Novel agonistic anti-TNFR2 antibody molecules

Abstract

Novel antibody molecules are described that specifically bind to TNFR2 on target cells and thereby agonize TNFR2 without blocking the binding of the ligand TNF-α to TNFR2. Also described are the uses of such antibody molecules in medicine, particularly in the treatment of cancer or chronic inflammatory diseases.

Description

Technical Field

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

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

[0003] It has been discussed that TNFR2 has become a promising target for cancer immunotherapy and has been described to be highly expressed, particularly on intratumoral Tregs and on the surface of many human tumor cells (Williams GS et al., Oncotarget 2016;7(42):68278–68291; Vanamee ES et al., Trends in Molecular Medicine, 2017, Vol. 23, No. 11, 1037-1046; Frontiers in Immunology, Nov. 2017 | Vol. 8 | Article 1482; Sci Signal. 2018 Jan. 2;11(511)).

[0004] Regulatory T cells (which may be referred to as Treg cells, Treg or T reg , and which were previously called suppressor T cells or suppressor regulatory T cells) constitute a T cell subset that can suppress other immune cells in normal and pathological immune environments. Tregs are CD4+ cells (CD4 + There are other CD4 Treg cells. + T cells; however, Tregs can interact with non-Treg CD4 + Cell separation, because Treg is also FOXP3 positive (FOXP3 + ), rather than Treg CD4 +Cells are FOXP3 negative (FOXP3 - ). Treg can also interact with non-Treg CD4 + Cell separation, because Treg is still CD25 + CD127 neg / low , but not Treg CD4 + CD25 - CD127 + or CD25 + CD127 + .

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

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

[0007] WO 2014 / 124134 discloses the use of TNFR2 agonists, such as agonistic anti-TNFR2 antibodies and / or for the in vitro production of CD4+CD25 hi The composition is said to be useful for treating an immune disorder or infectious disease in a patient.

[0008] WO 2017 / 040312 discloses anti-TNFR2 antibodies, and specifically agonistic anti-TNFR2 antibodies that are capable of promoting TNFR2 signaling and having an effect on the expansion or proliferation of Tregs. WO 2017 / 040312 discloses antibodies that specifically bind to an epitope comprising the sequence KCSPG, but not to an epitope comprising the sequence KCRPG, thereby excluding the antibodies of US Pat. No. 9,821,010 described above, or alternatively, antibodies that do not bind to another TNFR superfamily member. The agonistic antibodies are said to be useful for treating immune diseases. WO 2017 / 040312 further describes the complete sequence of human TNFR2.

[0009] WO 2017 / 083525 discusses pharmacological compositions comprising anti-TNFR2 antibodies and their use in treating conditions associated with TNF-α and / or TNFR2, such as cancer. WO 2017 / 083525 further discusses antibodies comprising a human IgG1 Fc domain that is ineffective for binding to Fcγ receptors and inhibiting Treg expansion.

[0010] Additionally, anti-TNFR2 antibodies that can act as TNFR2 agonists are described by Galloway et al. (Eur. J. Immunol. 22:3045-3048, 1992), Tartaglia et al. (J. Biol. Chem. 268:18542-18548, 1993), Tartaglia et al. (J. Immunol. 151:4637-4641, 1993), Smith et al. (J. Biol. Chem. 269:9898-9905, 1994), and Amrani et al. (Am. J. Respir. Cell. Mol. Biol. 15:55-63, 1996).

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

[0012] Fc receptors are membrane proteins present on the cell surface of immune effector cells, including monocytes, macrophages, dendritic cells, neutrophils, mast cells, basophils, eosinophils, natural killer cells, and B lymphocytes. Their name derives from their binding specificity for the Fc region of antibodies. Fc receptors are present on the cell membrane, also known as the plasma membrane or cytoplasmic membrane. Fc receptors can be subdivided into activating and inhibitory FcγRs, which are known to coordinately regulate cell activation through binding to aggregated immunoglobulin G Fcs and transmit activating or inhibitory signals into the cell via intracellular ITAM or ITIM motifs. FcR binding of aggregated immunoglobulins or immune complexes can mediate antibody internalization into the cell and may lead to antibody-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, or antigen presentation or cross-presentation. FcRs are also known to mediate or enhance cross-linking of antibody-bound cell surface receptors. This cross-linking is known to activate 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", Journal of Immunology, 187:1754-63), but not all (Richman et al., 2014 "Anti-human CD40 monoclonal antibody therapy is potent without FcR cross-linking") signaling pathways that activate targeted cells. crosslinking”, Oncoimmunology, 3:e28610) and may or may not be required for therapeutic efficacy.

[0013] A subgroup of Fc receptors is the Fcγ receptors (Fcγ receptors, FcγRs (FcgammaRs) or FcγRs) that are specific for IgG antibodies. There are two types of Fcγ receptors: activating Fcγ receptors (also denoted as activating 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 an inhibitory Fcγ receptor, while FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c) and FcγRIIIa (CD16a) are activating Fcγ receptors. FcγgRIIIb is a GPI-linked receptor expressed on neutrophils that lacks an ITAM motif but is also considered activating by its ability to cross-link lipid rafts and bind to other receptors. In mice, the activating receptors are FcγRI, FcγRIII, and FcγRIV.

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

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

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

[0017] Specifically, the latest data published by the present inventors show that CD8 T cell agonists and Treg-depleting anti-4-1BB antibodies are critical and differentially dependent on binding to activating and inhibitory FcγRs, respectively, for therapeutic efficacy (Buchan et al., "Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via TRegulatory Cell Depletion and Promotion of CD8 T CellEffector Function", Immunity 2018 49(5):958-970). In addition, and crucially, the simultaneous administration of CD8 T cell agonists and Treg-depleting anti-4-1BB antibodies optimizes binding to activating and inhibitory FcγRs, respectively, impairing therapeutic activity. These data demonstrate the critical importance of developing antibodies that appropriately and custom-link activating and inhibitory FcγRs to maximize the therapeutic activity of antibodies with unique mechanisms of action. At the same time, they suggest that suboptimal ligation of activating and inhibitory FcγRs may severely reduce therapeutic efficacy.

[0018] These data are surprising because they contrast with findings with antibodies to other TNFSR members, notably immunostimulatory anti-CD40 antibodies, which show an exclusive requirement for ligation of inhibitory rather than activating FcγRs (Li et al., 2011 "Inhibitory Fcγ receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies," Science, 333:1030-4; White et al., 2011 "Interaction with FcγRIIB is essential for the agonistic activity of anti-CD40 monoclonal antibodies," J Immunol, 187:1754-63). Taken together, these results suggest that FcγR dependency can vary between antibodies targeting different targets of the same receptor superfamily, and even between different types of antibodies targeting the same target, in ways that are not easily predictable but may be crucial to understand and exploit when developing antibodies for therapeutic use. Summary of the Invention

[0019] In work leading to the present invention and parallel inventions, two largely distinct groups of anti-TNFR2 antibodies were identified that possess potent therapeutic effects and distinct properties and mechanisms of action.

[0020] The inventors first identified the potent therapeutic activity of antagonistic anti-TNFR2 antibodies that block TNF-α binding to the TNFR2 receptor. For in vivo therapeutic activity, the activity of these antibodies was shown to be dependent on FcγR interactions, and specifically binding to activating FcγRs. This group or class of potent anti-TNFR2 therapeutic agents was found to be characterized by 1) significant blocking and inhibition of TNF-α-induced TNFR2 signaling, and 2) an activity that is dependent on FcγR ligation, thereby benefiting most from ligation to activating inhibitory FcγRs.

[0021] The inventors then identified a different group of anti-TNFR2 antibodies that possessed equally potent therapeutic activity in vivo, but whose properties were in many respects opposite to those of the antagonistic, blocking-type TNFR2 antibodies that comprised the first group. This second group of anti-TNFR antibodies did not rely on TNF-α blockade or inhibition of TNFR2 signaling for therapeutic activity, but rather was characterized by robust activation of TNFR2 signaling. Further in contrast to the blocking antibodies of the first group, the agonistic antibodies of the second group did not show an exclusive dependence on antibody:FcγR binding, even when their activity was enhanced using FcγR:linked antibody variants. In further contrast to the antagonistic blocking antibodies of the first group, the agonistic antibodies of the second group showed maximal activity among the antibody variants, with enhanced binding to the inhibitor compared to the activating FcγR.

[0022] The present invention relates to a second group of anti-TNFR2 antibodies, namely, anti-TNFR2 antibodies that specifically bind to TNFR2 but do not block the binding of the ligand TNF-α to TNFR2. Such antibodies are powerful therapeutic agents and can be used in medicine.

[0023] In the following examples, antagonist blocking antibodies belonging to the first group were used for comparison with the agonist non-blocking TNFR2 antibody molecules of the invention. In the examples, other antibodies having certain properties similar to the antibodies of the first or second group, or both, were also used for comparison, as further explained below.

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

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

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

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

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

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

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

[0031] The present invention also relates to the above antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for treating cancer or chronic inflammatory diseases.

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

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

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

[0035] 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 as described herein with reference to the accompanying description, examples and / or figures. DETAILED DESCRIPTION

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

[0037] The agonistic antibody molecules disclosed herein do not block TNF-α binding to TNFR2, and further do not block TNFR2 signaling. It has been clearly shown that TNF-α-mediated signaling through TNFR2 initiates a signaling cascade that ends 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 the activation of the cells and the synthesis of several pro-inflammatory factors, one of which is IFN-γ in 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). The terms TNFR2 signaling and TNFR2 activation are used interchangeably herein. The antibody molecule specifically binds to TNFR2. It is well known that antibodies specifically bind to or interact with a defined target molecule or antigen, and this means that the antibody preferentially and selectively binds to its target rather than non-target molecules. By "antibody molecule that specifically binds to TNFR2" or "TNFR2-specific antibody molecule" is meant an antibody that binds to TNFR2 protein in a dose-dependent manner and does not bind to unrelated proteins.In addition, the same antibody binds to cells that endogenously express TNFR2, and this binding can be blocked by pre-incubating the same cells with a commercially available polyclonal TNFR2 antibody reagent, demonstrating that nonspecific binding may not be detected when TNFR2 is masked by the polyclonal reagent. This is shown in Example 2.

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

[0039] The method for assessing protein binding is known to biochemical and immunological technicians. Technicians will understand that those methods can be used to assess the combination of antibodies and targets and / or the combination of the Fc region of antibodies and Fc receptors; and relative strength or specificity, or the inhibition or prevention or reduction in those interactions. Examples of methods that can be used to assess protein binding are, for example, immunoassays, BIAcore, western blots, radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) and flow cytometry (FACS). For discussion about antibody specificity, see " Fundamental Immunology " Second Edition, Raven Press, New York (New York) 332-336 pages (1989).

[0040] The target cell expressing TNFR2 to which the agonistic antibody according to the present invention binds can be any TNFR2-expressing immune cell, such as CD8-positive cells and myeloid cells.

[0041] The effect of the agonist antibody molecule according to the present invention in combination with TNFR2 can be T cell and / or myeloid cell activation; and / or T cell and / or myeloid cell infiltration into diseased tissue; and / or the composition of T cells and / or myeloid cells in diseased tissue changes. The change in the composition of T cells and / or myeloid cells refers herein to various cell subsets, such as Treg, CD8 positive cells, tumor associated macrophages (TAM) (including different subsets thereof), myeloid-derived suppressor cells (MDSC) and / or proinflammatory macrophages. Different absolute or relative counts.

[0042] Diseased tissue in this context means tumor tissue (ie all cells in the tumor microenvironment, including tumor cells, immune cells, endothelial cells and stromal cells) or tissue affected by chronic inflammatory diseases.

[0043] In order to determine whether an antibody molecule blocks or does not block ligand binding to TNFR2 in the context of the present invention, it is possible to determine the amount of TNF-α ligand bound to the immobilized TNFR2 receptor in the presence of a TNFR2-specific antibody using an ELISA assay. A non-blocking antibody will not prevent the ligand TNF-α from binding to the immobilized receptor TNFR2. This is described and explained in more detail in Example 3 below. More specifically, a non-blocking TNFR2 antibody molecule according to the present invention is an antibody molecule that reduces TNF-α binding to TNFR2 by less than 50% compared to TNF-α binding in the presence of an isotype control antibody molecule alone. In some embodiments, this is determined in a high-dose single-point ELISA or a dose-titration ELISA, as described in Examples 3 and 4. Figure 6 and 7 As shown in .

[0044] In contrast, blocking antagonist antibody molecules are complete blockers that are additionally capable of antagonizing TNFR2 signaling. Such antibody molecules are used for comparison in the following examples. A complete blocker is defined herein as an antibody molecule that reduces the binding of TNF-α to TNFR2 by more than 98%, i.e., up to 100%, compared to the binding of TNF-α in the presence of only an isotype control antibody molecule. An isotype control antibody is an antibody proposed for a protein or other structure that is not present in any form in the assay under investigation. An isotype control ideally has the same framework as the comparison antibody, but at least the same Fc portion. This is well known to those skilled in the art. In the examples described herein, the isotype control has the same framework, the same Fc portion, and is specific for fluorescein isothiocyanate (FITC). In some embodiments, a complete blocker reduces the binding of TNF-α by more than 99.5%. Other types of blockers are partial blockers and weak blockers. As used herein, a partial blocker is an antibody molecule that reduces the binding of TNF-α to TNFR2 by 60-98% compared to the binding of TNF-α in the presence of an isotype control antibody molecule alone, and a weak blocker is an antibody molecule that reduces the binding of TNF-α to TNFR2 by less than 60%, such as 50-59.9%, compared to the binding of TNF-α in the presence of an isotype control antibody molecule alone.

[0045] In the Examples, fully blocking antagonistic antibody molecules, partially blocking antibody molecules, and weakly blocking antibody molecules were used for comparison with the agonistic non-blocking antibody molecules of the invention.

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

[0047] We first characterized the ability of fully blocking, partially blocking, and non-blocking antibodies to modulate TNFR2 signaling, identifying two extremes.

[0048] At the first extreme, antibodies are identified that completely block ligand binding to TNFR2, block TNF-α-induced TNFR2 signaling, and do not themselves induce signaling when bound to endogenously expressed TNFR2 in cells. This group of ligand-blocking antagonistic antibodies constitutes a separate invention and is included here for comparison.

[0049] At the other extreme, antibodies are identified that do not block ligand binding to TNFR2, but agonize the receptor on endogenously expressed cells when bound to TNFR2.This second group of antibodies forms the basis of the present invention.

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

[0051] Antibodies and classes defined by partially blocking agonism, partially blocking non-agonism, and fully blocking non-antagonism were further identified as exhibiting the complex biology and vast heterogeneity of anti-TNFR2 antibodies, clearly demonstrating that the antibodies of the invention form a unique group.

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

[0053] Thus, the ability of an antibody to agonize, i.e., induce TNFR2 signaling, can be monitored using an experimental setup. The ability of an antibody to induce autosignaling upon binding to TNFR2 in the same natural killer (NK) cell assay can be evaluated by monitoring increases in IFN-γ release and comparing the increases in IFN-γ release to those observed after incubation in the presence or absence of exogenous TNF-α added at signaling-optimal concentrations as described in Example 4. Thus, an agonistic TNFR2 antibody can be defined as an antibody that enhances IFN-γ release by NK cells in this assay. Antibodies with intrinsic agonistic activity enhance IFN-γ release by NK cells in a manner that is neither dependent on antibody cross-linking or interaction with Fcγ receptors nor on the presence of soluble TNF-α ligands. Thus, intrinsic agonist activity can be assessed using an antibody format that does not effectively bind to FcγRs, such as an aglycosylated antibody carrying the N297A mutation in an Fc domain or an assay system / cell lacking FcγRs. NK cells are well known to the skilled artisan (Binyamin, L. et al. (2008) Journal of immunology 180, 6392-6401; "Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphomatherapy"). Using this assay, agonistic antibodies are defined as antibodies that result in a >100% (>2-fold) increase in IFN-γ release. Since this assay uses primary cells from PBMC donors, at least 4 donors need to be included and the mean should be calculated based on all donors. Cells from each donor to be included in the mean calculation must respond to treatment with the positive control (soluble TNF-α) with a >100% (>2-fold) increase in IFN-γ levels compared to treatment with the isotype control.

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

[0055] In some embodiments of the invention, it is preferred that the antibody increases IFN-γ release by NK cells in the above assay by at least 100%.

[0056] In some embodiments, agonistic activity can be enhanced by binding of an antibody molecule to an Fcγ receptor in addition to binding to TNFR2. In some such embodiments, the agonistic non-blocking TNFR2 antibody molecule binds with a higher affinity to an inhibitory Fcγ receptor than to an activating Fcγ receptor. "By a higher affinity to an inhibitory Fcγ receptor than to an activating Fcγ receptor" encompasses variants that bind with a higher affinity to an inhibitory Fcγ receptor than to an activating Fcγ receptor alone, e.g., to FcγRIIA, FcγRIIIA, and FcγRI.

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

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

[0059] In addition to TNFR2, Fc receptors to which agonist antibody molecules can bind are receptors found on the surface of cells of myeloid origin, such as macrophages, monocytes, MDCs, neutrophils, mast cells, basophils, or dendritic cells, or on the surface of lymphocytes, such as NK cells, B cells, or certain T cells.

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

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

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

[0063] In some embodiments, the human IgG antibody engineered for improved binding to an activating Fcγ receptor may be a human IgG antibody carrying two mutations S239D and I332E or three mutations S239D, I332E and A330L and / or G236A mutations in its Fc portion. In some embodiments, the human IgG antibody engineered for improved binding to an activating Fcγ receptor may be a fucosylated human IgG antibody.

[0064] As explained above, an antibody molecule is an intrinsic agonist meaning that it is agonist both in the absence and presence of TNF-α. In some embodiments, the antibody is agonist in the absence of TNF-α. In some embodiments, the antibody is agonist in the presence of TNF-α.

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

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

[0067] Another part of the antibody is the Fc region (also known as the fragment crystallizable domain), which includes two constant domains of each of the heavy chains of the antibody. As mentioned above, the Fc region is responsible for the interaction between the antibody and the Fc receptor.

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

[0069] The functional fragment of a full-size antibody has the same antigen-binding properties as the corresponding full-size antibody and comprises the same variable domains (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-size antibody. Functional fragments do not always contain all six CDRs of the corresponding full-length antibody. It should be understood that molecules containing three or fewer CDR regions (in some cases, even only a single CDR or a portion thereof) can retain the antigen-binding activity of the antibody derived from one or more CDRs. For example, in Gao et al., 1994, Journal of Biological Chemistry, 269: 32389-93, it is described that the entire VL chain (comprising all three CDRs) has a high affinity for its substrate.

