addition of an antagonist TNFR2 ligand blocking antibody may deplete Treg cells and thereby further enhance the immune system.

A person skilled in the art of medicine may be aware that drugs can be modified by various additives, for example, to change the rate at which the drug is absorbed into the body; and can be modified in various forms, for example, to provide for a particular route of administration into the body.

Therefore, this application includes the antagonist blocking antibody molecules, nucleotide sequences, plasmids, viruses and/or cells described herein may be combined with a pharmaceutically acceptable vehicle, carrier, diluent, vehicle and/or adjuvant into a pharmaceutical composition. In such context, the term "pharmaceutical composition" may be used interchangeably with the terms "pharmaceutical preparation", "pharmaceutical formulation", "therapeutic composition", "therapeutic preparation", "therapeutic formulation" and "therapeutic entity".

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

The pharmaceutical compositions described herein may, in some embodiments, consist of or contain plasmids comprising nucleotide sequences encoding the antibody molecules described above or comprising the nucleotide sequences described above.

In some embodiments, the pharmaceutical compositions may comprise nucleotide sequences encoding portions or a complete antibody molecule as described herein integrated into a cellular or viral genome or into a viriom. The pharmaceutical composition may then comprise a cell or virus as a vehicle for delivering an antibody of the invention (or a vehicle for delivering a nucleotide sequence encoding an antibody of the invention). For example, in an embodiment, the virus may be in the form of a therapeutic oncolytic virus comprising nucleotide sequences encoding at least one of the antibody molecules described herein. In some embodiments, such an oncolytic virus comprises nucleotide sequences encoding a full-length human IgG antibody.

Some embodiments of the invention relate to a virus comprising a nucleotide sequence as described herein or a plasmid as described herein. Preferably, the virus is an oncolytic virus, such as a therapeutic oncolytic virus. Such oncolytic viruses are known to those skilled in the art of medicine and virology.

In some embodiments, such an oncolytic virus comprises nucleotide sequences encoding an amino acid sequence having at least 80% identity to the sequence listed in Table 1 above. In some embodiments, such an oncolytic virus comprises an amino acid sequence having at least 85% identity to the sequence listed in Table 1 above. In some embodiments, such an oncolytic virus comprises an amino acid sequence having at least 90% identity to the sequence listed in Table 1 above. In some embodiments, such an oncolytic virus comprises an amino acid sequence having at least 95% identity to the sequence listed in Table 1 above.

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

Table 5. Example of nucleotide sequences encoding antibody 001-H10 - the parts of the sequences that are underlined in the table encode the VH and VL sequences of antibody 001-H10, respectively.

Some oncolytic viruses are able to accept DNA inserts large enough to allow integration of full-length human antibody sequences. Attenuated vaccinia and herpes simplex viruses are examples of therapeutic oncolytic viruses whose genome is large enough to integrate full-length IgG antibody sequences (Chan, W.M. 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 have been successfully integrated into the oncolytic Vaccinia virus, resulting in the expression and extracellular release (production) of full-length IgG antibodies upon infection of virus-sensitive host cells, such as cancer cells (Kleinpeter, P., 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 cellular infection (Marino, N., et al. 2017. 'Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes', PLoS One, 12: e0177810).

The invention also relates to pharmaceutical compositions comprising a virus, for example an oncolytic virus as discussed above, and a pharmaceutically acceptable diluent, carrier and/or adjuvant.

The invention also includes other therapeutic agents or "forms" of drugs, such as antibody-drug conjugates, fusion proteins, etc., and a pharmaceutical composition containing such therapeutic agents.

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 that may contain antioxidants and/or buffers and/or bacteriostats and/or solutes that form a composition isotonic with the blood of the intended recipient; and/or aqueous and/or non-aqueous sterile suspensions that 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 single or multiple dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (i.e., lyophilized) state requiring the addition of only a sterile liquid carrier, such as water for injection, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders and/or granules and/or tablets of the type previously described.

When administered parenterally to human patients, the daily dose level of the anti-TNFR2 antibody molecule is typically 1 mg/kg of the patient's body weight to 20 mg/kg, and in some cases even up to 100 mg/kg, administered in a single or divided dose. Lower doses may be used in special circumstances, such as in combination with continuous administration. In any case, the physician will determine the actual dosage that will be most suitable for each individual patient, which will vary depending on the age, weight and response of the individual patient. The doses given above are typical for the average case. Of course, there may be individual cases where higher or lower dosage ranges are justified, and such cases are within the scope of the present invention.

Typically, the pharmaceutical composition (or medicament) described herein comprising an antibody molecule will comprise the anti-TNFR2 antibody molecule at a concentration in the range of about 2 mg/mL to 150 mg/mL or about 2 mg/mL to 200 mg/mL.

In general, for humans, oral or parenteral administration of the antibody molecule, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein is the preferred and most convenient route. For veterinary use, the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein are administered in a suitable acceptable formulation in accordance with normal veterinary practice, and a veterinary surgeon will determine the dosage regimen and route of administration that will be most suitable for a particular animal. As a result, the present invention relates to a pharmaceutical composition comprising an amount of an antibody molecule, nucleotide sequence, plasmid, virus and/or cell of the present invention effective for the treatment of various conditions (as described above and further below). Preferably, the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein are adapted for delivery via a route selected from the group consisting of: intravenous (IV or i.v.); intratumoral (IM or i.m.); intramuscular (SC or i.m.) or subcutaneous.

The present invention also includes the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein, comprising pharmaceutically acceptable acid addition salts or base addition salts of the target binding molecules or portions thereof of the present invention. Acids that are used to prepare pharmaceutically acceptable acid addition salts of the above-mentioned basic compounds useful in the present invention are acids that form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as, in particular, the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, lactate, citrate, hydrogen citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1'-methylene bis-(2-hydroxy-3 naphthoate)] salts, among others. Pharmaceutically acceptable base addition salts can also be used to prepare pharmaceutically acceptable salt forms of the agents of the present invention. Chemical bases that can be used as reagents for the preparation of pharmaceutically acceptable base salts of the present agents that are acidic in nature are chemical bases that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, non-toxic base salts derived from such pharmacologically acceptable cations 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), as well as including lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines. The antibody molecules, nucleotide sequences, plasmids, viruses and/or cells described herein may be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, pellet drying) and/or resuspension methods may be used. Those skilled in the art will appreciate that lyophilization and resuspension may result in varying degrees of loss of antibody activity by the body (e.g., when using conventional immunoglobulins, IgM antibodies tend to lose more activity than IgG antibodies) and that application levels may be adjusted upward to compensate for this. In one embodiment of the invention, the lyophilized (freeze-dried) polypeptide-binding fragment loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (before lyophilization) upon rehydration.

The anti-TNFR2 antibody molecules, nucleotide sequences, and pharmaceutical compositions described herein can be used to treat cancer in a subject or patient. The terms "subject" and "patient" are used interchangeably herein.

In the context of this document, the term "patient" (or "subject") refers to an animal, including a human, diagnosed with a specific disease.

In some embodiments of the invention, the patient (or subject) is an animal, including a human, who has been diagnosed with cancer and/or exhibits symptoms of cancer.

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

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

It is contemplated that 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.

In the context of this document, the term “demonstrate” means that the patient demonstrates a symptom of cancer and/or a diagnostic marker of cancer; and/or the symptom of cancer and/or the diagnostic marker of cancer is measurable and/or assessable and/or quantifiable.

A medical professional will immediately understand what the symptoms of cancer and diagnostic markers of cancer are, as well as how to measure and/or assess and/or quantify whether there is a decrease or increase in the severity of cancer symptoms or a decrease or increase in diagnostic markers of cancer; and how these cancer symptoms and/or diagnostic markers of cancer can be used to form a prognosis for the course of cancer.

Cancer treatment is often given as a course of treatment, meaning the drug is given over a period of time. The length of the course of treatment will depend on a number of factors, which may include, but are not limited to, the type of drug being given; the type of cancer being treated; the severity of the cancer being treated; and the age and health of the patient.

In the context of this document, the expression "during treatment" means that the patient is currently undergoing treatment and/or receiving a medicinal product and/or is receiving a course of medicinal product.

In some embodiments, the cancer to be treated in accordance with the present invention is a solid tumor.

Each of the above types of cancer is well known, and the symptoms and diagnostic markers of cancer are well described, as are the drugs used to treat these types of cancer. Accordingly, the symptoms, diagnostic markers of cancer, and drugs used to treat the above types of cancer are known to medical professionals.

Clinical determinations of the diagnosis, prognosis and progression of a large number of cancers are based on specific classifications known as cancer staging. These cancer staging systems work by comparing a number of different diagnostic markers of cancer and symptoms of cancer to provide a diagnosis and/or prognosis and/or progression of cancer. A specialist in oncology will know how to assess the diagnosis and/or prognosis and/or progression of cancer using a cancer staging system and which diagnostic markers of cancer and symptoms of cancer should be used to do so.

In the context of this document, the expression "staging of cancer" means staging of cancer according to the Rai classification, which includes stage 0, stage I, stage II, stage III and stage IV, and/or according to the Binet classification, which includes stage A, stage B and stage C, and/or according to the Ann Arbor classification, which includes stage I, stage II, stage III and stage IV.

It is known that cancer can cause abnormalities in cell morphology. These abnormalities often recur in certain types of cancer, meaning that the study of these changes in morphology (also known as histology) can be used in the diagnosis or prognosis of cancer. Methods for imaging samples to study cell morphology and prepare samples for imaging are well known in the art; for example, light microscopy or confocal microscopy.

