FC binding fragment comprising CD137 antigen binding site

Abstract

The present invention relates to specific binding members that bind to CD137. The present invention relates to specific binding members comprising a CD137 antigen binding site located in a constant domain of the specific binding member, which are useful, for example, in the treatment of cancer and infectious diseases.

Description

[0001] This application is a divisional application of the invention patent application with the application date of July 12, 2019, application number 201980059925.4, and invention name “FC binding fragment comprising CD137 antigen binding site”. Field of the Invention

[0002] The present invention relates to specific binding members that bind to CD137. The specific binding members comprise a CD137 antigen binding site located in the constant domain of the specific binding member and can be used, for example, to treat cancer and infectious diseases. Background of the Invention

[0003] Cell signaling is an essential component of all biological life and typically involves cell surface receptors interacting with soluble or surface-expressed ligands. This interaction results in changes in the receptor, the ligand, or both. For example, ligand binding can induce conformational changes in receptors, causing them to aggregate into dimers or oligomers. This aggregation effect then leads to the activation of intracellular signaling pathways. There are many receptors that are activated in this way, including members of the tumor necrosis factor receptor superfamily (TNFRSF), such as CD137.

[0004] CD137 (4-1BB; TNFRSF9) is a co-stimulatory molecule of the tumor necrosis factor receptor superfamily (TNFRSF). + It is upregulated on T cells and can also be upregulated on activated CD4 + CD137 is expressed on helper T cells, B cells, regulatory T cells, natural killer (NK) cells, natural killer T (NKT) cells, and dendritic cells (DCs) (Bartkowiak and Curran, 2015). The primary functional role of CD137 in enhancing T cell cytotoxicity was first described in 1997 (Shuford et al., 1997), and anti-CD137 mAbs were proposed as anticancer therapeutics shortly thereafter.

[0005] CD137 is a transmembrane protein with four extracellular cysteine-rich domains, called CRD1-4, and a cytoplasmic region responsible for CD137 signaling. The ligand of CD137 is CD137L. Although there is no crystal structure of the CD137 / CD137L complex, it can be predicted that CD137 forms a trimer / trimer complex with CD137L (Won et al., 2010). The binding of CD137L leads to the formation of receptor trimers and the subsequent aggregation of multiple receptor trimers, leading to the activation of the CD137 signaling cascade. This signaling cascade provides T cells with a survival signal against activation-induced cell death (Hurtado et al., 1997), thereby playing a vital role in maintaining effective T cell immune responses and generating immune memory (Bartkowiak and Curran, 2015).

[0006] The role of CD137 in leukocyte biology is well understood, and its role in tumor immunology has a clear biological basis. CD137 is expressed by activated T cells and has been used to identify antigen-specific CD4 + and CD8 + T cell marker. Usually, CD137 is found in CD8 + Higher expression on T cells than CD4 + T cells (Wen et al., 2002). + For T cells, proliferation, survival, and cytotoxic effector functions through the production of interferon gamma and interleukin 2 are attributed to CD137 aggregation. CD137 aggregation also contributes to memory CD8 + Differentiation and maintenance of T cells. + In certain subsets of T cells, CD137 accumulation similarly leads to proliferation and activation, and to the release of cytokines such as interleukin-2 (Makkouk et al., 2016).

[0007] Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells via tumor-targeting monoclonal antibodies has been shown to be enhanced in vitro and in vivo by stimulation of CD137 with agonistic anti-CD137 monoclonal antibodies (Bartkowiak and Curran, 2015). NK cells bind antibodies via their Fc receptors, which, depending on the antibody isotype, can lead to NK cell activation, cytotoxic granule release, and target cell lysis (Kohrt et al., 2012). Kohrt and colleagues demonstrated that combining anti-CD137 agonist antibodies with the therapeutic drugs rituximab, trastuzumab, and cetuximab can enhance their antitumor activity by enhancing ADCC (Kohrt et al., 2014; Kohrt et al., 2011). Furthermore, human NK cells upregulate CD137 expression following binding of cell-bound antibodies via FcγRs. Subsequent stimulation of these NK cells with anti-CD137 antibodies has been shown to enhance their ADCC against tumor cells (Chester et al., 2015; Chester et al., 2016).

[0008] B lymphocytes also express CD137 upon activation. Binding of the CD137 ligand to CD137 enhances B cell proliferation, survival, and cytokine production. CD137 expression is also induced on normal and malignant human B cells following binding of CD40 to its ligand, CD154 (CD40 ligand), leading to increased B cell survival if CD137 is subsequently activated (Vinay and Kwon, 2011).

[0009] It has also been shown that CD137 is expressed on tumor-reactive subtypes of tumor-infiltrating lymphocytes (TILs). CD137 monotherapy has been shown to be effective in several preclinical immunogenic tumor models (e.g., MC38, CT26, and B-cell lymphomas). Combining CD137 with other anticancer drugs (e.g., chemotherapy, cytokines, and other checkpoint regulators) has been shown to increase the growth reduction of existing tumors. Specifically, in various preclinical xenograft models, combinations of anti-CD137 antibodies with anti-CD20, anti-EGFR, and anti-HER-2 antibodies have shown a synergistic effect in reducing tumor growth (Kohrt et al., 2014; Kohrt et al., 2012; Kohrt et al., 2011).

[0010] The combination of tumor-targeted monoclonal antibody therapy and treatment with anti-CD137 agonist antibodies has shown encouraging results in preclinical models of lymphoma (Kohrt et al., 2011), head and neck cancer, colorectal cancer (Kohrt et al., 2014), and breast cancer (Kohrt et al., 2012). A variety of tumor-targeted monoclonal antibodies have also been tested in combination with CD137 agonist antibodies in the clinic, including anti-CD20 mAb rituximab (NCT01307267, NCT02951156), anti-EGFR mAb cetuximab (NCT02110082), and anti-CS1 mAb elozumab (NCT02252263). However, clinical development has been slowed by the dose-limiting high liver inflammation associated with CD137 agonist antibody treatment. Urelumab (BMS-663513), a non-ligand-blocking human IgG4 isotype antibody (Chester et al., 2018), was the first anti-CD137 antibody to enter clinical trials, but these were discontinued after significant on-target, dose-dependent hepatotoxicity was observed (Chester et al., 2018). Recently, the use of urelumab has been proposed in clinical trials for the treatment of solid cancers, in which urelumab treatment is combined with radiation therapy (NCT03431948) or other therapeutic antibodies, such as rituximab (NCT01775631), cetuximab (NCT02110082), the anti-PD-1 antibody nivolumab (NCT02253992, NCT02534506, NCT02845323), and the combination of nivolumab and the anti-LAG-3 antibody BMS986016 (NCT02658981). However, to reduce the hepatotoxicity associated with elumab treatment, the dose of elumab had to be limited in these trials, and the efficacy was disappointing (Chester et al., 2018).

[0011] Pfizer's anti-CD137 antibody, utomilumab (PF-05082566), is a human IgG2 isotype antibody that has been tested in phase I clinical trials for advanced cancers at doses ranging from 0.03 mg / kg to 10 mg / kg, with no dose-limiting toxicities observed (Chester et al., 2016; Segal et al., 2018). However, the overall objective response rate in patients with solid tumors with this antibody was only 3.8%, which may indicate that utomilumab has less potency and clinical efficacy than utomilumab, while having a more favorable safety profile (Chester et al., 2018; Segal et al., 2018). Etomituzumab is being tested in combination with radiation therapy (NCT03217747) or chemotherapy, and in combination with other antibody therapies, including the anti-PD-L1 antibody avelumab (NCT02554812) and the anti-PD-1 antibody pembrolizumab (NCT02179918), to evaluate the safety, tolerability, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and efficacy of different treatment combinations. These trials are ongoing, and early results show no DLTs at doses up to 5 mg / kg, and a 26% response rate in patients receiving the combination of etomituzumab and pembrolizumab. Triple combinations of etomituzumab and avelumab are also being tested, along with other immuno-oncology therapies (NCT02554812, NCT03217747).

[0012] Numerous bispecific molecules targeting CD137 are also in early stages of development, many of which are derived from non-antibody-based scaffold or fusion protein technologies. Methods for developing bispecific molecules targeting CD137 and FAPalpha using DARPin scaffold protein-based technologies have been reported (Link et al., 2018; Reichen et al., 2018). Tumor-targeting CD137 activation of T cells using DART molecules targeting HER2 and EphA2 has also been demonstrated (Liu et al., 2017). CD137L fusion proteins targeting solid tumors and lymphomas via FAPalpha or CD19 are also being developed. The most advanced CD137 bispecific molecule in clinical practice (and the only one containing a full-length antibody) is PRS-343, a CD137 / HER2 bispecific molecule. In this molecule, CD137 is linked to the Fc portion of the HER2-targeting antibody trastuzumab in an IgG4 format via an artificial binding protein (anticardiolipin). PRS-343 has been reported to provide tumor target-dependent activation of CD137 on lymphocytes at sites of HER2 overexpression in a humanized mouse model, but no improvement in tumor growth inhibition was observed compared to trastuzumab alone (Hinner et al., 2016 and WO 2016 / 177802 A1). PRS-343 has recently entered a Phase I clinical trial for the treatment of a range of solid tumors to evaluate its safety, tolerability, and efficacy (NCT03330561).

[0013] Description of the Invention

[0014] As described in the Background section above, clinical development of CD137 agonist molecules has been hampered by treatment being associated with dose-limiting severe inflammatory conditions (etulumab) or low clinical efficacy (etomilumab).

[0015] The present inventors have recognized that there is a need in the art for CD137 agonist molecules that exhibit high activity but are not associated with dose-limiting hepatitis. Such molecules can be administered to individuals at doses that optimize the potency and efficacy of the molecule and can be used, for example, as immunotherapeutic agents for the treatment of cancer or in the treatment of infectious diseases.

[0016] Without wishing to be bound by theory, it is believed that T cells present in the liver may have the potential to be activated by anti-CD137 agonist molecules, thereby leading to liver inflammation. Following sepsis / viral infection, CD8+ T cells can promote liver inflammation and apoptosis (Wesche-Soldato et al., 2007). Anti-CD137 agonist antibody therapy in mice has been shown to result in CD137-dependent T cell infiltration into the liver (Dubrot J et al., 2010). The results of these studies taken together suggest that highly active anti-CD137 agonist antibodies (such as levolumab) may result in activated CD8+ T cells infiltrating the liver, leading to liver inflammation. Alternatively, the dose-limiting hepatotoxicity observed with levolumab treatment may be due to the specific epitope bound by the antibody.

[0017] The inventors performed an extensive selection and affinity maturation program to isolate a group of antigen-binding Fc fragments (also referred to herein as "Fcabs") that contain the CD137 binding site in their CH3 domain and bind dimeric CD137 with a higher affinity than monomeric CD137.

[0018] As used herein, "affinity" may refer to the strength of the binding interaction between an antibody molecule and its cognate antigen, measured by K D It will be apparent to those skilled in the art that where an antibody molecule is capable of forming multiple binding interactions with an antigen (e.g., where the antibody molecule is capable of bivalently binding to an antigen and optionally the antigen is a dimer), the K D Measured affinity may also be affected by avidity, which refers to the overall strength of the antibody-antigen complex.

[0019] After activation, CD137 expression of T cells is upregulated. Without wishing to be bound by theory, it is believed that due to the high expression of CD137 on activated T cells, CD137 will exist on the surface of such cells in the form of dimers, trimers and higher order multimers. In contrast, immature immune cells, such as immature T cells, express low or negligible levels of CD137 on their cell surfaces, so any CD137 present is likely to be in monomeric form. Therefore, in contrast to, for example, immature immune cells, it is expected that Fcabs with a higher affinity for binding to dimeric CD137 than to monomeric CD137 will preferentially bind to activated immune cells, such as activated T cells.

[0020] The Fcabs of the present invention are also capable of binding to dimeric cynomolgus monkey CD137. This is beneficial because it allows for toxicology and efficacy testing in cynomolgus monkeys during preclinical development. This is particularly advantageous given the liver inflammation observed with certain anti-CD137 antibodies, especially when the antibody molecule binds to CD137. Two isolated Fcabs, FS22-053-014 and FS22-053-017, also bind to mouse CD137. This is advantageous because it allows for testing the same Fcab in mice before administration to cynomolgus monkeys or humans. Normally, an Fcab that binds to mouse CD137 is required.

[0021] The present inventors unexpectedly discovered that isolated anti-CD137 Fcab molecules that preferentially bind to dimeric rather than monomeric CD137 and are capable of affinity maturation all contain a PPY motif and a five-amino acid insertion in the AB loop of their CH3 domains. Another anti-CD137 Fcab lineage isolated after initial library screening lacked these features and could not undergo affinity maturation, thus not being studied further. Without wishing to be bound by theory, it is believed that due to the limited flexibility of proline residues, the presence of the PPY motif promotes the formation of an extended antigen-binding region by creating a more rigid or exposed loop structure. Alternatively, the PPY sequence may represent a specific conserved motif involved in binding to CD137, as proline-rich sequences have been shown to bind to aromatic sequences, such as in SH3 domain proteins. Furthermore, since the conserved PPY sequence has been independently selected in two separate Fcab lineages, it may be important for epitope binding on CD137. In addition, there is a conserved LE or LD sequence in the EF loop of the CH3 domain of most isolated Fcabs, suggesting that this amino acid sequence may also be important for CD137 binding.

[0022] As described in the Background section above, the initial binding of the CD137 ligand to its receptor, CD137, triggers a cascade of events that leads to CD137 trimerization, followed by receptor clustering, activation of the NFkB intracellular signaling pathway, and subsequent immune cell activation. For therapeutic agents to effectively activate CD137, several CD137 monomers need to be bridged together in a manner that mimics the trimeric ligand.

[0023] Etomitomilumab is an IgG2 molecule whose agonist activity depends on cross-linking through Fcγ receptors. Urolumab is a constitutively active IgG4 molecule, so its activity does not require cross-linking through Fcγ receptors, although its agonist activity is enhanced upon cross-linking of certain Fcγ receptors. Fcγ receptors are distributed throughout the body. Therefore, the immune cell-activating activity of etomilumab and urolumab is not limited to specific sites in the body but may also occur in the liver or other sites in the body.

[0024] The present inventors have demonstrated that the Fcabs of the present invention require cross-linking to aggregate and activate CD137. However, it should be noted that this is not an inherent characteristic of Fcabs that bind to CD137. Rather, many Fcabs isolated in the screening program bind to CD137 but do not require cross-linking for CD137 aggregation and activation, or induce limited CD137 aggregation and activation in the absence of cross-linking.

[0025] As described above, Fcγ receptor-mediated cross-linking has the disadvantage that Fcγ receptors are found throughout the human body, so CD137 activation is not limited to a specific site. Therefore, the present inventors introduced mutations into the CH2 domain of Fcab to reduce or eliminate Fcγ receptor binding. Therefore, in the absence of cross-linking via agents other than Fcγ receptors, the Fcab of the present invention does not exhibit CD137 agonist activity and is therefore not expected to induce hepatic inflammation.

[0026] The present inventors have recognized that the anti-CD137 Fcabs of the present invention can be used to prepare multispecific, e.g., bispecific, molecules that bind to a second antigen other than CD137, such as a tumor antigen. Preferably, the multispecific molecule binds the second antigen bivalently, although it is expected that in cases where the second antigen is a cell-bound tumor antigen, monovalent binding of the antigen will also be sufficient to crosslink the specific binding members / antibody molecules and induce CD137 aggregation and activation. Specifically, the present inventors have prepared antibody molecules comprising the anti-CD137 Fcabs of the present invention that bind bivalently to the second antigen via their Fab regions. The present inventors have shown that such bispecific antibody molecules are capable of conditionally activating CD137 in the presence of the second antigen, without requiring, for example, Fcγ receptor-mediated crosslinking required for conventional antibody molecules. It is believed that binding of the antibody molecule to the second antigen causes crosslinking of the antibody molecule at the antigenic site, which in turn leads to aggregation and activation of CD137 on the surface of T cells. Therefore, the agonistic activity of the antibody molecule depends on the presence of both the second antigen and CD137. In other words, the agonistic activity depends on the presence of both antigens. Furthermore, it is believed that cross-linking of the antibody in the presence of a second antigen facilitates the aggregation of CD137 bound by the constant domain antigen-binding site of the antibody molecule. Thus, where the second antigen is a disease antigen, such as a tumor antigen, the antibody molecule is expected to be able to activate immune cells, for example, in a disease-dependent manner, in the tumor microenvironment. For example, it is expected that such targeted activation of immune cells will be beneficial in avoiding liver inflammation seen with leukemia treatment.

[0027] The inventors have also demonstrated that bispecific antibody molecules comprising an anti-CD137 Fcab of the present invention are capable of inhibiting tumor growth in vivo, wherein the second antigen bound by the antibody molecule is an immune cell antigen, a tumor antigen, or an antigen expressed on both tumor cells and immune cells. Furthermore, more effective tumor growth inhibition was observed with the bispecific antibody molecule compared to a combination of two monospecific antibody molecules, wherein one of the two antibody molecules comprises the same constant domain as the bispecific molecule and the other antibody molecule comprises the same variable domain binding site as the bispecific molecule, indicating that when the two binding sites are present in the same molecule, enhanced CD137 aggregation and signaling, as well as the resulting T cell activation and corresponding anti-tumor effects, can be observed.

[0028] The antibody molecule comprising the anti-CD137 Fcab of the present invention and the Fab region having specificity for the second antigen preferably binds to CD137 and the second antigen bivalently. This is advantageous because it is expected that the bivalent binding of the two targets will make the bridge between the T cells expressing CD137 and the second antigen more stable, thereby prolonging the time that the T cells are located in a specific site (such as a tumor microenvironment) and can play a role in diseases (such as tumors). This is different from the vast majority of conventional bispecific antibody formats, which are heterodimers and bind to each target antigen monovalently through one Fab arm. It is expected that such monovalent interactions are not only unstable, but also in many cases are not sufficient to induce TNF receptors, such as CD137 aggregation, in the first position.