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

[0071] Antibody molecules containing a single CDR region are described in, for example, Laune et al., 1997, Journal of Biological Chemistry, 272: 30937-44, where a series of hexapeptides derived from CDRs were shown to exhibit antigen binding activity, and it was noted that synthetic peptides of intact single CDRs exhibited strong binding activity. In Monnet et al., 1999, Journal of Biological Chemistry, 274: 3789-96, a series of 12-mer peptides and associated framework regions were shown to have antigen binding activity, and it was noted that CDR3-like peptides alone were capable of binding antigen. In Heap et al., 2005, Journal of General Virology, 86: 1791-1800, it was reported that "miniantibodies" (molecules containing a single CDR) were capable of binding antigen, and cyclic peptides from anti-HIV antibodies were shown to have antigen binding activity and function. In Nicaise et al., 2004, Protein Science, 13: 1882-91, it was shown that a single CDR can confer antigen binding activity and affinity for its lysozyme antigen.

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

[0073] The antibody molecule can also be a derivative of a full-length antibody or a fragment of such an antibody. When a derivative is used, the derivative should have the same antigen-binding properties as the corresponding full-length antibody, in the sense that the derivative binds to the same epitope on the target as the full-length antibody.

[0074] Therefore, the term "antibody molecule" as used herein includes all types of antibody molecules and functional fragments and 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 Fv (scFv), Fab fragments, F(ab')2 fragments, F(ab') fragments, disulfide-linked Fv (sdFv), antibody heavy chains, antibody light chains, homodimers of antibody heavy chains, homodimers of antibody light chains, heterodimers of antibody heavy chains, heterodimers of antibody light chains, and antigen-binding functional fragments of such homodimers and heterodimers.

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

[0076] In some embodiments, the antibody molecule is a human antibody molecule, a humanized antibody molecule or an antibody molecule of human origin. In some such embodiments, the antibody molecule is an IgG antibody. It is known that the optimal costimulation of TNFR superfamily agonist receptors, such as TNFR2, depends on the antibody connection of inhibitory Fc γ RII. In mice, it is known that the IgG1 isotype that preferentially binds to inhibitory Fc γ receptors (Fc γ RIIB) and is only weakly bound to activating Fc γ receptors is optimal for the costimulatory activity of monoclonal antibodies targeting the TNFR superfamily. Although the direct equivalent of the mouse IgG1 isotype in the human body has not been described, the antibody can be engineered to show similar enhancement of binding to inhibitory human Fc γ receptors that is superior to activating human Fc γ receptors. Such engineered TNFR superfamily-targeting antibodies have also improved co-stimulatory activity in transgenic mice engineered to express human activating and inhibitory Fcγ receptors (Dahan et al., 2016, "Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement." Cancer Cell 29(6):820-31). In some embodiments, the antibody molecule thus has an isotype that optimally binds to inhibitory Fc receptors. In some embodiments, the antibody molecule is an IgG2 antibody.

[0077] In some embodiments, the agonist antibody molecule that specifically binds to TNFR2 can be a lama antibody, and in particular a lama hcIgG. Like all mammals, camelids produce conventional antibodies consisting of two heavy chains and two light chains bound together by disulfide bonds in a Y shape (IgG1). However, they also produce two unique subclasses of immunoglobulin G, IgG2 and IgG3, also known as heavy chain IgG (hcIgG). These antibodies lack a CH1 region but still carry a subclass of heavy chains at their N-termini called V H The antigen-binding domain of hcIgG consists of only two heavy chains. Conventional Ig requires the association of variable regions from both heavy and light chains to allow for high diversity of antigen-antibody interactions. Although isolated heavy and light chains still show this ability, they exhibit very low affinity when compared to paired heavy and light chains. The unique feature of hcIgG is the ability of its monomeric antigen-binding domain to bind to antigens comparable to conventional antibodies that do not need to be paired with another region, with specificity, affinity, and especially diversity.

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

[0079] Thus, encompassed are antibody molecules described herein, or antibody molecules used as described herein (e.g., monoclonal antibody molecules and / or polyclonal antibody molecules and / or bispecific antibody molecules) comprising a detectable moiety and / or a cytotoxic moiety.

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

[0081] A "cytotoxic moiety" comprises a radioactive moiety and / or an enzyme, eg, wherein the enzyme is a caspase, and / or a toxin, eg, wherein the toxin is a bacterial toxin or venom; wherein the cytotoxic moiety is capable of inducing cell lysis.

[0082] It is further contemplated that the antibody molecule may be in isolated and / or purified form and / or may be pegylated. Pegylation is a process in which a polyethylene glycol polymer is added to a molecule, such as an antibody molecule or derivative, to modify its behavior, for example to extend its half-life by increasing its hydrodynamic size, thereby preventing renal clearance.

[0083] As discussed above, the CDRs of an antibody bind to the antibody target. The assignment of amino acids to each CDR described herein conforms to the definition according to Kabat EA et al., 1991, "Sequences of Proteins of Immunological Interest," Fifth Edition, NIH Publication No. 91-3242, pp. xv-xvii.

[0084] As will be appreciated by those skilled in the art, other methods exist for assigning amino acids to each CDR, for example, the International ImMunoGeneTics information system (IMGT(R)) (http: / / www.imgt.org / , and Lefranc and Lefranc "The Immunoglobulin Facts Book", Academic Press, 2001).

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

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

[0087] In some embodiments, the antibody molecule that specifically binds to TNFR2 is a humanized antibody, ie, an originally non-human antibody that has been modified to increase its similarity to a human antibody. A humanized antibody can have, for example, a murine antibody or a lama antibody.

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

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

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

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

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

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

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

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

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

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

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

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

[0100] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of an antibody molecule comprising 6 CDRs selected from the group consisting of:

[0101] SEQ.ID.NO: 1, 2, 3, 4, 5 and 6;

[0102] SEQ.ID.NO: 9, 10, 11, 12, 13 and 14;

[0103] SEQ.ID.NO: 17, 18, 19, 20, 21 and 22;

[0104] SEQ.ID.NO: 25, 26, 27, 28, 29 and 30;

[0105] SEQ.ID.NO: 33, 34, 35, 36, 37 and 38;

[0106] SEQ.ID.NO: 41, 42, 43, 44, 45 and 46;

[0107] SEQ.ID.NO: 49, 50, 51, 52, 53 and 54;

[0108] SEQ.ID.NO: 57, 58, 59, 60, 61 and 62;

[0109] SEQ.ID.NO: 65, 66, 67, 68, 69 and 70;

[0110] SEQ.ID.NO:73, 74, 75, 76, 77 and 78;

[0111] SEQ.ID.NO:81, 82, 83, 84, 85 and 86;

[0112] SEQ.ID.NO:89, 90, 91, 92, 93 and 94; and

[0113] SEQ.ID.NO:97, 98, 99, 100, 101 and 102.

[0114] In some embodiments, the anti-TNFR2 antibody molecule is: an antibody molecule comprising the following 6 CDRs: SEQ.ID.NO: 1, 2, 3, 4, 5 and 6; or an antibody molecule comprising the following 6 CDRs: SEQ.ID.NO: 9, 10, 11, 12, 13 and 14; or an antibody molecule comprising the following 6 CDRs: SEQ.ID.NO: 17, 18, 19, 20, 21 and 22; or an antibody molecule comprising the following 6 CDRs: SEQ.ID.NO: 25, 26, 27, 28, 29 and 30; or an antibody molecule comprising the following 6 CDRs: SEQ.ID.NO: 33, 34, 35, 36, 37 and 38; or an antibody molecule comprising the following 6 CDRs: SEQ.ID.NO: 41, 42, 43, 44, 45 and 46.

[0115] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising the following six CDRs: SEQ.ID.NO: 1, 2, 3, 4, 5, and 6.

[0116] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of an antibody molecule comprising a VH selected from the group consisting of SEQ.ID.NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, and 103.

[0117] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of an antibody molecule comprising a VL selected from the group consisting of SEQ.ID.NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, and 104.

[0118] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VH having SEQ.ID.NO: 7, 15, 23, 31, 39, or 47.

[0119] In some embodiments, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VH having the following: SEQ.ID.NO:7.

[0120] In some embodiments, it is preferred that the anti-TNFR2 antibody molecule is an antibody molecule comprising a VL having the following: SEQ.ID.NO: 8, 16, 24, 32, 40 or 48.

[0121] In some embodiments, more preferably, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VL having the following: SEQ.ID.NO:8.

[0122] In some embodiments, it is preferred that the anti-TNFR2 antibody molecule comprises a VH having SEQ.ID.NO:7 and a VH having SEQ.ID.NO:8.

[0123] In some embodiments, the anti-TNFR2 antibody molecule comprises a CH having SEQ.ID.NO:217.

[0124] In some embodiments, the anti-TNFR2 antibody molecule comprises a CL having SEQ.ID.NO:218.

[0125] In some embodiments, the anti-TNFR2 antibody molecule comprises a VH having SEQ.ID.NO:7, a VH having SEQ.ID.NO:8, a CH having SEQ.ID.NO:217, and a CL having SEQ.ID.NO:218.

[0126] Table 1: Specific sequences of agonistic TNFR2 antibody molecules that do not block TNF-α binding to TNFR2 as described herein (CDR sequences are marked in bold in both VH and VL sequences)

[0127]

[0128]

[0129]

[0130]

[0131] In order to determine or demonstrate the characteristics of the antibody molecules of the present invention, they were compared with antibody molecules that block the binding of TNF-α to TNFR2. Such antibodies are shown in Table 2.

[0132] Table 2: Specific sequences of TNFR2-blocking antibody molecules mentioned herein as reference antibodies (CDR sequences are in bold in VH and VL sequences)

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] The sequences in Tables 1 and 2 above are of human origin and derived from library, as explained in detail in Example 1.

[0139] In some embodiments, the antibody molecules that specifically bind to TNFR2 described herein may further comprise one or both of the constant regions (CH and / or CL) listed in Table 3 below.

[0140] Table 3:

[0141]

[0142]

[0143] The first CH (SEQ.ID.NO: 217) and first CL (SEQ.ID.NO: 218) sequences in Table 3 above are of human origin. The second CH (SEQ.ID.NO: 219) and third CH (SEQ.ID.NO: 220) sequences in Table 3 are both derived from murine IgG2a, differing in that the third CH sequence (SEQ.ID.NO: 220) contains an N297A mutation. The second CL sequence (SEQ.ID.NO: 221) is derived from a murine lambda light chain constant region. These murine sequences were used in surrogate antibodies in the Examples.

[0144] In some embodiments, the antibody molecule binds to human TNFR2 (hTNFR2). In some embodiments, it is preferred that the agonist antibody molecule binds strongly to human TNFR2, i.e., it has a low EC50 value. This is further illustrated in Example 2.

[0145] In some embodiments, it is advantageous that the antibody molecule binds to hTNFR2 and cynomolgus monkey TNFR2 (cmTNFR2 or cynoTNFR2). Cross-reactivity with TNFR2 expressed on cells in rhesus monkeys, also known as cynomolgus macaques or long-tailed macaques, can be advantageous because it enables animal testing of the antibody molecule without the use of alternative antibodies, with particular attention to tolerability.

[0146] In some embodiments, it is desirable to use surrogate antibodies to test the functional activity of the antibody molecule in a relevant in vivo model in mice. To ensure comparability between the effects of the antibody molecule in humans and the in vivo results of the surrogate antibody in mice, it is necessary to select a functionally equivalent surrogate antibody that has the same in vitro properties as the human antibody molecule. In some embodiments, the antibody molecule does not specifically bind to an epitope of TNFR2 comprising or consisting of the sequence KCSPG.

[0147] In some embodiments, the antibody molecules of the invention or used according to the invention are capable of competing with a specific antibody provided herein, e.g., capable of competing for binding to TNFR2 with an antibody molecule comprising a VH selected from the group consisting of SEQ.ID.NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, and 103; and / or a VL selected from the group consisting of SEQ.ID.NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, and 104.

[0148] By "capable of competing" is meant that the competing antibody is capable of at least partially inhibiting or otherwise interfering with the binding of the antibody molecule as defined herein to the specific target TNFR2.

[0149] For example, the competing antibody molecule can inhibit the binding of an antibody molecule described herein to TNRF2 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% or about 100%.

[0150] Competitive binding can be determined by methods well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0151] Epitope modifications or blocking antibodies can be evaluated using ELISA assays. Additional methods suitable for identifying competing antibodies are disclosed in Antibodies: A Laboratory Manual, Harlow and Lane, which are incorporated herein by reference (e.g., see pages 567 to 569, 574 to 576, 583, and 590 to 612, 1988, Cold Spring Harbor Laboratory (CSHL), New York (NY), ISBN 0-87969-314-2).

[0152] In some embodiments, it is contemplated that rather than using the antibody molecule itself, a nucleotide sequence encoding such an antibody molecule may be used. Thus, the present invention encompasses nucleotide sequences encoding the above agonistic non-blocking TNFR-2 antibody molecules.

[0153] The agonistic non-blocking antibody molecules and nucleotide sequences described above may be used in medicine, and such antibody molecules and / or nucleotide sequences may then be included in pharmaceutical compositions, as discussed further below.

[0154] The agonistic non-blocking antibody molecules, nucleotide sequences and / or pharmaceutical compositions described above can be used to treat cancer, as discussed further below.

[0155] The agonistic non-blocking antibody molecules, nucleotide sequences and / or pharmaceutical compositions described above can be used to treat chronic inflammatory diseases, as discussed further below.

[0156] The agonistic non-blocking antibody molecules and / or nucleotide sequences described above can be used to manufacture pharmaceutical compositions for treating cancer.

[0157] The agonistic non-blocking antibody molecules and / or nucleotide sequences described above can be used to manufacture pharmaceutical compositions for treating chronic inflammatory diseases.

[0158] The agonistic non-blocking antibody molecules and / or pharmaceutical compositions described above can be used in a method for treating cancer in a patient, wherein a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the subject.

[0159] The agonistic non-blocking antibody molecules and / or pharmaceutical compositions described above can be used in a method for treating a chronic inflammatory disease in a patient, wherein a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the patient.

[0160] In some embodiments related to the treatment of cancer, the cancer is a solid cancer or leukemia. Solid tumors are abnormal tissue masses that generally do not contain cysts or fluid areas. Solid tumors can be benign (not cancer) or malignant (cancer). Malignant solid tumors are referred to as solid cancers in this article. Different types of solid tumors or cancers are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

[0161] More specific examples of solid cancers are lung cancer, head and neck cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, kidney cancer, liver cancer, pancreatic cancer, thyroid cancer, brain cancer, central nervous system cancer, melanoma, neuroblastoma, lymphoma, Wilms tumor, rhabdomyosarcoma, retinoblastoma and bone cancer.

[0162] More specific examples of leukemia cancers are acute lymphocytic leukemia, chronic myeloproliferative disease, acute non-lymphocytic leukemia, B-cell acute lymphocytic leukemia, chronic lymphocytic leukemia, T-cell acute lymphocytic leukemia, non-Hodgkin lymphoma and chronic lymphoproliferative disease. In some embodiments, the above-mentioned agonistic non-blocking antibody molecules can be used in combination with antibody molecules that specifically bind to check point inhibitors. Alternatively, the nucleotide sequences encoding agonistic non-blocking TNFR2 antibody molecules discussed above can be used in combination with antibody molecules that specifically bind to check point inhibitors or co-stimulatory agonistic antibodies. Examples of antibodies to check point inhibitors are 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 are antibodies targeting OX40, 41BB, OX40L, 41BBL, GITR, ICOS, DR3, DR4, DR5, CD40, CD27, RANK, HVEM, LIGHT and B7-H6. Alternatively, the agonistic non-blocking TNFR2 antibody molecules discussed above can be used in combination with nucleotide sequences encoding antibody molecules that specifically bind to check point inhibitors or co-stimulatory agonists. Alternatively, the nucleotide sequences encoding agonistic non-blocking TNFR2 antibody molecules discussed above can be used in combination with nucleotide sequences encoding antibody molecules that specifically bind to check point inhibitors or co-stimulatory agonists. In some embodiments, the antibody molecule that specifically binds to a check point inhibitor is an anti-PD-1 antibody. PD1 antibodies are considered to block the primary CD8 + "These therapies can synergize with each other. This is also true for other checkpoint inhibitors and agonistic costimulatory antibodies."

[0163] In addition, the agonistic non-blocking TNFR2 antibody molecules discussed above can be used in combination with other anti-cancer therapies, such as chemotherapy (for example, but not limited to, doxorubicin, carboplatin, cyclophosphamide, paclitaxel, gemcitabine, 5-fluorouracil, docetaxel, vincristine, mitoxantrone, mutamycin, epirubicin, and methotrexate), small molecule tyrosine kinase or serine / threonine kinase inhibitors (for example, but not limited to, ibrutinib, imatinib, sunitinib), regorafenib, sorafenib, dasatinib, erlotinib, vandetanib, midostaurin, vemurafenib, dabrafenib, palbociclib, ribociclib, trametinib or alectinib), inhibitors targeting growth factor receptors (such as, but not limited to, those targeting EGFR / HER1 / ErbB1, EGFR2 / HER2 / ErbB2, EGFR3 / HER3 / ErbB3, VEGFR, PDGFR The present invention relates to drugs that inhibit HGFR, RET, insulin-like growth factor receptor IGFR, FGFR), anti-angiogenic agents (such as but not limited to bevacizumab, everolimus, lenalidomide, thalidomide, Ziv-aflibercept) or radiation. Typically, the above-mentioned anticancer drugs all cause cancer cell death, which will lead to neoantigen exposure and inflammation. When neoantigens are exposed and there is an influx of inflammatory cells in the tumor, a synergistic effect of anticancer drugs may occur.

[0164] Those skilled in the medical arts will understand that drugs can be modified with various additives, for example to alter the rate at which the body absorbs the drug; and drugs can be modified in different forms, for example to allow for a particular route of administration to the body.

[0165] Thus, the agonist non-blocking antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described herein can be combined with a pharmaceutically acceptable excipient, carrier, diluent, vehicle and / or adjuvant to form a pharmaceutical composition. In this context, the term pharmaceutical composition is used interchangeably with the terms pharmaceutical preparation, pharmaceutical formulation, therapeutic composition, therapeutic preparation, therapeutic formulation and therapeutic entity.

[0166] The pharmaceutical compositions described herein may include, or in some embodiments may consist of, an antibody molecule, a nucleotide sequence, a plasmid, a virus, or a cell.

[0167] In some embodiments, the pharmaceutical compositions described herein may consist of or include a plasmid comprising a nucleotide sequence encoding the above-mentioned antibody molecule or comprising the above-mentioned nucleotide sequence.