In the context of this document, the term "histological examination" means the presence of small mature lymphocytes and/or the presence of small mature lymphocytes with a narrow rim of cytoplasm, the presence of small mature lymphocytes with dense nuclei devoid of clearly visible nucleoli, and/or the presence of small mature lymphocytes with a narrow rim of cytoplasm and/or with dense nuclei devoid of clearly visible nucleoli, and/or the presence of atypical cells and/or cleaved cells and/or prolymphocytes.

It is well known that cancer results from mutations in the DNA of a cell, which can cause the cell to escape cell death or to proliferate uncontrollably. Therefore, the study of these mutations (also known as cytogenetic testing) can be a useful tool in assessing the diagnosis and/or prognosis of cancer. An example of this is the deletion of the chromosomal location 13q14.1, which is characteristic of chronic lymphocytic leukemia. Methods for studying mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

In the context of this document, the term "cytogenetic testing" means the testing of DNA in a cell and, in particular, in a chromosome. Cytogenetic testing can be used to detect changes in DNA that may be associated with the presence of refractory cancer and/or recurrent cancer. Such DNA alterations may include: deletions in the long arm of chromosome 13, and/or deletions in the 13q14.1 locus of chromosome 12, and/or trisomy of chromosome 12, and/or deletions in the long arm of chromosome 12, and/or deletions in the long arm of chromosome 11, and/or deletion of 11q, and/or deletions in the long arm of chromosome 6, and/or deletion of 6q, and/or deletions in the short arm of chromosome 17, and/or deletion in 17p, and/or t(11:14) translocation, and/or (q13:q32) translocation, and/or antigen receptor gene rearrangement, and/or BCL2 rearrangements, and/or BCL6 rearrangements, and/or t(14:18) translocations, and/or t(11:14) translocations, and/or (q13:q32), and/or translocations (3:v), and/or translocations (8:14), and/or translocations (8:v), and/or translocations t(11:14) and (q13:q32).

Cancer patients are known to exhibit certain physical symptoms that are often a result of the burden of cancer on the body. These symptoms often recur in the same cancer and therefore may be characteristic of the diagnosis and/or prognosis and/or disease progression. The health care professional should understand which physical symptoms are associated with which cancers and how the assessment of these physical systems may correlate with the diagnosis and/or prognosis and/or disease progression. For the purposes of this document, the term “physical symptoms” means hepatomegaly and/or splenomegaly.

Brief description of graphic materials

The following shapes are used in the examples below:

Fig. 1 shows that the antibodies of the present invention bind TNFR2. Fig. 1 A-D: Using ELISA assay, human antibodies were shown to bind to human TNFR2 protein in a dose-dependent manner, generating different EC50 values. Fig. 1 E: Murine antibodies 3-F10 and 5-A05 bind to mTNFR2 with similar affinity.

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 spleen-derived CD4 ⁺ T cells (Figure 2E) were activated with IL-2 and CD3/CD28 Dynabeads®. The affinity of TNFR2-specific n-CoDeR® antibodies to activated cells was analyzed by FACS at concentrations ranging from 0.002-267 nM (human) and 0.00003-133 nM (mouse). The curves show the mean fluorescence intensity (MFI) after subtraction of isotype control background (Fig. 2A (complete and partial blockers), Fig. 2B (partial blockers), Figs. C and D (non-blockers), Fig. 2E (mouse complete blocker (3-F10) and non-blocker (5-A05)).

While human TNFR2 antibodies bind with variable affinity to in vitro activated CD4 (EC50 values ranging from 0.59 to 53 nM), mouse TNFR2 antibodies bind with similar affinity (EC50 values ranging from 0.072 to 0.11 nM).

Figure 3 shows that TNFR2 n-CoDeR® antibodies specifically bind to TNFR2. Human blood-derived CD4 ⁺ T cells (Figure 3A) and mouse spleen-derived CD4 ⁺ T cells (Figure 3B) were activated for 3 days with recombinant IL-2 and CD3/CD28 activating beads. In vitro activated cells were blocked with anti-TNFR2 polyclonal antibody (gray line) or maintained in PBS (black line) for 30 min before staining with suboptimal concentrations of various TNFR2 n-CoDeR® antibodies or isotype control (dashed line) for 15 min. Cells were then washed and incubated with antigen-presenting cell-conjugated APC secondary antibody for 30 min before flow cytometric analysis.

All antibodies could be blocked by the TNFR2 polyclonal antibody, therefore, the TNFR2 n-CoDeR® antibodies (human and mouse) were shown to be specific for TNFR2.

Figure 4 shows the cross-reactivity of human TNFR2-specific n-CoDeR® antibodies to cynomolgus. CD4 ⁺ T cells were isolated from cynomolgus monkey blood and stimulated with PMA and ionomycin. After 2 days, cells were labeled with 0.1, 1, or 10 μg/ml TNFR2-specific n-CoDeR® antibodies or isotype control, followed by incubation with APC-conjugated human α secondary antibody. Cells were analyzed by flow cytometry. The figure shows the percentage of TNFR2 ⁺ T cells for individual antibodies compared to the isotype control. Results represent the mean and standard deviation (SD) of 2-3 separate experiments.

Most anti-TNFR2 antibodies exhibit cross-reactive binding to cynomolgus cells.

Figure 5 shows that all of the TNFR2-specific n-CoDeR® antibodies described herein bind different epitopes on the TNFR2 protein than the MR2-1 TNFR2 clone. Human blood-derived CD4 ⁺ T cells 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 bars) or maintained in PBS (Figure 5A, gray bars) for 30 min, then TNFR2-specific n-CoDeR®/polyclonal TNFR2 (pTNFR2) antibodies were added and the cells were incubated for 15 min. The percentage of bound TNFR2 n-CoDeR® antibodies was analyzed by FACS after incubation with APC-conjugated secondary antibodies. In Fig. 5B, activated CD4 ⁺ T cells were blocked with 40 μg/ml TNFR2-specific n-CoDeR®/pTNFR2 antibodies (black bars) or left in PBS (gray bar) and then incubated for 15 min with PE-conjugated MR2-1 antibodies. Cells were then analyzed by FACS.

The MR2-1 antibody did not interfere with the binding of TNFR2 specific n-CoDeR® antibodies, and n-CoDeR® antibodies did not affect MR2-1 binding to activated cells, indicating that all n-CoDeR® antibodies bind different domains of the TNFR2 protein than the MR2-1 antibody.

Figure 6 shows the ligand blocking activity of anti-human TNFR2 antibodies. Blocking ELISA was performed with n-CoDeR® mAb specific for hTNFR2 to evaluate the ligand blocking properties. Figure 6 A: All antibodies were incubated at 10 μg/mL. All antibodies that reduced the signal achieved by the isotype control by more than 50% (indicated by the dotted line) were then dosed to further study the ligand blocking potential. Figure 6 B shows the complete blocking mAbs, Figures 6 C and D show the partially blocking mAbs, and Figure 6 E shows the weak blocking mAbs. All other mAbs are considered non-blocking mAbs.

Figure 7 shows the ligand blocking activity of anti-mouse TNFR2 antibodies. Blocking ELISA was performed with n-CoDeR® mAb specific for mTNFR2 to evaluate the ligand blocking properties. Figure 7 A: All antibodies were incubated at 10 μg/mL. All antibodies that reduced the signal achieved by the isotype control by more than 50% (indicated by the dotted line) were then dosed to further examine the ligand blocking potential. Figure 7 B shows the fully blocking mAbs, Figures 7 C and D show the partially blocking mAbs, and Figure 7 E shows the weak blocking mAbs. All other mAbs are considered non-blocking mAbs. Based on this, antibodies 3-F10 and 5-A05 were selected to represent fully blocking antibodies and non-blocking antibodies, respectively.

Fig. 8. Categorization of TNFR2-specific n-CoDeR® antibodies according to their ability to agonize/antagonize TNFR2 signaling and their ability to block TNF-α binding to TNFR2. The ability of TNFR2-specific n-CoDeR® antibodies to increase or decrease IFN-γ production was monitored using IL-2- and IL-12-stimulated NK cells and plotted as a function of the ability of the antibodies to block TNF-α ligand binding to TNFR2 as described above. Fig. 8A: Human blood-derived NK cells were stimulated with 20 ng/ml rhIL-2 and 20 ng/ml rhIL-12 supplemented with 10 μg/ml TNFR2-specific n-CoDeR® antibody, isotype control, or 100 ng/ml rhTNF-α for 24 h. The amount of IFN-γ in culture supernatants was measured by MSD. The amount of IFN-γ was normalized to the isotype control (IFN-γ isotype control values = 1 in the figure) and is shown in Fig. 8A. Human antibodies that had an EC50 value 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 mean TNF-α levels of two donors). Cell culture supernatants were collected and the amount of IFN-γ produced was analyzed by MSD. Results are normalized to isotype control. IFN-γ results represent the mean of 3 donors in 2 independent experiments. The results reveal two extreme groups characterized by 1) antibodies with completely blocking and antagonist properties and 2) antibodies with non-blocking agonist properties, respectively. Agonist non-blocking antibodies are agonists and increase IFN-γ production from cytokine-stimulated NK cells, while blocking antibodies are antagonists and inhibit IFN-γ release. In Fig. 8C shows that IFN-γ release is TNF-α dependent upon neutralization of soluble TNF-α, decreasing IFN-γ, with addition of exogenous TNF-α increasing IFN-γ. Fig. 8D shows that addition of a blocking anti-TNF-α antibody results in dose-dependent neutralization of soluble TNF-α. At 1 μg/mL, soluble TNF-α is not detectable in the supernatant.