[0029] In an alternative preferred embodiment, the antibody molecule comprises an anti-CD137 Fcab of the present invention that is capable of bivalently binding to CD137 and a specific monovalent binding site for a second antigen (e.g., a single Fab domain). The monovalent binding site can bind, for example, a tumor-associated antigen. In the context of such molecules, monovalent binding of the second antigen is expected to cause the antibody molecule to pack more tightly on the cell surface, thereby enhancing the aggregation of CD137, thereby leading to T cell activation.

[0030] Another feature of the antibody molecule comprising the anti-CD137 Fcab of the present invention is that both antigen binding sites of CD137 and the second antigen are contained within the antibody structure itself. In particular, the antibody molecule does not need to fuse other proteins to the antibody molecule via a linker or other means to produce a molecule that is bivalently bound to both targets. This has many advantages. Specifically, a method similar to that used to produce standard antibodies can be used to produce antibody molecules because they do not contain any additional fusion parts. Since the linker may degrade over time, resulting in a heterogeneous population of antibody molecules, this structure is also expected to improve the stability of the antibody. Those antibodies in a group with only one fusion protein may not be able to induce conditional excitement of TNF receptors (such as CD137) as effectively as those antibodies with two fusion proteins. The cleavage / degradation of the linker can be performed before or after the therapeutic agent is administered to the patient (for example, by enzymatic cleavage or the patient's in vivo pH), thereby causing its effectiveness to decrease while circulating in the patient's body. Since there is no linker in the antibody molecule, it is expected that the antibody molecule retains the same number of binding sites before and after administration. Furthermore, from the viewpoint of the immunogenicity of the molecule, the structure of the antibody molecule is also preferred, because the introduction of a fusion protein or a linker or both may induce immunogenicity when the molecule is administered to a patient, resulting in a reduction in the therapeutic effect.

[0031] The inventors have further demonstrated that the rigid positioning and / or close proximity of the CD137 antigen binding site resulting from the rigid structure of the Fcab molecules of the present invention facilitates the induction of CD137 aggregation, compared to molecules in which the CD137 binding site is not part of the antibody structure, but is provided for attachment to, e.g., an antibody molecule or part thereof via binding, e.g., via a flexible linker.

[0032] Therefore, the present invention provides:

[0033] [1] A specific binding member that binds to CD137 and comprises a CD137 antigen binding site located in the CH3 domain of the specific binding member, the CD137 antigen binding site comprising a first sequence located in the AB structural loop of the CH3 domain, wherein the sequence comprises the sequence PPY (SEQ ID NO: 10).

[0034] [2] The specific binding member according to [1], wherein the specific binding member comprises an insertion in the AB structural loop.

[0035] [3] The specific binding member of [2], wherein the insertion is 1 to 10 amino acids in length.

[0036] [4] The specific binding member of [3], wherein the insertion is 4 to 6 amino acids in length.

[0037] [5] The specific binding member of [4], wherein the insertion is 5 amino acids in length.

[0038] [6] The specific binding member of any one of [2] to [5], wherein the insertion is located between positions 10 and 19 of the CH3 domain of the specific binding member, wherein the amino acid residues are numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0039] [7] The specific binding member of [6], wherein the insertion is located between positions 14 and 17 of the CH3 domain of the specific binding member.

[0040] [8] The specific binding member of [7], wherein the insertion is located between positions 16 and 17 of the CH3 domain of the specific binding member.

[0041] [9] The specific binding member of any one of [2] to [8], wherein the insertion is located at positions 16.5 to 16.1 of the CH3 domain of the specific binding member, wherein the amino acid residues are numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0042]

[10] The specific binding member of any one of [1] to [9], wherein the PPY sequence is located between positions 15 and 17 of the CH3 domain, and wherein the amino acid residues are numbered according to the IMGT numbering scheme.

[0043]

[11] The specific binding member of any one of [1] to

[10] , wherein the PPY sequence is located between positions 16 and 17 of the CH3 domain, and wherein the amino acid residues are numbered according to the IMGT numbering scheme.

[0044]

[12] The specific binding member according to

[11] , wherein the PPY sequence is located at positions 16.3, 16.2 and 16.1 of the CH3 domain.

[0045]

[13] The specific binding member according to any one of [1] to

[12] , wherein the first sequence is the first sequence of the following specific binding member:

[0046] (i) FS22-172-003 shown in SEQ ID NO: 138;

[0047] (ii) FS22-172-002 shown in SEQ ID NO: 129;

[0048] (iii) FS22-172-004 shown in SEQ ID NO: 147:

[0049] (iv) FS22-172-001 shown in SEQ ID NO: 120:

[0050] (v) FS22-172-005 shown in SEQ ID NO: 156.

[0051] (vi) FS22-172-006 shown in SEQ ID NO: 110; or

[0052] (vii) FS22-172 shown in SEQ ID NO:110.

[0053]

[14] The specific binding member according to

[13] , wherein the first sequence is the first sequence of the following specific binding member:

[0054] (i) FS22-172-003 shown in SEQ ID NO: 138;

[0055] (ii) FS22-172-002 shown in SEQ ID NO: 129;

[0056] (iii) FS22-172-004 shown in SEQ ID NO: 147:

[0057] (iv) FS22-172-001 shown in SEQ ID NO: 120:

[0058] (v) FS22-172-005 shown in SEQ ID NO: 156. Or

[0059] (vi) FS22-172-006 shown in SEQ ID NO:110.

[0060]

[15] The specific binding member according to

[14] , wherein the first sequence is the first sequence of the following specific binding member:

[0061] (i) FS22-172-003 shown in SEQ ID NO: 138;

[0062] (ii) FS22-172-002 set forth in SEQ ID NO: 129; or

[0063] (iii) FS22-172-004 shown in SEQ ID NO: 147.

[0064]

[16] The specific binding member according to

[15] , wherein the first sequence is the first sequence of the specific binding member FS22-172-003 shown in SEQ ID NO: 138.

[0065]

[17] The specific binding member according to

[10] , wherein the PPY sequence is located at positions 16, 16.5 and 16.4 of the CH3 domain.

[0066]

[18] The specific binding member according to any one of [1] to

[10] or

[17] , wherein the first sequence is the first sequence of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053, preferably the specific binding member FS22-053-008, as shown in SEQ ID NO: 19.

[0067]

[19] The specific binding member of any one of

[13] to

[16] or

[18] , wherein the first sequence is located between positions 14 and 17 of the CH3 domain of the specific binding member, wherein the amino acid residues are numbered according to the IMGT numbering scheme.

[0068]

[20] The specific binding member of

[19] , wherein the first sequence is located at positions 15, 16, 16.5, 16.4, 16.3, 16.2 and 16.1 of the CH3 domain of the specific binding member.

[0069]

[21] The specific binding member according to any one of [1] to

[20] , wherein the specific binding member further comprises a second sequence located in the EF structural loop of the CH3 domain.

[0070]

[22] The specific binding member according to

[21] , wherein the second sequence is the second sequence of the specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172, preferably the specific binding member FS22-172-003, as shown in SEQ ID NO: 111.

[0071]

[23] The specific binding member according to

[21] , wherein the second sequence is the second sequence of the following specific binding member:

[0072] (i) FS22-053-008 shown in SEQ ID NO: 20;

[0073] (ii) FS22-053-009 shown in SEQ ID NO: 29;

[0074] (iii) FS22-053-011 shown in SEQ ID NO: 47;

[0075] (iv) FS22-053-017 shown in SEQ ID NO: 101:

[0076] (v) FS22-053-014 shown in SEQ ID NO: 74;

[0077] (vi) FS22-053-010 shown in SEQ ID NO: 38;

[0078] (vii) FS22-053-012 shown in SEQ ID NO: 56;

[0079] (viii) FS22-053-013 shown in SEQ ID NO: 66;

[0080] (ix) FS22-053-015 shown in SEQ ID NO: 83;

[0081] (x) FS22-053-016 set forth in SEQ ID NO: 92; or

[0082] (xi) FS22-053 shown in SEQ ID NO: 174.

[0083]

[24] The specific binding member according to

[23] , wherein the second sequence is the second sequence of the following specific binding member:

[0084] (i) FS22-053-008 shown in SEQ ID NO: 20;

[0085] (ii) FS22-053-009 shown in SEQ ID NO: 29;

[0086] (iii) FS22-053-011 shown in SEQ ID NO: 47;

[0087] (iv) FS22-053-017 shown in SEQ ID NO: 101:

[0088] (v) FS22-053-014 shown in SEQ ID NO: 74.

[0089]

[25] The specific binding member according to

[24] , wherein the second sequence is the second sequence of the following specific binding member:

[0090] (i) FS22-053-008 shown in SEQ ID NO: 20;

[0091] (ii) FS22-053-009 shown in SEQ ID NO: 29;

[0092] (iii) FS22-053-011 shown in SEQ ID NO: 47; or

[0093] (iv) FS22-053-017 shown in SEQ ID NO: 101.

[0094]

[26] The specific binding member according to

[25] , wherein the second sequence is the second sequence of the specific binding member FS22-053-008 shown in SEQ ID NO: 20.

[0095]

[27] The specific binding member of any one of

[21] to

[26] , wherein the second sequence is located at positions 92-98 of the CH3 domain of the specific binding member, wherein the amino acid residues are numbered according to the IMGT numbering scheme.

[0096]

[28] The specific binding member according to any one of [1] to

[27] , wherein the specific binding member further comprises a third sequence located in the CD structural loop of the CH3 domain.

[0097]

[29] The specific binding member of

[28] , wherein the third sequence is located at positions 43 to 78 of the specific binding member, wherein the amino acid residues are numbered according to the IMGT numbering scheme.

[0098]

[30] The specific binding member according to any one of

[28] to

[29] , wherein the third sequence has the sequence shown in SEQ ID NO: 2.

[0099]

[31] The specific binding member according to any one of [1] to

[30] , wherein the CH3 domain is a human IgG1 CH3 domain.

[0100]

[32] The specific binding member of any one of [1] to

[16] ,

[19] to

[22] and

[27] to

[31] , wherein the specific binding member comprises the CH3 domain sequence of the following specific binding member:

[0101] (i) FS22-172-003 shown in SEQ ID NO: 139;

[0102] (ii) FS22-172-002 shown in SEQ ID NO: 130;

[0103] (iii) FS22-172-004 shown in SEQ ID NO: 148;

[0104] (iv) FS22-172-001 shown in SEQ ID NO: 121;

[0105] (v) FS22-172-005 shown in SEQ ID NO: 157;

[0106] (vi) FS22-172-006 shown in SEQ ID NO: 165; or

[0107] (vii) FS22-172 shown in SEQ ID NO:112.

[0108]

[33] The specific binding member of

[32] , wherein the specific binding member comprises the CH3 domain sequence of the following specific binding member:

[0109] (i) FS22-172-003 shown in SEQ ID NO: 139;

[0110] (ii) FS22-172-002 shown in SEQ ID NO: 130;

[0111] (iii) FS22-172-004 shown in SEQ ID NO: 148;

[0112] (iv) FS22-172-001 shown in SEQ ID NO: 121;

[0113] (v) FS22-172-005 set forth in SEQ ID NO: 157; or

[0114] (vi) FS22-172-006 shown in SEQ ID NO:165.

[0115]

[34] The specific binding member according to

[33] , wherein the specific binding member comprises the CH3 domain sequence of the following specific binding member:

[0116] (i) FS22-172-003 shown in SEQ ID NO: 139;

[0117] (ii) FS22-172-002 set forth in SEQ ID NO: 130; or

[0118] (iii) FS22-172-004 shown in SEQ ID NO: 148.

[0119]

[35] The specific binding member according to

[34] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding member FS22-172-003 shown in SEQ ID NO: 139.

[0120]

[36] The specific binding member according to any one of [1] to

[10] ,

[17] to

[18] ,

[19] to

[21] and

[23] to

[31] , wherein the specific binding member comprises the CH3 domain sequence of the following specific binding member:

[0121] (i) FS22-053-008 shown in SEQ ID NO: 21;

[0122] (ii) FS22-053-009 shown in SEQ ID NO: 30;

[0123] (iii) FS22-053-011 shown in SEQ ID NO: 48;

[0124] (iv) FS22-053-017 shown in SEQ ID NO: 102;

[0125] (v) FS22-053-014 shown in SEQ ID NO: 75;

[0126] (vi) FS22-053-010 shown in SEQ ID NO: 39;

[0127] (vii) FS22-053-012 shown in SEQ ID NO: 57;

[0128] (viii) FS22-053-013 shown in SEQ ID NO: 66;

[0129] (ix) FS22-053-015 shown in SEQ ID NO: 84;

[0130] (x) FS22-053-016 set forth in SEQ ID NO: 93; or

[0131] (xi) FS22-053 shown in SEQ ID NO:175.

[0132]

[37] The specific binding member of

[36] , wherein the specific binding member comprises the CH3 domain sequence of the following specific binding member:

[0133] (i) FS22-053-008 shown in SEQ ID NO: 21;

[0134] (ii) FS22-053-009 shown in SEQ ID NO: 30;

[0135] (iii) FS22-053-011 shown in SEQ ID NO: 48;

[0136] (iv) FS22-053-017 shown in SEQ ID NO: 102; or

[0137] (v) FS22-053-014 shown in SEQ ID NO:75.

[0138]

[38] The specific binding member of

[37] , wherein the specific binding member comprises the CH3 domain sequence of the following specific binding member:

[0139] (i) FS22-053-008 shown in SEQ ID NO: 21;

[0140] (ii) FS22-053-009 shown in SEQ ID NO: 30;

[0141] (iii) FS22-053-011 shown in SEQ ID NO: 48; or

[0142] (iv) FS22-053-017 shown in SEQ ID NO: 102.

[0143]

[39] The specific binding member according to

[38] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding member FS22-053-008 shown in SEQ ID NO: 21.

[0144]

[40] The specific binding member according to

[37] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding member FS22-053-017 shown in SEQ ID NO: 102.

[0145]

[41] The specific binding member according to

[37] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding member FS22-053-014 shown in SEQ ID NO: 75.

[0146]

[42] The specific binding member according to any one of [1] to

[41] , wherein the specific binding member further comprises a CH2 domain, preferably a CH2 domain of human IgG1.

[0147]

[43] The specific binding member according to any one of [1] to

[42] , wherein the specific binding member is a dimer of two identical polypeptide chains, each comprising a CH2 and a CH3 domain.

[0148]

[44] The specific binding member according to

[42] or

[43] , wherein the CH2 domain has the sequence shown in SEQ ID NO: 6 or 5.

[0149]

[45] The specific binding member according to any one of

[42] to

[44] , which further comprises an immunoglobulin hinge region or a portion thereof, preferably a human IgG1 hinge region or a portion thereof, at the N-terminus of the CH2 domain.

[0150]

[46] The specific binding member according to

[45] , wherein the hinge region has the sequence shown in SEQ ID NO: 179 or a fragment thereof.

[0151]

[47] The specific binding member according to

[46] , wherein the hinge region has the sequence shown in SEQ ID NO: 7.

[0152]

[48] ​​A specific binding member according to any one of [1] to

[16] ,

[19] to

[22] ,

[27] to

[35] and

[42] to

[47] , wherein the specific binding member comprises the sequence of the specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172 as shown in SEQ ID NO: 141, 132, 150, 123, 159, 167 and 114, respectively.

[0153]

[49] The specific binding member of

[48] , wherein the specific binding member comprises the sequence of the specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005 or FS22-172-006 as shown in SEQ ID NO: 141, 132, 150, 123, 159 and 167, respectively.

[0154]

[50] The specific binding member of

[49] , wherein the specific binding member comprises the sequence of the specific binding member FS22-172-003, FS22-172-002 or FS22-172-004 as shown in SEQ ID NO: 141, 132 and 150, respectively.

[0155]

[51] The specific binding member according to

[50] , wherein the specific binding member comprises the sequence of specific binding member FS22-172-003 shown in SEQ ID NO: 141.

[0156]

[52] A specific binding member according to any one of [1] to

[10] ,

[17] to

[18] ,

[19] to

[21] ,

[23] to

[31] and

[36] to

[47] , wherein the specific binding member comprises the sequence of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 as shown in SEQ ID NO: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95 and 175.

[0157]

[53] The specific binding member of

[52] , wherein the specific binding member comprises the sequence of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017 or FS22-053-014 as shown in SEQ ID NOs: 23, 32, 50, 104 and 77, respectively.

[0158]

[54] The specific binding member of

[53] , wherein the specific binding member comprises the sequence of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011 or FS22-053-017 as shown in SEQ ID NO: 23, 32, 50 and 103, respectively.

[0159]

[55] The specific binding member according to

[54] , wherein the specific binding member comprises the sequence of specific binding member FS22-053-008 shown in SEQ ID NO: 23.

[0160]

[56] The specific binding member of any one of [1] to

[55] , wherein the specific binding member binds to human CD137.

[0161]

[57] The specific binding member according to

[56] , wherein the human CD137 has, comprises or consists of the sequence shown in SEQ ID NO: 181.

[0162]

[58] The specific binding member of any one of [1] to

[55] , wherein the specific binding member binds to dimeric CD137 with a higher affinity than to monomeric CD137.

[0163]

[59] The specific binding member of any one of [1] to

[58] , wherein the specific binding member binds to cynomolgus monkey CD137.

[0164]

[60] The specific binding member or antibody molecule according to

[59] , wherein the cynomolgus monkey CD137 has, comprises or consists of the sequence shown in SEQ ID NO: 183.

[0165]

[61] The specific binding member of any one of [1] to

[60] , wherein the specific binding member further comprises a second antigen binding site.

[0166]

[62] The specific binding member of

[61] , wherein the specific binding member is a multispecific molecule.

[0167]

[63] The specific binding member of

[62] , wherein the specific binding member is a bispecific, trispecific or tetraspecific molecule.

[0168]

[64] The specific binding member of

[63] , wherein the specific binding member is a bispecific molecule.

[0169]

[65] A specific binding member according to any one of

[61] to

[64] , wherein the second antigen binding site is a CDR-based antigen binding site.

[0170]

[66] The specific binding member according to

[65] , wherein the second antigen binding site comprises the heavy chain variable domain CDR1, CDR2 and CDR3, and the light chain variable domain CDR1, CDR2 and CDR3.