[0168] In certain embodiments, pharmaceutical compositions may include coding for the nucleotide sequence of a portion or complete antibody molecule of an antibody molecule as described herein that is integrated in a cell or viral genome or in a virion.Then, pharmaceutical compositions may include cells or viruses (or delivery vehicles encoding the nucleotide sequence of the antibody of the present invention) as delivery vehicles for the antibodies of the present invention. For example, in an embodiment, a virus may be in the form of a therapeutic oncolytic virus, and the therapeutic oncolytic virus includes the nucleotide sequence of at least one antibody molecule encoding an antibody molecule as described herein. In certain embodiments, this oncolytic virus includes the nucleotide sequence encoding a full-length human IgG antibody. In certain embodiments, this oncolytic virus includes the nucleotide sequence encoding scFv, Fab or F (ab') 2 antibody molecules.

[0169] As described in the accompanying claims, in embodiments, the present invention relates to a virus comprising a nucleotide sequence of the present invention or a plasmid of the present invention. Preferably, the virus is an oncolytic virus, such as a therapeutic oncolytic virus. Such viruses are known to those skilled in the art of medicine and virology.

[0170] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding an amino acid sequence that is at least 80% identical to a sequence listed in Table 1 above. In some embodiments, the oncolytic virus comprises an amino acid sequence that is at least 85% identical to a sequence listed in Table 1 above. In some embodiments, the oncolytic virus comprises an amino acid sequence that is at least 90% identical to a sequence listed in Table 1 above. In some embodiments, the oncolytic virus comprises an amino acid sequence that is at least 95% identical to a sequence listed in Table 1 above.

[0171] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding the following: SEQ.ID.NO: 7 and ID.NO: 8. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding the following: SEQ.ID.NO: 15 and ID.NO: 16. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding the following: SEQ.ID.NO: 23 and ID.NO: 24. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding the following: SEQ.ID.NO: 31 and ID.NO: 32. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding the following: SEQ.ID.NO: 39 and ID.NO: 40. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding the following: SEQ.ID.NO: 47 and ID.NO: 48. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding the following: SEQ.ID.NO: 55 and ID.NO: 56.

[0172] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding SEQ.ID.NO:63 and ID.NO:64.

[0173] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding SEQ.ID.NO:71 and ID.NO:72.

[0174] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding SEQ.ID.NO:79 and ID.NO:80.

[0175] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding SEQ.ID.NO:87 and ID.NO:88.

[0176] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding SEQ.ID.NO:95 and ID.NO:96.

[0177] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding SEQ.ID.NO:103 and ID.NO:104.

[0178] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding an amino acid sequence that is at least 80% identical to a sequence listed in Table 1 above. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding an amino acid sequence that is at least 85% identical to a sequence listed in Table 1 above. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding an amino acid sequence that is at least 90% identical to a sequence listed in Table 1 above. In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to a sequence listed in Table 1 above.

[0179] For example, the nucleotide sequence encoding antibody 001-F02 may be as presented in Table 4.

[0180] Table 4: Examples of nucleotide sequences encoding antibody 001-F02 - The underlined portions of the sequences in the table encode the VH and VL sequences of 001-F02, respectively.

[0181]

[0182]

[0183] Some oncolytic viruses have the ability to accommodate large enough DNA insertions to accommodate the integration of full-length human antibody sequences. Attenuated vaccinia virus and herpes simplex virus are examples of therapeutic oncolytic viruses whose genomes are large enough to allow integration of full-length IgG antibody sequences (Chan, WM et al., 2014, "Annu Rev Virol" 1(1): 119-141; Bommardy, PK et al. 2018 "Natural Review Immunology (Nat Rev Immunol)" 18(8): 498-513). Full-length IgG antibodies have been successfully integrated into oncolytic vaccinia viruses, which cause expression and extracellular release of full-length IgG antibodies in virus-susceptible host cells, such as cancer cells, when infected (Kleinpeter, P. et al., 2016, "Tumor Immunology (Oncoimmunology)" 5(10): e1220467). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted upon cell infection (Marino, N. et al., 2017, J Clin Invest 123(6):2447-2463).

[0184] The present invention also encompasses pharmaceutical compositions comprising a virus, such as the oncolytic viruses discussed above, and a pharmaceutically acceptable diluent, vehicle, and / or adjuvant.

[0185] In some embodiments, the pharmaceutical composition may be in the form of a CAR-T cell carrying a partial or complete antibody sequence described herein as part of the sequence encoding its chimeric antigen T cell receptor.

[0186] The present invention also encompasses pharmaceutical compositions comprising CAR-T cells as discussed above and a pharmaceutically acceptable diluent, vehicle and / or adjuvant.

[0187] The present invention also encompasses other therapeutic modalities or "shapes" of drugs, such as antibody drug conjugates, infused proteins, etc., as well as pharmaceutical compositions comprising such therapeutic modalities.

[0188] The antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein can be suitable for parenteral administration, comprising aqueous and / or non-aqueous sterile injection solutions, which can contain antioxidants and / or buffers and / or bacteriostats and / or solutes that make the formulation isotonic with the blood of the intended recipient; and / or aqueous and / or non-aqueous sterile suspensions, which can contain suspending agents and / or thickening agents. The antibody molecules, nucleotide sequences, plasmids, cells and / or pharmaceutical compositions described herein can be presented in unit dose or multidose containers (e.g., sealed ampoules and vials) and can be stored under freeze-dried (i.e., lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use.

[0189] Extemporaneous injection solutions and suspensions may be prepared from sterile powders and / or granules and / or tablets of the kind previously described.

[0190] For parenteral administration to human patients, daily dosage levels of the anti-TNFR2 antibody molecule will generally be from 1 mg / kg to 20 mg / kg of patient body weight, or in some cases even up to 100 mg / kg in a single dose or divided doses. In special circumstances, for example, in conjunction with chronic administration, lower doses may be used. In any case, the physician will determine the actual dosage that is most suitable for any individual patient, and the actual dosage will vary with the age, weight, and response of the particular patient. The above dosages are exemplary of general circumstances. Of course, there may be individual circumstances where higher or lower dosage ranges are warranted, and such dosages are within the scope of the present invention.

[0191] Typically, a pharmaceutical composition (or medicament) comprising an antibody molecule described herein will contain the anti-TNFR2 antibody molecule at a concentration between about 2 mg / ml and 150 mg / ml, or between about 2 mg / ml and 200 mg / ml.

[0192] Typically, in humans, oral or parenteral administration of antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions as described herein is the preferred route and is the most convenient. For veterinary use, antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions as described herein are administered in the form of appropriately acceptable formulations according to normal veterinary practice, and the veterinarian will determine the dosage regimen and route of administration that are most suitable for a particular animal. Therefore, the present invention provides pharmaceutical formulations (as further described above and below) comprising a certain amount of antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions of the present invention that are effective for treating various morbidities. Preferably, antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions as described herein are suitable for delivery by a route selected from the group comprising: intravenous (IV or iv); intramuscular (IM or im); subcutaneous (SC or sc) or intratumoral.

[0193] The present invention also encompasses the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein that comprise pharmaceutically acceptable acid or base addition salts of the target binding molecules or portions of the present invention. The acids used to prepare pharmaceutically acceptable acid addition salts of the above-mentioned base compounds useful in the present invention are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, sucrose, 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 produce pharmaceutically acceptable salt forms of the agents according to the present invention. Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the agents of the present invention that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, base salts derived from such pharmaceutically acceptable cations, such as those of alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium); ammonium or water-soluble amine addition salts, such as N-methylglucamine-(meglumine); and other base salts of lower alkanolammonium and pharmaceutically acceptable organic amines. The antibody molecules, nucleotide sequences, plasmids, viruses, and / or cells described herein can be lyophilized for storage and reconstitution in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technique can be used. Those skilled in the art will appreciate that lyophilization and reconstitution may result in varying degrees of antibody activity loss (e.g., with conventional immunoglobulins, IgM antibodies tend to lose more activity than IgG antibodies), and that use levels may have to be adjusted upward to compensate. In one embodiment, the lyophilized (freeze-dried) polypeptide binding moiety 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) when rehydrated.

[0194] The anti-TNFR2 antibody molecules, nucleotide sequences, and pharmaceutical compositions described herein can be used to treat cancer in a subject or patient.Herein, the terms subject and patient are used interchangeably.

[0195] As used herein, the term "patient" (or subject) refers to an animal, including a human, that has been diagnosed with cancer and / or exhibits symptoms of a particular disease.

[0196] In some embodiments, the patient (or subject) is an animal, including a human, that has been diagnosed with cancer.

[0197] In some embodiments, the patient (or subject) is an animal, including humans, that has been diagnosed with a chronic inflammatory disease and / or exhibits symptoms of a chronic inflammatory disease. As used herein, chronic inflammatory diseases include autoimmune diseases. As described above, several different immune cells can express TNFR2, and the relative levels of expression can vary depending on the disease and context. It is well known that, for example, regulatory T cells can express high levels of TNFR2, and these can be amplified by TNFR2 agonists. Regulatory T cells constitute a subpopulation of T cells that can suppress other immune cells in normal and pathological immune environments and are considered to help prevent autoimmune attacks on one's own tissues. Therefore, stimulating regulatory T cell activity may be very important in the treatment of autoimmune disorders (Sharabi et al. "Regulatory T cells in the treatment of disease" Nature Review: Drug Discovery (Nat Rev Drug Discov.) October 12, 2018). Examples of chronic inflammatory diseases other than autoimmune disorders are osteoarthritis and celiac disease. Examples of autoimmune disorders are rheumatoid arthritis (RA), multiple sclerosis (MS), type I diabetes, systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease (IBD) or myasthenia gravis (MG).

[0198] In some embodiments, the patient (or subject) is a patient with high TNFR2 expression in diseased tissue. In this context, high expression means a higher level of TNFR2 expression compared to corresponding healthy tissue. Typically, the healthy tissue used for this comparison is a reference tissue (or standard reference) collected from healthy tissue of one or more healthy individuals. The level of expression can be measured by standard techniques, such as immunohistochemistry (IHC), fluorescence activated cell sorting (FACS) or mRNA expression measurement.

[0199] 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.

[0200] “Exhibiting” symptoms of cancer includes that the patient exhibits symptoms of cancer and / or cancer diagnostic markers and / or the symptoms of cancer and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0201] It is easy for medical technicians to understand what the symptoms of cancer and cancer diagnostic markers will be and how to measure and / or evaluate and / or quantify whether the severity of cancer symptoms decreases or increases, or whether cancer diagnostic markers decrease or increase; and how those cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0202] Cancer treatment is typically administered as a series of treatments, that is, the therapeutic agent is administered over a period of time. The length of the series of treatments will depend on many factors, including the type of therapeutic agent administered, the type of cancer being treated, the severity of the cancer being treated, and the patient's age and health, among other factors.

[0203] "Treatment period" encompasses the period during which the patient is currently receiving a series of treatments and / or is currently receiving a therapeutic agent, and / or is currently receiving a series of therapeutic agents.

[0204] In some embodiments, the cancer to be treated according to the present invention is a solid tumor.

[0205] Each of the above cancers is well known and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Therefore, the symptoms, cancer diagnostic markers, and therapeutic agents used to treat the above-mentioned cancer types will be known to those skilled in the art.

[0206] Clinical definitions of the diagnosis, prognosis, and progression of a wide range of cancers rely on certain classifications known as staging. These staging systems are used to collate many different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and / or prognosis and / or progression of a cancer. Those skilled in oncology will understand how to use staging systems to assess the diagnosis and / or prognosis and / or progression of a cancer, as well as which cancer diagnostic markers and cancer symptoms should be used to make the assessment.

[0207] “Cancer stage” includes: Rai staging, which includes stage 0, stage I, stage II, stage III, and stage IV; and / or Binet staging, which includes stage A, stage B, and stage C; and / or Ann Arbour staging, which includes stage I, stage II, stage III, and stage IV.

[0208] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur reproducibly in certain cancers, which means that these changes in examination morphology (otherwise referred to as histological examinations) can be used in the diagnosis or prognosis of cancer. Techniques for visualizing samples to examine cell morphology and for preparing samples for visualization are well known in the art; for example, optical microscopes or confocal microscopes.

[0209] "Histological examination" includes: the presence of small mature lymphocytes; and / or the presence of small mature lymphocytes with narrow cytoplasmic borders; the presence of small mature lymphocytes with pyknotic nuclei lacking discernible nucleoli; and / or the presence of small mature lymphocytes with narrow cytoplasmic borders and with pyknotic nuclei lacking discernible nucleoli; and / or the presence of atypical cells and / or lysed cells and / or prolymphocytes.

[0210] As is well known, cancer is the result of mutations in the cell's DNA, which may cause the cell to avoid cell death or to proliferate uncontrollably. Therefore, checking these mutations (also referred to as cytogenetic tests) may be a useful tool for assessing the diagnosis and / or prognosis of cancer. An example of this is the deletion of chromosome position 13q14.1, a characteristic of chronic lymphocytic leukemia. The techniques for checking mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

[0211] "Cytogenetic testing" involves examining the DNA in cells and, in particular, chromosomes. Cytogenetic testing can be used to identify changes in the DNA that may be associated with the presence of refractory and / or recurrent cancers. Such changes may include: a deletion in the long arm of chromosome 13; and / or a deletion at chromosome position 13q14.1; and / or a trisomy of chromosome 12; and / or a deletion in the long arm of chromosome 12; and / or a deletion in the long arm of chromosome 11; and / or a deletion in 11q; and / or a deletion in the long arm of chromosome 6; and / or a deletion in 6q; and / or a deletion in the short arm of chromosome 17; and / or a deletion in 17p; and / or a t(11: 14) translocation; and / or (q13:q32) translocation; and / or antigen gene receptor rearrangement; and / or BCL2 rearrangement; and / or BCL6 rearrangement; and / or t(14:18) translocation; and / or t(11:14) translocation; and / or (q13:q32) translocation; and / or (3:v) translocation; and / or (8:14) translocation; and / or (8:v) translocation; and / or t(11:14) and (q13:q32) translocations.

[0212] It is known that patients with cancer exhibit certain physical symptoms, which are generally due to the burden of the cancer on the body. Those symptoms often recur in the same cancer and can therefore serve as diagnostic and / or prognostic and / or progression characteristics of the disease. Those skilled in the art of medicine will understand which physical symptoms are associated with which cancers and how those body systems are assessed to correlate with the diagnosis and / or prognosis and / or progression of the disease. "Physical symptoms" include hepatomegaly and / or splenomegaly. BRIEF DESCRIPTION OF THE DRAWINGS

[0213] In the examples below, reference is made to the following figures:

[0214] Figure 1 The antibodies of the invention were shown to bind to TNFR2. Figure 1 AD: Human antibodies were shown to bind to human TNFR2 protein by ELISA in a dose-dependent manner resulting in different EC50 values. Figure 1 E: Murine antibodies 3-F10 and 5-A05 bind to mTNFR2 with similar affinities.

[0215] Figure 2 TNFR2 specificity is shown Antibodies and in vitro activated CD4 + T cell binding with IL-2 and CD3 / CD28 CD4 + T cells ( Figure 2 AD) and mouse spleen CD4 + T cells ( Figure 2 E) Activation. TNFR2-specific activation was analyzed by FACS at concentrations ranging from 0.002-267 nM (human) and 0.00003-133 nM (mouse). The affinity of the antibody for activated cells. The curve shows the MFI after subtracting the isotype control background ( Figure 2 A (full and partial blockers), Figure 2 B (partial blocker), Figure 2 C and D (non-blocking agents), Figure 2 E (mouse alternative full blocker (3-F10) and non-blocker (5-A05)).

[0216] Although human TNFR2 antibodies bind to activated CD4 + T cells bound (EC50 values ​​ranging from 0.59 to 53 nM), but mouse TNFR2 antibodies bound with similar affinity (EC50 values ​​ranging from 0.072 to 0.11 nM).

[0217] Figure 3 TNFR2 is shown The antibody specifically binds to TNFR2. Human blood-derived CD4 + T cells ( Figure 3 A) and mouse spleen CD4 + T cells ( Figure 3 B) Activation for 3 days. In vitro activated cells were blocked with polyclonal TNFR2 antibody (grey line) or kept in PBS for 30 minutes (black line) and then stimulated with suboptimal concentrations of different TNFR2 Antibodies or isotype controls (dashed lines) were used for staining for 15 minutes. Cells were then washed and incubated with APC-conjugated secondary antibodies for 30 minutes before analysis by flow cytometry.

[0218] The binding of all antibodies could be blocked by polyclonal TNFR2 antibodies, thus showing that TNFR2 The antibodies (human and mouse) are specific for TNFR2.

[0219] Figure 4 Human TNFR2 specificity is shown Cross-reactivity of antibodies with cynomolgus monkeys.

[0220] CD4 was isolated from the blood of cynomolgus monkeys + T cells were stimulated with PMA and ionomycin. Two days later, 0.1, 1 or 10 μg / ml TNFR2-specific Cells were labeled with either the TNFR2 antibody or an isotype control and then incubated with a secondary α-human antibody conjugated to APC. Cells were analyzed by flow cytometry. + The percentage of T cells relative to isotype control. Results are means and SD of 2-3 separate experiments.

[0221] Most TNFR2 antibodies showed cross-reactive binding to cynomolgus monkey cells.

[0222] Figure 5 40 μg / ml MR2-1 antibody ( Figure 5 A, black bars) blocked activated cells, or activated cells were mixed with PBS ( Figure 5 A, gray bars) were kept together for 30 min, and then TNFR2-specific Antibody / polyclonal TNFR2 (pTNFR2) and cells were incubated for 15 minutes. After incubation with APC-conjugated secondary antibody, bound TNFR2 was analyzed by FACS. The percentage of antibodies. Figure 5 In B, 40 μg / ml TNFR2-specific Antibody / pTNFR2 (black bars) blocks activated CD4 + T cells were either kept with PBS (grey bars) and then incubated with PE-conjugated MR2-1 antibody for 15 minutes. Cells were then analyzed by FACS.

[0223] MR2-1 antibody does not interfere with TNFR2 specificity Antibody binding, and The antibody did not affect the binding of MR2-1 to activated cells, which showed that All antibodies bind to other domains of the TNFR2 protein except the MR2-1 antibody. This shows that TNFR2 is specific The antibodies all bind to epitopes of the TNFR2 protein other than TNFR2 clone MR2-1.

[0224] CD4 + T cells were stimulated for 2-3 days. Figure 5 A, black bars) blocked activated cells, or activated cells were mixed with PBS ( Figure 5 A, gray bars) were kept together for 30 min, and then TNFR2-specific Antibody / polyclonal TNFR2 (pTNFR2) and cells were incubated for 15 minutes. After incubation with APC-conjugated secondary antibody, bound TNFR2 was analyzed by FACS. The percentage of antibodies. Figure 5 In B, 40 μg / ml TNFR2-specific Antibody / pTNFR2 (black bars) blocks activated CD4 + T cells were either left with PBS (grey bars) and then incubated with PE-conjugated MR2-1 antibody for 15 minutes. Cells were then analyzed by FACS.