Figure 9 shows that non-blocking agonist but not blocking antagonist TNFR2-specific n-CoDeR® antibodies increase the proportion of CD25 ⁺ cells in the memory CD4 ⁺ T cell population. Human blood-derived CD4 ⁺ T cells (Figure 9A) and mouse spleen-derived CD4 ⁺ T cells (Figure 9B) were activated with recombinant IL-2 and TNFR2-specific n-CoDeR® antibodies, isotype control, or recombinant TNF-α. After 3 days in culture, cells were stained for CD25 and CD45RO (human)/CD44 and CD62L (mouse) and analyzed by flow cytometry. Results show the percentage of CD25-expressing cells in the memory population (CD45RO ⁺ (human)/CD44 ^{+ CD62L−} (mouse)) compared with the percentage of CD25 ⁺ cells isolated in isotype control cultures. Results represent the mean and SEM for 7 donors (Fig. 9A, human) and 3 mice (in 2 independent experiments, Fig. 9B). In both human and mouse cultures, non-blocking TNFR2 antibodies induced a percentage of CD25 ⁺ memory cells, whereas blocking antibodies had no effect on the memory population. For both human (Fig. 9A) and mouse (Fig. 9B) cultures, addition of exogenous TNF-α increased the CD25 ⁺ memory T cell population * = p < 0.05 as calculated by one-way ANOVA.

Figure 10A shows that ligand-blocking antagonist antibodies have the most potent antitumor activity, as does mIgG2a, an isotype that preferentially engages activating Fc receptors. Balb/c mice were injected subcutaneously with 1 × 10 ⁶ CT26 cells. After 8 days, when tumors averaged 3 × 3 mm, mice were treated intraperitoneally with 10 mg/kg antibody twice weekly as shown in the figures. Tumors were measured twice weekly until they reached 15 mm in diameter, at which time the mice were sacrificed. The top figure shows tumor growth in mice treated with an isotype control, then the two figures below, on the left panel, show a ligand-blocking antagonist antibody (middle figure) and a non-ligand-blocking agonist antibody (lower figure), in the FcγR-deficient Ig format. The middle panel shows the same antibodies in the mouse IgG2a format, which primarily engages activating FcγR, and the right panel shows the antibodies in the mouse IgG1 format, which primarily engages inhibitory FcγRIIb. In Fig. 10B, surviving mice were followed for 70 days. As can be seen in the figures, the blocking antagonist antibody is most effective in treating tumors in the IgG2a format, which primarily engages activating FcγR, and has no effect in the FcγR binding-deficient format. On the other hand, the non-blocking agonist antibody is most effective in treating tumors in the IgG1 format, preferentially engaging inhibitory FcγR. In addition, the agonist antibody has an intrinsic, FcγR-independent, antitumor effect, as seen using the N297A antibody format. *** = p < 0.001 compared to isotype control calculated using Mantel-Cox log-rank test

Figure 11 shows that ligand=blocking antagonist antibodies are effective as antitumor treatment in combination with anti-PD1. C57/BL6 mice were injected subcutaneously with 1 × 10 ⁶ MC38 cells. At an average tumor size of 3 × 3 mm, mice were treated twice weekly with 10 mg/kg antibody intraperitoneally as shown in the figures. The figures show tumor growth curves in individual mice. Figure 11 A: isotype control, Figure 11 B: targeted PD-1 antibody, Figure 11 C: 3-F10 antibody (surrogate antibody, ligand blocker, antagonist), Figure 11 D: combination of 3-F10 and PD1. Tumors were measured twice weekly until they reached a diameter of 15 mm, at which time the mice were sacrificed. In Figure 11 11E shows survival curves for four different treatment groups, * = p < 0.01, *** = p < 0.001 compared with isotype control, calculated using the Mantel-Cox log-rank test.

Figure 12 shows that blocking antagonist antibodies are effective as antitumor treatment in combination with anti-PD-L1. C57/BL6 mice were injected subcutaneously with 1 × 10 ⁶ MC38 cells. At an average tumor size of 5 × 5 mm, mice were treated twice with isotype control antibody or 3F10 (days 1 and 4), or four consecutive days with anti-PD-L1 followed by 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 at a dose of 10 mg/kg intraperitoneally. The figure shows mean tumor growth +/- SEM, n = 10/group. * = p < 0.05, *** = p < 0.001, as calculated using one-way ANOVA.

Fig. 13. C57/BL6 mice were injected subcutaneously with 1 × 10 ⁶ B16.F10 cells. Three days later, the mice were injected intraperitoneally with 10 mg/kg antibody twice a week as shown in the figures. Tumors were measured twice a week until they reached a diameter of 15 mm, after which the mice were sacrificed. Fig. 13 A: isotype control, Fig. 13 B: 3-F10 antibody (surrogate antibody, ligand blocker, antagonist). Fig. 13 C shows the survival curves for the two different treatment groups. * = p < 0.05 compared with isotype control calculated by the Mantel-Cox log-rank test.

Figure 14 shows 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 treated with antibodies as indicated after tumors reached approximately 7 x 7 mm in size after 3 injections. At day 8 after treatment initiation, mice were sacrificed and tumors were harvested. Single cell tumor suspensions were analyzed for immune cell content by FACS. Figure 14 A: The ligand blocking antagonist surrogate antibody 3F10 induces Treg depletion, and Figure 14 B: CD8 influx or expansion⁺T cells. This causes a shift in the ratio CD8⁺/Treg T cells, as shown in Fig. 14 C. Fig. 14 D shows that not only the number of T cells, but also the number of myeloid cells, in this case tumor-associated macrophages (TAMs, defined as CD11b⁺F4/80⁺MHCII⁺, but negative for both Ly6G and Ly6C) is very significantly reduced. The non-blocking agonist surrogate antibody 5A05 also modulates TAM numbers, but still differs significantly from the ligand-blocking antibody 3F10.

Figure 15 shows that T cells in human tumors express levels of TNFR2 similar to T cells derived from PBMCs from reconstituted NOG mice. Briefly, NOG mice were injected i.v. with 15-20 × ¹⁰⁶ PBMCs. After 10-12 days, the mice were spleen-removed, single-cell suspensions were prepared, and TNFR2 expression was assessed by FACS. TNFR2 expression was previously assessed on T cells derived from blood and tumor samples from 3 or 9 cancer patients, respectively. As shown in the figure, TNFR2 expression on Tregs and CD8 ⁺ T cells is highly comparable between human T cells expanded and activated in vivo in NOG mice and T cells from human tumors.

Figure 16 shows that the ligand blocking antagonist antibody 1-H10 depletes Tregs in vivo in an FcγR-dependent manner. NOG mice were injected i.v. with 15–20 × 10⁶PBMC cells. After 10-12 days, the mice's spleens were removed, a suspension of single cells was prepared, and then injected intraperitoneally into SCID mice. (10-15 x 10⁶/mouse). After 1 hour, mice were injected with 10 mg/kg of antibody intraperitoneally, and 24 hours after antibody injection, intraperitoneal fluid was collected from the mice and the cells in this fluid were analyzed by FACS. Figure 16A shows the average percentage of stained Tregs (defined as CD45⁺CD3⁺CD4⁺CD25⁺CD127^low/neg) in the CD45 population⁺human and showed that Tregs were significantly depleted by the blocking antibody 1-H10. Fig. 16B shows the mean percentage of CD8+ T from CD45⁺human and shows that 1-H10 significantly increases the population CD8⁺T cells. Fig. 16C shows that the ratio of CD8⁺T cell to Treg depletion was significantly increased by 1-H10. Data in Fig. 16A-C are shown as the average of four different experiments, where each point represents one mouse. Yervoy and a commercially available anti-CD25 antibody were used as positive controls. All data were normalized to isotype control, so that isotype control is set to 100% in Fig. 16A and B and to 1 in Fig. 16C. Figure 16D shows a separate experiment in which the FcγR binding-deficient 1-H10 IgG1N297Q antibody (labeled 1-H10NQ in the figure) was used to assess the dependence of FcγR binding on Treg cell depletion. As shown in the figure, depletion was most effective with wild-type IgG1 (1-H10) compared to Fc-deficient (1-H10NQ).

Figure 17 shows that non-TNFR2 ligand blocking antagonist antibodies do not induce cytokine release in vitro. IFN-γ release induced by different TNFR2 specific antibodies was measured in three different in vitro systems. Anti-CD3 antibody = OKT3, anti-CD52 antibody = alemtuzumab, and anti-CD28 antibody were used as positive controls. Isotype control was used as negative control. Each point represents PBMC from a single human donor. Figure 17A shows the results for high-density cell cultures where PBMC were cultured at a concentration of 1 × 10 ⁷ cells/ml. After 48 h, 10 μg/ml antibody was added and incubated for 24 h. As can be seen in the figure, both alemtuzumab and OKT3 induced significant IFN-γ release but did not induce any TNFR2-specific antibodies. Figure 17B shows in vitro solid phase cultures prepared by coating wells of a 96-well plate with antibodies prior to addition of PBMC. Again, both alemtuzumab and OKT3 induced significant IFN-γ release along with some TNFR2-specific antibodies. However, the complete blocking antibody 1-H10 did not induce cytokine release above the isotype control antibody. Figure 17C shows whole blood stimulation with antibody, and here alemtuzumab induced significant IFN-γ release but not any TNFR2-specific antibodies.