[0171]

[67] A specific binding member according to any one of

[61] to

[66] , wherein the second antigen binding site comprises a heavy chain variable domain and a light chain variable domain.

[0172]

[68] The specific binding member of any one of

[61] to

[67] , wherein the specific binding member is an antibody molecule.

[0173]

[69] The antibody molecule according to

[68] , wherein the antibody molecule is a human IgG1 molecule.

[0174]

[70] The antibody molecule of any one of

[65] to

[69] , wherein the CDR-based antigen binding site of the antibody molecule binds to a second antigen selected from the group consisting of an immune cell antigen, and a disease antigen.

[0175]

[71] The antibody molecule according to

[70] , wherein the disease antigen is a tumor antigen or a pathogen antigen.

[0176]

[72] The antibody molecule of

[70] , wherein the immune cell antigen is an immunoregulatory molecule, such as PD-L1.

[0177]

[73] The antibody molecule of

[70] , wherein the immune cell antigen is a member of the tumor necrosis factor receptor superfamily (TNFRSF).

[0178]

[74] The antibody molecule according to

[71] , wherein the tumor antigen is a tumor-associated antigen (TAA).

[0179]

[75] The antibody molecule according to

[71] or

[74] , wherein the tumor antigen is a cell surface antigen on cancer cells.

[0180]

[76] The antibody molecule according to

[71] , wherein the tumor antigen is a soluble multimer.

[0181]

[77] The antibody molecule according to

[76] , wherein the soluble multimer is at least a dimer.

[0182]

[78] The antibody molecule according to

[77] , wherein the soluble multimer is at least a trimer.

[0183]

[79] The antibody molecule according to

[71] , wherein the pathogen antigen is a bacterial or viral antigen.

[0184]

[80] The antibody molecule according to any one of

[70] to

[79] , wherein the antibody molecule is capable of activating CD137 present on immune cells in the presence of a second antigen.

[0185]

[81] The antibody molecule according to any one of

[70] to

[80] , wherein binding of the antibody molecule to CD137 and a second antigen causes aggregation of CD137 on immune cells.

[0186]

[82] The antibody molecule according to

[80] or

[81] , wherein the immune cell is a T cell.

[0187]

[83] The specific binding member or antibody molecule of any one of [1] to

[82] , wherein the specific binding member or antibody molecule has been modified to reduce or eliminate binding of the CH2 domain of the specific binding member or antibody molecule to one or more Fcγ receptors.

[0188]

[84] The specific binding member or antibody molecule according to any one of [1] to

[83] , wherein the specific binding member or antibody molecule does not activate Fcγ receptors.

[0189]

[85] The specific binding member or antibody molecule of

[83] or

[84] , wherein the Fcγ receptor is selected from the group consisting of FcγRI, FcγRIIa, FcγRI Ib and FcγRIII.

[0190]

[86] The specific binding member or antibody molecule according to any one of [1] to

[85] , wherein the specific binding member or antibody molecule is conjugated to a biologically active molecule.

[0191]

[87] The specific binding member or antibody molecule according to any one of [1] to

[85] , wherein the specific binding member or antibody molecule is conjugated to a detectable label.

[0192]

[88] A nucleic acid molecule encoding the specific binding member or antibody molecule of any one of [1] to

[85] .

[0193]

[89] The nucleic acid molecule according to

[88] , wherein the nucleic acid molecule comprises:

[0194] (i) the CH3 domain nucleic acid sequence of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 as shown in SEQ ID NO: 22, 31, 49, 103, 76, 40, 58, 67, 85, 94 and 176, respectively; or

[0195] (ii) the CH3 domain nucleic acid sequence of the specific binding members FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172 as shown in SEQ ID NO: 140, 131, 149, 122, 158, 166 and 113, respectively.

[0196]

[90] The nucleic acid molecule according to

[88] or

[89] , wherein the nucleic acid molecule comprises the following nucleic acid sequence: a specific binding member:

[0197] (i) FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 as set forth in SEQ ID NOs: 24, 33, 51, 105, 78, 42, 60, 69, 87, 96 and 177, respectively; or

[0198] (ii) FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172 as shown in SEQ ID NO: 142, 133, 151, 124, 160, 168 and 115, respectively.

[0199]

[91] The nucleic acid molecule according to

[89] or

[90] , wherein the nucleic acid molecule comprises the CH3 domain nucleic acid sequence or nucleic acid sequence of the following specific binding member:

[0200] (i) FS22-172-003; or

[0201] (ii)FS22-053-008.

[0202]

[92] A vector comprising the nucleic acid described in any one of

[88] to

[91] .

[0203]

[93] A recombinant host cell comprising the nucleic acid of any one of

[88] to

[91] or the vector of

[92] .

[0204]

[94] A method for producing a specific binding member or antibody molecule according to any one of [1] to

[85] , comprising culturing the recombinant host cell of

[93] under conditions that produce the specific binding member or antibody molecule.

[0205]

[95]

[94] The method further comprises isolating and / or purifying the specific binding member or antibody molecule.

[0206]

[96] A pharmaceutical composition comprising the specific binding member or antibody molecule according to any one of [1] to

[87] and a pharmaceutically acceptable excipient.

[0207]

[97] A specific binding member or antibody molecule according to any one of [1] to

[87] for use in a method of treating the human or animal body by therapy.

[0208]

[98] A method of treating a disease or condition in an individual, comprising administering to the individual a therapeutically effective amount of a specific binding member or antibody molecule according to any one of [1] to

[87] .

[0209]

[99] A specific binding member or antibody molecule for use according to

[97] , or a method according to

[98] , wherein the treatment is treatment of cancer or an infectious disease in an individual.

[0210]

[10] A specific binding member or antibody molecule for use according to

[97] or

[99] , or a method according to

[98] or

[99] , wherein the treatment method comprises administering the specific binding member or antibody molecule to the individual in combination with a second therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0211] Figure 1: Figure 1A , A and C show the sequence alignment of Fcab FS22-053, FS22-053-008, FS22-053-009, FS22-053-010, FS22-053-011, FS22-053-012, FS22-053-013, FS22-053-014, FS22-053-015, FS22-053-016, FS22-053-017, FS22-172, FS22-172-001, FS22-172-002, FS22-172-003, FS22-172-004, FS22-172-005 and FS22-172-006, and the wild-type (WT) Fcab CH3 domain. Residue numbers are given according to the IMGT, IMGT exon (serial numbering), EU and Kabat numbering systems. Figure 1D Shown are the percentage sequence identities of the CH3 domains of clones FS22-053-008 to FS22-053-016 and FS22-053-017 (see Example 10.1) compared to the parental FS22-053 CH3 domain. Figure 1E Shown are the percentage sequence identities of the CH3 domains of clones FS22-172-001 to FS22-172-006 compared to the parental FS22-053 CH3 domain.

[0212] Figure 2 NF-κB signaling as determined by luminescence values ​​is shown as a measure of luciferase production resulting from CD137 aggregation and activation. HelD1.3 mimic mAb 2 The anti-human CD137 parental Fcabs FS22-053 and FS22-172 drive limited CD137 aggregation and NF-κB signaling in the HEK hCD137 NF-κB reporter gene assay (open and filled circles) when cross-linked to protein L. 2 Affinity-matured anti-human CD137 Fcabs FS22-053-008, -009, -011 (open squares, triangles, and diamonds) and FS22-172-002, -003, -004 (filled squares, triangles, and diamonds) drive stronger CD137 aggregation and NF-kB signaling in a HEK hCD137 NF-kB reporter assay compared to the parental clones when cross-linked to Protein L. The positive control anti-human CD137 mAb (20H4.9) showed enhanced luminescence and a smaller EC50 value when cross-linked to Protein L (dashed line) compared to the affinity-matured anti-human CD137 Fcabs.

[0213] Figure 3 Shown is the release of IL-2 in a T cell activation assay in the presence of anti-human CD137 Fcab. PD-L1(S70 LALA) mAb 2 Formats FS22-053-008, FS22-053-009, FS22-053-011 and FS22-172-002, FS22-172-003, FS22-172-004 only drove CD137 aggregation and activation when cross-linked to HEK cells overexpressing PD-L1 (hollow and solid black symbols), resulting in the release of mouse interleukin 2 (mIL-2) in a DO11.10 T cell activation assay. At increasing concentrations, the positive control anti-human CD137 mAb (20H4.9) showed increased mIL-2 release (dashed line), but the maximum release was significantly lower than that of the anti-human CD137 Fcab. Compared to when cross-linked (hollow and solid black symbols), the mAb 2 All PD-L1-type anti-human CD137 Fcabs in this format showed significantly lower mIL-2 release when not cross-linked to HEK cells overexpressing PD-L1 (solid grey symbols).

[0214] Figure 4 showed that in primary human CD8 + Human IL-2 (hIL-2) release in T cell activation assays. 2Affinity-matured PD-L1 mAb when cross-linked by HEK cells overexpressing PD-L1 2 Anti-human CD137 Fcab FS22-053-008, FS22-053-009, FS22-053-011, FS22-172-002, FS22-172-003 and FS22-172-004, driving CD8 + CD137 activation of T cells leads to the release of human IL-2. The affinity-matured Fcab showed better activity than the parental Fcabs FS22-053 (open circles) and FS22-172 (filled circles). The positive control anti-human CD137 mAb (20H4.9) (dashed line) showed increased hIL-2 release and greater IL-2 release than with the Fcab, but the EC50 value was greater than that of some anti-human CD137 Fcab antibodies, indicating that better activity was achieved with some anti-human CD137 Fcab antibodies.

[0215] Figure 5 NF-κB signaling as determined by luminescence as a measure of luciferase production resulting from CD137 aggregation and activation is shown. In the HEK mCD137 NF-κB reporter assay, HelD1.3 mimic mAb was activated when cross-linked to protein L. 2 The anti-mouse CD137 Fcab FS22m-063 in a 1:1 format drives CD137 aggregation and NF-kB signaling. At concentrations above 100 nM and when not cross-linked to protein L, FS22m-063 drives some CD137 aggregation and NF-kB signaling, but at levels much lower than when cross-linked (open circles). The positive control anti-mouse CD137 mAb (Lob12.3) showed increased luminescence when cross-linked to protein L (closed squares) compared to when not cross-linked (open squares), but the maximum response obtained at the highest antibody concentration was significantly lower than the maximum response of the anti-mouse CD137 Fcab. As expected, the isotype control showed no activity.

[0216] Figure 6 The results show that the patients were treated with G1-AA / 4420 (IgG control), G1-AA / S70 (PD-L1 positive control), G1-AA / Lob12.3 (CD137 positive control), the combination of G1-AA / S70 plus G1-AA / Lob12.3 and FS22m-063-AA / S70 (PD-L1 mAb) 2 Tumor volume measurements of a MC38 syngeneic tumor model grown subcutaneously in C57BL6 mice treated with an anti-mouse CD137 Fcab FS22m-063 (Figure 5—figure supplement 1). Mean tumor volumes [mm 3] plus or minus 95% confidence intervals. FS22m-063-AA / S70 significantly reduced tumor growth in the MC38 syngeneic tumor model compared to mice treated with an IgG control, mice treated with an anti-PD-L1 positive control mAb, and mice treated with an anti-CD137 positive control. Statistical significance for growth rates is shown pairwise across the study period using mixed model analysis. ****≤0.0001 p-value

[0217] Figure 7 : A shows tumor volume measurements of a MC38 syngeneic tumor model grown subcutaneously in C57BL / 6 mice treated with G1-AA / 4420 (IgG control), G1-AA / F2 (PD-1 positive control), G1 / Lob12.3 (CD137 positive control), a combination of G1-AA / F2 plus G1-AA / Lob12.3, and FS22m-063-AA / F2 (anti-mouse CD137 Fcab FS22m-063 in the form of PD-L1 mAb2). Mean tumor volume [mm 3 ] plus or minus 95% confidence intervals. FS22m-063-AA / F2 significantly reduced tumor growth in the MC38 syngeneic tumor model compared to mice treated with IgG control. Statistical significance of growth rates is shown pairwise across the study period using mixed model analysis. ****≤0.0001 p-value. B shows the difference between the two groups. Figure 7 A. Same data, except that tumor volumes are shown separately for each treated mouse. This highlights that all but one mouse treated with FS22m-063-AA / F2 showed no tumor regrowth during the study. In contrast, tumor regrowth was observed in mice treated with G1-AA / Lob12.3 and G1-AA / F2 at the end of the study.

[0218] Figure 8 Shown are mean tumor volume measurements in a subcutaneous mesothelin-positive CT26 syngeneic tumor model (Balb / c). 2 Mice treated with FS22m-063-AA / FS28m-228 showed significant tumor growth inhibition compared to mice treated with IgG control. The mean tumor volume [mm 3 ] plus or minus 95% confidence intervals. Statistical significance of growth rates across the study period is shown pairwise using mixed model analysis. ****≤0.0001 p-value

[0219] Figure 9The results show that the expression of G1-AA / HelD1.3 (human IgG1 control), FS22m-063-AA / HelD1.3 (mock mAb 2 anti-mouse CD137 Fcab), FS22m-063-AA / 4420 (mock mAb 2 The combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN mAb) and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN mAb) were tested. 2 ) in a CT26.G10 syngeneic tumor model treated with FS22m-063-AA / FS28m-228-010. FS22m-063-AA / FS28m-228-010 demonstrated reduced tumor growth compared to isotype controls and other treatment groups.

[0220] Figure 10 The results show that the expression of G1-AA / HelD1.3 (IgG control), FS22m-063-AA / HelD1.3 (mAb 2 Anti-mouse CD137 Fcab), FS22m-063-AA / 4420 (mAb 2 The combination of G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN Fab) and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN Fab) were used. 2 Kaplan Meier survival curves of the CT26.G10 syngeneic tumor model treated with FS22m-063-AA / FS28m-228-010. The results show that treatment with FS22m-063-AA / FS28m-228-010 significantly improved survival compared to the isotype control group or other treatment groups.

[0221] Figure 11 Shown is the release of mouse IL-2 in a DO11.10 human CD137 T cell activation assay testing mouse and human cross-reactive mimetic mAbs cross-linked to protein L. 2The CD137 Fcabs FS22-053-014 and FS22-053-017 were tested in vitro. Both Fcabs were able to activate CD137 when cross-linked with protein L. The positive control anti-human CD137 mAb (20H4.9) showed increased mIL-2 release compared to the mock mAb. 2 Compared with the cross-linked FS22-053-017, the simulated mAb not cross-linked with protein L 2 Both anti-human CD137 Fcab formats showed negligible mIL-2 release (filled symbols).

[0222] Figure 12 Shown is mouse IL-2 release in a DO11.10 mouse CD137 T cell activation assay testing mouse and human cross-reactive mimetic mAbs when cross-linked to protein L. 2 FS22-053-014 and FS22-053-017, as well as anti-mouse CD137 Fcab FS22m-063, are HelD1.3 mimetic mAbs. 2 The positive control anti-mouse CD137 mAb Lob12.3 showed an expected increase in mIL-2 release compared to when cross-linked. 2 Both anti-CD137 Fcabs showed a significant reduction in mIL-2 release. FS22-053-017 was less active in this assay (EC50 8-fold lower than FS22-053-014), but still showed activity in the assay.

[0223] Figure 13 Shown is a homology model of the FS22 Fcab compared to a structural template containing the wild-type human IgG1 Fc CH3 domain (PDB ID 5JII). Images show 5JII Fc (A), as well as representative structures of the FS22-053-008 (B) and FS-172-003 (C) Fcabs. One CH3 domain from each structure has been rendered using gray ribbon diagram representation. The AB, CD, and EF loops are indicated by black shading of the ribbon and are labeled accordingly. For comparison purposes, the predicted structure of the AB loop is highlighted with a dashed line, showing the bulge caused by the PPY motif, which may play a key role in binding.

[0224] Figure 14 mAb 2The homodimeric Fcab (FS22-172-003-AA) of the form (i.e., containing two CD137-binding CH3 domains) was compared to the mAb 2 Sensorgrams showing the binding kinetics of a heterodimeric Fcab (containing one CD137-binding CH3 domain and one wild-type CH3 domain) in the form of a β-catenin. These sensorgrams demonstrate that both molecules bind to CD137, even though only one binding chain is involved. Compared to the heterodimeric Fcab, the dissociation rates differ, with the homodimeric Fcab exhibiting a much slower dissociation curve. These sensorgrams demonstrate that anti-CD137 Fcabs can bivalently bind to CD137.

[0225] Figure 15 Simulated mAb is shown 2 The anti-human CD137 Fcab (FS22-172-003-AA / HelD1.3) positive control antibody (G1-AA / 20H4.9) and isotype control antibody (G1-AA / HelD1.3) were used in combination with cells expressing a range of CD137 levels, including a negative control cell line (hCD137 neg Binding of G1-AA / 20H4.9 to the DO11.10 cell line (A). Binding was determined by flow cytometry. The results show that while G1-AA / 20H4.9 binds to all cell lines expressing CD137 (BE), FS22-172-003-AA / HelD1.3 does not bind to the DO11.10 cell line, which expresses low levels of CD137 (B), and the fold difference in binding compared to the positive control is inversely proportional to the amount of CD137 expressed by the cell line. Detailed description

[0226] The present invention relates to specific binding members that bind to CD137. CD137 is also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9) or 4-1BB. The specific binding member preferably binds to human CD137, more preferably human and cynomolgus monkey CD137, more preferably dimeric human and cynomolgus monkey CD137. The portion of CD137 bound by the specific binding member is preferably the extracellular domain of CD137. The extracellular domains of human and cynomolgus monkey CD137 may comprise or consist of the sequences listed in SEQ ID NO: 181 and 183, respectively. The specific binding member is preferably capable of binding to CD137 expressed on the surface of a cell. The cell is preferably an immune cell, such as CD8 + or CD4 + T cells or regulatory T (Treg) cells, preferably CD8 + T cells, or B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DCs), or tumor infiltrating lymphocytes (TILs).

[0227] Specific binding members preferably specifically bind to CD137. The term "specificity" may refer to a situation in which a specific binding member does not show any significant binding to a molecule other than its specific binding partner, here CD137. The term "specificity" also applies to situations in which a specific binding member is specific for a particular epitope (e.g., an epitope on CD137) carried by many antigens, in which case the specific binding member will be able to bind to a variety of antigens carrying the epitope. Specific binding members preferably do not bind to or show any significant binding to CD40, OX40, and / or GITR.