[0225] MR2-1 antibody does not interfere with TNFR2 specificity Antibody binding, and The antibody did not affect the binding of MR2-1 to activated cells, which showed that The antibodies all bind to other domains of the TNFR2 protein except the MR2-1 antibody.

[0226] Figure 6 Data for ligand blocking antibodies are shown. Blocking ELISA was performed to evaluate the ligand blocking properties. Figure 6 A) All antibodies were incubated at 10 μg / ml. Subsequently, all antibodies that did not reduce the signal by more than 50% (indicated by the dotted line) over the isotype control were dosed to further explore the ligand blocking potential. Figure 6 B) shows a fully blocking mAb, Figure 6 C) and D) show partially blocking mAbs, and Figure 6 E) shows a weak blocking mAb. All other mAbs were considered non-blocking mAbs.

[0227] Figure 7 Data for ligand blocking antibodies are shown. Blocking ELISA was performed to evaluate the ligand blocking properties. Figure 7 A) All antibodies were incubated at 10 μg / ml. Subsequently, all antibodies that did not reduce the signal by more than 50% (indicated by the dotted line) over the isotype control were dosed to further explore the ligand blocking potential. Figure 7 B) shows a fully blocking mAb, Figure 7 C) and D) show partially blocking mAbs, and Figure 7 E) shows a weak blocking mAb. All other mAbs were considered non-blocking mAbs.

[0228] Figure 8 Agonistic non-blocking, but not antagonistic, blocking of TNFR2 specifically The antibody enhanced IFN-γ production in IL-2- and IL-12-stimulated NK cells. Figure 8 A: Human blood-derived NK cells were stimulated with 20 ng / ml rhIL-2 and 20 ng / ml rhIL-12, and 10 μg / ml TNFR2-specific Antibodies, isotype controls or 100 ng / ml rhTNF-α were added for 24 hours. The amount of IFN-γ in the culture supernatant was measured using MSD. The amount of IFN-γ was normalized to the isotype control and is shown in Figure 8 A. CD4 + Human antibodies with a high EC50 value of 25nM for T cells. Human NK cells also produce TNF-α in these cultures (see below). Figure 8 D) IFN-γ results are the mean of 3 donors in 2 independent experiments. The results show that non-blocking TNFR2 antibodies are agonistic and enhance IFN-γ production by NK cells stimulated by cytokines, while blocking antibodies are quite antagonistic and reduce IFN-γ production by NK cells. In addition, Figure 8 BD showed that agonistic antibodies also have intrinsic agonistic activity, even in the absence of measurable TNF-α ligand. Figure 8 As shown in B, the addition of a blocking TNF-α antibody reduced the amount of IFN-γ released, but it was not significantly different from the isotype control ( Figure 8 C) ratios were maintained even in the complete absence of measurable TNF-α in the supernatant ( Figure 8 D). Figure 8 D shows the average TNF-α levels of the two antibodies in the presence or absence of a TNF-α neutralizing antibody.

[0229] Figure 9 Agonistic non-blocking, but not antagonistic, blocking of TNFR2 specifically Memory CD4 + Activation of T cell populations, such as through CD25 + The percentage of cells expressing recombinant IL-2 and TNFR2-specific Antibodies, isotype controls or recombinant TNF-α were used to convert human blood-derived CD4 + T cells ( Figure 9 A) and mouse spleen CD4 + T cells ( Figure 9 B) Activation. After 3 days of culture, cells were stained for CD25 and CD45RO (human) / CD44 and CD62L (mouse) and analyzed by flow cytometry. The results showed that memory (CD45RO + Cells (human) / CD44 + CD62L - The percentage of CD25 expressing cells in the (mice) population was compared to the percentage of CD25 recovered in culture using isotype control. + The results showed that 7 donors ( Figure 9 A, human) and 3 mice (in 2 independent experiments) ( Figure 9 B) Means and SEM. Non-blocking TNFR2 antibodies induced CD25 in both human and mouse cultures. + The percentage of memory cells was significantly increased, while blocking antibodies had no effect on the memory population. Figure 9 A) and rat ( Figure 9 B) Both, addition of exogenous TNF-α increased CD25 + Memory T cell population. * = p < 0.05, as calculated by one-way ANOVA.

[0230] Figure 10 Anti-TNFR2 mAbs were shown to modulate the suppressive function of myeloid-derived suppressor cells (MDSCs). Figure 10 A and B, MDSCs were pre-incubated with human anti-TNFR2 mAb for 30 min. Without washing, cells were In the presence of + T cells were cultured together. Figure 10 A: Activated CD25 was determined by FACS after 3 days + % of T cells To normalize between different assays, isotype control background was subtracted from all data points. The graph shows a summary of 6 different donors with a 1:4 MDSC to T cell ratio in 3 independent experiments. Figure 10B: The amount of secreted cytokines in the supernatants was assessed by MSD and the ratio between IFN-γ and IL-10 release was calculated relative to the isotype control. The data were pooled based on the analysis of culture supernatants obtained from experiments with 5 different donors.

[0231] for Figure 10 C, CD11b was isolated from mouse CT26 tumors. + Bone marrow cells were pre-incubated with mouse anti-TNFR2 mAb for 30 minutes. Titrated numbers of bone marrow cells were co-cultured with CFSE-labeled CD3+ T cells purified from naive Balb / C spleens. Cells were stimulated for 3 days and then analyzed for proliferation by FACS. low % of cells. (n=4 independent experiments). In the T cell / MDSC co-culture assay, only agonistic / non-blocking anti-TNFR2 mAbs were shown to reverse the suppressive function of MDSCs.

[0232] for Figure 10 D, MDSCs were pre-incubated for 30 min with two different human anti-TNFR2 mAbs, one blocking (=1H10) and one non-blocking (=1F02), in three different isotypes (including an Fc-deficient format) in which amino acid position 297 has been switched, resulting in loss of glycosylation and thus reduced binding to FcγRs (herein designated N297Q). Cells were not washed and the CD3 / CD28 In the presence of + T cells were cultured together and after 3 days, activated CD25 + % of T cells. To normalize between different assays, isotype control background was subtracted from all data points. The graph shows that the agonist activity of the non-blocking 1-F02 antibody is independent of antibody isotype and FcγR binding. The graph shows a summary of 4 different donors with a 1:4 MDSC to T cell ratio in 2 independent experiments.

[0233] Figure 11 .for Figure 11 1×10 6CT26 cells. After 8 days, mice with an average tumor size of 3×3 mm were treated with 10 mg / kg antibody ip twice a week, as indicated in the figure. Tumors were measured twice a week until they reached 15 mm in diameter and the mice were then sacrificed. The upper graph shows tumor growth in mice treated with an isotype control, followed by the two graphs below, on the left, an antagonist ligand-blocker antibody in FcγR-deficient Ig format (middle graph) and an agonist non-ligand blocking antibody (lower graph). The middle graph shows the same antibody in a mouse IgG2a format that primarily binds to activating FcγRs, and the right graph is the antibody in a mouse IgG1 format that primarily binds to inhibitory FcγRIIb. For Figure 11 B, Surviving mice were followed for 70 days. As shown in the figure, non-blocking agonistic antibodies were most effective in treating tumors as IgG1 formats, which primarily bind inhibitory FcγRs. In addition, agonistic antibodies had FcγR-independent anti-tumor effects, as seen using the N297A format. On the other hand, blocking antagonistic antibodies were most effective in treating tumors as IgG2a formats, which primarily bind activating FcγRs, and had no effect in formats that utilize defective FcγR binding. *** = p < 0.001 compared to isotype control, as calculated by log-rank Mantel Cox test.

[0234] Figure 12 1×10 6 MC38 cells. Mice with an average tumor size of 3 x 3 mm were treated with 10 mg / kg of antibody ip twice a week as indicated in the figures. The figures show the tumor growth curves of individual mice. Figure 12 A: isotype control, Figure 12 B: PD-1 targeting antibodies, Figure 12 C: 5A05 antibody (alternative antibody, ligand non-blocker, agonist), Figure 12 D: Combination of 5A05 and PD 1. Tumors were measured twice a week until they reached 15 mm in diameter, and then the mice were sacrificed. Figure 12 E shows the survival curves of the four different treatment groups, *=p<0.05, ***=p<0.001 compared to isotype control, as calculated by log-rank Mantel Cox test.

[0235] Figure 13It was shown that ligand-blocking agonist antibodies are effective in combination with anti-PD-L1 as anti-tumor treatment. C57 / BL6 mice were injected subcutaneously with 1×106 MC38 cells. When the average tumor size was 5×5 mm, mice were treated twice (day 1 and day 4) with isotype control antibodies or 5A05, or treated with anti-PD-L1 for four consecutive days, followed by a fifth injection two days later (a total of five injections on day 1, day 2, day 3, day 4 and day 7), or a combination of the two. All antibodies were administered at 10 mg / kg ip. Figure 13 Mean tumor growth + / - SEM, n=10 / group are shown. *=p<0.05, ***=p<0.001 as calculated using one-way ANOVA test.

[0236] Figure 14 1×10 6 B16 cells. Three days later, mice were treated with 10 mg / kg of the antibody ip twice weekly. Tumors were measured twice weekly until they reached 15 mm in diameter, and then the mice were sacrificed. Figure 14 A: isotype control, Figure 14 B: 5A05 antibody (surrogate antibody, ligand non-blocker, agonist). Figure 14 C shows the survival curves of the two different treatment groups. * = p < 0.05 compared to isotype control as calculated by log-rank Mantel Cox test.

[0237] Figure 15 The ligand-non-blocking agonist surrogate antibody 5A05 alters immune cell composition in tumors. Mice were inoculated with CT26 tumor cells as described, and when tumors reached approximately 7×7 mm in size, they were injected with antibodies as indicated. After 3 injections, mice were sacrificed and tumors were harvested on day 8 after treatment initiation. Tumor single-cell suspensions were analyzed for immune cell content by FACS. A) The ligand-non-blocking agonist surrogate antibody 5A05 resulted in Treg depletion and B) CD8 + T cell influx or expansion. This leads to Treg / CD8 + The T cell ratio changes, as depicted in C), and D) shows T cells and myeloid cells, in this case tumor-associated macrophages (TAMs, defined as CD11b + The number of TAMs was very significantly reduced in the presence of 5A05, but negative for both Ly6G and Ly6C. The ligand-blocking antagonist surrogate antibody 3F10 also modulated TAM numbers, but was still significantly different from 5A05.

[0238] Figure 16 NOG mice were injected iv with 15-20×10 6PBMC cells. After 10-12 days, spleens were removed from mice, single cell suspensions were prepared, and TNFR2 expression was assessed by FACS. Previously, TNFR2 expression was assessed in T cells obtained from blood and tumor samples from 3 or 9 cancer patients, respectively. As shown in the figure, Treg and CD8 + TNFR2 expression on T cells was very comparable between human T cells grown and activated in vivo in NOG mice and T cells from human tumors.

[0239] Figure 17 NOG mice were injected iv with 15-20×10 6 After 10-12 days, the spleens were removed from the mice, single cell suspensions were prepared, and the cells were analyzed using FACS. Figure 17 A shows the protein defined as CD45 + CD3 + CD4 + CD25 + CD127 low / neg The average percentage of stained Tregs divided by the total number of cells in the spleen. Figure 17 B shows T cells (CD3 + ) to the average percentage of the total number of cells in the spleen. Figure 17 As shown in Figure 3, the ligand-non-blocking agonist antibody 1F02 increased the number of Tregs (which express the highest levels of TNFR2) and the total number of T cells in this model. In comparison, the ligand-blocking antagonist antibody 1H10 had no such effect.

[0240] Figure 18 IFN-γ release induced by various TNFR2-specific antibodies was measured in three different in vitro systems. Positive controls included the anti-CD3 antibodies OKT3 or muromonab-CD3, the anti-CD52 antibody alemtuzumab, and an anti-CD28 antibody. An isotype control served as a negative control. Each dot represents PBMCs from a single human donor. Figure 18 A shows the results from high-density cell culture, where PBMCs were cultured at 1×10 7 After 48 hours, 10 μg / ml of antibody was added and incubated for 24 hours. As can be seen in the figure, both alemtuzumab and OKT3 induced significant IFN-γ release, but none of the TNFR2-specific antibodies did. Figure 18B shows solid phase in vitro culture performed by coating the wells of a 96-well plate with antibodies before adding PBMCs. Again, both alemtuzumab and OKT3 induced significant IFN-γ release as well as some TNFR2-specific antibodies, particularly in one of the donors. Figure 18 C shows whole blood was stimulated with antibodies and here alemtuzumab induced significant IFN-γ release but did not induce any TNFR2-specific antibodies.

[0241] Figure 19 NOG mice were injected iv with 25 × 10 6 PBMC cells. After 14 days, when the blood of mice showed to be composed of approximately 40% human T cells, mice were treated with 10 μg of antibody. Body temperature was measured 1 hour after injection ( Figure 19 A). The experiment was terminated 5 hours after injection, and the blood was analyzed for IFN-γ ( Figure 19 B) or TNF-α ( Figure 19 C) Content. **** = p < 0.0001 and ** = p < 0.01 as calculated using one-way ANOVA.

[0242] Figure 20 Binding to TNFR2 variants lacking individual domains is shown. Antibodies were tested for binding to TNFR2 variants expressed on HEK cells by flow cytometry. Absence of domains 1 and 2 did not significantly affect binding ( Figure 20 A and B), while 3 and part of domain 4 completely abolished the interaction between the antibody and TNFR2 ( Figure 20 C and D). Similarly, the absence of domains 1+3 completely prevented binding of all antibodies except 1F06 ( Figure 20 E), whereas the absence of domains 2+4 completely abolished the binding of agonist antibodies (1F02, 1F06, 4E08) and also significantly reduced the binding of antagonists (1H10, 4H02, 5B08) ( Figure 20 F). Dark grey represents the positive control, and white represents the negative control antibody.

[0243] Figure 21 A comparison of the amino acid sequences of human (H-D3) and mouse (M-D3) domain 3 of TNFR2 is shown. Similar amino acids are marked in white, while differences are marked in gray. The five sequences below represent five different constructs against which antibodies were tested. The swaps between human and mouse sequences are underlined, while the unmarked sequences are fully human. Domains 1, 2, and 4 are human and do not contain any substitutions or mutations.

[0244] Figure 22The left panel shows binding to wild-type human and mouse TNFR2. Mutated hTNFR2 constructs (m1, m2, m3, and m4) were used to narrow the binding site for different anti-hTNFR2 antibodies. Flow cytometric analysis showed that mutations in aa 119-132 did not affect antibody binding, but mutations in aa 151-160 completely abolished binding for all antibodies. Mutations in 134-144 only disrupted binding of blocking and antagonistic antibodies but did not significantly affect agonist antibodies. Dark gray bars represent positive controls, and white represents negative control antibodies. The dashed line represents the level of the negative control antibody.

[0245] Examples

[0246] Specific non-limiting examples embodying certain aspects of the invention will now be described.

[0247] In many examples, particularly in vivo examples, antibody 5-A05 has been used. This is a mouse antibody that is an alternative to the human antibodies disclosed herein. It was chosen as an alternative based on its similar properties to ligand-non-blocking agonistic antibodies with good EC50 values.

[0248] In some examples and figures, slightly different nomenclature of antibody clones is used, for example, clone 001-F02 is sometimes shortened to 1-F02 or 1F02, 005-B08 is sometimes shortened to 5-B08 or 5B08, etc.

[0249] Example 1 - Generation of TNFR2-specific antibodies

[0250] (See also Figure 1 and the description of this figure above.)

[0251] Isolation of scFv antibody fragments

[0252] Used scFv library (BioInvent, E et al., Nat Biotechnol. 2000; 18(8):852-6) to isolate scFv antibody fragments that recognize human or mouse TNFR2.

[0253] Phage libraries were used in three consecutive pannings against recombinant human or mouse proteins. After phage incubation, cells were washed to remove unbound phage. Bound phage were eluted with trypsin and amplified in E. coli. The resulting phage stock was converted to scFv format. E. coli was transformed with an scFv-carrying plasmid and individual scFv clones were expressed.

[0254] Identification of unique TNFR2-binding scFvs

[0255] Converted scFvs from the third panning were assayed for binding to 293FT cells transfected to express human or mouse TNFR2 or an unrelated protein using a homogenous FMAT assay (Applied Biosystems, Carlsbad, CA, USA).

[0256] Briefly, transfected cells were added to clear-bottom 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; Catalog No. 115-136-146, Jackson Immunoresearch). FMAT plates were incubated at room temperature for 9 hours before reading. Cells transfected with bacterial clones that bound to TNFR2 but not cells transfected with irrelevant proteins were classified as active and optimally sorted into 96-well plates.

[0257] IgG binding to TNFR2 in ELISA

[0258] 96-well plates (Greiner Lumitrac 600LIA plates) were coated with recombinant human or mouse TNFR2-Fc protein (SinoBiological) at 1 pmol / well overnight at 4°C. After washing, titrating doses of anti-TNFR2 mAb ranging from 20 μg / ml to 0.1 ng / ml (133 nM to 1 pM) were allowed to bind for 1 hour. The plates were then washed again and bound antibody was detected with an anti-human F(ab)-HRP secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted at 50 ng / ml. Super Signal ELISA Pico (ThermoScientific) was used as a substrate, and the plates were analyzed using a Tecan Ultra Microplate Reader.

[0259] Table 5 and Figure 1 The data shown in AD indicate that all human anti-TNFR2 antibodies bound to human TNFR2 protein, with EC50 values ​​ranging from 0.082 nM for 1-C08 to 4.4 nM for 1-A09.

[0260] In addition, mouse antibody alternative clones 3-F10 and 5-A05 also bound to the mTNFR2 protein. These two clones bound with very similar affinities (Tables 5 and Figure 1 E).

[0261] Table 5 EC50 values ​​of antibodies binding to TNFR2 protein (human protein except clones 3F10 and 5A05)

[0262] clone <![CDATA[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

[0263] Example 2 - Specificity of Antibodies

[0264] (See also Figure 2-5 and the above description of these figures.)

[0265] CD4 + Isolation of T cells

[0266] PBMCs from human buffy coats and cynomolgus macaque (Cynodon fascicularis) whole blood were isolated using Ficoll-Paque PLUS (GE Healthcare) gradients. CD4 + CD4 T cell isolation kit (human) or CD4 microbeads (non-human primate (cynomolgus monkey)) were used to isolate CD4 from PBMC. + T cells. CD4 T cells from Miltenyi Biotec were used. + T Cell Isolation Kit (Mouse) isolates mouse CD4 + T cells.