Figure 18 shows that non-TNFR2 ligand blocking antagonist antibodies do not induce cytokine release in vivo. NOG mice were injected i.v. with 25 x 10 ⁶ PBMC cells. After 14 days, when the blood of mice was shown to contain approximately 40% human T cells, the mice were treated with 10 μg of antibody. Body temperature was measured 1 h after injection (Fig. 18 A). The experiment was terminated 5 h after injection, and blood was analyzed for IFN-γ (Fig. 18 B) or TNF-α (Fig. 18 C). **** = p < 0.0001 and ** = p < 0.01, as calculated by one-way ANOVA.

Figure 19 shows binding to TNFR2 variants lacking individual domains. Antibody binding to TNFR2 variants expressed on HEK cells was tested by flow cytometry. The absence of domains 1 and 2 does not significantly affect binding (Figure 19 A and B), whereas 3 and part of 4 completely abolish the interaction between the antibody and TNFR2 (Figure 19 C and D). Similarly, the absence of domain 1+3 completely prevents binding of all antibodies (except 1F06) (Figure 19 E), whereas the absence of domain 2+4 completely abolishes binding of agonist antibodies (1F02, 1F06, 4E08) and significantly reduces binding also to antagonists (1H10, 4H02, 5B08) (Figure 19 F). Dark gray indicates positive control and white indicates negative control antibodies.

Figure 20 shows a comparison of the amino acid sequence of domain 3 of human (H-D3) and mouse (M-D3) TNFR2. Similar amino acids are highlighted in white and differences are highlighted in gray. The five sequences below represent the five different constructs against which antibodies are tested. Sequence exchanges from human to mouse are underlined, while the unmarked sequence is fully human. Domains 1, 2, and 4 are human and contain no substitutions or mutations.

Figure 21 shows binding to wild-type human and mouse TNFR2 (left panel). Mutated hTNFR2 constructs (m1, m2, m3, and m4) were used to narrow the binding site for different anti-hTNFR2 antibodies. Flow cytometry analysis showed that mutations in amino acids 119–132 did not affect antibody binding, whereas mutations in amino acids 151–160 completely abolished binding of all antibodies. Mutations in 134–144 impaired binding of only blocking and antagonist antibodies but had no significant effect on agonist antibodies. Dark gray bars indicate positive controls and white bars indicate negative controls. The dotted line indicates the level of negative control antibody.

Examples

The following are specific, non-limiting examples that illustrate certain aspects of the invention.

In many examples, particularly in vivo examples, antibody 3-F10 was used. This is a murine antibody that is a surrogate antibody for the human antibodies described herein. It was selected based on its ability to bind murine TNFR2, block the binding of its murine TNF-α ligand to TNFR2, and its antagonist activity in a murine T cell activation assay as described in Example 4. In some examples, antibody 3-F10 was tested and compared in various antibody formats associated with strong and preferential binding to activating over inhibitory Fc gamma receptors (mIgG2a), strong and preferential binding to inhibitory murine FcγR (mIgG1), or defective binding to murine FcγR (mIgG2a N297A).

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

In some examples and figures, slightly different names are used for antibody clones, for example, clone 001-H10 is sometimes shortened to 1-H10 or 1H10, 005-B08 is sometimes shortened to 5-B08 or 5B08, etc.

Example 1. Production of antibodies specific to TNFR2

(See also Fig. 1 and the above description of this figure.)

Isolation of scFv antibody fragments

The n- ^Coder® scFv library (BioInvent; Söderlind E, et al. Nat Biotechnol. 2000; 18(8):852-6) was used to isolate scFv antibody fragments recognizing human or mouse TNFR2.

The phage library was used in three consecutive pannings against recombinant human or mouse protein (Sino Biological). After phage incubation, cells were washed to remove unbound phage. Binding phage were eluted with trypsin and amplified in E. coli. The resulting phage stock was converted to scFv format. E. coli was transformed with plasmids carrying scFv, and individual scFv clones were expressed.

Identification of a unique TNFR2 binding scFv

Converted scFvs from the third panning were analyzed using the FMAT homogeneity assay (Applied Biosystems, Carlsbad, CA, USA) for binding to 293 FT cells transfected to express human or mouse TNFR2 or an unrelated protein.

Briefly, transfected cells were added to clear-bottomed plates along with scFv-containing supernatant 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; cat. no. 115-136-146, Jackson Immunoresearch). FMAT plates were incubated at room temperature for 9 h before reading. Bacterial clones binding to TNFR2-transfected cells but not to cells transfected with an unrelated protein were classified as active and selectively collected into a 96-well plate.

IgG binding to TNFR2 in ELISA

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

The data presented in Table 6 and Figs. 1 A-D show that all human anti-TNFR2 antibodies bind to the human TNFR2 protein. EC50 values range from 0.082 nM for 1-C08 to 4.4 nM for 1-A09.

In addition, surrogate mouse antibody clones 3-F10 and 5-A05 also bind to the mTNFR2 protein. These two clones bind with very similar affinity (Table 6 and Fig. 1E).

Table 6. EC50 values of antibody binding to TNFR2 protein (human protein, except clone 3F10 and 5A05)

Clone EC ₅₀ (nM) 1-C08 0.082 1-E06 0.20 1-G10 0.29 1-H10 0.29 4-H02 0.20 5-B02 0.15 5-B08 0.17 1-G04 1.7 1-H09 0.30 1-D01 0.37 5-F10 0.22 1-B11 0.25 1-C07 0.26 1-B05 0.23 1-F02 0.31 1-F06 0.15 4-E08 0.38 1-G05 0.54 1-A09 4.4 1-B09 0.18 1-C03 0.75 1-C05 0.38 3-F10 (mouse) 0.97 5-A05 (mouse) 1.4

Example 2. Antibody specificity

(See also Figs. 2-5 and the above description of these figures.)

CD4 ⁺ T cell isolation

PBMCs from human buffy coat and cynomolgus macaque (M. fascicularis) whole blood were isolated using Ficoll-Paque PLUS gradients (GE Healthcare). CD4 ⁺ T cells were isolated from PBMCs by magnetic cell sorting using the CD4 ⁺ T Cell Isolation Kit (Human) or CD4 MicroBeads, non-human primate (cynomolgus macaque), both from Miltenyi. Mouse CD4 ⁺ T cells were isolated from spleen using the CD4 ⁺ T Cell Isolation Kit (Mouse) from Miltenyi.

Titration of TNFR2-specific antibodies n-CoDeR®

The ability and affinity of the TNFR2 n-CoDeR® antibody to bind TNFR2 expressed on cells were determined using in vitro activated CD4 ⁺ T cells. Human CD4 ⁺ T cells were stimulated with 50 ng/ml hrIL-2 (R&D Systems) and Dynabeads® CD3/CD28 T-Activator, for T cell activation and expansion (Gibco), for 2-3 days at 37°C. The in vitro activated cells were labeled with increasing amounts of n-CoDeR® antibodies specific for TNFR2 or isotype control, ranging from 0.002-267 nM. The cells were then incubated with a-human IgG secondary antibody conjugated to APC (Jackson), followed by flow cytometric analysis (FACSVerse, BD). The resulting titration curves are shown in Fig. 2 AD. Murine CD4 ⁺ T cells were stimulated with 135 U/ml rmIL-2 (R&D systems) and Dynabeads® CD3/CD28 T activator for T cell expansion 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 0.00003-133 nM. Cells were then incubated with APC-conjugated a-mouse IgG secondary antibody (Jackson) followed by flow cytometry analysis (FACSVerse, BD). Titration curves are shown in Fig. 2E. The EC50 values for the titration curves were calculated in Microsoft Excel and are shown in Table 7. For human antibodies, the EC50 values ranged from 0.6 nM (4-H02) to 52.7 nM (1-CO3). Mouse antibodies bound to in vitro activated cells with similar affinity (0.072 nM (3-F10) and 0.11 nM (5-A05)).

Specificity of TNFR2 n-CoDeR® antibodies

The specificity of TNFR2 antibodies to TNFR2 was obtained in FACS blocking experiments with commercial polyclonal TNFR2 antibodies (R&D systems). CD4 ⁺ T cells (mouse and human) stimulated for 2-3 days with 50 ng/ml rhIL-2 (R&D systems) (human)/135 U/ml rm IL-2 (R&D systems) (mouse) and Dynabeads® CD3/CD28 T Activator for T Cell Expansion and Activation (Gibco) were blocked with 40 μg/ml TNFR2 polyclonal antibody (R&D systems) for 30 min followed by 15 min incubation with TNFR2 n-CoDeR® antibodies or isotype control. The concentration of n-CoDeR® antibodies used was based on titration curves for individual n-CoDeR® TNFR2 antibodies and a suboptimal concentration was selected for each antibody. Cells were then washed and incubated for 30 min with APC-conjugated secondary antibody (Jackson). Cells were analyzed by flow cytometry (FACSVerse, BD). All binding of TNFR2-specific n-CoDeR® antibodies (both human and mouse) could be blocked by the anti-TNFR2 polyclonal antibody as shown in Fig. 3. These results confirm that TNFR2 n-CoDeR® antibodies specifically bind TNFR2 on CD4 ⁺ T cells activated in vitro.

Epitope mapping of TNFR2-specific n-CoDeR® antibodies against TNFR2 antibody clone MR2-1

The TNFR2 antibody clone MR2-1 (Invitrogen) binds a specific domain of the TNFR2 protein. If the TNFR2-specific n-CoDeR® antibodies bind to the same domain as MR2-1, they were tested by FACS blocking experiments.