[0228] As described in the Background section above, treatment of patients with the anti-CD137 antibody umelatumab is associated with dose-limiting high-grade hepatitis. Without wishing to be bound by theory, it is believed that the liver inflammation seen with umelatumab treatment may be due to activation of T cells present in the liver, or infiltration and accumulation of activated T cells in the patient's liver. To select molecules with the potential to reduce or not cause liver inflammation, the inventors selected Fcabs with high affinity for CD137. Specifically, the inventors selected Fcabs that bind to dimeric CD137 with higher affinity than to monomeric CD137. Upon priming and activation, CD137 expression on T cells is upregulated. It is believed that due to the higher expression of CD137 on activated T cells, CD137 will be present on the surface of such cells in the form of dimers, trimers, and higher-order multimers. In contrast, inactive T cells express low or even undetectable levels of CD137. Therefore, it is believed that even if CD137 is fully expressed on the surface of such T cells, it is likely to be in monomeric form. Thus, it is believed that Fcabs that bind to CD137 with high affinity preferentially bind to activated T cells, as opposed to inactive T cells, such as those present in the liver, thereby exhibiting reduced or absent liver inflammation.

[0229] The specific binding member preferably binds with an affinity (K of 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM or 2 nM. D ) or higher affinity binds to dimeric human CD137.

[0230] In a preferred embodiment, the specific binding member binds to dimeric CD137 with a higher affinity than to monomeric CD137. In a preferred embodiment, the specific binding member binds to dimeric CD137 with an affinity that is at least 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or 200-fold greater than the affinity of the specific binding member for monomeric CD137.

[0231] The human CD137 may, for example, have the sequence set forth in SEQ ID NO: 183. The sequence is the same whether the antigen is in monomeric or dimeric form.

[0232] Specific binding members of the FS22-53 and FS22-172 lineages have also been shown to bind to dimeric cynomolgus monkey CD 137. Binding to cynomolgus monkey CD 137 as well as human CD 137 is beneficial because it allows the efficacy and toxicity of specific binding members to be tested in cynomolgus monkeys before administration to humans.

[0233] In a preferred embodiment, the specific binding member binds to dimeric macaque CD137 with an affinity (K) of 250 nM, 200 nM, 150 nM, 140 nM, 120 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, or 2 nM. D ) or higher. Preferably, the specific binding member binds to cynomolgus macaque CD137 with an affinity (K) of 2 nM. D ) or higher.

[0234] The specific binding member can bind to dimeric human CD137 and dimeric cynomolgus monkey CD137 with similar affinity. It is believed that it is beneficial to conduct efficacy and toxicity studies with the specific binding member in cynomolgus monkeys, which can predict the efficacy and toxicity of the specific binding member in humans.

[0235] Thus, in a preferred embodiment, the affinity of the specific binding member binding to dimeric cynomolgus monkey CD137 is no more than 10-fold higher or lower than the affinity of the specific binding member binding to dimeric human CD137, preferably no more than 5-fold higher or lower.

[0236] The specific binding members can bind to dimeric mouse CD137, dimeric cynomolgus monkey CD137, and dimeric human CD137. This is believed to be beneficial because the same specific binding member can be used for initial efficacy studies in mice, toxicity studies in cynomolgus monkeys, and clinical trials in humans, potentially simplifying the path to the clinic. Specifically, it was surprisingly found that specific binding members FS22-053-014 and FS22-053-017 bind to dimeric mouse, human, and cynomolgus monkey CD137.

[0237] The binding affinity of a specific binding member to a cognate antigen, such as human or cynomolgus monkey CD137, can be determined by surface plasmon resonance (SPR), such as Biacore.

[0238] The term "specific binding member" describes an immunoglobulin or fragment thereof that comprises a constant domain comprising a CD137 antigen binding site. Thus, as used herein, the term "specific binding member" includes an antigen binding fragment provided that the antigen binding fragment comprises a CD137 antigen binding site located in a specific binding member constant domain. The constant domain may be a CL, CH1, CH2, CH3, or CH4 domain, preferably a CH1, CH2, or CH3 domain, more preferably a CH2 or CH3 domain, and most preferably a CH3 domain. Specific binding members may be partially or fully synthetically produced.

[0239] Preferably, the specific binding member comprises a CH2 and a CH3 domain, wherein the CH2 or CH3 domain, preferably the CH3 domain, comprises a CD137 antigen binding site. The specific binding member is preferably a dimer of two (identical) polypeptide chains, each comprising a CH2 and a CH3 domain. In a preferred embodiment, the specific binding member further comprises an immunoglobulin hinge region or a portion thereof at the N-terminus of the CH2 domain. Such molecules are also referred to herein as antigen-binding Fc fragments or Fcab TM The hinge region may comprise or consist of the sequence shown in SEQ ID NO: 179 or a fragment thereof. Preferably, the fragment is a C-terminal fragment of the sequence shown in SEQ ID NO: 179. The length of the fragment may be at most 20, at most 10, at most 8 or at most 6 amino acids. The length of the fragment may be at least 3, at least 4, at least 5 or at least 6 amino acids. In a preferred embodiment, the hinge region has the sequence shown in SEQ ID NO: 7.

[0240] In a preferred embodiment, the specific binding member is an antibody molecule, preferably a monoclonal antibody or a fragment thereof. The antibody molecule is preferably human or humanized. The antibody molecule can be an immunoglobulin G molecule, such as an IgG1, IgG2, IgG3 or IgG4 molecule, preferably an IgG1, IgG2 or IgG4 molecule, more preferably an IgG1 molecule or a fragment thereof.

[0241] Since antibodies can be modified in a variety of ways, the term "antibody molecule" should be interpreted as covering antibody fragments, derivatives, functional equivalents and homologs of antibodies, whether natural or wholly or partially synthetic. An example of an antibody fragment comprising a CH3 domain is the Fc domain of an antibody. An example of an antibody fragment comprising CDR sequences and a CH3 domain is a minibody, which comprises a scFv linked to a CH3 domain (Hu et al., (1996), Cancer Res., 56(13):3055-61).

[0242] The specific binding member comprises a CD137 antigen binding site. The CD137 antigen binding site is located in the constant domain of the specific binding member, preferably the CH3 domain. The CD137 antigen binding site comprises one or more modified structural loops in the constant domain of the specific binding member. The engineering antibody constant domain structural loops for generating the antigen binding site of the target antigen are known in the art and are described, for example, in Wozniak-Knopp G et al. (2010); WO2006 / 072620 and WO2009 / 132876.

[0243] After extensive selection and affinity maturation procedures, the inventors isolated two groups of Fcabs that preferentially bind to dimeric rather than monomeric human CD137. Surprisingly, all of the isolated Fcabs contained the sequence PPY and a five-amino acid insertion in their AB loop. The two groups of Fcabs were independently selected from separate selection campaigns, each using a library containing a five-amino acid insertion in the AB loop. Thus, the PPY sequence was independently selected twice, demonstrating the importance of this sequence for CD137 binding. Anti-CD137 Fcabs isolated from the Fcab library did not contain the five-amino acid insertion in the AB loop and were therefore not further studied, for example because the selected Fcabs could not be affinity matured. This suggests that the amino acid insertion present in the AB loop may also be important for CD137 binding. Therefore, the presence of the PPY sequence and the five-amino acid insertion in the AB loop may be important for CD137 binding, with the PPY sequence optionally being present in whole or in part within the five-amino acid insertion.

[0244] Thus, the CD137 antigen binding site of the specific binding member may comprise a first and / or second sequence, preferably a first and a second sequence, wherein the first and the second sequence are located in the AB and EF structural loops, respectively, of the constant domain, preferably the CH3 domain, of the specific binding member.

[0245] In a preferred embodiment, the residues at positions 95 and 96 of the CH3 domain of the specific binding member are wild-type, i.e., preferably arginine (R) and tryptophan (W), respectively. Both residues are located in the EF structural loop. Unless otherwise indicated, amino acid residue positions herein are numbered according to the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., 2005.

[0246] The first sequence preferably comprises the sequence PPY (SEQ ID NO: 10).

[0247] The PPY sequence may be located between positions 10 and 19, preferably between positions 15 and 17, of the CH3 domain of the specific binding member. In a preferred embodiment, the PPY sequence is located at positions 16, 16.5, and 16.4 of the CH3 domain. Alternatively, the PPY sequence may be located between positions 16 and 17 of the CH3 domain. In another preferred embodiment, the PPY sequence is located at positions 16.3, 16.2, and 16.1 of the CH3 domain. In the IMGT numbering scheme, inserted residues are numbered according to the direction of the loop in which they are located. If the loop direction is "upward," the inserted residue takes the number of the residue immediately preceding the insertion, and the number of residues inserted in the sequence is represented in ascending decimal order, for example, 16, 16.1, 16.2, 16.3, where there are three mutations after residue 16. If the loop direction is “downward,” the inserted residue is the residue number immediately preceding the insertion, and the residue number inserted in the sequence is expressed in descending decimal order, for example, 16, 16.3, 16.2, 16.1, where there are three more mutations after residue 16 (LeFranc et al., 2005; LeFranc et al., 2015).

[0248] In a preferred embodiment, the AB structural loop comprises an amino acid insertion. The insertion length can be 1 to 10, 2 to 9, 3 to 7, 4 to 6 or 5 amino acids. Preferably, the insertion length is 5 amino acids.

[0249] The insertion may be located between positions 10 and 19 of the CH3 domain of the specific binding member, preferably between positions 14 and 17, more preferably between positions 16 and 17. In a preferred embodiment, the insertion is located between positions 16.5 and 16.1 of the CH3 domain of the specific binding member, as shown in FIG1 .

[0250] Most specific binding members identified after affinity maturation include a leucine (L) residue at position 97 of the CH3 domain. Many specific binding members also include aspartic acid (D) residues or glutamic acid (E) residues at position 98 of the CH3 domain of the specific binding member. Both of these amino acid changes are located in the EF structural loop. These results indicate that one or both of these residues may be important for CD137 binding. Therefore, the second sequence preferably includes sequence LD or LE, wherein the LD or LE sequence is preferably located at positions 97 and 98 of the CH3 domain of the specific binding member.

[0251] The first sequence and the second sequence can be the first and second sequences of the CH3 domain of the following: specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172, preferably the specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005 or FS22-172-006, more preferably the specific binding member FS22-172-003, FS22-172-002, or FS22-172-004, and most preferably the specific binding member FS22-172-003.

[0252] Alternatively, the first sequence and the second sequence may be the first and second sequences of the CH3 domain of the following specific binding members: FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053 -014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015 or FS22-053-016, or FS22-053, preferably the specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, or FS22-053-014, more preferably the specific binding member FS22-053-008, FS22-053-009, FS22-053-011, or FS22-053-017, and most preferably the specific binding member FS22-053-008. In an alternative preferred embodiment, the first sequence and the second sequence are the first and second sequences of the CH3 domain of the specific binding member FS22-053-017 or FS22-053-014, more preferably the specific binding member FS22-053-017.

[0253] The CH3 domain sequences of the specific binding members FS22-053, FS22-053-008, FS22-053-009, FS22-053-010, FS22-053-011, FS22-053-012, FS22-053-013, FS22-053-014, FS22-053-015, FS22-053-016, FS22-053-017, FS22-172, FS22-172-001, FS22-172-002, FS22-172-003, FS22-172-004, FS22-172-005 and FS22-172-006 are shown in SEQ ID NO: NO: 175, 21, 30, 39, 49, 58, 66, 75, 84, 93, 102, 112, 121, 130, 139, 148, 157 and 166.

[0254] The first and second sequences of the specific binding members FS22-053, FS22-053-008, FS22-053-009, FS22-053-010, FS22-053-011, FS22-053-012, FS22-053-013, FS22-053-014, FS22-053-015, FS22-053-016, FS22-053-017, FS22-172, FS22-172-001, FS22-172-002, FS22-172-003, FS22-172-004, FS22-172-005 and FS22-172-006 can be specific binding members FS 004, FS22-172-005, FS22-172-006, FS22-172-007, FS22-172-008, FS22-053-009, FS22-053-010, FS22-053-011, FS22-053-012, FS22-053-013, FS22-053-014, FS22-053-015, FS22-053-016, FS22-053-017, FS22-172, FS22-172-001, FS22-172-002, FS22-172-003, FS22-172-004, FS22-172-005, and FS22-172-006.

[0255] Alternatively, the first and second sequences of the specific binding members FS22-053-008, FS22-053-010, FS22-053-011, FS22-053-012, and FS22-053-016 can be the sequences between positions 14 and 17, and positions 92 and 99, of the CH3 domain of the specific binding members FS22-053-008, FS22-053-010, FS22-053-011, FS22-053-012, and FS22-053-016, respectively.

[0256] The first and second sequence of the specific binding member FS22-053-015 may alternatively be the sequence between positions 14 and 17, and positions 92 and 98, respectively, of the CH3 domain of the specific binding member FS22-053-015.

[0257] The CD loop sequence of the specific binding member is preferably unmodified, i.e., wild-type. Thus, the CD loop sequence preferably has the sequence shown in SEQ ID NO: 2. The CD loop sequence is preferably located at positions 43 to 78 of the CH3 domain of the specific binding member.

[0258] The first sequence and the second sequence can be the complete AB and EF structural loop sequences of the specific binding members FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, FS22-172, FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016, or FS22-053, respectively. Determining the positions of the AB, CD, and EF loops within the CH3 domain sequence is within the skill of the art, for example according to the IMGT, IMGT exon, EU, or Kabat numbering systems, and is described in Hasenhindl et al. (2013). In a preferred embodiment, the AB, CD, and EF loops according to the IMGT numbering system are located between positions 10 and 19, 42 and 79, and 91 and 102, respectively, of the CH3 domain of the specific binding member. Thus, in a preferred embodiment, the first, second, and third sequences are FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, The sequence between positions 10 and 19, 42 and 79, and 91 and 102 of the CH3 domain of FS22-053-016, FS22-053, FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172.

[0259] In a preferred embodiment, the specific binding member comprises the following first and / or second sequence, preferably the first and second sequence, of the specific binding member:

[0260] (i) FS22-172-003 shown in SEQ ID NO: 141;

[0261] (ii) FS22-172-002 shown in SEQ ID NO: 132;

[0262] (iii) FS22-172-004 shown in SEQ ID NO: 150;

[0263] (iv) FS22-172-001 shown in SEQ ID NO: 123;

[0264] (v) FS22-172-005 shown in SEQ ID NO: 159;

[0265] (vi) FS22-172-006 set forth in SEQ ID NO: 167; or

[0266] (vii) FS22-172 as shown in SEQ ID NO: 114; wherein the first and second sequences are preferably located between positions 14 and 17 and 91 and 99, respectively, of the CH3 domain of the specific binding member.

[0267] In a more preferred embodiment, the specific binding member comprises the following first and / or second sequence, preferably the first and second sequence, of the specific binding member:

[0268] (i) FS22-172-003 shown in SEQ ID NO: 141;

[0269] (ii) FS22-172-002 shown in SEQ ID NO: 132;

[0270] (iii) FS22-172-004 shown in SEQ ID NO: 150;

[0271] (iv) FS22-172-001 shown in SEQ ID NO: 123;

[0272] (v) FS22-172-005 set forth in SEQ ID NO: 159; or

[0273] (vi) FS22-172-006 shown in SEQ ID NO:167.

[0274] In yet another preferred embodiment, the specific binding member comprises the following first and / or second sequence, preferably the first and second sequence, of the specific binding member:

[0275] (i) FS22-172-003 shown in SEQ ID NO: 141;

[0276] (ii) FS22-172-002 set forth in SEQ ID NO: 132; or

[0277] (iii) FS22-172-004 shown in SEQ ID NO: 150.

[0278] In a more preferred embodiment, the specific binding member comprises the first and / or second sequence, preferably the first and second sequence, of the specific binding member FS22-172-003 as shown in SEQ ID NO: 141.

[0279] In a preferred embodiment, the specific binding member, in particular the specific binding member comprising the first and / or second sequence, preferably the first and second sequence, of the specific binding member FS22-172-006 may comprise a leucine (L) at position 19 of the CH3 domain of the specific binding member.

[0280] In an alternative preferred embodiment, the specific binding member comprises the following first and / or second sequence, preferably the first and second sequence, of specific binding member:

[0281] (i) FS22-053-008 shown in SEQ ID NO: 23;

[0282] (ii) FS22-053-009 shown in SEQ ID NO: 32;

[0283] (iii) FS22-053-011 shown in SEQ ID NO: 50;

[0284] (iv) FS22-053-017 shown in SEQ ID NO: 104;

[0285] (v) FS22-053-014 shown in SEQ ID NO: 77;

[0286] (vi) FS22-053-010 shown in SEQ ID NO: 41;

[0287] (vii) FS22-053-012 shown in SEQ ID NO: 59;

[0288] (viii) FS22-053-013 shown in SEQ ID NO: 68;

[0289] (ix) FS22-053-015 shown in SEQ ID NO: 86;

[0290] (x) FS22-053-016 set forth in SEQ ID NO:95; or

[0291] (xi) FS22-053 as shown in SEQ ID NO: 15; wherein the first and second sequences are preferably located between positions 14 and 17 and 91 and 99, respectively, of the CH3 domain of the specific binding member.

[0292] In a more preferred embodiment, the specific binding member comprises the following first and / or second sequence, preferably the first and second sequence, of the specific binding member:

[0293] (i) FS22-053-008 shown in SEQ ID NO: 23;

[0294] (ii) FS22-053-009 shown in SEQ ID NO: 32;

[0295] (iii) FS22-053-011 shown in SEQ ID NO: 50;

[0296] (iv) FS22-053-017 shown in SEQ ID NO: 104; or

[0297] (v) FS22-053-014 shown in SEQ ID NO: 77.