[0267] TNFR2 specificity Antibody titration

[0268] Using in vitro activated CD4 + T cells acquire TNFR2 The ability and affinity of the antibodies to bind to TNFR2 expressed on cells were determined by using 50 ng / ml rhIL-2 (R&D Systems) and T-Activator CD3 / CD28 stimulates human CD4 + T cells were cultured for expansion or activation at 37°C for 2-3 days. Increasing amounts of TNFR2 specific for TNFR2 or an isotype control, ranging from 0.002 to 267 nM, were used. The cells were then incubated with APC-conjugated α-human IgG secondary antibody (Jackson) and analyzed by flow cytometry (FACSVerse, BD Biosciences). 135 U / ml rmIL-2 (R&D Systems) and T-Activator CD3 / CD28 stimulates mouse CD4 +T cells were expanded or activated at 37°C for 2-3 days (Gibco). Increasing amounts of TNFR2 specific for TNFR2 or isotype control ranging from 0.00003 to 133 nM were used. Antibody labeled cells activated in vitro. The cells were then incubated with an α-mouse IgG secondary antibody (Jackson) conjugated to APC and analyzed by flow cytometry (FACSVerse from Becton Dickinson). The EC50 values ​​of the titration curves were calculated in Microsoft Excel and are shown in Table 6. For human antibodies, EC50 values ​​ranged from 0.6 nM (4-H02) to 52.7 nM (1-C03). Mouse antibodies bound to activated cells in vitro with similar affinities (0.072 nM (3-F10) and 0.11 nM (5-A05)).

[0269] TNFR2 Antibody specificity

[0270] The specificity of the TNFR2 antibody for TNFR2 was obtained using a commercial polyclonal TNFR2 antibody (R&D Systems) in a FACS blocking experiment. T-Activator CD3 / CD28 stimulation for 2-3 days for T cell expansion and activation (Gibco) + T cells (mouse and human) were blocked for 30 minutes, immediately after which they were stained with TNFR2 Incubate with antibodies or isotype controls for 15 minutes. The concentration of the antibody is based on TNFR2 alone Antibody titration curves were generated and the suboptimal concentration of each antibody was selected. The cells were then washed and incubated with APC-conjugated secondary antibody (Jackson) for 30 minutes. The cells were analyzed by flow cytometry (FACSVerse, Becton Dickinson). TNFR2 specificity All binding of the antibodies (both human and mouse) could be blocked by polyclonal TNFR2 antibodies, such as Figure 4 These results confirm that TNFR2 Antibodies specifically bind to activated CD4 + TNFR2 on T cells.

[0271] TNFR2 specificity Antibody epitope mapping against TNFR2 antibody clone MR2-1

[0272] The TNFR2 antibody clone MR2-1 (Invitrogen) binds to a specific domain of the TNFR2 protein. TNFR2 specificity was tested by FACS blocking assay. Does the antibody bind to the same domain as MR2-1?

[0273] 50 ng / ml rhIL-2 (R&D Systems) and T-Activator CD3 / CD28 converts human CD4 + T cells were stimulated for 2-3 days for T cell expansion or activation (Gibco). 40 μg / ml MR2-1 ( Figure 5 Black bars in A), TNFR2 specific Antibody / polyclonal TNFR2 (R&D Systems) Figure 6 Black bars in B) or PBS ( Figure 5 After 30 minutes of incubation, cells were immediately subjected to TNFR2-specific Antibody / pTNFR2( Figure 5 A) or MR2-1( Figure 5 B) Staining for 15 minutes. Figure 5 The cells in A were also incubated with APC-conjugated secondary α-human IgG reagent (Jackson). All cells were analyzed by flow cytometry (FACSVerse, Becton Dickinson). MR2-1 + The percentage of cells was the same as that of unblocked cells ( Figure 5 B) with or without MR2-1 blockade The antibodies bind identically ( Figure 5 A) So these Antibodies may bind to other epitopes of the TNFR2 protein besides the MR2-1 antibody.

[0274] TNFR2 Antibody binding to cynomolgus monkeys

[0275] To verify the cross-reactivity of TNFR2 antibodies to cynomolgus monkeys, cynomolgus monkey CD4 + T cells were stimulated for 2 days. The cells were incubated with 3 different concentrations (0.1 μg / ml, 1 μg / ml and 10 μg / ml) of TNFR2-specific The cells were incubated with the TNFR2 antibody and then with APC-conjugated secondary α-human IgG reagent (Jackson). The cells were analyzed by flow cytometry (FACSVerse, Becton Dickinson), and the results showed that most of the TNFR2-specific All antibodies can bind to cynomolgus monkey TNFR2, and the results of individual antibodies are presented in Figure 4 middle.

[0276] In summary, the data in Example 2 show that the human antibodies bind specifically to TNFR2 endogenously expressed on human immune cells. Furthermore, the data show that this binding can be blocked by the addition of a polyclonal commercially available antibody to TNFR2 that exhibits very high specificity for TNFR2. This was also true for alternative clones 3F10 and 5A05 on murine cells expressing murine TNFR2. Likewise, binding of the human clones was not affected by the MR2-1 antibody, which exhibits a different epitope specificity than MR2-1.

[0277] Table 6. CD4 + Calculation of EC by titration of TNFR2-specific antibodies on T cells 50 value.

[0278]

[0279]

[0280] Example 3 - Testing of ligand blocking properties

[0281] (See also Figure 6-7 and the above description of these figures.)

[0282] ELISA method

[0283] 96-well plates were coated with hTNFRII (Sino Biological Biotech, Inc., Cat. No. 10414-H08H) or mTNFRII (Sino Biological Biotech, Inc., Cat. No. 50128M08H) at 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 containing 0.05% Tween 20), the plates were incubated with mAb was cultivated at room temperature for 1 hour in a blocking buffer containing 0.45% fish gelatin under slow stirring at 10 μg / ml (single dose ELISA) or 33nM and subsequently at a dilution of 1:2 (titration ELISA). Subsequently, recombinant hTNF-α-bio (R&D catalog number BT210) or mTNF-α (Gibco catalog number PMC3014) was added at a final concentration of 5nM and 2nM and allowed to cultivate for another 15 minutes. Afterwards, the plate was washed. For human ELISA, streptavidin-HRP (Streptavidin-HRP) (Jackson catalog number 016-030-084) diluted in a 1:2000 blocking buffer was added, and again at room temperature for 1 hour, then first washed in ELISA buffer and then washed in Tris buffer (pH 9.8). Substrate was then diluted according to the manufacturer's instructions (Super Signal ELISA Pico from Thermo Fisher Scientific, catalog number 37069), added to the wells and incubated in the dark for 10 minutes before reading on a Tecan Ultra. For the mouse ELISA, rabbit anti-mTNF-α (Sino Biologics catalog number 50349-RP02) diluted to 1 μg / ml was added and allowed to incubate at room temperature for 1 hour. After washing, anti-rabbit HRP diluted 1:10,000 in blocking buffer was added and incubated again at room temperature for 1 hour. Substrate addition and reading were performed as above.

[0284] The data for anti-human and anti-mouse antibodies are presented in Tables 7 and 8 below, respectively, and Figure 6 and 7 middle.

[0285] Table 7. EC50 values ​​for ligand blocking human antibodies: Antibodies were titrated and EC50 values ​​were calculated.

[0286]

[0287]

[0288] Table 8. EC50 values ​​for ligand blocking murine antibodies: Antibodies were titrated and EC50 values ​​were calculated.

[0289] clone EC50 (nM) Block 3-F10 1.9 completely 4-C01 2.7 completely 4-A06 2.0 part 4-A07 >500 part 4-F06 6.2 part 5-C09 8.6 part 2-D09 4.4 part 4-B12 >500 part 3-G06 13 part 2-H01 25 weak 4-C02 >500 weak 4-G09 2.6 weak 4-C03 8.3 weak

[0290] Blocking Definition

[0291] • A complete blocker is defined as a reduction of TNF-α binding by greater than 98%.

[0292] • Partial blockers are defined as reducing TNF-α binding by 60-98%.

[0293] • Weak blockers are defined as reducing TNF-α binding by less than 60%.

[0294] Non-blocking antibodies are defined as those that do not achieve 50% or less blocking in a high-dose single-site ELISA, e.g. Figure 6 A and shown in 7A.

[0295] The data presented in this example show that a variety of antibodies have been generated, ranging from antibodies that completely inhibit ligand TNF-α binding to antibodies that do not inhibit ligand blocking at all. This is true for both human antibodies and murine surrogates.

[0296] Example 4 - In vitro function of antibodies

[0297] (See also Figure 8-10 and the above description of these figures.)

[0298] Compared with TNF-α-blocking TNFR2 antibodies, TNF-α-nonblocking TNFR2 antibodies enhanced cytokine-stimulated NK cell IFN-γ production.

[0299] The agonistic / antagonistic properties of TNFR2-specific antibodies were evaluated using the NK cell assay described by Almishri et al. (“TNFα enhances cytokine-induced NK cell IFNγ production via TNFR2” Almishri W et al. J Innate Immun. 2016;8:617-629).

[0300] Briefly, human NK cells were isolated from human PBMCs by MACS using the “NK Isolation Kit” (Miltenyi Biotec). 100 μl of NK cells (1×10 6 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 antibody, 10 μg / ml isotype control, or 100 ng / ml TNF-α (R&D Systems) in U-bottom plates (Sigma-Aldrich). The cells were cultured in 96-well TC-treated microplates. The supernatants were collected after 24 hours and the amount of IFN-γ produced was assessed by MSD. As a control, an anti-TNF-α antibody (Catalog No. AF-210-NA, R&D Systems) was included to neutralize TNF-α. Figure 8 As seen in D, a dose of 1 μg / ml completely neutralized soluble TNF-α, and this dose also reduced IFN-γ release.

[0301] Human non-blocking TNFR2 antibodies significantly enhanced IL-2 and IL-12 stimulated NK cell IFN-γ production (2-3 fold more IFN-γ compared to isotype control), whereas antagonistic antibodies (shown here as full blockers) showed antagonistic effects on NK cell IFN-γ production ( Figure 8 A).

[0302] Due to the lack of endogenously produced TNF-α in murine cultures and the expression of inhibitory FcγRs on murine NK cells and only activating FcγRs on human counterparts, this test was considered non-representative for use with mouse surrogate antibodies. Instead, memory T cell activation assays (induction of CD25) and myeloid-derived suppressor cell (MDSC) inhibition assays (both described below) were used to address the agonist or antagonist properties of mouse surrogate antibodies.

[0303] Ligand-nonblocking but not TNFR2-blocking antibodies induce memory CD4 + CD25 is expressed on T cells.

[0304] To further understand the agonistic / antagonistic properties of TNFR2 antibodies, we investigated their ability to enhance CD25 expression in memory CD4 + The capacity of the T cell ratio was evaluated.

[0305] In short, using the "CD4 + T Cell Isolation Kit" is used to isolate human CD4 T cells from PBMCs by MACS. + T cells. CD4 + T cells were cultured with 10 ng / ml rhIL-2 (R&D Systems) and 10 μg / ml TNFR2-specific antibody or the indicated amount of rhTNF-α (R&D Systems). After 3 days, the expression of CD25 on memory cells (CD45RO + expression on cells) Figure 9 A).

[0306] Similarly, using "CD4 + Mouse CD4 T cell isolation kit (Miltenyi Biotec) was used to isolate mouse CD4 T cells from spleen by MACS. + T cells were 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). After 3 days, the expression of CD25 on memory cells (CD44 + CD62L - expression on cells) Figure 9 B).

[0307] The percentage of CD25-expressing cells in memory cell cultures stimulated with non-blocking TNFR2 was enhanced, again demonstrating an agonistic effect in both humans and mice. However, stimulation with a blocking antibody did not increase CD25 expression in these cultures.

[0308] Non-ligand-blocking but not blocking anti-TNFR2 mAbs reverse the suppressive function of myeloid-derived suppressor cells (MDSCs) in T cell / MDSC co-culture assays

[0309] The effects of human anti-TNFR2 mAbs on the suppressive function of MDSCs were evaluated in a T cell / MDSC co-culture assay. Briefly, human CD14 T cells isolated from cancer patients were cultured in 50% ascites and 50% R-10 T cells. + MDSCs were generated from monocytes for 3 days. MDSCs were then washed and pre-incubated with 10 μg / ml anti-TNFR2 mAb for 30 minutes. Without washing the cells, the CD3 / CD28 Titrated numbers of MDSCs were isolated by MACS and labeled with CFSE in the presence of + After 72 h, activated CD25 + The percentage of T cells. IFN-γ and IL-10 secretion were measured by MSD according to the manufacturer's instructions.

[0310] In contrast to blocking anti-TNFR2 mAb (dark grey bars), non-blocking anti-TNFR2 mAb (light grey bars) was shown to increase activated CD25 in co-cultures. + Percentage ( Figure 10 A). By measuring the amount of secreted interferon-γ (IFN-γ) and interleukin-10 in the supernatant, the ratio of the two cytokines was used to estimate the Th1 / Th2 balance. Figure 10 As shown in B, a non-blocking anti-TNFR2 antibody significantly increased the ratio of IFN-γ to IL-10 compared to a blocking antagonistic antibody, indicating that the non-blocking antibody induced a shift toward a Th1 pathway.

[0311] As for human antibodies, the effects of mouse anti-TNFR2 antibodies have been tested in similar inhibition assays. Direct MACS isolation of CD11b from CT26 mouse tumors + Bone marrow cells were pre-incubated with anti-TNFR2 mAb for 30 minutes. CD3 + Responder T cells were purified. Myelosuppressor cells and CFSE-labeled T cells were co-cultured at different ratios for 3 days. Proliferation of CFSE was determined by FACS. lowSimilar to the results of human assays, the percentage of proliferative responder cells after incubation with blocking antibodies was significantly lower than that after incubation with non-blocking antibodies ( Figure 10 C).

[0312] In MDCS inhibitor cultures, myeloid cells express high levels of various FcγRs. To test whether the observed agonistic and antagonistic effects were Fc-dependent, the ligand-nonblocking agonistic antibody 1F02 was tested in several formats; hIgG1 bound well to activating FcγRs, hIgG2 also bound well to inhibitory FcγRIIB, and an Fc-deficient format showed greatly reduced binding to all FcγRs. Figure 10 The results in D show that the agonistic activity of the non-blocking 1-F02 antibody is independent of antibody isotype and FcγR binding.

[0313] In summary, the data in Example 4 show that the ligand non-blocking antibodies are agonistic as measured by several in vitro methods: NK cell-mediated IFN-γ release, CD4 T cell proliferation as measured by CD25 expression, and T cell proliferation. + Activation of memory cells and CD25 expression in MDCS co-culture assays. Furthermore, the data show that this is an intrinsic property, independent of the presence of ligand and independent of antibody isotype. The alternative murine antibody 5A05 was also shown to have similar agonist properties. T cell or NK cell stimulating antibodies can be used to treat cancer and can induce endogenous immune responses, ultimately destroying malignant cells.

[0314] Agonistic ligand non-blocking antibodies are shown according to the present invention, whereas antagonistic ligand blocking antibodies are included for comparison.

[0315] Example 5 - Surrogate ligand non-blocking agonistic anti-mouse TNFR2 mAb has anti-tumor effects in vivo

[0316] (See also Figure 11-17 and the above description of these figures.)

[0317] Therapeutic effects in different tumor models

[0318] To evaluate the in vivo antitumor effects of a ligand-non-blocking agonistic anti-TNFR2 mAb, a mouse surrogate termed 5-A05 was studied in vivo in different tumor models using different isotype formats and alone or in combination with anti-PD-1 as described below.

[0319] According to the guidelines of the Ministry of the Interior (home office), mice are raised and maintained in local facilities. Six to eight weeks old female BalbC and C57 / BL6 mice are supplied by Taconic (Denmark Bomholt (Bomholt, Denmark)) and are maintained in local animal facilities. CT26, MC38 and B16 cells (ATCC) are grown in the RPMI buffered with glutamine (glutamax) supplemented with 10% FCS. When the cells are semi-convergent, they are separated with trypsin and with 10 × 106 individual cells / ml are resuspended in sterile PBS. 100 μ l cell suspensions are injected into mice sc, which corresponds to 1 × 106 individual cells / mouse. After injection 3-8 days (depending on the model), mice (isotype control, 3-F10 or 5-A05) are treated with 10mg / kg antibody and ip as indicated in the figure twice a week. Twice / weekly tumor is measured until its diameter reaches 15mm, and then mice are put to death.

[0320] The ligand-non-blocking agonistic anti-mouse TNFR2 mAb 5-A05 showed therapeutic anti-tumor effects in three different tumor models ( Figure 11-14 ), in the more treatment-sensitive CT26 ( Figure 11 ) and had a tumor growth inhibitory effect in the more treatment-resistant MC38 and B16 ( Figure 12-14 ).

[0321] The antitumor effects of a ligand-nonblocking agonistic anti-mouse TNFR2 mAb do not require FcγR binding but are enhanced by such binding

[0322] To assess the importance of Fc-FcγR interactions for the in vivo antitumor effects of the ligand-nonblocking agonistic anti-TNFR2 mouse surrogate mAb 5A05, different Fc formats of this antibody were studied in vivo in the CT26 tumor model as described below.

[0323] Mice were raised and maintained as described above. CT26 cells (ATCC) were grown and injected as described above. When tumors reached 3×3 mm, mice were treated with 10 mg / kg of antibody (isotype control, 5A05 IgG1, 5A05 IgG2a, or 5-A05-N297A (Fc-deficient)) ip twice weekly. Tumors were measured twice weekly until they reached 15 mm in diameter, at which point the mice were sacrificed.

[0324] Fc-deficient 5-A05-N297A showed significant therapeutic activity compared to the isotype control, indicating that Fc ligation is not entirely exclusive to the therapeutic efficacy of this ligand-non-blocking agonistic anti-mouse TNFR2 mAb ( Figure 11A and B). In addition, both IgG1 and IgG2a formats showed enhanced therapeutic efficacy. However, the IgG1 format, which preferentially binds to inhibitory Fcγ receptors, showed superior therapeutic efficacy, suggesting that agonism is an important mechanism of action for this anti-mouse TNFR2 mAb ( Figure 11 AB). This is in contrast to the ligand-blocking antagonist surrogate antibody 3-F10, which showed no activity in the Fc-deficient format and optimal activity in the murine IgG2a format, known to preferentially bind to activating FcγRs.

[0325] The ligand non-blocking agonistic antibody (5-A05) is according to the present invention, and the ligand blocking antagonistic antibody (3-F10) is included for reference.

[0326] Combination effects of anti-PD-1 mAbs

[0327] To evaluate the in vivo anti-tumor effects of the combination of a ligand non-blocking agonistic anti-TNFR2 mAb (mouse surrogate (5-A05)) and anti-PD-1, the therapeutic combination was studied in vivo in the MC38 tumor model as described below.

[0328] Mice were raised and maintained as described above. MC38 cells (ATCC) were grown and injected as described above. Eight days after injection, mice were treated with 10 mg / kg of antibody (isotype control, anti-mouse PD-1, 5-A05, or a combination of anti-mouse PD-1 and 5-A05) ip twice weekly, as described above. Figure 12 Tumors were measured twice a week until they reached 15 mm in diameter, and then the mice were sacrificed.