Human CD4 ⁺ T cells were stimulated for 2–3 days with 50 ng/ml rhIL-2 (R&D systems) and Dynabeads® CD3/CD28 T cell activator for T cell expansion and activation (Gibco). Activated cells were blocked with 40 μg/ml MR2-1 (black bars in Fig. 5 A) or PBS (gray bars in Fig. 5 A). After 30 min of incubation, cells were immediately stained with TNFR2-specific antibody n-CoDeR® or polyclonal TNFR2 (pTNFR2) for 15 min. After incubation with APC-conjugated anti-human IgG secondary reagent (Jackson), cells were analyzed by flow cytometry (FACSVerse, BD). In Fig. 5B, activated CD4 ⁺ T cells were blocked with 40 μg/ml TNFR2-specific n-CoDeR® or pTNFR2 antibodies (black bars) or left in PBS (gray bar) and then incubated for 15 min with PE-conjugated MR2-1 antibody. Again, cells were analyzed by FACS. Since the percentage of MR2-1 ⁺ cells was the same for both n-CoDeR®-blocked and unblocked cells (Fig. 5B), and n-CoDeR® antibody binding was the same with or without MR2-1 blocker (Fig. 5A), these data demonstrate that n-CoDeR® antibodies bind different epitopes of the TNFR2 protein than the MR2-1 antibody.

Binding of TNFR2 n-CoDeR® antibodies to cynomolgus monkey

To confirm the cross-reactivity of TNFR2 antibodies to cynomolgus macaque, cynomolgus macaque CD4 ⁺ T cells were stimulated for 2 days with 50 ng/ml PMA (Sigma) and 100 ng/ml ionomycin (Sigma) to induce TNFR2 activation. The cells were incubated with TNFR2-specific n-CoDeR® antibodies at 3 different concentrations (0.1, 1, and 10 μg/ml) and then incubated with APC-conjugated secondary a-human IgG reagent (Jackson). The cells were analyzed by flow cytometry (FACSVerse, BD), and the result showed that most of the n-CoDeR® antibodies specific for human TNFR2 could bind cynomolgus macaque TNFR2, the results for individual antibodies are shown in Fig. 4.

Thus, the data in Example 2 demonstrate that human antibodies bind specifically to TNFR2 endogenously expressed on human immune cells. Furthermore, the data demonstrate that this binding can be blocked by the addition of a commercially available polyclonal anti-TNFR2 antibody, indicating a very high specificity for TNFR2. The same is true for the surrogate clones 3F10 and 5A05 against mouse cells expressing mouse TNFR2. Furthermore, binding of the human clones is not dependent on MR2-1 antibodies, which exhibit a different epitope specificity than MR2-1.

Table 7. EC ₅₀ values calculated from titration of TNFR2-specific antibodies to in vitro activated CD4 ⁺ T cells.

Clone EC ₅₀ (nM) 1-C08 2.6 1-E06 4.1 1-G10 3.3 1-H10 1,1 4-H02 0.59 5-B02 0.80 5-B08 1,2 1-G04 18 1-H09 16 1-D01 3.9 5-F10 32 1-B11 27 1-C07 36 1-B05 1.5 1-F02 0.79 1-F06 2.5 4-E08 2,3 1-G05 0.66 1-A09 48 1-B09 29 1-C03 53 1-C05 12 3-F10 (mouse) 0.072 5-A05 (mouse) 0.11

Example 3. Testing the ligand blocking properties

(See also Figs. 6-7 and the above description of these figures.)

ELISA method

96-well plates were coated with hTNFR2 (Sinobiology Cat. No. 10414-H08H) or mTNFR2 (Sinobiology Cat. No. 50128 M08H) at a concentration of 2.5 pmol/well in ELISA coating buffer (0.1 M sodium carbonate, pH 9.5) and incubated overnight at 4°C. After washing in ELISA wash buffer (PBS with 0.05% Tween20), the plates were incubated with slow shaking for 1 h at room temperature with n-CoDeR® monoclonal antibodies at a concentration of 10 μg/ml (single-dose ELISA) or 33 nM, followed by a 1:2 dilution (ELISA titration) in blocking buffer containing 0.45% fish gelatin. Subsequently, recombinant hTNF-α-bio (R&D, Cat. No. BT210) or mTNF-α (Gibco Cat. No. PMC3014) was added at a final concentration of 5 nM and 2 nM, respectively, and incubated for an additional 15 min. The plates were then washed. For human ELISA, streptavidin-HRP (Jackson Cat. No. 016-030-084) was diluted 1:2000 in blocking buffer and incubated again for 1 h at room temperature, followed by washes first in ELISA buffer and then in Tris buffer (pH 9.8). The substrate (Super Signal ELISA Pico from Thermo Scientific cat. no. 37069) was then diluted according to the manufacturer's instructions, added to the wells and incubated in the dark for 10 min before reading on a Tecan Ultra. For the mouse ELISA assay, rabbit anti-mTNF-α (Synobiology cat. no. 50349-RP02) was diluted to 1 μg/ml and allowed to incubate for 1 h at room temperature. After washing, anti-rabbit-HRP diluted 1:10,000 in blocking buffer was added and incubated again for 1 h at room temperature. Substrate addition and reading were performed as above.

The data are presented below in Tables 8 and 9, as well as in Figs. 6 and 7.

Table 8. EC50 values of human ligand blocking antibodies: Antibodies were titrated and EC50 values were calculated

Clone EC50 (nM) Blocking 001-H10 0.9 Complete 004-H02 0.4 Complete 005-B08 0.3 Complete 005-B02 0.2 Complete 001-E06 0.3 Partial 001-G10 1.6 Partial 001-C08 1,1 Partial 001-H09 1.4 Partial 005-F10 0.03 Partial 001-G04 3.2 Partial 001-B11 1.0 Weak 001-C07 0.8 Weak 001-D01 1.4 Weak

Table 9. EC50 values of mouse ligand blocking antibodies: Antibodies were titrated and EC50 values were calculated

Clone EC50 (nM) Blocking 3-F10 1.9 Complete 4-C01 2.7 Complete 4-A06 2.0 Partial 4-A07 >500 Partial 4-F06 6.2 Partial 5-C09 8.6 Partial 2-D09 4.4 Partial 4-B12 >500 Partial 3-G06 13 Partial 2-H01 25 Weak 4-C02 >500 Weak 4-G09 2.6 Weak 4-C03 8.3 Weak

Definitions of blockers

٠ Complete blockers are defined as reducing TNF-α binding by greater than 98%

٠ Partial blockers are defined as reducing TNF-α binding by 60-98%

٠ Weak blockers are defined as those that reduce TNF-α binding by less than 60%

٠ Non-blocking antibodies are defined as those not achieving greater than 50% blocking in the high-dose single-point ELISA assay, as shown in Figs. 6A and 7A.

The data presented in this example show that a variety of antibodies have been generated, ranging from antibodies that completely inhibit TNF-α ligand from binding to antibodies that do not inhibit ligand blocking at all. This is true for both human antibodies and mouse surrogates.

Example 4. In vitro functionality of antibodies

(See also Figs. 8-9 and the above description of these figures.)

The ability of TNFR2 antibodies to regulate cytokine-stimulated IFN-γ production by NK cells

The agonist/antagonist properties of TNFR2-specific antibodies were assessed using the 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).

Briefly, human NK cells were isolated from human PBMC by MACS using the “NK Isolation Kit” (Miltenyi). 100 μl of NK cells (1 x ¹⁰⁶ cells/ml) were cultured with 20 ng/ml rhIL-2 (R&D systems) and 20 ng/ml rhIL-12 (R&D systems) together with 10 μg/ml TNFR2-specific antibodies, 10 μg/ml isotype control or 100 ng/ml TNF-α (R&D systems) in U-bottom plates (96-well Corning® TC-treated microplates, Sigma-Aldrich). Supernatants were collected after 24 h and the amount of IFN-γ produced was estimated by MSD.

As a control, anti-TNF-α antibodies neutralizing TNF-α (Cat. No. AF-210-NA, R&D systems) were used. As shown in Fig. 8D, the 1 μg/ml dose completely neutralized soluble TNF-α and this dose also reduced the release of IFN-γ.

Non-blocking human TNFR2 antibodies markedly enhanced IFN-γ production by IL-2 and IL-12 stimulated NK cells (2-3 times more IFN-γ than isotype control), whereas antagonist antibodies (shown here with full blockers) exhibited antagonistic effects on NK cells and reduced IFN-γ production (Fig. 8A).

This assay was considered unrepresentative for use with mouse surrogate antibodies due to the lack of endogenously produced TNF-α in mouse cultures, as well as the expression of inhibitory FcγR on mouse NK cells, whereas only activating FcγR was expressed on the human counterpart. Instead, a memory T cell activation assay (CD25 induction) was used, as described below, to examine the agonist or antagonist properties of the mouse surrogate antibodies.

Induction of CD25-expressing CD4 ⁺ memory T cells by anti-TNFR2 antibodies

To further understand the agonist/antagonist properties of TNFR2 antibodies, their ability to increase the proportion of CD25 expressing memory CD4 ⁺ T cells was assessed.

Briefly, human CD4 ⁺ T cells were isolated from PBMC by MACS using the “CD4 ⁺ T cell isolation kit” from Miltenyi. CD4 were cultured with 10 ng/ml rhIL-2 (R&D systems) and 10 μg/ml TNFR2 specific antibodies or the indicated amount of rhTNF-α (R&D systems). After 3 days, CD25 expression on memory cells (CD45RO ⁺ cells) was analyzed by FACS (Fig. 9A).