[0298] In a more preferred embodiment, the specific binding member comprises the first and / or second sequence, preferably the first and second sequence, of the CH3 domain of the following specific binding member:

[0299] (i) FS22-053-008 shown in SEQ ID NO: 23;

[0300] (ii) FS22-053-009 shown in SEQ ID NO: 32;

[0301] (iii) FS22-053-011 shown in SEQ ID NO: 50; or

[0302] (iv) FS22-053-017 shown in SEQ ID NO: 104.

[0303] In a more preferred embodiment, the specific binding member comprises the first and / or second sequence, preferably the first and second sequence, of the specific binding member FS22-053-008 as shown in SEQ ID NO: 23.

[0304] As an alternative to IMGT numbering, amino acid residue positions, including amino acid sequences, substitutions, deletions, and insertions described herein, can be numbered according to IMGT exon numbering (also known as sequential numbering), EU numbering, or Kabat numbering. The consistency between IMGT numbering, IMGT exon numbering, EU numbering, and Kabat numbering for residue positions in the CH3 domain is shown in Figure 1. Thus, for example, in the present application, a reference to a first sequence located between positions 14 and 17 of the CH3 domain of a specific binding member, respectively, wherein the residue positions are numbered according to the IMGT numbering scheme, is a reference to a first sequence located between positions 18 and 21 of the CH3 domain, wherein the residue positions are numbered according to the IMGT exon numbering scheme, as shown in Figure 1. Alternatively, as described herein, the positions of amino acid residues in the CH3 domain, including the positions of amino acid sequences, substitutions, deletions, and insertions, can be defined by reference to their positions in the wild-type CH3 domain sequence set forth in SEQ ID NO: 4. The consistency between IMGT numbering and the wild-type CH3 domain sequence is also shown in Figure 1.

[0305] In a preferred embodiment, the specific binding member comprises a CH3 domain, which comprises, has or consists of the CH3 domain sequence of the specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172, preferably the specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172. -005, or the CH3 domain sequence of FS22-172-006, more preferably the CH3 domain sequence of the specific binding member FS22-172-003, FS22-172-002 or FS22-172-004, and most preferably the CH3 domain sequence of the specific binding member FS22-172-003.

[0306] In an alternative preferred embodiment, the specific binding member comprises a CH3 domain comprising, having or consisting of the CH3 domain of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 Sequence composition, preferably the CH3 domain sequence of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017 or FS22-053-014, more preferably the CH3 domain sequence of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011 or FS22-053-017, and most preferably the CH3 domain sequence of the specific binding member FS22-053-008.

[0307] The CH3 domain of the specific binding member may optionally comprise an additional lysine residue (K) immediately C-terminal to the CH3 domain sequence.

[0308] Monoclonal antibodies and other antibodies can be adopted, and the technology of recombinant DNA technology is used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. This technology can be related to CDR or variable region being introduced into different immunoglobulins. For example, a method for introducing the CDR of a kind of immunoglobulin into another kind of immunoglobulin is described in EP-A-184187, GB 2188638A and EP-A-239400. Similar technology can be adopted to introduce the constant domain sequence of the CD137 antigen binding site of the specific binding members of the present invention into constant domain, for example, the CH3 domain of another specific binding member, thereby resulting in the specific binding members comprising the CD137 antigen binding site in its constant domain. Alternatively, the whole constant domain sequence of the specific binding members can be replaced with the constant domain sequence of the specific binding members of the present invention, to prepare the specific binding members comprising the CD137 antigen binding site in its constant domain. Similarly, the fragment of the constant domain sequence of the specific binding members of the present invention can be replaced with the corresponding fragment of the constant domain sequence of the specific binding members of the present invention comprising the CD137 antigen binding site.

[0309] The CH2 domain of specific binding members may include one or more mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIII and / or complement). The inventors assume that reducing or eliminating the binding to Fcγ receptors will reduce or eliminate ADCC mediated by specific binding members. Similarly, it is expected that reducing or eliminating the binding to complement will reduce or eliminate CDC mediated by specific binding members. Mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors and / or complement are known in the art (Wang et al., 2018). These mutations are included in Bruhns et al., 2009 and Hezareh et al., "LALA mutations" described in 2001, which involve replacing the leucine residues (L1.3A and L1.2A) at positions 1.3 and 1.2 of the CH2 domain with alanine. Alternatively, it is also known to generate aglycosyl antibodies by mutation of conserved N-linked glycosylation sites (by mutating the asparagine (N) at position 84.4 of the CH2 domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q)) to reduce IgG1 effector function (Wang et al., 2018). Alternatively, it is known that complement activation (C1q binding) and ADCC can be reduced by mutating the proline at position 114 of the CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can also be combined to generate specific binding members with further reduced or no ADCC or CDC activity.

[0310] Thus, the specific binding member may comprise a CH2 domain, wherein the CH2 domain preferably comprises:

[0311] (i) alanine residues at positions 1.3 and 1.2; and / or

[0312] (ii) alanine or glycine at position 114; and / or

[0313] (iii) alanine, glutamine or glycine at position 84.4;

[0314] The amino acid residues are numbered according to the IMGT numbering scheme.

[0315] In a preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain preferably comprises:

[0316] (i) alanine residues at positions 1.3 and 1.2; and / or

[0317] (ii) alanine or glycine at position 114;

[0318] The amino acid residues are numbered according to the IMGT numbering scheme.

[0319] In another preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain comprises:

[0320] (i) an alanine residue at position 1.3; and

[0321] (ii) an alanine residue at position 1.2;

[0322] The amino acid residues are numbered according to the IMGT numbering scheme.

[0323] For example, the CH2 domain may have the sequence set forth in SEQ ID NO: 6. [LALA]

[0324] In an alternative preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain comprises:

[0325] (i) an alanine residue at position 1.3;

[0326] (ii) an alanine residue at position 1.2; and

[0327] (iii) alanine at position 114;

[0328] The amino acid residues are numbered according to the IMGT numbering scheme.

[0329] For example, the CH2 domain may have the sequence set forth in SEQ ID NO: 5. [LALA-PA]

[0330] In a preferred embodiment, the specific binding member comprises, has or consists of the CH2 and CH3 domain sequences of the specific binding members FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172, preferably the CH2 and CH3 domain sequences of the specific binding members FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005 or FS22-172-006. The CH2 and CH3 domain sequences, more preferably the CH2 and CH3 domain sequences of the specific binding members FS22-172-003, FS22-172-002 or FS22-172-004, most preferably the CH2 and CH3 domain sequences of the specific binding member FS22-172-003, wherein the CH2 and CH3 domain sequences of the specific binding members FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 and FS22-172 are shown in SEQ ID NOs: 141, 132, 150, 123, 159, 167 and 114, respectively, starting from amino acid 7.

[0331] In an alternative preferred embodiment, the specific binding member comprises, has or consists of the CH2 and CH3 domain sequences of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053, preferably the CH2 and CH3 domain sequences of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017 or FS22-053-014, More preferably, the CH2 and CH3 domain sequences of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011 or FS22-053-017 are used, and most preferably, the CH2 and CH3 domain sequences of the specific binding members FS22-053-008, wherein the CH2 and CH3 domain sequences of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 and FS22-053 are shown in SEQ ID NO: 1 ID NO: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95 and 15, starting from amino acid 7.

[0332] In an alternative preferred embodiment, the specific binding member comprises, has or consists of the sequence of specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053, preferably specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017 or FS22-053-014. 14, more preferably the sequence of the specific binding member FS22-053-008, FS22-053-009, FS22-053-011 or FS22-053-017, most preferably the sequence of the specific binding member FS22-053-008, wherein the sequences of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 and FS22-053 are respectively in SEQ Listed in ID NO: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95 and 15.

[0333] In a preferred embodiment, in addition to the CD137 antigen binding site located in the constant domain of the specific binding member, the specific binding member may also comprise one or more additional antigen binding sites that bind to one or more additional antigens. The one or more additional antigen binding sites preferably specifically bind to their cognate antigens.

[0334] The one or more additional antigen binding sites can bind to CD137 or another antigen. Therefore, the specific binding member can be a multispecific molecule, such as a bispecific, trispecific or tetraspecific molecule, preferably a bispecific molecule. In a preferred embodiment, the specific binding member is capable of simultaneously binding to CD137 and one or more other antigens.

[0335] Antibody molecules are known to have a modular structure comprising discrete domains that can be combined in a variety of different ways to produce multispecificity, such as bispecific, trispecific or tetraspecific antibody formats. Exemplary multispecific antibody formats are described in, for example, Spiess et al. (2015) and Kontermann (2012). Specific binding members of the present invention can be used in this multispecific antibody format. This has the added advantage of introducing additional antigen binding sites into this multispecific antibody format in the presence of antigen binding site constant domains (e.g., the CH3 domains of specific binding members).

[0336] For example, the specific binding members of the present invention can be a heterodimeric antibody molecule, such as a heterodimeric complete immunoglobulin molecule or a fragment thereof. In this case, a part of an antibody molecule will have one or more sequences as described herein. For example, in the case where the specific binding members of the present invention are bispecific heterodimeric antibody molecules, the specific binding members can include a heavy chain containing a CH3 domain as described herein, which is paired with a heavy chain in combination with an antigen other than CD137. The technology for preparing heterodimeric antibodies is known in the art, including knobs-into-holes (KIHs) technology, which involves modifying the CH3 domains of an antibody molecule to produce a "knob" or "hole" to promote chain heterodimerization. Alternatively, heterodimeric antibodies can be prepared by introducing charge pairs into antibody molecules to avoid homodimerization of the CH3 domains due to electrostatic repulsion, and to guide heterodimerization by electrostatic attraction. The example of a heterodimeric antibody form includes CrossMab, mAb-Fv, SEED antibodies, and KIH IgG.

[0337] Alternatively, the multispecific specific binding members of the present invention may comprise a complete immunoglobulin molecule or a fragment thereof and one or more additional antigen binding moieties. The antigen binding moiety may, for example, be an Fv, scFv or single domain antibody and may be fused to a complete immunoglobulin molecule or a fragment thereof. Examples of multispecific antibody molecules comprising additional antigen binding moieties fused to a complete immunoglobulin molecule include DVD-IgG, DVI-IgG, scFv4-IgG, IgG-scFv and scFv-IgG molecules (Spiess et al., 2015; Figure 1). Examples of multispecific antibody molecules comprising additional antigen binding moieties fused to an immunoglobulin fragment comprising a CH3 domain include, for example, scDiabody-CH3, Diabody-CH3 and scFv-CH3 KIH (Spiess et al., 2015; Figure 1).

[0338] Other suitable multispecific formats will be apparent to the skilled artisan.

[0339] In a preferred embodiment, the specific binding member comprises a second antigen binding site that binds to a second antigen, wherein the second antigen binding site is preferably an antigen binding site based on CDRs. The antigen binding site based on CDRs is the antigen binding site in the antibody variable region. The antigen binding site based on CDRs is composed of six CDRs; three light chain variable domains (VL) CDRs and three heavy chain variable domains (VH) CDRs.

[0340] The preparation of antibody molecules for given antigens and the determination of the CDR sequences of such antibody molecules are well known, and many suitable techniques are known in the art. CDR sequences can be determined, for example, according to Kabat et al., 1991 or the International ImMunoGeneTics Information System (IMGT) (Lefranc et al., 2015).

[0341] For example, the specific binding member can be a mAb 2 (TM) Bispecific Antibody. As referred to herein, mAb 2 Bispecific antibodies are IgG immunoglobulins that contain a CDR-based antigen binding site in each of their variable regions and at least one antigen binding site in the constant domain. 2 In this format, the specific binding member thus comprises, in addition to the CD137 antigen binding site in its constant domain, a CDR-based antigen binding site in each of its variable regions.

[0342] The three VH domain CDRs of the antigen binding site can be located within an immunoglobulin VH domain, and the three VL domain CDRs can be located within an immunoglobulin VL domain. For example, a CDR-based antigen binding site can be located in an antibody variable region.

[0343] The specific binding member may have one or preferably more than one, for example two, CDR-based antigen binding sites for the second antigen. Thus, the specific binding member may comprise one VH and one VL domain, but preferably comprises two VH and two VL domains, i.e. two VH / VL domain pairs, as in the case of naturally occurring IgG molecules.

[0344] In some preferred embodiments, the specific binding member may be an immunoglobulin comprising two variable regions, each comprising a CDR-based antigen binding site for a second antigen.

[0345] In a preferred embodiment, the antibody is therefore an antibody molecule that binds CD137 and a second antigen, the antibody molecule comprising:

[0346] (i) two antigen-binding sites for CD137 located in the two CH3 domains of the antibody molecule; and

[0347] (ii) Two CDR-based antigen-binding sites for a second antigen, each formed by an immunoglobulin VH domain and an immunoglobulin VL domain.

[0348] In a more preferred embodiment, the antibody is an intact immunoglobulin molecule, for example, an intact IgG1 molecule, which binds to CD137 and a second antigen, and the antibody molecule comprises:

[0349] (i) two antigen-binding sites for CD137 located in the two CH3 domains of the antibody molecule; and

[0350] (ii) two CDR-based antigen-binding sites for a second antigen, each formed by an immunoglobulin VH domain and an immunoglobulin VL domain; and

[0351] The immunoglobulin molecule further comprises CH1, CH2 and CL domains.

[0352] Activation of CD137 requires CD137 to aggregate on the surface of immune cells, such as T cells, thereby stimulating intracellular signaling pathways and immune cell activation. In the absence of cross-linking of the specific binding member, binding of the specific binding member to CD137 on the surface of an immune cell may not result in CD137 aggregation and, therefore, may not result in immune cell activation.

[0353] The inventors have shown that in the absence of cross-linking of the specific binding members, specific binding members of the FS22-53 and FS22-172 lineages do not cause T cell activation (see Example 5).

[0354] As described above, crosslinking antibody molecules by binding to Fcγ receptors is both inefficient and incapable of targeting specific sites, such as disease sites, because cells expressing Fcγ receptors are present throughout the human body. Therefore, the second antigen bound by the second antigen-binding site is preferably not an Fcγ receptor.

[0355] In a preferred embodiment, the specific binding member of the invention thus comprises a second antigen binding site that binds a second antigen, wherein the second antigen is capable of binding to and cross-linking multiple specific binding members.

[0356] For example, the present inventors have shown that, when the second antigen is a multimeric molecule, the combination of specific binding members and the second antigen causes or enhances T cell activation. Therefore, the second antigen is preferably a multimeric antigen, such as a dimer, trimer or a higher order polymer, and therefore can cross-link several specific binding members.

[0357] The present inventors also showed that using CD137 / secondary antigen mAb 2 In some embodiments, the second antigen is a surface antigen, such as a cell surface antigen, which can be a monomer or a polymer, and is present in high concentrations and / or aggregated on the surface, such as on the cell surface, and the combination of the antibody molecule and the second antigen causes or enhances T cell activation. Without wishing to be bound by theory, it is believed that the combination of, for example, an antibody molecule and an abundant cell surface antigen causes a high concentration of antibody molecules to be combined on the cell surface, which makes the antibody molecule close enough to drive CD137 aggregation and immune cell activation. Therefore, in a preferred embodiment, the second antigen is a surface antigen, which is expressed in high concentration on a surface such as a cell surface.

[0358] A specific binding member comprising a second antigen binding site, the second antigen binding site is in conjunction with the second antigen as described herein, and only when in conjunction with the second antigen, immune cells are activated, such as T cells, or when bound to the second antigen, its immune cell activation activity is enhanced, also referred to as a conditional agonist. This immune cell activation activity combined with the second antigen is independent of the combination of the specific binding member and Fc γ receptors and / or external cross-linking agents (such as protein A or G or a second antibody), and therefore allows specific binding members to target the conditional agonist activity of the site where the second antigen is present. For example, when the second antigen is a disease antigen, the specific binding member can selectively activate immune cells at the site of the disease rather than at other sites of the individual.

[0359] In addition, specific binding members that rely solely on binding to a second antigen to activate immune cells (e.g., T cells) preferably have increased immune cell activation activity compared to specific binding members that rely on cross-linking by other mechanisms (e.g., external cross-linking agents or cross-linking by Fcγ receptor interactions). Because activation of CD137 is more efficient, activation of immune cells can be achieved at lower concentrations of the specific binding members described herein relative to other specific binding members.

[0360] Thus, the specific binding members of the invention preferably induce enhanced immune cell activation, such as T cell activation, when the specific binding member is cross-linked (e.g. by binding to a second antigen) than when the specific binding member is not cross-linked.

[0361] The ability of an antibody molecule or specific binding member to activate T cells can be measured using a T cell activation assay. T cells release IL-2 upon activation. Therefore, a T cell activation assay can measure the release of IL-2 to determine the level of T cell activation induced by the antibody molecule or specific binding member.

[0362] For example, the ability of an antibody molecule or specific binding member to activate T cells can be determined by measuring the concentration of the antibody molecule or specific binding member required to achieve half-maximal release of IL-2 from T cells in a T cell activation assay when the specific binding member or antibody molecule is cross-linked. This is hereinafter referred to as the EC of the antibody molecule or specific binding member. 50 Lower EC 50 This indicates that a lower concentration of the antibody molecule or specific binding member is required to achieve half-maximal release of IL-2 from T cells in a T cell activation assay, and therefore the antibody molecule or specific binding member has higher T cell activation activity. For example, an anti-CH2 antibody can be used to crosslink the specific binding member or antibody molecule.

[0363] In a preferred embodiment, the antibody molecule or specific binding member is activated in the EC of a T cell 50 is the EC of FS22-172-003 / HelD1.3 (containing the LALA mutation) in the same assay 50 The heavy chain of FS22-172-003 / HelD1.3(LALA) is within 10-fold, 5-fold, 4-fold, 3-fold or 2-fold of that of FS22-172-003 / HelD1.3(LALA), wherein FS22-172-003 / HelD1.3(LALA) comprises or consists of the heavy chain of SEQ ID NO: 145 and the light chain of SEQ ID NO: 173.

[0364] In a preferred embodiment, the antibody molecule or specific binding member is activated in the EC of a T cell 50 is the EC of FS22-053-008 / HelD1.3 (containing the LALA mutation) in the same assay 50 The heavy chain of FS22-053-008 / HelD1.3(LALA) is within 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold of that of FS22-053-008 / HelD1.3(LALA), wherein FS22-053-008 / HelD1.3(LALA) comprises or consists of the heavy chain of SEQ ID NO: 27 and the light chain of SEQ ID NO: 173.