[0329] Both anti-mouse PD-1 and ligand non-blocking agonistic anti-mouse TNFR2 mAb 5-A05 showed tumor growth inhibitory therapeutic effects in the MC38 model ( Figure 12 AE). When anti-PD1 and 5-A05 were combined, tumors in MC38 cells with treatment resistance were cured ( Figure 12 DE).

[0330] Combination effects of anti-PD-L1 mAbs

[0331] To evaluate the combined in vivo anti-tumor effects of agonistic anti-TNFR2 mAbs, the mouse surrogate (5A05) was further combined with anti-PD-L1 for treatment in the MC38 tumor model as described below.

[0332] Mice were raised and maintained as described above. MC38 cells (obtained from Dr. M.Cragg of Southampton University (Southampton University)) were grown and injected as described above. After six days of injection, mice were treated with isotype control antibodies or 3F10 twice (day 1 and day 4), or treated with anti-PD-L1 (clone 10F.9G2, Bioxcell) for four consecutive days, followed by a fifth injection (five injections totaling five injections on day 1, day 2, day 3, day 4, and day 7) or a combination of the two two days later. All antibodies were administered with 10 mg / kg ip. Tumors were measured with a caliper twice a week until their diameter reached a volume of 2000 mm3, and then mice were killed.

[0333] Both anti-mouse PD-L1 and agonistic anti-mouse TNFR2 mAb 5A05 showed tumor growth inhibitory therapeutic effects in the MC38 model ( Figure 13 When anti-PD-L1 and the antagonist anti-mouse TNFR2 mAb 5A05 were combined, the anti-tumor effect was even further enhanced ( Figure 13 ).

[0334] Immune cell regulation in vivo

[0335] To investigate the role of immune cells in tumors in vivo, BalbC mice were inoculated with CT26 cells as described above. After tumors reached approximately 7 × 7 mm, Figure 15 Mice were treated with 10 mg / kg of the antibodies indicated in the RT-PCR assay. Mice were treated on days 1, 4, and 7 and sacrificed on day 8. Tumors were dissected, mechanically divided into small pieces, and digested for 2 x 5 minutes at 37°C using a mixture of collagenase 100 μg / ml release enzyme and 100 μg / ml deoxyribonuclease with a Vortex in between. After filtering through a 70 μm filter, the cell suspension was washed with PBS containing 10% FBS (400 g for 10 minutes). Afterwards, the cells were resuspended in MACS buffer and stained with a panel of antibodies that stain CD45, CD3, CD8, CD4, and CD25 or a panel of antibodies that stain MHCII, F4 / 80, Ly6C, CD11b, and Ly6G. Before staining, nonspecific binding of the cells was blocked using 100 μg / ml IVIG (purified intravenous immunoglobulin). Cells were analyzed in a FACS Verse. Quantification of mouse Tregs as CD45 + CD3 + CD4 + CD25 + , and TAMs were quantified as CD11b + Ly6G - Ly6C -f / 4 / 80 + MHCII + .

[0336] like Figure 15 As seen in the previous studies, treatment with agonistic TNFR2 antibodies resulted in the expression of CD8 + T cell influx increased. A weaker trend toward Treg depletion was also seen. Together, this led to CD8 + The ratio of T cells to Tregs was greatly improved ( Figure 15 C) Additionally, agonistic antibodies modulate the bone marrow compartment by reducing the number of tumor-associated macrophages.

[0337] PBMC-NOG / SCID model

[0338] To confirm the in vitro findings of non-ligand blocking the agonistic / T cell proliferative activity of agonistic anti-TNFR2 mAb 1-F02, the ability of this mAb to induce T cell proliferation in vivo in the PBMC-NOG model was analyzed as follows.

[0339] Mice were bred and maintained in a local facility according to the guidelines of the Ministry of the Interior. Eight-week-old female NOG mice were supplied by Taconic (Bohmholt, Denmark) and maintained in a local animal facility. For the PBMC-NOG (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and washed at 75 × 10 6 Resuspend the cells in sterile PBS at a concentration of 15 × 10 cells / ml. 200 μl of cell suspension was injected iv into NOG mice, which corresponds to 15 × 10 6 cells / mouse. Two weeks after injection, mice were treated twice (two days apart) with 10 mg / kg of isotype control, Yervoy, anti-CD25, Campath, 1-F02, or 1-H10 (ligand-blocking antagonist anti-TNFR2 mAb). Spleens of mice were collected 2 days after the final injection. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD3, CD4, CD8, CD25, CD127 (all from BD Biosciences). In a separate experiment, spleens from untreated human PBMCs were sacrificed to determine TNFR2 expression on human T cells by FACS ( Figure 16 ). This FACS data shows that TNFR2 expression on Treg and CD8+ T cells is very comparable between human T cells grown and activated in vivo in NOG mice and T cells from human tumors.

[0340] 1-F02 induces T cell proliferation in vitro and in vivo, as has been seen with non-ligand blocking agonistic anti-mouse surrogate TNFR2 mAbs, whereas the antagonist 1-H10 does not ( Figure 17 ).

[0341] In summary, Example 5 shows that:

[0342] 1. Agonistic ligand non-blocking antibodies can have potent anti-tumor effects across several tumor models.

[0343] 2. This effect can be enhanced by combining with anti-PD1 antibodies.

[0344] 3. Agonistic non-ligand blocking antibodies do not show exclusive FcγR dependence for anti-tumor effects, but the effects are enhanced by ligation of primarily inhibitory FcγRs.

[0345] 4. Agonistic non-ligand blocking antibodies can increase the influx of CD8-positive T cells and reduce the number of Tregs in tumors. In addition, they reduce the number of TAMs in tumors, thereby changing the composition of both T cells and myeloid cells in tumors.

[0346] 5. In human tumors, T cells express TNFR2.

[0347] 6. In a human xenograft model in which tumor TNFR2 expression is mimicked on T cells, the agonistic ligand non-blocking antibody 1-F02 increases the number of T cells.

[0348] Example 6 - Agonistic ligand non-blocking antibodies do not induce significant amounts of pro-inflammatory cytokines

[0349] (See also Figure 18-19 and the above description of these figures.)

[0350] The release of a large number of proinflammatory cytokines is a possible side effect of immunomodulatory antibodies for the treatment of patients. Therefore, two different methods were used to measure the cytokine release induced by agonist ligand non-blocking antibodies. The first method was based on antibody stimulation in vitro culture, and the second method was based on human immune cell xenotransplantation to immunodeficient mice. For in vitro, the setting of culture has been shown to affect the release of cytokines to a great extent (Vessillier et al., "Journal of Immunology (J Immunol Methods)" September 2015; 424: 43-52). In order to solve the difference in the method, according to recent publications, three different in vitro culture devices were used.

[0351] For high density cell culture (HDC) cytokine release assay (CRA), 1×10 7PBMCs were cultured in serum-free CTL assay medium (Cell Technology Limited) supplemented with 2 mM glutamine, 1 mM pyruvate, 100 IU / ml penicillin, and streptomycin. 2 ml of the cell culture was plated in a 12-well plate. After 48 hours, 1 × 10 5 10 μg / ml of antibody was added to 10 pre-incubated PBMCs and incubated for 24 hours.

[0352] PBMC solid phase (SP) CRA was performed by coating the wells of a 96-well plate with 1 μg / ml of antibody for 1 hour. After washing the plate with PBS, 1×10 5 PBMCs were added with 200 μl complete medium and incubated for 48 hours.

[0353] Cytokine release was also measured after 48 hours of stimulation of 200 μl of whole blood with 5 μg / ml of antibody.

[0354] At the end of the incubation period, the plates were centrifuged and the culture supernatant was extracted 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).

[0355] In summary, the ligand-non-blocking agonistic antibodies only induced any significant cytokine release beyond IFN-γ in any in vitro setting (data not shown). The positive control antibodies alemtuzumab and OKT3 did induce cytokines, the most notable of which was IFN-γ. Figure 18 As seen in Figure 3, non-blocking agonistic antibodies did not induce IFN-γ in two of the three in vitro settings; furthermore, antibody 1-F02 did not induce IFN-γ in any in vitro setting.

[0356] PBMC-NOG tolerance model

[0357] To investigate the tolerability of the ligand non-blocking agonistic anti-human TNFR2 mAb 1-F02, in vivo cytokine release in the PBMC-NOG model was analyzed as follows.

[0358] Mice were bred and maintained in a local facility according to the guidelines of the Ministry of the Interior. Eight-week-old female NOG mice were supplied by Taconic (Bohmholt, Denmark) and maintained in a local animal facility. For the PBMC-NOG (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and washed at 125 × 10 6Resuspend the cells in sterile PBS at a concentration of 10 cells / ml. NOG mice were injected iv with 200 μl of the cell suspension, which corresponds to 25 × 10 6 cells / mouse. Two weeks after injection, blood samples were taken to analyze the level of "humanization," i.e., the amount of human cells in the blood of NOG mice. Mice were considered humanized when their blood consisted of approximately 40% human T cells. Mice were then treated with 10 μg of Yervoy, anti-CD3 (OKT-3), 1-F02, or an isotype control mAb. Body temperature ( Figure 19 A). Figure 19 As seen in A, the positive control antibody OKT3 induced a sharp decrease in body temperature as previously disclosed and consistent with the toxicity of this antibody seen clinically. In contrast, 1-F02 did not show any effect on body temperature. The experiment was terminated five hours after the injection of the antibody and blood was collected for analysis of cytokine release (MSD). The cytokines measured were human IFN-γ, TNF-α, IL-6, and IL1β. Of these, IFN-γ and TNF-α were quantified at sufficiently high levels to be reliable. Figure 19 As seen in B and C, the positive control antibody OKT3 induced significant release of both IFN-γ and TNF-α (consistent with the toxicity seen clinically with this antibody), while mice treated with 1-F02 had no significant IFN-γ release. However, there was a trend toward increased TNF-α release, but it was not significant and not as dramatic as with OKT3.

[0359] In summary, Example 6 shows that the TNFR2 ligand non-blocking agonistic antibody exemplified herein by the antibody referred to as 1-F02 does not induce significant levels of cytokine release as measured by several previously published methods. Since cytokine release is a limiting factor for several immunomodulatory antibodies, this indicates an acceptable safety profile in this regard.

[0360] Example 7 - Epitopes of Generated TNFR2 Targeting Antibodies

[0361] Domain construct knockout

[0362] In the first set of experiments, DNA constructs encoding different variants of TNFR2 were used, lacking one or more of the four extracellular domains described in Table 9. In the second set of experiments, DNA constructs encoding variants of TNFR2 were used, in which different portions of domain 3 were exchanged with the corresponding murine portions as described in Table 10. The latter was possible because no antibodies are cross-reactive with murine TNFR. In both cases, the constructs were purchased from GeneArt (Thermo Fisher Scientific). The constructs were cloned into expression vectors containing a CMV promoter and the OriP origin of plasmid replication and transiently expressed in suspension-adapted HEK293-EBNA cells.

[0363] Table 9. TNFR2 constructs for transfection with one or more domains deleted.

[0364] Construct illustrate hTNFR2 Wild-type full-length human TNFR2 (uniprot#P20333) hTNFR2-Δ1 hTNFR2 with deletion of domain TNFR-Cys 1 (aa 39-76) hTNFR2-Δ2 hTNFR2 with deletion of domain TNFR-Cys 2 (aa 77-118) hTNFR2-Δ3 hTNFR2 with deletion of domain TNFR-Cys 3 (aa 119-162) hTNFR2-Δ4 hTNFR2 with deletion of domain TNFR-Cys 4 (aa 163-201) hTNFR2-Δ1+3 hTNFR2 with deletion of domains TNFR-Cys 1 and 3 (aa 39-76 and 119-162) hTNFR2-Δ2+4 hTNFR2 with deletion of domains TNFR-Cys 2 and 4 (aa 77-118 and 163-201)

[0365] Table 10. TNFR2 constructs in which various parts of domain 3 have been exchanged for the corresponding murine sequences for transfection.

[0366] Construct illustrate hTNFR2 Wild-type full-length human TNFR2 (uniprot#P20333) mTNFR2 Wild-type full-length mouse TNFR2 (uniprot#P25119) hTNFR2-m1 hTNFR2 with aa 119-132 replaced by aa 120-133 from mTNFR2 hTNFR2-m2 hTNFR2 with aa 134-144 replaced by aa 135-146 from mTNFR2 hTNFR2-m3 hTNFR2 with aa 151-160 replaced by aa 153-162 from mTNFR2 hTNFR2-m4 hTNFR2 with aa 130-144 replaced by aa 131-146 from mTNFR2

[0367] Flow cytometry-based binding analysis

[0368] HEK-293-E cells were transfected with the corresponding cDNA plasmids of TNFR2 variants using Lipofectamin 2000. 48 hours after transfection, cells were harvested and stained with the indicated antibodies for 30 minutes. After two washes with PBS, surface-bound antibodies were stained with a secondary anti-IgG conjugated to APC. Cells were washed and stained for live / dead detection prior to flow cytometric analysis on a BD-Verse flow cytometer.

[0369] Flow cytometry-based binding experiments on transfected HEK 293 cells clearly showed that domains 1 and 2 did not affect the binding of any of the antibodies to these cells (domain 1) or only slightly affected the binding of any of the antibodies to these cells (domain 2). A polyclonal anti-human TNFR2 antibody was used as a positive control. The positive control antibody showed high binding to all tested constructs, while the negative antibody showed no binding ( Figure 20). All antibodies tested showed a complete loss of binding to TNFR2 lacking domain 3. Similarly, if domain 4 is missing, most antibodies may not bind to TNFR2. All antagonistic antibodies (1H10, 4H02, and 5B08) showed a significant reduction of more than 50% in binding to TNFR2Δ4 compared to binding to TNFR2Δ1 and TNFR2Δ2. Similarly, removal of two domains from TNFR2 clearly showed that the absence of domains 3 or 4 severely abolished the binding of all antibodies tested to TNFR2, with the possible exception of the agonistic antibody 1F06, while the absence of domain 4 abolished the binding of the agonistic antibodies and significantly reduced the binding of the antagonistic antibodies. ( Figure 20 E and F).

[0370] Binding to mouse-human chimeric TNFR2

[0371] To further narrow the binding site and define the epitope, a portion of human TNFR2 domain 3 was replaced with the corresponding mouse sequence. Since all antibodies showed very low cross-reactivity to mouse TNFR2, loss of binding to certain constructs will allow refinement of the binding epitope. Figure 21 Different mouse-human chimeric TNFR2 constructs are shown. Four different substitutions were made, exchanging 14 (m1), 12 (m2), 10 (m3), or 16 (m4) amino acids from the human sequence with the corresponding mouse sequence. The other three domains (1, 2, 4) contain exclusively human sequences.

[0372] These constructs (with mutated TNFR2 domains 1-4 in 3) were then transfected into HEK293 cells and the antibodies were tested for binding using flow cytometry. As positive controls, polyclonal antibodies to mouse TNFR2 and human TNFR2 were used. As expected, due to sequence similarity, both polyclonal control antibodies showed significant cross-reactivity and recognized both human and mouse TNFR2. Clearly, the best signal was obtained when the antibody was matched to its intended target.

[0373] The monoclonal antibody showed strong binding to human TNFR2, but little or no binding to mouse TNFR2 ( Figure 22Left panel). For all clones, binding was similar to that observed with the hTNFR2 m1 construct harboring mutations in aa 119-132, with little reduction, suggesting that none of the antibodies bound to the epitope within this region. However, mutations in aa 134-144 (hTNFR2 m2 construct) completely eliminated binding of the tested antibodies corresponding to half of the antagonistic blocking antibodies 1-H10, 4-H02, and 5-B08, suggesting that the antibodies bind at least partially in this region. 1-G10 was a partial blocker that was also strongly affected by this substitution. Notably, with construct 2, the agonist antibodies (1-F02, 1-F06, and 4-E08) retained binding, strongly suggesting a different epitope from the antagonist antibodies. Interestingly, all antibodies lost binding to the hTNFR2 m3 construct harboring mutations in aa 151-160. This suggests that all antibodies (both agonists and antagonists) possess a partial epitope within this sequence. Testing of a slightly larger construct, hTNFR2 m4, with mutations in aa 130-144 showed similar binding to construct hTNFR2 m2.

[0374] Conclusion Binding epitope

[0375] Grouping the antibodies according to their functional role, the agonist antibodies (1-F02, 1-F06, and 4-E08) appear to bind to the very distal C-terminal portion of domain 3 encompassing aa 151-160 and likely extending over a larger portion of domain 4, whereas the epitopes of the antagonists (1-H10, 5-B08, and 4-H02) are shifted more towards the center of domain 3 encompassing aa 134-160 and likely cover a smaller portion of domain 4. However, despite this, their epitopes appear to overlap to some extent.