Similarly, mouse CD4 ⁺ T cells were isolated from the spleen by MACS using the “CD4 ⁺ T cell isolation kit” (Miltenyi) and cultured with 10 ng/ml rmIL-2 (R&D systems) and 10 μg/ml TNFR2-specific antibody or the indicated amount of rmTNF-α (R&D systems). CD25 expression on memory cells (CD44 ⁺ CD62L ⁻ cells) was analyzed by FACS after 3 days (Fig. 9B).

The percentage of cells expressing CD25 was increased in memory cell cultures stimulated with non-blocking TNFR2 in both humans and mice. However, stimulation with blocking antibodies did not increase CD25 expression in these cultures, but rather decreased it.

Thus, the data in Example 4 show that the ligand blocking antibodies are antagonistic as measured by several in vitro methods: inhibition of NK cell-mediated IFN-γ release and activation of CD4 ⁺ memory cells as measured by CD25 expression. In accordance with the invention, ligand blocking antagonist antibodies are shown, while non-ligand blocking agonist antibodies are included for comparison.

Example 5. Surrogate ligand blocking antagonist anti-mouse TNFR2 mAb has antitumor effect in vivo

(See also Figs. 10-16 and the description of these figures above.)

Therapeutic effect on different tumor models

To evaluate the in vivo antitumor effect of ligand blocking antagonist monoclonal anti-TNFR2 antibodies, a mouse surrogate named 3F10 was tested in vivo in various tumor models using different isotype formats and alone or in combination with anti-PD-1, as described below.

Mice were bred and maintained at local facilities according to home office recommendations. Six- to eight-week-old female BalbC and C57/BL6 mice were provided by Taconic (Bomholt, Denmark) and maintained at the local animal facilities. CT26, MC38, and B16.F10 cells (ATCC) were grown in glutamax-buffered RPMI medium supplemented with 10% FCS. When the cells were semiconfluent, they were detached with trypsin and resuspended in sterile PBS at 10 × 10 ⁶ cells/mL. Mice were injected s.c. with 100 μL of the cell suspension, corresponding to 1 × 10 ⁶ cells/mouse. Three to eight days after injection, depending on the model, mice were treated twice weekly with 10 mg/kg antibody intraperitoneally (isotype control, 3-F10 or 5-A05) and as indicated in the figures. Tumors were measured twice weekly until they reached a diameter of 15 mm, at which time mice were sacrificed.

The ligand blocking antagonist anti-murine TNFR2 mAb 3-F10 exhibited therapeutic antitumor effects in three different tumor models (Figs. 10–13) with a therapeutic effect in the more treatment-sensitive CT26 (Fig. 10) and a tumor growth inhibitory effect in the more treatment-resistant MC38 and B16 (Figs. 11–13).

Antitumor effect of ligand-blocking, antagonistic murine TNFR2 mAbs is Fc:FcγR-dependent

To assess the importance of the Fc-FcγR interaction on the in vivo antitumor effect of a ligand-blocking antagonist anti-TNFR2 murine surrogate mAb, different Fc formats of this antibody were studied in vivo in the CT26 tumor model as described below.

Mice were bred and maintained as described above. CT26 cells (ATCC) were grown and injected as described above. When tumors reached 3 x 3 mm, mice were injected intraperitoneally with 10 mg/kg antibody twice weekly (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, at which time mice were sacrificed.

Fc-deficient 3-F10-N297A exhibits minimal or no therapeutic activity compared to the isotype control, indicating that Fc engagement is critical for the therapeutic efficacy of this ligand-blocking antagonist anti-mouse TNFR2 mAb (Fig. 10A and B). Both the IgG1 and IgG2a formats exhibit significant therapeutic efficacy. However, the IgG2a format, which preferentially binds to activating Fcγ receptors, exhibits superior therapeutic effect, indicative of Treg cell depletion/phagocytosis, which is one of the important mechanisms of action of this ligand-blocking antagonist anti-mouse TNFR2 mAb (Fig. 10A-B). This contrasts with the non-blocking agonist surrogate antibody 5A05, which exhibits some activity in the defective Fc format and exhibits better activity in the mouse IgG1 format, which is known to preferentially bind the inhibitory FcγR. The ligand blocking antagonist antibody (3F10) of the present invention, and the non-blocking ligand agonist antibody (5A05) is included as a reference.

Combined effect with anti-PD-1 mAb

To evaluate the combined in vivo antitumor effect of the ligand-blocking antagonist anti-TNFR2 surrogate murine mAb (3-F10) with anti-PD-1, the treatment combination was tested in vivo in the MC38 tumor model as described below.

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 twice weekly with 10 mg/kg antibody intraperitoneally (isotype control, anti-mouse PD-1, 3-F10, or a combination of anti-mouse PD-1 and 3-F10) as shown in Fig. 11A–E. Tumors were measured twice weekly until they reached 15 mm in diameter, at which time mice were sacrificed.

Both anti-mouse PD-1 and ligand-blocking antagonist anti-mouse TNFR2 mAb 3-F10 exhibited therapeutic tumor growth inhibition effects in the MC38 model (Fig. 11 A-E). When anti-PD1 and antagonist anti-mouse TNFR2 mAb 3-F10 were combined, tumors were cured in treatment-resistant MC38 (Fig. 11 D-E).

Combined effect with anti-PD-L1 mAb

To evaluate the combined antitumor effect in vivo of ligand blocking antagonist anti-TNFR2 mAbs, we further combined a murine surrogate (3F10) with anti-PD-L1 for treatment in the MC38 tumor model as described below.

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 twice with isotype control antibody or 3F10 (days 1 and 4) or four consecutive days with anti-PD-L1 (clone 10F.9G2, Bioxcell) followed by 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 at 10 mg/kg intraperitoneally. Tumours were measured with calipers twice weekly until they reached a volume of 2000 mm ³ , at which time mice were sacrificed.

Both anti-mouse PD-L1 and ligand blocking antagonist anti-mouse TNFR2 mAb 3-F10 showed therapeutic tumor growth inhibition effect in MC38 model (Fig. 12). When anti-PD-L1 and antagonist anti-mouse TNFR2 mAb 3-F10 were combined, the antitumor effect was further enhanced (Fig. 12).

Modulation of immune cells in vivo

To investigate the effect of immune cells on tumor in vivo, BalbC mice were inoculated with CT26 cells as described above. After tumors reached approximately 7 x 7 mm, mice were treated with antibodies at 10 mg/kg intraperitoneally as indicated in the figures. Mice were treated on days 1, 4, and 7 and stopped on day 8. Tumors were excised, mechanically divided into small pieces and digested using a mixture of collagenase, 100 μg/ml liberase and 100 μg/ml DNase at 37°C for 2 x 5 min with vortex in between. After filtration through a 70 μm filter, the cell suspension was washed (400 g for 10 min) with PBS containing 10% FBS. The cells were then resuspended in MACS buffer and stained with a panel of antibodies staining for CD45, CD3, CD8, CD4, and CD25 or a panel of antibodies staining for MHCII, F4/80, Ly6C, CD11b, and Ly6G. Before staining, the cells were blocked for nonspecific binding with 100 g/ml purified intravenous immunoglobulin (IVIG). The cells were analyzed on a FACS Verse. Murine Treg cells were quantified as CD45 ⁺ CD3 ⁺ CD4 ⁺ CD25 ⁺ and TAMs as CD11b ⁺ Ly6G ^– Ly6C ^– F4/80 ⁺ MHCII ⁺ . The results are shown in Fig. 14.

As shown in Fig. 14, treatment with TNFR2 blocking ligand/antagonist antibody resulted in depletion of Treg cells in the tumor. There was also a weaker trend towards increased influx CD8⁺T cells. Together, this results in a significant improvement in the CD8 ratio⁺T cells to Treg cells (Fig. 14 C). In addition, the antagonist antibody modulates the myeloid compartment by reducing the number of tumor-associated macrophages (Fig. 14 D).

Model PBMC-NOG/SCID

To confirm the in vivo data on the depleting activity of the ligand blocking antagonist surrogate anti-mouse TNFR2 mAb, we analyzed the depleting ability of the ligand blocking antagonist anti-human TNFR2 mAb 1-H10 in the PBMC-NOG/SCID model in vivo as described below.

Mice were bred and maintained at local facilities according to home office recommendations. Eight-week-old female SCID and NOG mice were purchased from Taconic (Bomholt, Denmark) and maintained at local animal facilities. For the PBMC-NOG/SCID (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and after washing, the cells were resuspended in sterile PBS at 75 × 10 ⁶ cells/mL. NOG mice were injected i.v. with 200 μL of the cell suspension, corresponding to 15 × 10 ⁶ cells/mouse. Two weeks after injection, spleens were isolated and converted to a single-cell suspension. A small sample was then taken to determine TNFR2 expression on human T cells by FACS (Fig. 15). These FACS showed that TNFR2 expression on Tregs and CD8+ T cells was highly comparable between human T cells expanded and activated in vivo in NOG mice and T cells from human tumors. Most cells were resuspended in sterile PBS at a concentration of 50 × 10 ⁶ cells/mL. SCID mice were injected i.p. with 200 μL of the suspension, corresponding to 10 × 10 ⁶ cells/mouse. After 1 h, mice were treated with 10 mg/kg of either Yervoy, anti-CD25, 1-H10, 1-H10-N297Q (1-H10 Fc-deficient version), or an isotype control mAb. The intraperitoneal fluid of mice 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).