[0365] For example, the EC of an antibody molecule or specific binding member in a T cell activation assay 50 It may be 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, or 0.5 nM or less.

[0366] Additionally or alternatively, the ability of the antibody molecule or specific binding member to activate T cells can be determined by measuring the maximum IL-2 concentration released by T cells in a T cell activation assay in the presence of the antibody molecule or specific binding member, wherein the antibody molecule or specific binding member is cross-linked.

[0367] In a preferred embodiment, the maximum concentration of IL-2 released by T cells in a T cell activation assay in the presence of the antibody molecule or specific binding member in the presence of cross-linking is within 3-fold, 2-fold or 1.5-fold the maximum concentration of IL-2 released by T cells in the presence of FS22-053-008 / HelD1.3 (comprising the LALA mutation) or FS22-172-003 / HelD1.3 (comprising the LALA mutation).

[0368] The T cell activation assay can be a T cell assay as described herein, such as a CD8+ T cell assay as described in this example, see, for example, Example 5.4.

[0369] For example, T cell activation experiments can be based on the IL-2 release assay of CD8+T cells separated from human peripheral blood mononuclear cells (PBMC). For example, T cell activation experiments can include separating human PBMC from leucocyte depletion cones. The method of separating PBMC is known in the art and is described in the present embodiment. CD8+T cells can then be isolated from PBMC. The method of separating CD8+T cells from PBMC is known in the art and is described in the present embodiment.

[0370] CD8+ T cells can then be added to a plate coated with an anti-human CD3 antibody. Suitable dilutions of each test antibody molecule or specific binding member can be prepared and added to the wells. T cells can then be incubated with the test antibody at 37°C, 5% CO2 for 24 hours. The supernatant can be collected and assayed to determine the concentration of IL-2 in the supernatant. Methods for determining the concentration of IL-2 in the solution are known in the art and are described in the present embodiment. The logarithmic concentration of the concentration relative antibody molecule or specific binding member of human IL-2 can be plotted. The logarithmic (agonist) relative response equation can be used to fit the resulting curve.

[0371] The second antigen bound by the second antigen binding site of the specific binding member can be an immune cell antigen or a disease antigen. Disease antigens include pathogenic antigens and tumor antigens.

[0372] The immune cell antigen bound by the specific binding member may be present on the same or a different immune cell as CD137.

[0373] The immune cell antigen may be a member of the tumor necrosis factor receptor superfamily (TNFRSF) other than CD 137. The TNFRSF receptor is a membrane-bound cytokine receptor that contains an extracellular cysteine-rich domain that binds one or more ligands of the tumor necrosis factor superfamily (TNFSF).

[0374] TNFRSF receptors can be located on the surface of immune cells. After binding to TNFRSF ligands, TNFRSF receptors form clusters on the surface of immune cells, thereby activating immune cells. For example, TNFRSF receptors bound to ligands can form multimers (e.g., trimers), or multimer clusters. The presence of ligand-bound TNFRSF receptor clusters stimulates intracellular signaling pathways that activate immune cells.

[0375] Without wishing to be bound by theory, it is believed that by binding to CD137 and a second TNFRSF receptor on the surface of an immune cell, the specific binding member will cause CD137 and a second TNFRSF receptor to aggregate and activate the immune cell. In other words, when both targets are bound, the specific binding member will act as a TNFRSF receptor agonist.

[0376] TNFRSF receptors include CD27, CD40, EDA2R, EDA, FAS, LTBR, ​​RELT, TNFRSF1A, TNFRSF1B, TNFRSF4, TNFRSF6B, TNFRSF8, TNFRSF10A-10D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, and TNFRSF25.

[0377] CD27 (TNFRSF7: Gene ID 939) has a reference amino acid sequence of NP_001233.1 and may be encoded by a reference nucleotide sequence of NM_001242.4. CD40 (TNFRSF5: Gene ID 958) has a reference amino acid sequence of NP_001241.1 and may be encoded by a reference nucleotide sequence of NM_001250.5. EDA2R (TNFRSF27: Gene ID 60401) has a reference amino acid sequence of NP_001186616.1 and may be encoded by a reference nucleotide sequence of NM_001199687.2. EDAR (Gene ID 10913) has a reference amino acid sequence of NP_071731.1 and may be encoded by a reference nucleotide sequence of NM_022336.3. FAS (TNFRSF6: Gene ID 355) has a reference amino acid sequence of NP_000034.1 and may be encoded by a reference nucleotide sequence of NM_000043.5. LTBR (TNFRSF3: Gene ID 4055) has a reference amino acid sequence of NP_001257916.1 and may be encoded by a reference nucleotide sequence of NM_001270987.1. RELT (TNFRSF19L: Gene ID 84957) has a reference amino acid sequence of NP_116260.2 and may be encoded by a reference nucleotide sequence of NM_032871.3. TNFRSF1A (Gene ID 7132) has a reference amino acid sequence of NP_001056.1 and may be encoded by a reference nucleotide sequence of NM_001065.3. TNFRSF1B (Gene ID 7133) has a reference amino acid sequence of NP_001057.1 and can be encoded by a reference nucleotide sequence of NM_001066.2.

[0378] TNFRSF4 (Gene ID 7293) has a reference amino acid sequence of NP_003318 and may be encoded by a reference nucleotide sequence of NM_003327. TNFRSF6B (Gene ID 8771) has a reference amino acid sequence of NP_003814.1 and may be encoded by a reference nucleotide sequence of NM_003823.3. TNFRSF8 (Gene ID 943) has a reference amino acid sequence of NP_001234.3 and may be encoded by a reference nucleotide sequence of NM_001243.4. TNFRSF10A (Gene ID 8797) has a reference amino acid sequence of NP_003835.3 and may be encoded by a reference nucleotide sequence of NM_003844.3. TNFRSF10B (Gene ID 8795) has a reference amino acid sequence of NP_003833.4 and may be encoded by a reference nucleotide sequence of NM_003842.4. TNFRSF10C (Gene ID 8794) has a reference amino acid sequence of NP_003832.2 and may be encoded by a reference nucleotide sequence of NM_003841.4. TNFRSF10D (Gene ID 8793) has a reference amino acid sequence of NP_003831.2 and may be encoded by a reference nucleotide sequence of NM_003840.4. TNFRSF11A (Gene ID 8792) has a reference amino acid sequence of XP_011524547.1 and may be encoded by a reference nucleotide sequence of XM_11526245.2. TNFRSF11B (Gene ID 4982) has a reference amino acid sequence of NP_002537.3 and may be encoded by a reference nucleotide sequence of NM_002546.3. TNFRSF12A (Gene ID 51330) has a reference amino acid sequence of NP_057723.1 and may be encoded by a reference nucleotide sequence of NM_016639.2. TNFRSF13B (Gene ID 23495) has a reference amino acid sequence of NP_0036584.1 and may be encoded by a reference nucleotide sequence of NM_012452.2. TNFRSF13C (Gene ID 115650) has a reference amino acid sequence of NP_443177.1 and may be encoded by a reference nucleotide sequence of NM_052945.3. TNFRSF14 (Gene ID 8764) has a reference amino acid sequence of NP_001284534.1 and may be encoded by a reference nucleotide sequence of NM_001297605.1. TNFRSF17 (Gene ID 608) has a reference amino acid sequence of NP_001183.2 and can be encoded by a reference nucleotide sequence of NM_001192.2.TNFRSF18 (Gene ID 8784) has a reference amino acid sequence of NP_004195.2 and can be encoded by a reference nucleotide sequence of NM_004186.1. TNFRSF19 (Gene ID 55504) has a reference amino acid sequence of NP_001191387.1 and can be encoded by a reference nucleotide sequence of NM_001204458.1. NFRSF21 (Gene ID 27242) has a reference amino acid sequence of NP_055267.1 and can be encoded by a reference nucleotide sequence of NM_014452.4. TNFRSF25 (DR3: Gene ID 8718) binds to the ligand TNFSF15 (TL1A), has a reference amino acid sequence of NP_001034753.1, and can be encoded by a reference nucleotide sequence of NM_001039664.1.

[0379] Alternatively, the immune cell antigen bound by the second antigen binding site can be a molecule with a regulatory function in the immune system, rather than a TNFRSF member, such as an immune co-stimulatory molecule or an inhibitory checkpoint molecule. Examples of such immunomodulatory molecules include ICOS (CD278), LAG3, PD1, PD-L1, PD-L2, B7H3, B7H4, CTLA4, TIGIT, BTLA, HVEM, T cell immunoglobulin, mucin domain containing 3 (TIM-3), CD47, CD73, A2aR, CD200, CD200R, colony stimulating factor 1 receptor (CSF-1R), VISTA CD28, CD80, LLT1, galectin-9, NKG2A, NKG2D, and KIR.

[0380] The immune cell on which the immune cell antigen is presented may belong to any immune cell subpopulation, and may be a T cell, a tumor-infiltrating leukocyte (TIL), a myeloid lineage cell (e.g., an antigen-presenting cell (APC), a NK cell, and / or a B cell). When the immune cell antigen is a TNFRSF receptor, the immune cell on which the TNFRSF receptor is presented is preferably a T cell.

[0381] Alternatively, the second antigen binding site can bind to the disease antigen as described above. Without wishing to be bound by theory, it is believed that the binding of a specific binding member to CD137 and a disease antigen will result in activation of T cells near the disease. The activated T cells can then initiate, promote, or participate in an immune response, such as an immune response against a pathogen or cancer cell. Chen and Mellman (2013) provide a review of the role of the immune system in identifying and eliminating cancer cells.

[0382] In a preferred embodiment, the disease antigen is a tumor antigen. Tumor antigens are antigens that are primarily present in the tumor environment and are not prevalent elsewhere in the individual. For example, tumor antigens may be present on the surface of tumor cells or on other stromal cells in the tumor microenvironment or in biological fluids near the tumor. Therefore, tumor antigens are markers of the location of tumor cells in an individual.

[0383] In some embodiments, the tumor antigen may be an antigen located on the surface of a cancer cell. Preferably, the tumor antigen is upregulated or overexpressed on the tumor cell, while in the absence of a tumor, the corresponding normal somatic cell from the same tissue does not express it in large amounts.

[0384] In some embodiments, the tumor antigen is upregulated or overexpressed on stromal cells of the tumor microenvironment compared to stromal cells of corresponding normal tissue in the absence of a tumor.

[0385] Preferred tumor antigens are present on the cell surface and are not rapidly internalized.

[0386] Tumor antigens suitable for targeting by specific binding members can be identified using methods known in the art. For example, specific binding members that target the CD137 receptor and a tumor antigen can be used in an assay in which cells expressing CD137 are co-cultured with cells expressing the tumor antigen and activation of the CD137-expressing cells is measured, for example, by a T cell activation assay, a proliferation assay, or a cytotoxicity assay.

[0387] Cell surface tumor antigens can be tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).

[0388] Tumor antigens expressed by cancer cells can include, for example, cancer-testis (CT) antigens encoded by cancer germline genes, such as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2, (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 / CT7, MAGE-C2, NY-ESO-I, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and XAGE, and immunogenic fragments or variants thereof (Simpson et al., 2005; Gure et al., 2005; Velazquez et al., 2007; Andrade et al., 2008; Tinguely et al., 2008; Napoletano et al., 2008).

[0389] Other cell surface tumor antigens include, for example, AFP, α v β3 (vitronectin receptor), α vβ6, B cell maturation agent (BCMA), CA125 (MUC16), CD4, CD20, CD22, CD33, CD52, CD56, CD66e, CD80, CD140b, CD227 (MUC1), EGFR (HER1), EpCAM, GD3 ganglioside, HER2, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), CD5, CD19, CD21, CD25, CD37, CD30 , CD33, CD45, HLA-DR, anti-idiotypic carcinoembryonic antigen (CEA) (e.g., carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)), TAG-72, folate binding protein, A33, G250, ferritin, glycolipids (e.g., gangliosides), carbohydrates (e.g., CA-125), IL-2 receptor, fibroblast activation protein (FAP), IGF1R, B7H3, B7H4, PD-L1, CD200, EphA2, and mesothelin or its variants. These and other cell surface tumor antigens are described in Carter et al., 2004; Scott and Renner, 2001; and Cheever et al., 2009; Tai and Anderson, 2015; and Podojil and Miller, 2017.

[0390] Other tumor antigens include out-of-frame peptide-MHC complexes generated by non-AUG translation initiation machinery employed by stressed cancer cells (Malarkannan et al., 1999).

[0391] Other tumor antigens include peptide-MHC complexes on the surface of tumor cells or tumor microenvironment cells, wherein the peptide-MHC complexes comprise tumor-specific neoantigen peptide fragments of mutated intracellular tumor antigens, wherein the peptide neoantigens comprise one or more tumor-specific mutations (Gubin et al., 2015). Other tumor antigens are well known in the art (see, for example, WO00 / 20581; Cancer Vaccines and Immunotherapy (2000), edited by Stern, Beverley and Carroll, Cambridge University Press, Cambridge). The sequences of these tumor antigens are readily available from public databases, but can also be found in WO1992 / 020356A1, WO1994 / 005304A1, WO1994 / 023031A1, WO1995 / 020974A1, WO1995 / 023874A1 and WO1996 / 026214A1.

[0392] Preferred tumor antigens include HER2, FAP, EpCAM, CEACAM5, CD20, CD73, PSMA, mesothelin, EphA2, IGF1R, CD200, α vβ6, BCMA, PD-L1, B7H3, B7H4 and EGFR.

[0393] In a more preferred embodiment, the tumor antigen is mesothelin (MSLN).

[0394] In another more preferred embodiment, the tumor antigen is PD-L1.

[0395] HER2 (ERBB2; Gene ID 2064) may have a reference amino acid sequence of NP_001005862.1 and may be encoded by a reference nucleotide sequence of NM_001005862.2. FAP (Gene ID 2191) may have a reference amino acid sequence of NP_001278736.1 and may be encoded by a reference nucleotide sequence of NM_001291807.1. EpCAM (Gene ID 4072) may have a reference amino acid sequence of NP_002345.2 and may be encoded by a reference nucleotide sequence of NM_002354.2.

[0396] CEACAM5 (Gene ID 1048) may have a reference amino acid sequence of NP_001278413.1 and may be encoded by a reference nucleotide sequence of NM_001291484.2. CD20 (MS4A1; Gene ID 931) may have a reference amino acid sequence of NP_068769.2 and may be encoded by a reference nucleotide sequence of NM_021950.3. CD73 (NT5E; Gene ID 4907) may have a reference amino acid sequence of NP_001191742.1 and may be encoded by a reference nucleotide sequence of NM_001204813.1. PSMA (FOLH1; Gene ID 2346) may have a reference amino acid sequence of NP_001014986.1 and may be encoded by a reference nucleotide sequence of NM_001014986.1. Mesothelin (MSLN; Gene ID 10232) may have a reference amino acid sequence of NP_001170826.1 and may be encoded by a reference nucleotide sequence of NM_001177355.2. EphA2 (Gene ID 1969) may have a reference amino acid sequence of NP_001316019.1 and may be encoded by a reference nucleotide sequence of NM_001329090.1. IGF1R (Gene ID 3480) may have a reference amino acid sequence of NP_000866.1 and may be encoded by a reference nucleotide sequence of NM_000875.4. CD200 (Gene ID 4345) may have a reference amino acid sequence of NP_001004196.2 and may be encoded by a reference nucleotide sequence of NM_001004196.3.v β6 is a heterodimer consisting of integrin subunit αV and integrin subunit β6. Integrin subunit αV (ITGAV; Gene ID 3685) may have a reference amino acid sequence of NP_001138471.1 and may be encoded by a reference nucleotide sequence of NM_001144999.2. Integrin subunit β6 (ITGB6; Gene ID 3694) may have a reference amino acid sequence of NP_000879.2 and may be encoded by a reference nucleotide sequence of NM_000888.4. BCMA (TNFRSF17; Gene ID 608) may have a reference amino acid sequence of NP_001183.2 and may be encoded by a reference nucleotide sequence of NM_001192.2. PD-L1 (CD274; Gene ID 29126) may have a reference amino acid sequence of NP_001254635.1 and may be encoded by a reference nucleotide sequence of NM_001267706.1. B7H3 (CD276; Gene ID 80381) may have a reference amino acid sequence of NP_001019907.1 and may be encoded by a reference nucleotide sequence of NM_001024736.1. B7H4 (VTCN1; Gene ID 79679) may have a reference amino acid sequence of NP_001240778.1 and may be encoded by a reference nucleotide sequence of NM_001253849.1. EGFR (Gene ID 1956) may have a reference amino acid sequence of NP_001333826.1 and may be encoded by a reference nucleotide sequence of NM_001346897.1.

[0397] In other embodiments, the tumor antigen may be a soluble tumor antigen, such as a growth factor produced by or in response to a cancer cell. Soluble factors may be upregulated or overexpressed in biological fluids near the tumor. Soluble tumor antigens may be multimers, such as dimers or trimers. The concentration of soluble tumor antigens in the tumor site or tumor microenvironment may be higher than in other parts of the individual's body. The tumor microenvironment and related soluble tumor antigens are described in more detail in Bhome et al. (2015).

[0398] Suitable soluble tumor antigens include VEGF, HGF, SDF1, and TGF-β, such as TGF-β-1, TGF-β-2, TGF-β-3, and TGF-β-4.

[0399] VEGF (VEGFA; Gene ID 7422) has a reference amino acid sequence of NP_001020537.2 and may be encoded by a reference nucleotide sequence of NM_001025366.2. HGF (Gene ID 3082) has a reference amino acid sequence of NP_000592.3 and may be encoded by a reference nucleotide sequence of NM_000601.5. SDF1 (CXCL12; Gene ID 6387) has a reference amino acid sequence of NP_000600.1 and may be encoded by a reference nucleotide sequence of NM_000609.6. TGF-β-1 (TGFB1; Gene ID 7040) may have a reference amino acid sequence of NP_000651.3 and may be encoded by a reference nucleotide sequence of NM_000660.6. TGF-β-2 (TGFB2; Gene ID 7042) may have a reference amino acid sequence of NP_001129071.1 and may be encoded by a reference nucleotide sequence of NM_001135599.3. TGF-β-3 (TGFB3; Gene ID 7043) may have a reference amino acid sequence of NP_001316867.1 and may be encoded by a reference nucleotide sequence of NM_001329938.1. TGF-β-4 (LEFTY2; Gene ID 7044) may have a reference amino acid sequence of NP_001165896.1 and may be encoded by a reference nucleotide sequence of NM_001172425.2.