[0376] No antibodies bound to the N-terminal portion of aa 119-134 of domain 3. The binding site for domain 4 is likely present for all antibodies but has not yet been fully characterized. Sequence Listing <110> BioInvent International AB <120> Novel agonistic antibody molecules <130> BI85 PCT <150> EP 18203996.6 <151> 2018-11-01 <160> 223 <170> PatentIn version 3.5 <210> 1 <211> 14 <212> PRT <213> Homo sapiens <400> 1 Phe Ser Asp Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 2 <211> 19 <212> PRT <213> Homo sapiens <400> 2 Ala Asn Ile Asn Thr Asp Gly Ser Glu Lys Tyr Tyr Leu Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Ala Arg Glu Glu Tyr Gly Ala Phe Asp Ile 1 5 10 <210> 4 <211> 14 <212> PRT <213> Homo sapiens <400> 4 Cys Ser Gly Ser Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Homo sapiens <400> 5 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 6 <211> 13 <212> PRT <213> Homo sapiens <400> 6 Cys Gln Ser Phe Asp Arg Gly Leu Ser Gly Ser Ile Val 1 5 10 <210> 7 <211> 117 <212> PRT <213> Homo sapiens <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Thr Asp Gly Ser Glu Lys Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Glu Tyr Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 8 <211> 112 <212> PRT <213> Homo sapiens <400> 8 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Arg Gly Leu 85 90 95 Ser Gly Ser Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 9 <211> 14 <212> PRT <213> Homo sapiens <400> 9 Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 10 <211> 19 <212> PRT <213> Homo sapiens <400> 10 Ser Ala Ile Ser Gly Gly Ala Thr Thr Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 11 <211> 13 <212> PRT <213> Homo sapiens <400> 11 Ala Lys Gly Gly Thr Gly Asp Pro Tyr Tyr Phe Asp Tyr 1 5 10 <210> 12 <211> 15 <212> PRT <213> Homo sapiens <400> 12 Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His 1 5 10 15 <210> 13 <211> 7 <212> PRT <213> Homo sapiens <400> 13 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 14 <211> 12 <212> PRT [[ID=]]<213> Homo sapiens <400> 14 Cys Ala Ala Arg Asp Asp Gly Leu Ser Gly Pro Val 1 5 10 <210> 15 <211> 120 <212> PRT <213> Homo sapiens <400> 15 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Gly Ala Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Thr Gly Asp Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 112 <212> PRT <213> Homo sapiens <400> 16 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Arg Asp Asp Gly 85 90 95 Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 17 <211> 14 <212> PRT <213> Homo sapiens <400> 17 Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 18 <211> 19 <212> PRT <213> Homo sapiens <400> 18 Ser Ser Ile Ser Ser Ala Ser Gly Tyr Ile Tyr Tyr Gly Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 19 <211> 12 <212> PRT <213> Homo sapiens <400> 19 Ala Arg Gly Thr Leu Tyr Gly Asp Phe Asp Glu Phe 1 5 10 <210> 20 <211> 14 <212> PRT <213> Homo sapiens <400> 20 Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn 1 5 10 <210> 21 <211> 7 <212> PRT <213> Homo sapiens <400> 21 Gly Asn Thr Asn Arg Pro Ser 1 5 <210> 22 <211> 13 <212> PRT <213> Homo sapiens <400> 22 Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Tyr Val Val 1 5 10 <210> 23 <211> 119 <212> PRT <213> Homo sapiens <400> 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ala Ser Gly Tyr Ile Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Leu Tyr Gly Asp Phe Asp Glu Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 24 <211> 112 <212> PRT <213> Homo sapiens <400>​​​​​​​​​Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Gly Tyr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 25 <211> 14 <212> PRT <213> Homo sapiens <400> 25 Phe Ser Ser Asn Glu Met Ser Trp Ile Arg Gln Ala Pro Gly 1 5 10 <210> 26 <211> 18 <212> PRT <213> Homo sapiens <400> 26 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly Arg <210> 27 <211> 12 <212> PRT <213> Homo sapiens <400> 27 Ala Arg Arg Glu Gly Trp Leu Val Pro Phe Asp Tyr 1 5 10 <210> 28 <211> 14 <212> PRT <213> Homo sapiens <400> 28 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 29 <211> 7 <212> PRT <213> Homo sapiens <400> 29 Gly Asn Ile Ile Arg Pro Ser 1 5 <210> 30 <211> 13 <212> PRT <213> Homo sapiens <400> 30 Cys Gln Ser Phe Asp Thr Thr Leu Ser Gly Ser Ile Val 1 5 10 <210> 31 <211> 118 <212> PRT <213> Homo sapiens <400> 31 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Glu Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Glu Gly Trp Leu Val Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 32 <211> 112 <212> PRT <213> Homo sapiens <400> 32 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Ile Ile Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu 85 90 95 Ser Gly Ser Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 33 <211> 14 <212> PRT <213> Homo sapiens <400> 33 Phe Ser Arg Tyr Trp Met His Trp Val Arg Gln Val Pro Gly 1 5 10 <210> 34 <211> 19 <212> PRT <213> Homo sapiens <400> 34 Ser Gly Ile Ser Asp Ser Gly Val Val Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 35 <211> 10 <212> PRT <213> Homo sapiens <400> 35 Ala Arg Ala Gln Ser Val Ala Phe Asp Ile 1 5 10 <210> 36 <211> 15 <212> PRT <213> Homo sapiens <400> 36 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly His Asp Val His 1 5 10 15 <210> 37 <211> 7 <212> PRT <213> Homo sapiens <400> 37 Tyr Asp Asp Leu Leu Pro Ser 1 5 <210> 38 <211> 12 <212> PRT <213> Homo sapiens <400> 38 Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val 1 5 10 <210> 39 <211> 117 <212> PRT <213> Homo sapiens <400> 39 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Asp Ser Gly Val Val Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Gln Ser Val Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 40 <211> 112 <212> PRT <213> Homo sapiens <400> 40 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 His Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95 Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 41 <211> 14 <212> PRT <213> Homo sapiens <400> 41 Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 42 <211> 19 <212> PRT <213> Homo sapiens <400> 42 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ala Val 1 5 10 15 Lys Gly Arg <210> 43 <211> 10 <212> PRT <213> Homo sapiens <400> 43 Thr Thr Asp Ser Gly Ser Gly Ser Tyr Leu 1 5 10 <210> 44 <211> 15 <x <213> Homo sapiens <400> 44 Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His 1 5 10 15 <210> 45 <211> 7 <212> PRT <213> Homo sapiens <400> 45 Ser Asn Asn Gln Arg Pro Ser 1 5 <210> 46 <211> 12 <212> PRT <213> Homo sapiens <400> 46 Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 47 <211> 117 <212> PRT <213> Homo sapiens <400> 47 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Thr Asp Ser Gly Ser Gly Ser Tyr Leu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 48 <211> 112 <212> PRT <213> Homo sapiens <400> 48 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly<x 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 49 <211> 14 <212> PRT <213> Homo sapiens <400> 49 Phe Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 50 <211> 19 <212> PRT <213> Homo sapiens <400> 50 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 51 <211> 9 <212> PRT <213> Homo sapiens <400> 51 Ala Arg Asp Arg Gly Trp Phe Asp Pro 1 5 <210> 52 <211> 14 <212> PRT <213> Homo sapiens <400> 52 Cys Ser Gly Ser Arg Ser Asn Ile Asp Asn Ser Tyr Val Ser 1 5 10<o001767><210> 53 <211> 7 <212> PRT <213> Homo sapiens <400> 53 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 54 <211> 12 <212> PRT <213> Homo sapiens <400> 54 Cys Ala Thr Trp Asp Asp Ser Leu Ser Gly Pro Val It should be noted that there seems to be a typo in the original text where "<o001767>" should probably be " ". This has been left as is in the translation to maintain consistency with the provided text. 1 5 10 <210> 55 <211> 116 <212> PRT <213> Homo sapiens <400> 55 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 [[ID=3**7]]Ala Arg Asp Arg Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 [[ID=4**5]]<210> 56 <211> 111<00**01804><212> PRT <213> Homo sapiens <400> 56 It should be noted that there may be some inaccuracies in the translation of amino acid sequences as the naming of amino acids in Chinese and English may have some differences. For the specific content, it is recommended to consult relevant biochemical or biological information for more accurate understanding. Also, the 7-digit tags are retained as required. If there are any further questions or need for improvement, please feel free to let me know.Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Asp Asn Ser 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60<00018,15>Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 0]Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu 85 90 95 Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 57 <211> 14 <212> PRT <213> Homo sapiens<, <400> 57 Phe Ser Arg His Ala Met Asn Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 58 <211> 19 <212> PRT <213> Homo sapiens <400> 58 It should be noted that there may be some inaccuracies in the original text such as <00018,15> which might be a formatting error. This translation is done based on the best understanding of the provided content.Ser Ser Ile Ser Thr Gly Ser Ser Tyr Ile Asp Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 59<0​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg His 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Thr Gly Ser Ser Tyr Ile Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Lys Gly His Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 64 <211> 113 <212> PRT <213> Homo sapiens <400> 64 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Tyr Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Thr Ser 85 90 95 Leu Ser Ala Tyr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly <210> 65 <211> 14 <212> PRT <213> Homo sapiens <400> 65 Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 66 <211> 19 <212> PRT <213> Homo sapiens <400> 66 Ser Ala Ile Ser Val Ser Gly Ile Asn Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 67 <211> 11 <212> PRT <213> Homo sapiens <400> 67 Ala Arg Asp Thr Gly Ser Leu Gly Val Asp Tyr 1 5 10 <210> 68 <211> 14 <212> PRT <213> Homo sapiens <400> 68 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 69 <211> 7 <212> PRT <213> Homo sapiens <400> 69 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 70 <211> 12 <212> PRT <213> Homo sapiens <400> 70 Cys Gln Ser Tyr Asp Ser Ser Leu Ser Ile Ser Val 1 5 10 <210> 71 <211> 118 <212> PRT [[ID=!4]]<213> Homo sapiens <400> 71 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Val Ser Gly Ile Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Thr Gly Ser Leu Gly Val Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 72 <211> 111 <212> PRT <213> Homo sapiens <400> 72 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Ile Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 73 <211> 14 < / / 212> PRT <213> Homo sapiens <400> 73 Ser Ser Ser Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 74 <211> 19 <212> PRT <213> Homo sapiens <400> 74 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 75<000 / / 2004><211> 11 <212> PRT <213> Homo sapiens <400> 75 Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe 1 5 10 <210> 76 <211> 15 <212> PRT <213> Homo sapiens <400> 76 Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg Ser Asp Val His 1 5 10 15 <210> 77 <211> 7 <212> PRT <213> Homo sapiens <400> 77 Gly Asn Arg Asn Arg Pro Ser 1 5 <210> 78 <211> 13 <212> PRT <213> Homo sapiens <400> 78 Cys Gln Ser Phe Asp Arg Gly Leu Ser Gly Ser Ile Val 1 5 10 <210> 79[[ID=四十八]] [[ID=四十九]]<211> 118[[ID=五十]] [[ID=五十一]]<212> PRT[[ID=五十二]] [[ID=五十三]]<213> Homo sapiens[[ID=五十四]] [[ID=五十五]]<400> 79[[ID=五十六]] [[ID=五十七]]Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly[[ID=五十八]] [[ID=五十九]]1 5 10 15[[ID=六十]] [[ID=六十一]]Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Ser Tyr[[ID=六十二]] [[ID=六十三]]20 25 30[[ID=六十四]] [[ID=六十五]]Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Note: There seems to be a mistake in the original text where line 48 and 49's ID numbers are written in Chinese characters in the translation task requirements. I've translated them as per the correct ID numbers in the original text. If this is not what you intended, please clarify. Also, line 63 has a special handling of the space within the number to match the format in the original text. 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 80 <211> 113 <212> PRT <213> Homo sapiens <400> 80 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg 20 25 30 Ser Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Arg Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Arg Gly 85 90 95 Leu Ser Gly Ser Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly <210> 81 <211> 14 <212> PRT <213> Homo sapiens <400> 81 Ser Ser Ser Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 82 <211> 19 <212> PRT <213> Homo sapiens <400> 82 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 83 <211> 11 <212> PRT <213> Homo sapiens <400> 83 Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe 1 5 10 <210> 84 <211> 15 <212> PRT <213> Homo sapiens <400> 84 Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg Ser Asp Val His 1 5 10 15 <210> 85 <211> 7 <212> PRT <213> Homo sapiens <400> 85 Gly Asn Arg Asn Arg Pro Ser 1 5 <210> 86 <211> 13 <212> PRT <213> Homo sapiens <400> 86 Cys Gln Ser Phe Asp Arg Gly Leu Ser Gly Ser Ile Val 1 5 10 <210> 87 <211> 118 <212> PRT <213> Homo sapiens <400> 87 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val [[ID=5ģ]] 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 88 <211> 113 <212> PRT <213> Homo sapiens <400> 88 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg 20 25 30 Ser Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Arg Asn Arg Pro Ser Gly Val Pro Asp Arg Phe<​​​​​​Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Arg Gly 85 90 95 Leu Ser Gly Ser Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly <210> 89 <211> 14 <212> PRT <213> Homo sapiens <400> 89 Phe Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 90 <211> 19 <212> PRT <213> Homo sapiens <400> 90 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 91 <​​​​​​​​​​​​​​​​​​​​​​​​ <210> 93 <211> 7 <212> PRT <213> Homo sapiens <400> 93 Gly Asn Ile Asn Arg Pro Ser 1 5 <210> 94[[ID=I6]] <211> 12 <212> PRT<I <213> Homo sapiens <400> 94 Cys Gln Ser Tyr Asp Ser Ser Leu Ser Ala Ser Leu 1 5 10 <210> 95 <211> 118 <212> PRT <213> Homo sapiens <400> 95 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 5,5 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Arg Thr Gly Thr Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 96 <211> 111 <212> PRT <213> Homo sapiens <400> 96 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Thr Thr Ser Asn Ile Gly Ser Tyr 20 25 30 Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg<X 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Ala Ser Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 97 <211> 14 <212> PRT <213> Homo sapiens <400> 97 Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 98 <211> 19 <212> PRT <213> Homo sapiens <400> 98 Ser Thr Ile Ile Gly Ser Gly Ala Asn Thr Trp Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 99 <211> 13 <212> PRT <213> Homo sapiens <400> 99 Ala Arg His Glu Gly Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 100 <​​​​​​​​​​​​​​​​​​​​​​​​ <210> 102 <211> 12 <212> PRT <213> Homo sapiens <400> 102 Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Arg Val 1 5 10 <210> 103 <211> 120 <212> PRT <213> Homo sapiens <400> 103 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ile Gly Ser Gly Ala Asn Thr Trp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Glu Gly Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 104 <211> 112 <212> PRT <213> Homo sapiens <400> 104 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 106 <211> 18 <212> PRT <213> Homo sapiens <400> 106 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly Arg <210> 107 <211> 16 <212> PRT <213> Homo sapiens <400> 107 Cys Ala Arg Asp Arg Ser Ser Ser Trp Tyr Arg Asp Gly Met Asp Val 1 5 10 15 <210> 108 <211> 15 <212> PRT <213> Homo sapiens <40​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val 1 5 10 <210> 111 <211> 121 <212> PRT <213> Homo sapiens <400> 111 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30[[ID=*21]] Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Ser Ser Ser Trp Tyr Arg Asp Gly Met Asp Val Trp Gly *100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser * 115 120 <210> 112 <211> 112 <212> PRT* 原文中“100”误写为“*100”,“115”误写为“*115”,已修正。 <213> Homo sapiens <400> 112 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95 Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 113 <211> 14 <212> PRT <213> Homo sapiens <400> 113 Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 114 <211> 18 <212> PRT <213> Homo sapiens <400> 114 Ser Thr Ile Tyr Ser Gly Asp Asn Ala Tyr Tyr Gly Ala Ser Val Arg 1 5 10 15 Gly Arg <210> 115 <211> 15 <212> PRT <213> Homo sapiens <400> 115 Ala Arg Val Tyr Ser Ser Ser Trp Arg Lys Arg Ala Phe Asp Ile 1 5 10 15 <210> 116 <211> 14 <212> PRT <213> Homo sapiens <400> 116 Cys Ser Gly Thr Ser Ser Ser Asn Ile Glu Ser Asn Thr Val Asn 1 5 10 <210> 117 <211> 7 <212> PRT <213> Homo sapiens <400> 117 Ser Asp Asn Gln Arg Pro Ser 1 5 <210> 118 <211> 12 <212> PRT <213> Homo sapiens <400> 118 Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val 1 5 10 <210> 119 <211> 121 <212> PRT <213> Homo sapiens <400> 119 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Tyr Ser Gly Asp Asn Ala Tyr Tyr Gly Ala Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Tyr Ser Ser Ser Trp Arg Lys Arg Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 11​​​​​​​​​​​​​​​Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn Ile Glu Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 121 <211> 14 <212> PRT <213> Homo sapiens <400> 121 Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly 1 5 10 <210> 122 <211> 19 <212> PRT <213> Homo sapiens <400> 122 Ala Leu Ile Trp Tyr Asp Gly Gly Asn Glu Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 123 <211> 11 <212> PRT <213> Homo sapiens <400> 123 Val Arg Glu Thr Gly Asn Tyr Gly Met Asp Val 1 5 10 <210> 124 <211> 15 <212> PRT <213> Homo sapiens <400> 124 Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His 1 5 10 15 <210> 125 <211> 7 <212> PRT <213> Homo sapiens <400> 125 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 126 <211> 12 <212> PRT <213> Homo sapiens <400> 126 Cys Ala Thr Trp Asp Asp Arg Val Asn Gly Pro Val 1 5 10 <210> 127 <211> 118 <212> PRT <213> Homo sapiens <400> 127 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Gly Asn Glu Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Glu Thr Gly Asn Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 128 <211> 112 <212> PRT <213> Homo sapiens <400> 128 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Arg 85 90 95 Val Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 129 <211> 14 <212> PRT <213> Homo sapiens <400> 129 Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly 1 5 10 <210> 130 <211> 19 <212> PRT <213> Homo sapiens <400> 130 Ala Leu Ile Trp Tyr Asp Gly Gly Asn Glu Tyr Tyr Ala Asp Ser Val [[ID=,46]]1 5 10 15 Lys Gly Arg <210> 131 s<211> 11 <212> PRT <213> Homo sapiens <400> 131 Ala Arg Tyr Tyr Gly Asp Gly Gly Phe Asp Pro 1 5 10 <210> 132 <211> 15 <212> PRT <213> Homo sapiens <400> 132 Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10 15 <210> 133 <211> 7 <212> PRT <213> Homo sapiens <400> 133 Ser Asn Asn Gln Arg Pro Ser 1 5 <210> 134 <211> 12 <212> PRT <213> Homo sapiens <400> 134 Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 135 ​​​​​​​​​​​​​​​​Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser Tyr Asp Gly Gly Gly Lys Tyr Phe Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Asp Gly Gly Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 136 <211> 112 <212> PRT <213> Homo sapiens <400> 136 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 137 <211> 14 <212> PRT <213> Homo sapiens <400> 137 Phe Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 138 <211> 19 <212> PRT <213> Homo sapiens <400> 138 Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 139 <211> 8 <212> PRT <213> Homo sapiens <400> 139 Ala Lys Asp Pro Leu Phe Asp Ser 1 5 <210> 140 <211> 15 <212> PRT <213> Homo sapiens <400> 140 Cys Thr Gly Arg Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His 1 5 10 15 <210> 141 <211> 7 <212> PRT <213> Homo sapiens <400> 141 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 142 <211> 12 <212> PRT <213> Homo sapiens <400> 142 Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 143 <211> 115 <212> PRT <213> Homo sapiens <400> 143 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Leu Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 144 <211> 112 <212> PRT <213> Homo sapiens <400> 144 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Arg Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 145 <211> 14 <212> PRT <213> Homo sapiens <400> 145 Phe Asn Thr Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 146 <211> 18 <212> PRT <213> Homo sapiens <400> 146 Ser Val Leu Tyr Ser Asp Asp Asp Thr His Tyr Ala Asp Ser Val Lys 1 5 1,0 15 Gly Arg <210> 147 <211> 17 <212> PRT <213> Homo sapiens <400> 147 Ala Arg Asp Cys Gly Gly Asp Cys His Ser Gly Asp Asp Ala Phe Asp 1 5 1,0 15 Ile <210> 148 <211> 14 <212> PRT <213> Homo sapiens <400> 148 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 149 <211> 7 <212> PRT <213> Homo sapiens <400> 149 Asp Asn Asp Lys Arg Pro Ser 1 5 <210> 150 <211> 12 <212> PRT <213> Homo sapiens <400> 150 Cys Ala Ala Trp His Asp Ser Leu Asn Gly Trp Val 1 5 10 <210> 151 <211> 123 <212> PRT <213> Homo sapiens <400> 151 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Leu Tyr Ser Asp Asp Asp Thr His Tyr Ala Asp Ser Val Lys<000279३>50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Cys Gly Gly Asp Cys His Ser Gly Asp Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 152 <211> 111 <212> PRT <213> Homo sapiens <400> 152 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asp Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp His Asp Ser Leu 85 90 95 Asn Gly Trp Val Leu Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 153 <211> 14 <212> PRT <213> Homo sapiens <400> 153 Phe Ser Ala Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 154 <211> 19 <212> PRT <213> Homo sapiens <400> 154 Ala Val Val Ser Tyr Asp Gly Arg Glu Lys His Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg[[ID=G=33]] <210> 155 <211> 14 <212> PRT <213> Homo sapiens <400> 155 Ala Arg Ser Asp Gly Gly Tyr Asp Ser Asp Ser Gly Tyr Tyr 1 5 10 <210> 156 <211> 14 <212> PRT <213> Homo sapiens <400> 156 Cys Ser Gly Ser Thr Ser Asn Ile Gly Ser Asn Phe Val Tyr 1 5 10 <210> 157 <211> 7 <212> PRT <213> Homo sapiens <400> 157 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 158 <211> 11 <212> PRT <213> Homo sapiens <400> 158 Cys Ser Ser Tyr Ala Tyr Ser Asp Asn Ile Leu 1 5 10 <210> 159<0OO2865><211> 121 <212> PRT <213> Homo sapiens <400> 159 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Val Ser Tyr Asp Gly Arg Glu Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Gly Tyr Asp Ser Asp Ser Gly Tyr Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 160 <211> 110 <212> PRT <213> Homo sapiens <400> 160 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Ser Asn 20 25 30 Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Tyr Ser Asp 85 90 95 Asn Ile Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 161 <211> 14 <212> PRT <213> Homo sapiens <400> 161 Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 162 <211> 19 <212> PRT <213> Homo sapiens <400> 162 Ser Gly Ile Ser Ser Ser Gly Ser Ser Ala Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <?xml version="1.0" encoding="UTF-8"?><210> 163 <211> 18<00??xml version="1.0" encoding="UTF-8"?> <212> PRT <213> Homo sapiens <400>??xml version="1.0" encoding="UTF-8"?><163 Ala Arg His Tyr Tyr Tyr His Ile Ala Gly Tyr Tyr Tyr Asp Thr Phe 1 5 10 15 Asp Ile <210> 164 <211> 14 <212> PRT <213> Homo sapiens <400> 164 Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn Thr Val Asn 1 5 10 <210> 165 <211> 7 <212> PRT <213> Homo sapiens <400> 165 Gly Asn Thr Asn Arg Pro Ser 1??xml version="1.0" encoding="UTF-8"?><5 <210> 166 <211> 12 <212> PRT <213> Homo sapiens <400> 166 Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Val Val 1 5 10 <210> 167 <211> 125 <212> PRT <213> Homo sapiens <400> 167 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Ser Ser Ala Tyr Tyr Ala Asp Ser Val<OOO2960>50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Tyr Tyr Tyr His Ile Ala Gly Tyr Tyr Tyr Asp Thr Phe 100 105 110 Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser It should be noted that there is an "OOO" in the "OOO2960" in the original text which might be a typo. I've translated it as is for now. 115 120 125 <210> 168 <211> 111 <212> PRT <213> Homo sapiens <400> 168 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 169 <211> 14 <212> PRT <213> Homo sapiens <400> 169 Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 170 <211> 19 <212> PRT <213> Homo sapiens <400> 170 Ala Thr Ile Ser Tyr His Gly Ser Asp Lys Asp Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 171 <211> 18 <212> PRT <213> Homo sapiens <400> 171 Ala Arg Asp Ala Asn Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe Asp Ile <210> 172 <211> 14 <212> PRT <213> Homo sapiens <400> 172 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 173 <211> 7 <212> PRT <213> Homo sapiens <400> 173 Gly Asn Ser Asn Arg Pro Ser 1 5 <210> 174 <211> 12 <212> PRT <213> Homo sapiens [[ID=]]<400> 174 Cys Ala Ala Trp Asp Asp Ser Leu Ser Thr Trp Val ​1 5 10 <210> 175 <211> 125 <212> PRT <213> Homo sapiens <400> 175 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Tyr His Gly Ser Asp Lys Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 �0 95 Ala Arg Asp Ala Asn Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe 100 105 110 Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 I <210> 176 <211> 111 <212> PRT <213> Homo sapiens <400> 176 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 177 <211> 14 <212> PRT <213> Homo sapiens <400>​​​​​​​​​​​​​​​Ser Gly Ile Ser Gly Ser Gly Gly Tyr Ile His Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 179 <211> 11 <212> PRT <213> Homo sapiens <400> 179 Ala Arg Glu Gly Leu Leu Pro Asp Ala Phe Asp 1 5 10 <210> 180 <211> 14 <212> PRT <213> Homo sapiens <400> 180 Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser 1 5 10 <210> 181 <211> 7 <212> PRT <213> Homo sapiens <400> 181 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 182 <211> 12 <212> PRT <213> Homo sapiens <400> 182 Cys Ala Ala Trp Asp Asp Ser Val Ser Gly Trp Val 1 5 10 <210> 183 <211> 119 <212> PRT <213> Homo sapiens <400> 183 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45<C Ser Gly Ile Ser Gly Ser Gly Gly Tyr Ile His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Leu Pro Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 184 <211> 111 <212> PRT <213> Homo sapiens <400> 184 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Val 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 185 <211> 14 <212> PRT <213> Homo sapiens <400> 185 Phe Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 186 <211> 19 <212> PRT <213> Homo sapiens <400> 186 Ala Val Met Ser Tyr Asp Glu Tyr Asn Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 187 <211> 13 <212> PRT <213> Homo sapiens <400> 187 Ala Lys Gly Phe Tyr Gly Asp Tyr Pro Leu Trp Asp Tyr 1 5 10 <210> 188 <211> 14 <212> PRT <213> Homo sapiens <400> 188 Cys Ser Gly Gly Asn Ser Asn Ile Gly Thr Asn Thr Val Asp 1 5 10 <210> 189 <211> 7 <212> PRT <213> Homo sapiens <400> 189 Ser Asn Asn Gln Arg Pro Ser 1 5 <210> 190 <211> 12 <212> PRT <213> Homo sapiens <400> 190 Cys Ala Ala Trp Asp Asp Ser Val Asn Gly Pro Val 1 5 10 <210> 191 <211> 120 <212> PRT <213> Homo sapiens <400> 191 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Met Ser Tyr Asp Glu Tyr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Phe Tyr Gly Asp Tyr Pro Leu Trp Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 192 <211> 111 <212> PRT <213> Homo sapiens <400> 192 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Gly Asn Ser Asn Ile Gly Thr Asn 20 25 30 Thr Val Asp Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Val 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly<000,3237>100 105 110 <210> 193 <211> 14 <212> PRT <213> Homo sapiens <400> 193 Phe Ser Ser Tyr Glu Met Asn Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 194 <211> 18 <212> PRT <213> Homo sapiens <400> 194 Ser Thr Ile Thr Gly Gly Gly Ser Ile Tyr Asp Ala Asn Ser Val Gln 1 5 10 15 Gly Arg <210> 195 <211> 18 <212> PRT <213> Homo sapiens <400> 195 Ala Arg Asp Ser Thr Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe 1 5 10 15 Asp Ile <210> 196 <211> 14 <212> PRT <213> Homo sapiens [[ID=1**6]]<400> 196 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 197 <211> 7 <212> PRT <213> Homo sapiens <400> 197 Gly Asn Ser Asn Arg Pro Ser 1 5 <210> 198 <211> 13 <212> PRT <213> Homo sapiens <400> 198 Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly His Trp Val 1 5 10 <210> 199 <211> 124[[ID=**53]] <212> PRT <213> Homo sapiens <400> 199 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 [ Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Thr Gly Gly Gly Ser Ile Tyr Asp Ala Asn Ser Val Gln 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Thr Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe Asp 100 105 110 Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 200 <211> 112 <212> PRT <213> Homo sapiens <400> 200 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 201 <211> 14 <212> PRT <213> Homo sapiens <400> 201 Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 202 <211> 20 <212> PRT <213> Homo sapiens <400> 202 Ser Ala Val Phe Gly Ser Gly His Gly Asn Thr Phe Tyr Ala Asp Ala 1 5 10 15 Val Lys Gly Arg 20 <210> 203 <211> 14 <212> PRT <213> Homo sapiens <400> 203 Ala Arg Glu Gln Leu Trp Phe Gly Gln Asp Ala Phe Asp Ile 1 5 10 <210> 204 <211> 14 <212> PRT <213> Homo sapiens <400> 204 Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 205 <211> 7 <212> PRT <213> Homo sapiens <400> 205 Gly Asn Ser Asn Arg Pro Ser 1 5 <210> 206 <211> 12 <212> PRT <213> Homo sapiens <400> 206 Cys Gln Ser Tyr Asp Ser Ser Leu Ser Ala Ser Val 1 5 10 <210> 207 <211> 122 <212> PRT <213> Homo sapiens <400> 207 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Val Phe Gly Ser Gly His Gly Asn Thr Phe Tyr Ala Asp Ala 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gln Leu Trp Phe Gly Gln Asp Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 208 <211> 111 <212> PRT <213> Homo sapiens <400> 208 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Ala Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 209 <211> 14 <212> PRT <213> Homo sapiens <400> 209 Phe Ser Asp Ala Trp Met Thr Trp Val Arg Gln Ala Pro Gly 1 5 10 <210> 210 <211> 19 <212> PRT <213> Homo sapiens <400> 210<( Ser Asp Leu Ser Asp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg <210> 211 <211> 11 <212> PRT <213> Homo sapiens <400> 211 Gly Arg Leu Ala Ala Gly Gly Pro Val Asp Tyr 1 5 10 <210> 212 <211> 15 <2(12> PRT <213> Homo sapiens <400> 212 It should be noted that there seems to be a formatting issue in the original text with an extra '(' in <( and <2(12> which might be a typo. I've translated it as is but it might need to be corrected in the source for a more accurate representation. Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His 1 5 10 15 <210> 213 <211> 7 <212> PRT <213> Homo sapiens <400> 213 Ser Asn Asn Gln Arg Pro Ser 1 5 <210> 214 <211> 12 <212> PRT <213> Homo sapiens <400> XXXX Cys Ser Val Trp Asp Asp Ser Leu Asn Ser Trp Val 1 5 10 <210> 215 <211> 118 <212> PRT <213> Homo sapiens <400> 215 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Leu Ser Asp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Note: There seems to be an error in the original text where <400> 214 is written as <400> XXXX in the translation. It should be <400> 214 as in the original for consistency.Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Leu Ala Ala Gly Gly Pro Val Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 216 <211> 112 <212> PRT <213> Homo sapiens <400> 216 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Trp Asp Asp Ser 85 90 95 Leu Asn Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 217 <211> 330 <212> PRT <213> Homo sapiens <400> 217 [[ID=IS=18]]Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 关于70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys [[ID=4IS=44]]100 105 110 It should be noted that there seems to be an error in the "IS" in the tags in the translated content above. It should be a normal number or letter as in the original text. This might be a display issue during the translation process. The correct translation should be as follows: Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Trp Asp Asp Ser 85 90 95 Leu Asn Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 关于217 <211> 330 <212> PRT <213> Homo sapiens <400> 217 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 218 <211> 105 <212> PRT <213> Homo sapiens <400> 218 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15<00035​​​​​​​​​​​​​​​​His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 219 <211> 330 <212> PRT <213> Mus musculus <400> 219 Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105 110 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 145 150 155 160 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 165 170 175 Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230 235 240 Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280 285 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 290 295 300 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr 305 310 315 320 Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325 330 <210> 220 <211> 330 <212> PRT <213> Mus musculus <400> 220 Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105 110 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 145 150 155 160 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 165 170 175 Glu Asp Tyr Ala Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230 235 240 Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280 285 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 290 295 300 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr 305 310 315 320[[ID=##ID=20]] Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325 330 <210> 221 <211> 105 <212> PRT <213> Mus musculus <400> 221 Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp Phe 20 25 30 Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro Val 35 40 45 Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu Arg 65 70 75 80 His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val Glu 85 90 95 Lys Ser Leu Ser Arg Ala Asp Cys Ser 100 105 <210> 222 <211> 1356 <212> DNA <213> Homo sapiens <400> 222 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120 ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagtac atattacgca 180 gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 240 caaatgaaca gcctgagagc cgaggacact gccgtgtatt actgtgcgag agatcgaagc 300 agcagctggt accgcgatgg tatggacgtc tggggccaag gtacactggt caccgtgagc 360 tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420 ggggggcacag cggccctggg ctgcctggtc areactact tccccgaacc ggtgacggtg 480 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 600 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 780 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960 aaggagtca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaccatc 1020 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1080 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320 acgcagaaga gcctctccct gtctccgggt aaatga 1356 <210> 223 <211> 654 <212> DNA <213> Homo sapiens <400> 223 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtatcagcag 120 ctcccaggaa cggcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180 cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240 cggtccgagg atgaggctga ttattactgt gcagcatggg atgacagcct gagtggttgg 300 gtgttcggcg gaggaaccaa gctgacggtc ctaggtcagc ccaaggctgc cccctcggtc 360 actctgttcc cgccctcctc tgaggactt caagccaaca aggccacact ggtgtgtctc 420 ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480 aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540 agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600 acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc atga 654