The Treg depletion activity of 1-H10 was superior to Yervoy and 1-H10N297Q (Fig. 16), confirming that the ligand blocking antagonist anti-TNFR2 mAb 1-H10 depletes Tregs and that Fc interaction is involved in this depletion (Fig. 16D).

Thus, example 5 shows that:

1. Ligand blocking antagonist antibodies can exert potent antitumor effects in several tumor models.

2. This effect can be enhanced by combination with anti-PD1 antibodies.

3. The effect depends on the coverage of activating FcγRs.

4. Treatment with a ligand blocking antagonist surrogate antibody significantly alters the T cell composition in tumors with an increased CD8 ⁺ T cell/Treg cell ratio and a decrease in tumor-associated macrophages.

5. In human tumors, the cells that express TNFR2 to the highest degree are Treg cells.

6. In a human xenograft model where tumor TNFR2 expression is mimicked on T cells, human Treg cells divide and CD8 ⁺ T cell levels increase.

7. This Treg deletion is most pronounced if the antibody can be targeted by activating FcγRs.

Example 6. Ligand blocking antagonist antibodies do not induce large amounts of proinflammatory cytokines

(See also Figs. 17-18 and the above description of these figures.)

Release of large amounts of proinflammatory cytokines is one of the possible side effects of immunomodulatory antibodies used to treat patients. Therefore, here we measured cytokine release induced by ligand blocking antagonist antibodies using two different methods. 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. For in vitro culture, the culture setup has been shown to significantly influence cytokine release (Vessillier et al., J Immunol Methods. 2015 Sep; 424: 43–52). To account for differences in methodologies, three different in vitro culture setups were used according to recent publications.

For cytokine release assay (CRA) in high density cell culture (HDC), PBMCs were cultured at 1 x ¹⁰⁷ cells/ml in CTL-Test serum-free medium (Cell Technology Limited) supplemented with 2 mM glutamine, 1 mM pyruvate, 100 IU/ml penicillin and streptomycin. 2 ml of cell culture was placed in a 12-well plate. After 48 h, 10 μg/ml antibody was added to 1 x ¹⁰⁵ pre-incubated PBMCs in a 96-well flat-bottom plate and incubated for 24 h.

Solid phase (SP) CRA of PBMC was performed by coating the wells of a 96-well plate with 1 μg/ml antibody for 1 h. After washing the plate with PBS, 1 × 10 ⁵ PBMC in 200 μl complete medium per well were added and incubated for 48 h.

Cytokine release was also measured after stimulation with 200 μl whole blood of 5 μg/ml antibody for 48 h.

At the end of the incubation period, the plates were centrifuged and the culture supernatant was collected and stored at -20°C. The 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).

Thus, the blocking antagonist antibody did not induce any significant cytokine release under any in vitro conditions. The positive control antibodies, alemtuzumab and OKT3, did induce cytokines, the most prominent of all being IFN-γ, but as seen in Fig. 17, the 1H10 antibody did not induce IFN-γ beyond the isotype control antibody. No other cytokine was elevated by 1H10 (data not shown).

PBMC-NOG Tolerability Model

To investigate the tolerability of the ligand-blocking antagonist mAb 1-H10 against human TNFR2, we analyzed cytokine release in vivo in the PBMC-NOG model as described below.

Mice were bred and maintained at local facilities according to home office recommendations. Eight-week-old female NOG mice were supplied by Taconic (Bomholt, Denmark) and maintained at the local animal facilities. For the PBMC-NOG (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and after washing, the cells were resuspended in sterile PBS at 125 × 10 ⁶ cells/mL. NOG mice were injected i.v. with 200 μL of the cell suspension, corresponding to 25 × 10 ⁶ cells/mouse. Two weeks after injection, blood samples were collected to analyze the level of “humanization”, meaning the number of human cells in the blood of NOG mice. The blood consisted of ∼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 an isotype control mAb. Body temperature was measured before antibody injection and 1 hour after injection. Fig. 18 A. As seen in Fig. 18 A, the positive control antibody OKT3 induced a dramatic decrease in body temperature, as previously published and consistent with the toxicity observed in the clinic with this antibody. In contrast, 1-H10 had no effect on body temperature. Five hours after antibody injection, experiments were stopped and blood was collected for cytokine release assay (MSD). The cytokines measured were human IFN-γ, TNF-α, IL-6, and IL1β. Of these, IFN-γ and TNF-α were quantified at a high enough level to be reliable. As seen in Fig. 18 B and C, the positive control antibody OKT3 induced both significant IFN-γ and TNF-α release (consistent with the toxicity observed in the clinic with this antibody), whereas 1H10-treated mice did not have significant IFN-γ release. However, there was a trend toward increased TNF-α release, although not significant and not as dramatic as for OKT3.

Thus, Example 6 shows that TNFR2 ligand blocking antibodies, in this case illustrated by an antibody called 1-H10, do not induce significant levels of cytokine release as measured by several previously published methods. Since cytokine release is a limiting factor for clinical development for some immunomodulatory antibodies, this indicates an acceptable safety profile in this regard.

Example 7. Epitopes of generated TNFR2-targeted antibodies

Knockouts in domain construction

In the first set of experiments, DNA constructs encoding various TNFR2 variants lacking one or more of the 4 extracellular domains described in Table 10 were used. In the second set of experiments, DNA constructs encoding TNFR2 variants in which various parts of domain 3 were replaced by the corresponding murine part as described in Table 11 were used. The latter is possible because none of the antibodies cross-reacts with murine TNFR. In both cases, the constructs were purchased from GeneArt (ThermoFisher). The constructs were cloned into an expression vector containing the CMV promoter and the OriP plasmid origin of replication and transiently expressed in suspension-adapted HEK293-EBNA cells.

Table 10. TNFR2 constructs used for transfection where one or more domains were deleted.

Design Description hTNFR2 wild type, human full length TNFR2 (uniprot#P20333) hTNFR2-Δ 1 hTNFR2 with TNFR-Cys 1 domain deleted (amino acid 39-76) hTNFR2- Δ 2 hTNFR2 with TNFR-Cys 2 domain deleted (amino acid 77-118) hTNFR2- Δ 3 hTNFR2 with TNFR domain deleted-Cys 3 (amino acid 119-162) hTNFR2- Δ 4 hTNFR2 with TNFR domain deleted-Cys 4 (amino acid 163-201) hTNFR2- Δ 1 + 3 hTNFR2 with deleted TNFR-Cys domain 1 and 3 (amino acid 39-76 and 119-162) hTNFR2- Δ 2 + 4 hTNFR2 with deleted TNFR-Cys domain 2 and 4 (amino acid 77-118 and 163-201)

Table 11. TNFR2 constructs used for transfection replaced various parts of domain 3 with the corresponding murine sequences.

Design Description hTNFR2 wild type, human full length TNFR2 (uniprot#P20333) mTNFR2 wild type, full length mouse TNFR2 (uniprot #P25119) hTNFR2-m1 hTNFR2 with amino acids 119-132 replaced by amino acids 120-133 from mTNFR2 hTNFR2-m2 hTNFR2 with amino acids 134-144 replaced by amino acids 135-146 from mTNFR2 hTNFR2-m3 hTNFR2 with amino acids 151-160 replaced by amino acids 153-162 from mTNFR2 hTNFR2-m4 hTNFR2 with amino acids 130-144 replaced by amino acids 131-146 from mTNFR2

Flow cytometry based binding assay

HEK-293-E cells were transfected with the corresponding TNFR2 variant cDNA plasmids using Lipofectamine 2000. At 48 h post-transfection, cells were harvested and stained with the indicated antibodies for 30 min. After 2 PBS wash steps, surface-bound antibodies were stained with secondary anti-APC-coupled IgG. Cells were washed and stained for live/dead cells prior to flow cytometry analysis on a BD-Verse flow cytometer.

Flow cytometry-based binding experiments on transfected HEK 293 cells clearly showed that domain 1 and domain 2 either did not affect binding (domain 1) or only marginally affected binding (domain 2) of any of the antibodies to these cells. A polyclonal antibody against human TNFR2 was used as a positive control. The positive control antibody showed high binding to all constructs tested, whereas the negative antibody showed no binding (Fig. 19). All antibodies tested showed complete loss of binding to TNFR2 lacking domain 3. Likewise, most antibodies failed to bind to TNFR2 if domain 4 was missing. All antagonist antibodies (1H10, 4H02, and 5B08) showed dramatically reduced binding to TNFR2 Δ4 by more than 50% compared to binding to TNFR2 Δ1 and TNFR2 Δ2. Similarly, deletion of two domains from TNFR2 clearly showed that the absence of domain 3 or 4 significantly abolished binding of all tested antibodies to TNFR2, with the possible exception of the agonist antibody 1F06, while the absence of domain 4 abolished binding of agonist antibodies and reduced binding of antagonist antibodies significantly (Fig. 19E and F).

Binding to chimeric mouse-human TNFR2

To further narrow the binding site and define epitopes, portions of domain 3 of human TNFR2 were replaced with the corresponding mouse sequence. Since all antibodies exhibit very little cross-reactivity with mouse TNFR2, loss of binding to certain constructs will improve the binding epitope. Figure 20 shows various mouse-human TNFR2 chimeric constructs. Four different substitutions were made, replacing 14 (m1), 12 (m2), 10 (m3), or 16 (m4) amino acids from the human sequence with the corresponding mouse sequence. The remaining three domains (1, 2, 4) contain exclusively human sequences.