[0400] In another preferred embodiment, the disease antigen is a pathogenic antigen.

[0401] Activation of immune cells, such as T cells, NK cells and / or macrophages by specific binding members near the site of an infectious disease is expected to be useful in treating infectious diseases. The infectious disease may be an acute or persistent infectious disease, but is preferably a persistent infectious disease.

[0402] Pathogenic antigens are preferably antigens expressed by human pathogens, such as viral, bacterial, fungal or parasitic antigens (e.g., protozoan antigens), preferably viral or bacterial antigens. Pathogenic antigens are antigens that are primarily present on pathogens or near sites of infectious diseases, rather than antigens that are prevalent in other parts of an individual.

[0403] For example, pathogenic antigens can be antigens present on the surface of viruses, bacteria, fungi or parasites, or soluble antigens expressed by viruses, bacteria, fungi or parasites. Viruses, bacteria, fungi or parasites can be viruses, bacteria, fungi or parasites mentioned in other parts of this paper.

[0404] In the case where the pathogenic antigen is a soluble antigen, the antigen may be upregulated or overexpressed in biological fluids near the site of the infectious disease. For example, the soluble pathogenic antigen may be present at a higher concentration at or near the site of the infectious disease than elsewhere in the individual's body. The soluble pathogenic antigen may be a multimer, such as a dimer or trimer.

[0405] Pathogenic antigens suitable for targeting by specific binding members can be identified using methods known in the art. For example, specific binding members that target CD137 and a pathogenic antigen can be used in assays where cells expressing CD137 are co-cultured with a pathogen or pathogenic antigen and activation of the OX40-expressing cells is measured, for example, by a T cell activation assay, a proliferation assay, or a cytotoxicity assay.

[0406] Many pathogenic antigens suitable for being targeted by specific binding members are further known in the art, and can be selected by technical personnel according to the infectious disease to be treated.The example of viral antigen includes protein p24, gp120 and gp41 expressed by human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg) expressed by hepatitis B virus (HBV) and hemagglutinin and neuraminidase expressed by influenza virus.The example of bacterial antigen includes Rv1733, Rv2389 and Rv2435n expressed by Mycobacterium tuberculosis.

[0407] Specific binding members can also include the first or second sequence as described herein, AB CD or EF structure loop sequence, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain and / or heavy chain sequence variant. Suitable variants can be obtained by sequence change or mutation and screening method. In a preferred embodiment, the specific binding members comprising one or more variant sequences retain one or more functional characteristics of the parent specific binding members, such as binding specificity and / or binding affinity to CD137. For example, the specific binding members comprising one or more variant sequences preferably bind CD137 with the same or higher affinity as (parent) specific binding members. Parent specific binding members are specific binding members that do not include amino acid substitutions, deletions and / or insertions incorporated into variant specific binding members.

[0408] For example, a specific binding member can comprise a first, second or third sequence, AB, CD or EF structural loop sequence, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain or heavy chain sequence disclosed herein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a first, second or third sequence, AB, CD or EF structural loop sequence, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain and / or heavy chain sequence disclosed herein.

[0409] In a preferred embodiment, the specific binding member has or comprises a CH3 domain sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity, preferably at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the CH3 domain sequence of SEQ ID NO: 21, 30, 48, 102, 75, 39, 57, 66, 84, 93, 175, 139, 130, 148, 121, 157, 165 or 112.

[0410] In another preferred embodiment, the specific binding member has or comprises a CH2 domain sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the CH2 domain sequence shown in SEQ ID NO: 5 or 6.

[0411] In another preferred embodiment, the specific binding member has, comprises, or consists of a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the Fcab sequence set forth in SEQ ID NO: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95, 15, 141, 132, 150, 123, 159, 167, 114, 25, 34, 52, 106, 79, 43, 61, 70, 88, 97, 16, 143, 134, 152, 125, 161, 169, or 116.

[0412] Sequence identity is usually defined with reference to algorithm GAP (Wisconsin GCG software package, Accelerys, Inc., San Diego, USA). GAP uses Needleman and Wunsch algorithm to compare two complete sequences, thereby increasing the number of matches and minimizing the number of gaps to the greatest extent. Usually, a gap creation penalty is equal to 12, and a gap extension penalty is equal to the default parameters of 4. GAP can preferably be used, but other algorithms can be used, such as BLAST (using Altschul et al., 1990 method), FASTA (using Pearson and Lipman, 1988 method) or Smith-Waterman algorithm (Smith and Waterman, 1981) or TBLASTN program, referring to Altschul et al., 1990, the same, usually using default parameters. Especially, psi-Blast algorithm can be used.

[0413] The specific binding member may comprise a first, second or third sequence, AB, CD or EF structural loop sequence, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain or heavy chain, which has one or more amino acid sequence changes (additions, deletions, substitutions and / or insertions of amino acid residues) compared to the first, second or third sequence, AB, CD or EF structural loop sequence, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain or heavy chain sequence disclosed herein, preferably 20 or less, 15 or less, 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 change.

[0414] In a preferred embodiment, the specific binding member may comprise a CH3 domain sequence having one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues), preferably 20 or less, 15 or less, 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 altered CH3 domain sequence compared to the CH3 domain sequence set forth in SEQ ID NO: 21, 30, 48, 102, 75, 39, 57, 66, 84, 93, 175, 139, 130, 148, 121, 157, 165 or 112.

[0415] In another preferred embodiment, the specific binding member comprises a CH2 domain sequence having one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues), preferably 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 altered CH2 domain sequence, compared to the CH2 domain set forth in SEQ ID NO: 5 or 6.

[0416] In a preferred embodiment, the specific binding member may comprise or consist of a sequence having one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues) compared to the Fcab sequence set forth in SEQ ID NO: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95, 15, 141, 132, 150, 123, 159, 167, 114, 25, 34, 52, 106, 79, 43, 61, 70, 88, 97, 16, 143, 134, 152, 125, 161, 169, or 116, preferably 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 alteration.

[0417] When the specific binding member comprises a variant of the first sequence, AB loop sequence, CH3 domain, Fcab or heavy chain sequence disclosed herein, the specific binding member preferably retains the sequence PPY between positions 11 and 19, preferably between positions 15 and 17, of the CH3 domain of the specific binding member. In addition, the specific binding member preferably retains an insertion, preferably a 5-amino acid insertion, between positions 16 and 17 of the CH3 domain of the specific binding member. In another preferred embodiment, the specific binding member preferably retains this sequence at positions 97 and 98 of the CH3 domain of the specific binding member.

[0418] In particular, the specific binding member may be (or the antibody molecule may comprise) a variant of specific binding member FS22-053, wherein the variant:

[0419] (i) comprises one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues), preferably 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer or 1 alteration, compared to the sequence of the specific binding member FS22-053 disclosed herein; or

[0420] (ii) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the sequence of the specific binding member FS22-053 disclosed herein; and

[0421] wherein the specific binding member or antibody molecule comprises the sequence PPY between positions 15 and 17, and optionally comprises a 5 amino acid insertion between positions 16 and 17, of the CH3 domain of the specific binding member or antibody molecule; and

[0422] The residue numbering is according to the IMGT residue numbering scheme.

[0423] Additionally or alternatively, when a specific binding member comprises a variant of a CH3 domain, CH2 and CH3 domain, Fcab, light chain, or heavy chain sequence disclosed herein, the variant preferably does not comprise any amino acid changes in the first, second, and third sequences located within the AB, CD, and EF loops of the CH3 domain of the specific binding member. For example, the variant may not comprise any amino acid changes in the AB, CD, and EF loops of the CH3 domain of the specific binding member.

[0424] In preferred embodiments where one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, such as those described in the table below. In some embodiments, amino acids of the same class in the middle column are substituted for one another, i.e., for example, a non-polar amino acid is substituted with another non-polar amino acid. In some embodiments, amino acids in the same row in the rightmost column are substituted for one another.

[0425]

[0426] In some embodiments, substitutions may be functionally conservative. That is, in some embodiments, a substitution may not affect (or may not substantially affect) one or more functional properties (e.g., binding affinity) of the specific binding member comprising the substitution, compared to an equivalent unsubstituted specific binding member.

[0427] Also contemplated is a specific binding member comprising a constant domain, preferably a CD137 antigen binding site, located in the CH3 domain of the specific binding member, and competing with the specific binding members of the present invention for binding to CD137, or binding to the same epitope on CD137 as the specific binding members of the present invention. Methods for determining that two specific binding members compete for an antigen are known in the art. For example, surface plasmon resonance, such as Biacore, can be used to determine competitive binding of two specific binding members to an antigen. Methods for mapping the epitope bound by a specific binding member are similarly known in the art.

[0428] In some embodiments, a specific binding member may not comprise a CDR-based antigen binding site.

[0429] In particular, the specific binding member may not comprise a CDR-based antigen binding site that binds PD-L1.

[0430] Additionally, or alternatively, the specific binding member may not comprise a CDR-based antigen binding site that binds mesothelin (MSLN).

[0431] For example, the specific binding member may not comprise a CDR-based antigen binding site that binds PD-L1 or MSLN, wherein the specific binding member comprises the first, second and third sequences located in the AB, CD and EF structural loops of the CH3 domain of the specific binding member FS22-53-008 or FS22-172-003, the complete AB and EF structural loop sequences of the CH3 domain of the specific binding member FS22-53-008 or FS22-172-003, and / or the CH3 domain sequence of the specific binding member FS22-53-008 or FS22-172-003.

[0432] In particular, the specific binding member may not comprise the CDRs, VH and / or VL domains, and / or heavy and / or light chain sequences of FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 as shown below.

[0433] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VH domain CDRs

[0434] HCDR1(IMGT)GYPFTSYG

[0435] HCDR1(Kabat)SYGIS

[0436] HCDR2(IMGT)ISAYSGGT

[0437] HCDR2(Kabat)WISAYSGGTNYAQKLQG

[0438] HCDR3(IMGT)ARDLFPTIFGVSYYYY

[0439] HCDR3(Kabat)DLFPTIFGVSYYYY

[0440] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VH domains

[0441]

[0442] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VL domain CDRs

[0443] LCDR1(IMGT)QSIGNR

[0444] LCDR1(Kabat)RASQSIGNRLA

[0445] LCDR2(IMGT)EAS

[0446] LCDR2(Kabat)EASTSET

[0447] LCDR3(IMGT)QQSYSTPYT

[0448] LCDR3(Kabat)QQSYSTPYT

[0449] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VL domains

[0450]

[0451] Heavy chain FS22-172-003-AA / E12v2

[0452]

[0453] Light chain FS22-172-003-AA / E12v2

[0454]

[0455] Heavy chain FS22-053-008-AA / E12v2

[0456]

[0457] Light chain FS22-053-008-AA / E12v2

[0458]

[0459] Additionally, or alternatively, the specific binding member may not comprise the CDRs and / or VH and / or VL domains of the anti-MSLN antibody FS28-256-271 listed below.

[0460] FS28-256-271 VH domain CDRs

[0461] HCDR1(AA)(IMGT)GFTFTHTY

[0462] HCDR1(AA)(Kabat)HTYMS

[0463] HCDR2(AA)(IMGT)ISPTYSTT

[0464] HCDR2(AA)Kabat)AISPTYSTTNYADSVKG

[0465] HCDR3(AA)(IMGT)ARYNAYHAALDY

[0466] HCDR3(AA)(Kabat)YNAYHAALDY

[0467] FS28-256-271 VH domain

[0468]

[0469] FS28-256-271 VL domain CDRs

[0470] LCDR1(AA)(IMGT)QSVSSSY

[0471] LCDR1(AA)(Kabat)RASQSVSSSYLA

[0472] LCDR2(AA)(IMGT)GAS

[0473] LCDR2(AA)(Kabat)GASSRAT

[0474] LCDR3(AA)(IMGT)QQTVPYPYT

[0475] LCDR3(AA)(Kabat)QQTVPYPYT

[0476] FS28-256-271 VL domain

[0477]

[0478] Specific binding members can be conjugated to biologically active molecules or detectable labels. In this case, the specific binding members can be referred to as conjugates. Such conjugates can be used to treat the diseases described herein.

[0479] For example, the bioactive molecule can be an immune system modulator, such as a cytokine, preferably a human cytokine. For example, the cytokine can be a cytokine that stimulates T cell activation and / or proliferation. Examples of cytokines conjugated to specific binding members include IL-2, IL-10, IL-12, IL-15, IL-21, GM-CSF, and IFN-γ.

[0480] Alternatively, the bioactive molecule may be a ligand trap, such as a ligand trap for a cytokine (eg, TGF-β or IL-6).

[0481] Suitable detectable labels that can be conjugated to specific binding members are known in the art and include radioisotopes, such as iodine-125, iodine-131, yttrium-90, indium-111, and technetium-99; and fluorescent dyes, such as fluorescein, rhodamine, phycoerythrin, Texas Red, and cyanine dye derivatives, such as Cy7 and Alexa750; chromogenic dyes, such as diaminobenzidine; latex microspheres; enzyme labels, such as horseradish peroxidase; fluorescent powders or laser dyes with spectrally isolated absorption or emission properties; and chemiluminescence, such as biotin, which can be detected by binding to a specific cognate detectable moiety, such as labeled avidin.

[0482] Specific binding members can be conjugated to the bioactive molecule or detectable label by any suitable covalent or non-covalent bond, such as a disulfide bond or a peptide bond. When the bioactive molecule is a cytokine, the cytokine can be linked to the specific binding member by a peptide linker. Suitable peptide linkers are known in the art and can be 5 to 25, 5 to 20, 5 to 15, 10 to 25, 10 to 20, or 10 to 15 amino acids in length.

[0483] In some embodiments, the bioactive molecule can be conjugated to the specific binding member via a cleavable linker. The linker allows release of the bioactive molecule from the specific binding member at the treatment site. The linker may comprise an amide bond (e.g., a peptide linker), a disulfide bond, or a hydrazone. For example, a peptide linker may be cleaved by a site-specific protease, a disulfide bond may be cleaved by the reducing environment of the cytoplasm, and a hydrazone may be cleaved by acid-mediated hydrolysis.

[0484] The conjugate may be a fusion protein comprising a specific binding member and a bioactive molecule. In this case, the bioactive molecule may be conjugated to the specific binding member via a peptide linker or peptide bond. When the specific binding member is a multi-chain molecule, for example, when the specific binding member is or comprises an FcAb or a mAb 2 When a bioactive molecule is conjugated to one or more chains of a specific binding member, the bioactive molecule can be conjugated to one or more chains of a specific binding member. For example, a bioactive molecule can be conjugated to a mAb 2The advantage of fusion proteins is that they are easy to produce and purify, which facilitates the production of clinical-grade materials.

[0485] The present invention also provides isolated nucleic acid molecules or molecules encoding specific binding members of the present invention. A skilled person will have no difficulty in preparing such nucleic acid molecules using methods well known in the art.

[0486] In a preferred embodiment, the nucleic acid molecule encodes the CH3 domain of the following specific binding members: FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172, preferably FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005 or FS22-172-006, more preferably FS22-172-003, FS22-172-002 or FS22-172-004, and even more preferably FS22-172-003.

[0487] In an alternative preferred embodiment, the nucleic acid molecule encodes the CH3 domain of the following specific binding members: FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS 22-053-016 or FS22-053, preferably FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017 or FS22-053-014, more preferably FS22-053-008, FS22-053-009, FS22-053-011 or FS22-053-017, even more preferably FS22-053-008.

[0488] The CH3 domain sequences of these specific binding members are described herein.

[0489] For example, a nucleic acid molecule encoding the CH3 domain of the following specific binding member:

[0490] (i) FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 as set forth in SEQ ID NOs: 22, 31, 49, 103, 76, 40, 58, 67, 85, 94 and 176, respectively; or

[0491] (ii) FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172 as shown in SEQ ID NO: 140, 131, 149, 122, 158, 166 and 113, respectively.

[0492] In a preferred embodiment, the nucleic acid molecule encodes the following specific binding member: FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172, preferably FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005 or FS22-172-006, more preferably FS22-172-003, FS22-172-002 or FS22-172-004, even more preferably FS22-172-003.

[0493] In an alternative preferred embodiment, the nucleic acid molecule encodes the following specific binding members: FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016, 53-016 or FS22-053, preferably FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017 or FS22-053-014, more preferably FS22-053-008, FS22-053-009, FS22-053-011 or FS22-053-017, even more preferably FS22-053-008.

[0494] For example, a nucleic acid molecule encoding the following specific binding member:

[0495] (i) FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 as set forth in SEQ ID NOs: 24, 33, 51, 105, 78, 42, 60, 69, 87, 96 and 177, respectively; or

[0496] (ii) FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172 as shown in SEQ ID NO: 142, 133, 151, 124, 160, 168 and 115, respectively.

[0497] The isolated nucleic acid molecules can be used to express the specific binding members of the present invention. The nucleic acid will generally be provided for expression in the form of a recombinant vector. Therefore, another aspect of the present invention provides a vector comprising the nucleic acid as described above. Suitable vectors can be selected or constructed, comprising suitable regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other appropriate sequences. Preferably, the vector comprises suitable regulatory sequences to drive the expression of the nucleic acid in the host cell. The vector may be a plasmid, a virus such as a phage or a phagemid, as the case may be.

[0498] Nucleic acid molecules or vectors as described herein can be introduced into host cells. The technology of introducing nucleic acid or vector into host cells is established in the art, and any suitable technology can be used. A series of host cells suitable for producing recombinant specific binding members are known in the art, including bacteria, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NSO or HEK cells, such as HEK293 cells. The most preferred host cell is CHO cell.