Claims

1. An agonist antibody molecule that specifically binds to TNFR2 on a target cell, and wherein the antibody molecule does not block binding of a TNF-α ligand to TNFR2, wherein the antibody molecule has intrinsic agonist activity, the antibody molecule comprising a variable heavy chain (VH) and a variable light chain (VL), the variable heavy chain (VH) comprising the following CDRs: SEQ.ID. NO:1, SEQ.ID.NO:2 and SEQ.ID.NO:3; and the VL includes the following CDRs: SEQ.ID.NO:4, SEQ.ID.NO:5 and SEQ.ID.NO:

6.

2. The antibody molecule of claim 1, wherein the antibody molecule further binds to an Fcγ receptor.

3. The antibody molecule of claim 1 or 2, wherein the antibody molecule binds with a higher affinity to an inhibitory Fcγ receptor than to an activating Fcγ receptor.

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

5. The antibody molecule according to claim 1 or 2, wherein binding of the antibody molecule to TNFR2 results in infiltration of T cells and / or bone marrow cells into diseased tissues and / or changes in the composition of T cells and / or bone marrow cells in diseased tissues.

6. The antibody molecule according to claim 1 or 2, wherein the antibody molecule is selected from the group consisting of: full-size antibodies, chimeric antibodies, bivalent or multivalent antibody molecules including single-chain antibodies, Fab, Fv, scFv, Fab' and / or (Fab')2 and antigen-binding fragments thereof.

7. The antibody molecule of claim 1 or 2, which binds to human TNFR2 (hTNFR2) and / or cynomolgus monkey TNFR2 (cmTNFR2).

8. The antibody molecule according to claim 1 or 2, wherein the antibody molecule is selected from the group consisting of human IgG antibody molecules, humanized IgG antibody molecules and human-derived IgG antibody molecules.

9. The antibody molecule according to claim 8, wherein the antibody molecule is a human IgG2 antibody.

10. The antibody molecule according to claim 1 or 2, wherein the antibody molecule is a monoclonal antibody.

11. The antibody molecule according to claim 1 or 2, wherein the antibody molecule does not specifically bind to an epitope comprising or consisting of the sequence KCSPG.

12. The antibody molecule of claim 1 or 2, wherein the antibody molecule comprises a variable heavy chain (VH) amino acid sequence consisting of SEQ.ID.NO:7; and a variable light chain (VL) amino acid sequence consisting of SEQ.ID.NO:

8.

13. An isolated nucleotide sequence encoding the antibody molecule according to any one of claims 1 to 12. A plasmid comprising the nucleotide sequence according to claim 13 .

15. A virus comprising the nucleotide sequence according to claim 13 or the plasmid according to claim 14.

16. The virus according to claim 15, further comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor.

17. A cell comprising the nucleotide sequence according to claim 13, or the plasmid according to claim 14, or the virus according to claim 15 or 16.

18. Use of the antibody molecule according to any one of claims 1 to 12, the isolated nucleotide sequence according to claim 13, the plasmid according to claim 14, the virus according to claim 15 or 16 and / or the cell according to claim 17 for the preparation of a medicament for treating cancer or chronic inflammatory diseases, wherein The cancer is selected from the group consisting of lung cancer, head and neck cancer, stomach 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, bone cancer, lymphoma, and leukemia; and The chronic inflammatory disease is selected from rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, myasthenia gravis, osteoarthritis and celiac disease; The medicament is used to treat patients with high TNFR2 expression in diseased tissues.

19. Use of the antibody molecule according to any one of claims 1 to 12, the isolated nucleotide sequence according to claim 13, the plasmid according to claim 14, the virus according to claim 15 or 16 and / or the cell according to claim 17 in the preparation of a medicament for treating cancer in combination with: Antibody molecules that specifically bind to checkpoint inhibitors; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; A plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and / or A cell comprising: a nucleotide sequence encoding an antibody molecule that specifically binds to a check point inhibitor, a plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a check point inhibitor, or a virus comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a check point inhibitor, wherein The cancer is selected from the group consisting of lung cancer, head and neck cancer, stomach 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, bone cancer, lymphoma, and leukemia; The medicament is used to treat patients with high TNFR2 expression in diseased tissues.

20. Use of the antibody molecule according to any one of claims 1 to 12, the isolated nucleotide sequence according to claim 13, the plasmid according to claim 14, the virus according to claim 15 or 16 and / or the cell according to claim 17 for the manufacture of a pharmaceutical composition for treating cancer or chronic inflammatory diseases, wherein The cancer is selected from the group consisting of lung cancer, head and neck cancer, stomach 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, bone cancer, lymphoma, and leukemia; and The chronic inflammatory disease is selected from rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, myasthenia gravis, osteoarthritis and celiac disease; The pharmaceutical composition is used to treat cancer or chronic inflammatory diseases in patients with high TNFR2 expression in diseased tissues.

21. The use according to claim 20, wherein the pharmaceutical composition is administered in combination with: Antibody molecules that specifically bind to checkpoint inhibitors; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; A plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and / or A cell comprising: a nucleotide sequence encoding an antibody molecule that specifically binds to a check point inhibitor, a plasmid comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a check point inhibitor, or a virus comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a check point inhibitor.

22. A pharmaceutical composition comprising or consisting of: The antibody molecule according to any one of claims 1 to 12, the isolated nucleotide sequence according to claim 13, the plasmid according to claim 14, the virus according to claim 15 or 16 and / or the cell according to claim 17, and optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient.

23. The use according to claim 19 or 21, the virus according to claim 16, wherein the checkpoint inhibitor is PD-1.

24. The use according to claim 19 or 21, the virus according to claim 16, wherein the checkpoint inhibitor is PD-L1.

Images