These constructs (TNFR2 domains 1-4 with mutations in 3) were then transfected into HEK293 cells and the antibodies were tested for binding using a flow cytometric approach. Polyclonal antibodies against mouse TNFR2 as well as against human TNFR2 were used as positive controls. As expected, due to sequence similarity, both polyclonal control antibodies showed significant cross-reactivity and recognized both human and mouse TNFR2. Clearly, the best signals were obtained when the antibodies were aligned with the intended target.

The monoclonal antibodies showed strong binding to human TNFR2 but no or very weak binding to murine TNFR2 (Fig. 21, left panel). Similar binding with very little restoration was observed for all clones of the hTNFR2 m1 construct with mutations at amino acids 119-132, indicating that none of the antibodies bind to an epitope in this region. However, mutations at amino acids 134-144 (hTNFR2 m2 construct) completely abolished binding for half of the antibodies tested, corresponding to the antagonist blocking antibodies 1-H10, 4-H02, and 5-B08, indicating that the antibodies bind at least partially within this region. 1-G10 is a partial blocker also severely affected by this substitution. Notably, 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 hTNFR2 m3 construct with mutations at amino acids 151–160, indicating that all antibodies, both agonists and antagonists, share at least a partial epitope in this sequence. Testing of the slightly larger hTNFR2 m4 construct with mutations at amino acids 130–144 showed similar binding to the hTNFR2 m2 construct.

Epitope binding conclusions

Grouping the antibodies by their functional role, the agonist antibodies (1-F02, 1-F06, and 4-E08) appear to bind the very distal C-terminal portion of domain 3, spanning amino acids 151-160 of domain 4, whereas the epitope for the antagonists (1-H10, 5-B08, and 4-H02) is shifted more toward the center of domain 3, spanning amino acids 134-160 and likely covering a smaller portion of domain 4. However, despite this, their epitopes appear to overlap to some extent.

None of the antibodies bind to the N-terminal portion of domain 3, amino acids 119-134. Domain 4 binding sites are likely for all antibodies but have not been fully identified.

Claims (72)

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 signaling through TNFR2, wherein said antibody molecule also binds to an Fcγ receptor via its Fc region, wherein the antibody molecule contains (i) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; or (ii) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; or (iii) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22; or (iv) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110. 2. An antibody molecule according to claim 1, characterized in that it binds with higher affinity to activating Fcγ receptors than to inhibitory Fcγ receptors. 3. An antibody molecule according to claims 1 and 2, characterized in that its binding to TNFR2 leads to a change in the number and/or frequency of cells expressing TNFR2 in the affected tissue. 4. An antibody molecule according to any one of claims 1-3, characterized in that its binding to TNFR2 leads to infiltration of T cells and/or myeloid cells into the affected tissue and/or a change in the composition of T cells and/or myeloid cells in the affected tissue. 5. An antibody molecule according to any one of claims 1-4, characterized in that it is selected from the group consisting of a full-length 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 its Fc region. 6. An antibody molecule according to any one of claims 1-5, which binds to human TNFR2 (hTNFR2) and/or cynomolgus macaque TNFR2 (cmTNFR2). 7. An antibody molecule according to any one of claims 1-6, characterized in that it 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. An antibody molecule according to claim 7, characterized in that it is a human IgG1 antibody. 9. An antibody molecule according to paragraphs 7 and 8, characterized in that it is designed for better binding to activating Fc-gamma receptors. 10. An antibody molecule according to any one of claims 1-9, characterized in that it is a monoclonal antibody. 11. An antibody molecule according to any one of claims 1-10, characterized in that it does not bind specifically to an epitope containing or consisting of the KCSPG sequence. 12. An antibody molecule according to any one of paragraphs 1-11, characterized in that it contains: (i) the variable heavy chain (VH) amino acid sequence of SEQ ID NO: 7 and the variable light chain (VL) amino acid sequence of SEQ ID NO: 8; or (ii) the variable heavy chain (VH) amino acid sequence of SEQ ID NO: 15 and the variable light chain (VL) amino acid sequence of SEQ ID NO: 16; or (iii) the variable heavy chain (VH) amino acid sequence of SEQ ID NO: 23 and the variable light chain (VL) amino acid sequence of SEQ ID NO: 24; or (iv) the variable heavy chain (VH) amino acid sequence of SEQ ID NO: 111 and the variable light chain (VL) amino acid sequence of SEQ ID NO: 112. 13. An antibody molecule according to any one of claims 1-11, characterized in that it is an antibody molecule that is capable of competing in binding to TNFR2 with the antibody molecule claimed in claim 12. 14. An isolated nucleotide sequence encoding an antibody molecule as claimed in any one of claims 1-13. 15. A plasmid for expressing an antibody according to any one of claims 1-13, containing the nucleotide sequence claimed in claim 14. 16. A virus for the expression and extracellular release of an antibody according to any one of claims 1-13, containing the nucleotide sequence claimed in claim 14 or the plasmid claimed in claim 15. 17. The virus according to claim 16, further comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 18. The virus according to claim 17, wherein the checkpoint inhibitor is PD-1. 19. The virus according to claim 17, wherein the checkpoint inhibitor is PD-L1. 20. A cell as a carrier for delivering an antibody according to any one of claims 1-13, containing the nucleotide sequence claimed according to claim 14, or the plasmid claimed according to claim 15, or the virus claimed according to any one of claims 16-19. 21. The use of an antibody molecule according to any one of claims 1-13, a nucleotide sequence according to claim 14, a plasmid according to claim 15, a virus according to any one of claims 16-19 and/or a cell according to claim 20 in medicine in the treatment of cancer. 22. The use according to claim 21, characterized in that the patient to be treated is a patient having high expression of TNFR2 in the affected tissue. 23. The use according to claim 21, wherein the cancer is a solid cancer. 24. The use of an antibody molecule according to any one of claims 1-13, a nucleotide sequence according to claim 14, a plasmid according to claim 15, a virus according to any one of claims 16-19 and/or a cell according to claim 20 in medicine in the treatment of an infection caused by an intracellular pathogen. 25. The use according to claim 24, characterized in that the patient to be treated is a patient having high expression of TNFR2 in the affected tissue. 26. The use of an antibody molecule according to any one of claims 1-13, a nucleotide sequence according to claim 14, a plasmid according to claim 15, a virus according to any one of claims 16-19 and/or a cell according to claim 20 in the treatment of cancer in combination with: 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 containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 27. The use according to claim 26, wherein the checkpoint inhibitor is PD-1. 28. The use according to claim 26, wherein the checkpoint inhibitor is PD-L1. 29. The use according to any one of claims 26-28, wherein the cancer is a solid cancer. 30. The use of an antibody molecule as claimed in any one of claims 1-13, a nucleotide sequence as claimed in claim 14, a plasmid as claimed in claim 15, a virus as claimed in any one of claims 16-19, and/or a cell as claimed in claim 20, for the production of a pharmaceutical composition used in the treatment of cancer or an infection caused by an intracellular pathogen. 31. Use according to claim 30, characterized in that said pharmaceutical composition is intended for use in the treatment of cancer or infection in a patient having high expression of TNFR2 in the affected tissue. 32. Use according to paragraphs 30, 31, characterized in that the said pharmaceutical composition is used in the treatment of cancer and is administered in combination with: 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 containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 33. The use according to claim 32, wherein the checkpoint inhibitor is PD-1. 34. The use according to claim 32, wherein the checkpoint inhibitor is PD-L1. 35. The use according to any one of claims 30-34, wherein the cancer is a solid cancer. 36. A pharmaceutical composition for treating cancer or an infection caused by an intracellular pathogen, comprising or consisting of an antibody molecule as claimed in any one of claims 1-13, a nucleotide sequence as claimed in claim 14, a plasmid as claimed in claim 15, a virus as claimed in claims 16-19, and/or a cell as claimed in claim 20, in an effective amount, and, optionally, a pharmaceutically acceptable diluent, carrier, filler and/or excipient. 37. A pharmaceutical composition according to claim 36 for use in the treatment of cancer in combination with a pharmaceutical composition containing: 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 containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 38. The composition according to claim 37, wherein the checkpoint inhibitor is PD-1. 39. The composition according to claim 37, wherein the checkpoint inhibitor is PD-L1. 40. The composition according to any one of claims 36, 37, wherein the cancer is a solid cancer. 41. A method of treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of an antibody molecule as claimed in any one of claims 1-13, a nucleotide sequence as claimed in claim 14, a plasmid as claimed in claim 15, a virus as claimed in claims 16-19, a cell as claimed in claim 20, or a pharmaceutical composition as claimed in claim 36. 42. The method according to claim 41, characterized in that the patient is a patient having high expression of TNFR2 in the affected tissue. 43. The method for treating cancer according to paragraphs 41, 42, characterized in that the patient is also administered a therapeutically effective amount of: 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 containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 44. The method according to claim 43, characterized in that the checkpoint inhibitor is PD-1. 45. The method according to claim 43, characterized in that the checkpoint inhibitor is PD-L1. 46. The method according to paragraphs 41-45, characterized in that the cancer is a solid cancer. 47. A method for treating an infection caused by an intracellular pathogen in a patient, comprising administering to the patient a therapeutically effective amount of an antibody molecule as claimed in any one of claims 1-13, a nucleotide sequence as claimed in claim 14, a plasmid as claimed in claim 15, a virus as claimed in claims 16-19, a cell as claimed in claim 20, or a pharmaceutical composition as claimed in claim 36. 48. The method according to claim 47, characterized in that the patient is a patient having high expression of TNFR2 in the affected tissue.