[0499] Another aspect of the present invention provides a method for producing a specific binding members of the present invention, the method comprising expressing a nucleic acid encoding the specific binding members in a host cell, and optionally isolating and / or purifying the specific binding members produced thereby. Methods for culturing host cells are well known in the art. The method may further comprise isolating and / or purifying the specific binding members. The technology for purifying recombinant specific binding members is well known in the art, including, for example, HPLC, FPLC or affinity chromatography, for example, using protein A or protein L. In some embodiments, the affinity tag on the specific binding members can be used for purification. The method may also comprise optionally formulating a pharmaceutical composition with a pharmaceutically acceptable excipient or other substance as described below.

[0500] As mentioned above, CD137 is expressed on cells of the immune system, including CD8 + T cells, CD4 + T cells, Treg cells, B cells, NK cells, NKT cells, dendritic cells and tumor infiltrating lymphocytes (TIL). CD137 activation has been shown to enhance CD8 + T cell proliferation, survival and cytotoxic effector function and CD8 + T cell differentiation and memory CD8 + CD137 plays a role in the maintenance of T cells. + The expression level on T cells is higher than that on CD8 + T cells are low, but have also been shown to be associated with induction of CD4 + The proliferation and activation of certain subsets of T cells are involved. Activation of CD137 has also been shown to enhance NK cell-mediated ADCC as well as B cell proliferation, survival, and cytokine production.

[0501] Given the immune response-enhancing activity of CD137, CD137 agonist molecules have been investigated in the treatment of cancer as well as chronic infections.

[0502] Thus, the specific binding members described herein may be used in therapeutic applications, particularly in the treatment of cancer. Additionally, it is contemplated that the specific binding members may be used to treat infectious diseases, such as persistent infectious diseases.

[0503] The specific binding members as described herein can be used in methods of treating the human or animal body. Related aspects of the invention provide:

[0504] (i) a specific binding member as described herein for use as a medicament,

[0505] (ii) a method for using a specific binding member as described herein to treat a disease or disorder,

[0506] (iii) use of a specific binding member described herein in the preparation of a medicament for treating a disease or condition; and,

[0507] (iv) a method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a specific binding member as described herein.

[0508] The individual may be a patient, preferably a human patient.

[0509] Treatment can be any treatment or therapy that achieves some desired therapeutic effect, e.g., inhibiting or slowing progression of a disease, and includes a reduction in the rate of progression, arrest of the rate of progression, improvement of the disease, cure or alleviation (whether partial or total) of the disease, prevention, amelioration, delay, alleviation or arrest of one or more conditions and / or symptoms, or prolonging the survival of an individual or patient beyond that expected in the absence of treatment.

[0510] Also included are treatments that are used as preventative measures (i.e., prophylaxis). For example, a subject who is susceptible to or likely to develop or reoccur a disease (e.g., cancer) may be treated as described herein. Such treatments may prevent or delay the onset or reoccurrence of a disease in the subject.

[0511] The described therapeutic methods may include administering at least one other treatment to an individual in addition to the specific binding members. Therefore, the specific binding members described herein may be administered to an individual individually or in combination with one or more other treatments. When a specific binding member is administered to an individual in combination with another treatment, an additional treatment may be administered to the individual sequentially or separately while the specific binding member is administered. In the case where a specific binding member is administered simultaneously with an additional treatment, the specific binding member and the additional treatment may be administered to the individual as a combined preparation. For example, an additional treatment may be a known therapy or therapeutic agent for the disease to be treated.

[0512] Although specific binding members can be administered alone, specific binding members will generally be administered in the form of a pharmaceutical composition that may contain at least one component in addition to the specific binding member. Therefore, another aspect of the invention provides a pharmaceutical composition comprising a specific binding member as described herein. Also provided is a method comprising formulating a specific binding member into a pharmaceutical composition.

[0513] In addition to specific binding members, pharmaceutical composition may also include pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms, which are suitable for contacting the tissue of a subject (e.g., people) without excessive toxicity, irritation, allergic reaction or other problems or complications, and are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. In the sense of being compatible with the other ingredients of the preparation, each carrier, excipient etc. must also be "acceptable". The exact nature of the carrier or other materials will depend on the route of administration, which can be carried out by infusion, injection or any other suitable route, as described below.

[0514] For parenteral, such as subcutaneous or intravenous administration, such as by injection, the pharmaceutical composition comprising specific binding members can be in the form of an aqueous solution acceptable for parenteral, which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can use, for example, isotonic vehicles, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection to prepare suitable solutions. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be used as needed, including buffers such as phosphate, citrate and other organic acids; Antioxidants, such as ascorbic acid and methionine; Preservatives (such as octadecyl dimethyl benzyl ammonium chloride; Hexamethylammonium chloride; Benzalkonium chloride; Benzethonium chloride; Phenol, butyl alcohol or benzyl alcohol; Alkyl parahydroxybenzoate, such as methyl parahydroxybenzoate or propyl parahydroxybenzoate; Catechol; Catechol; Resorcinol; Cyclohexanol; 3-pentanol; With metacresol); Low molecular weight amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0515] In some embodiments, the specific binding member can be provided in a lyophilized form for reconstitution prior to administration. For example, the lyophilized specific binding member can be reconstituted in sterile water and mixed with saline before administration to an individual.

[0516] Administration can be in a "therapeutically effective amount", which is sufficient to show benefit to the individual. The actual amount administered and the rate and time course of administration will depend on the nature and severity of the disease being treated, the specific individual being treated, the individual's clinical condition, the cause of the disease, the composition delivery site, the type of specific binding member, the method of administration, the dosing schedule and other factors known to the doctor. The determination of treatment prescriptions, such as dosages, etc., is the responsibility of general practitioners and other doctors and may depend on the severity of symptoms and / or the progression of the disease being treated. Appropriate doses of immunoglobulins are well known in the art (Ledermann et al., (1991) Int. J. Cancer 47: 659-664; and Bagshawe et al., (1991) Antibodies, Immunoconjugates & Radiopharmaceuticals 4: 915-922). The specific doses described herein or the doses applicable to the administered antibody molecules indicated in the Physician's Desk Reference (2003) can be used. For antibody molecules, the therapeutically effective amount or suitable dose of the specific binding member can be determined by comparing in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other experimental animals to humans are known.The precise dosage will depend on many factors, including the size and location of the area to be treated and the precise nature of the specific binding member.

[0517] For systemic administration, typical immunoglobulin doses range from 100 μg to 1 g, and for topical administration, typical immunoglobulin doses range from 1 μg to 1 mg. A higher loading dose may be administered initially, followed by one or more lower doses. This is a single-dose dose for adult individuals, and the dose can be adjusted proportionally for children and infants, as well as for other specific binding member formats based on molecular weight.

[0518] Treatment may be repeated daily, twice weekly, weekly or monthly intervals, at the physician's discretion. An individual's treatment regimen may depend on the pharmacokinetic and pharmacodynamic properties of the particular binding member composition, the route of administration and the nature of the disease being treated.

[0519] Treatment can be cyclical, and the time between two administrations can be about two weeks or longer, such as about three weeks or longer, about four weeks or longer, about once a month or longer, about five weeks or longer, or about six weeks or longer. For example, treatment can be every two to four weeks or every four to eight weeks. Suitable formulations and routes of administration are as described above.

[0520] In a preferred embodiment, the specific binding members described herein may be used in a method of treating cancer.

[0521] Cancer can be characterized by the abnormal proliferation of malignant cancer cells. When referring to a specific type of cancer, such as breast cancer, this refers to the abnormal proliferation of malignant cells in related tissues, such as breast tissue. Secondary cancers that are located in the breast but are the result of abnormal proliferation of malignant cells in another tissue, such as ovarian tissue, are not breast cancer as referred to herein, but rather ovarian cancer.

[0522] The cancer may be a primary or secondary cancer.Thus, the specific binding members described herein may be used in a method of treating cancer in an individual, wherein the cancer is a primary tumor and / or a tumor metastasis.

[0523] Tumors of cancers treated using the specific binding members described herein may contain TILs expressing CD137, for example, on their cell surfaces. In one embodiment, it may have been determined that the tumor contains TILs expressing CD137. Methods for determining antigen expression on the cell surface are known in the art and include, for example, flow cytometry.

[0524] For example, the cancer treated using the specific binding members described herein can be selected from the group consisting of leukemias, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma; solid cancers, such as sarcomas (e.g., soft tissue sarcomas), skin cancers (e.g., Merkel cell carcinomas), melanomas, bladder cancers (e.g., urothelial carcinomas), brain cancers (e.g., glioblastoma multiforme), breast cancers (e.g., leukemia), and ovarian cancers. Cancers of the pancreas, ovary, ovary, and uterus include: ovarian / endometrial cancer, ovarian cancer (e.g., ovarian serous cystadenoma), prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), colorectal cancer (e.g., colorectal adenocarcinoma), cervical cancer (e.g., cervical squamous cell carcinoma and cervical adenocarcinoma), liver cancer (e.g., hepatocellular carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), esophageal cancer, pancreatic cancer, kidney cancer (e.g., renal cell carcinoma), adrenal gland cancer, stomach cancer (e.g., gastric adenocarcinoma), testicular cancer, gallbladder cancer, and bile duct cancer (e.g., bile duct cancer), thyroid cancer, thymus cancer, bone cancer, and brain cancer.

[0525] In a preferred embodiment, the cancer treated using the specific binding members described herein is a solid cancer. More preferably, the cancer treated using the specific binding members described herein is a solid cancer selected from the group consisting of sarcoma, melanoma, bladder cancer, brain cancer, breast cancer, uterine / endometrial cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer, and gastric cancer.

[0526] In the case of cancer, the treatment may include inhibiting the growth of the cancer, including complete cancer remission, and / or inhibiting the metastasis of the cancer, and inhibiting the recurrence of the cancer. Cancer growth generally refers to any one of a number of indicators that indicate that the state within the cancer has progressed to a more advanced form. Thus, indicators for measuring the inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasonography or other imaging methods), a delay in tumor growth, destruction of tumor vasculature, an improvement in delayed hypersensitivity skin test performance, an increase in anti-cancer immune cell or other anti-cancer immune response activity, and a decrease in tumor-specific antigen levels. Activating or enhancing an immune response to a cancerous tumor in an individual can improve the individual's ability to resist cancer growth, particularly the growth of a cancer already present in the subject and / or reduce the tendency of cancer growth in the individual.

[0527] In the context of cancer treatment, specific binding members as described herein can be co-administered to an individual with another anti-cancer therapy or therapeutic agent (e.g., a suitable anti-cancer therapy or therapeutic agent has been shown to be, or is expected to be suitable for treating the cancer in question). For example, specific binding members can be co-administered with a chemotherapeutic agent, radiotherapy, immunotherapeutic agent, anti-tumor vaccine, oncolytic virus, adoptive cell transfer (ACT) therapy (e.g., adoptive NK cells) or chimeric antigen receptor (CAR) T cells, autologous tumor infiltrating lymphocytes (TIL) or γ / δ T cells or a therapeutic agent for hormone therapy to an individual.

[0528] Without wishing to be bound by theory, it is believed that the specific binding members described herein can act as adjuvants in anti-cancer therapy. In particular, it is believed that, for example, administering a specific binding member to an individual in combination with chemotherapy and / or radiotherapy, or in combination with an anti-tumor vaccine, will elicit a more robust immune response against the cancer than chemotherapy and / or radiotherapy alone, or the use of an anti-tumor vaccine.

[0529] The one or more chemotherapeutic agents administered in combination with the specific binding members described herein can be selected from the group consisting of taxanes, cytotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, B-Raf enzyme inhibitors, MEK inhibitors, c-MET inhibitors, VEGFR inhibitors, PDGFR inhibitors, alkylating agents, platinum analogs, nucleoside analogs, antifolates, thalidomide derivatives, anti-tumor chemotherapeutic agents, and the like. Taxanes include docetaxel, paclitaxel, and nab-paclitaxel; cytotoxic antibiotics include actinomycin, bleomycin, and anthracyclines such as doxorubicin, mitoxantrone, and valproate; tyrosine kinase inhibitors include erlotinib, gefitinib, axitinib, PLX3397, imatinib, cobemitinib, and trametinib; PARP inhibitors include piraparib; B-Raf enzyme inhibitors include vemurafenib and dabrafenib; alkylating agents include dacarbazine, cyclophosphamide, and temozolomide; platinum analogs include carboplatin, cisplatin, and oxaliplatin; nucleoside analogs include azacitidine, capecitabine, fludarabine, fluorouracil, and gemcitabine; antifolates include methotrexate and pemetrexed. Other chemotherapeutic agents suitable for use in the present invention include dafatinib, entinostat, eribulin, irinotecan, and vinblastine.

[0530] Preferred therapeutic agents for administration with the antibody molecules described herein are doxorubicin, mitoxantrone, cyclophosphamide, cisplatin, and oxaliplatin.

[0531] Radiation therapy for administration in combination with a specific binding member described herein may be external beam radiation therapy or brachytherapy.

[0532] The immunotherapeutic agent administered in combination with specific binding members as described herein can be a therapeutic antibody molecule, a nucleic acid cytokine or a cytokine-based therapy. For example, the therapeutic antibody molecule can be combined with immunomodulatory molecules, such as inhibitory checkpoint molecules or immune costimulatory molecules, or tumor antigens, such as cell surface tumor antigens or soluble tumor antigens. The example of the immunomodulatory molecules that the therapeutic antibody molecule can be combined includes CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, PD-L1, PD-1, CD47, CD73, CSF-1R, KIR, CD40, HVEM, IL-10 and CSF-1. The example of the receptor of the innate immune system that the therapeutic antibody molecule can be combined includes TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-1-like receptors (such as RIG-1 and MDA-5) and STING. The example of the tumor antigen that the therapeutic antibody molecule can be combined includes HER2, EGFR, CD20 and TGF-β.

[0533] The nucleic acid administered in combination with a specific binding member described herein may be siRNA.

[0534] The cytokine or cytokine-based therapy may be selected from the group consisting of IL-2, a prodrug conjugated to IL-2, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21, and type I interferons.

[0535] Antitumor vaccines for the treatment of cancer have been implemented in the clinic and are discussed in detail in the scientific literature (e.g., Rosenberg, S. 2000). This primarily involves strategies that use these cells as a vaccination method to prompt the immune system to respond to various cell markers expressed by autologous or allogeneic cancer cells, with or without granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF elicits a strong response in terms of antigen presentation and is particularly effective when used in conjunction with the above strategies.

[0536] The chemotherapeutic agent, radiotherapy, immunotherapy agent, anti-tumor vaccine, oncolytic virus, ACT therapy, or hormone therapy agent is preferably a chemotherapeutic agent, radiotherapy, immunotherapy agent, anti-tumor vaccine, oncolytic virus, ACT therapy, or hormone therapy agent for the cancer in question, i.e., the chemotherapeutic agent, radiotherapy, immunotherapy agent, anti-tumor vaccine, oncolytic virus, ACT therapy, or hormone therapy agent has been shown to be effective in the treatment of the cancer in question. It is entirely within the ability of a skilled person to select an appropriate chemotherapeutic agent, radiotherapy, immunotherapy agent, anti-tumor vaccine, oncolytic virus, ACT therapy, or hormone therapy agent that has been shown to be effective for the cancer.

[0537] In view of the immune response enhancing activity of CD137, it is expected that CD137 agonist molecules can be used to treat infectious diseases. Therefore, in another preferred embodiment, the antibody molecules described herein can be used to treat infectious diseases, such as acute or persistent infectious diseases.

[0538] Without wishing to be bound by theory, it is believed that CD137 agonist molecules may be able to induce the rapid infiltration and activation of innate immune cells such as neutrophils and monocytes, thereby helping to remove pathogens that cause acute infectious diseases. Therefore, in another embodiment, the antibody molecules as described herein can be used to treat acute infectious diseases, such as methods for acute bacterial diseases. In a preferred embodiment, the acute infectious disease is an acute bacterial disease caused by bacterial infection of Gram-positive bacteria such as Listeria, Streptococcus pneumoniae or Staphylococcus aureus.

[0539] Infectious diseases are usually cleared by the immune system, but some infections can persist for a long time, such as months or years, and are not effectively fought by the immune system. These infections are also called persistent or chronic infections.

[0540] Preferably, the antibody molecules described herein are used to treat persistent infectious diseases, such as persistent viral, bacterial, fungal or parasitic infections, preferably persistent viral or bacterial infections.

[0541] In a preferred embodiment, the persistent viral infection treated using the antibody molecules described herein is a persistent infection caused by human immunodeficiency virus (HIV), Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, or varicella-zoster virus.

[0542] In a preferred embodiment, the persistent bacterial infection treated using the antibody molecules described herein is a persistent infection with Staphylococcus aureus, Hemophilus influenzae, Mycobacterium tuberculosis, Mycobacterium leprae, Salmonella typhi, Helicobacter pylori, Treponema pallidum, or Streptococcus pneumoniae.

[0543] It has been described that CD137 agonism is beneficial in the context of treating Gram-positive bacterial infections. Therefore, in a preferred embodiment, the persistent bacterial infection treated using the antibody molecules described herein is a persistent infection caused by Gram-positive bacteria. In a more preferred embodiment, the persistent bacterial infection is a persistent infection caused by a Gram-positive bacteria selected from the group consisting of Staphylococcus aureus, Mycobacterium leprae, and Streptococcus pneumoniae.

[0544] In a preferred embodiment, the persistent fungal infection treated using the specific binding members described herein is a persistent infection with Candida (e.g., Candida albicans), Cryptococcus (e.g., Cryptococcus gattii or Cryptococcus neoformans), Talaromyces (e.g., Talaromyces marneffe), Microsporum (e.g., Microsporum audouinii), or Trichophyton tonsurans.

[0545] In a preferred embodiment, the persistent parasitic infection treated using the specific binding members described herein is a persistent infection with a Plasmodium (eg Plasmodium falciparum) or a Leishmania (eg Leishmania donovani).

[0546] In the treatment of a persistent infectious disease, treatment can include eliminating the infection, reducing the pathogenic load of the individual, and preventing recurrence of the infection. For example, treatment can include preventing, ameliorating, delaying, alleviating, or stopping one or more symptoms and / or signs of a persistent infection. Alternatively, treatment can include preventing an infectious disease.

[0547] In the case of treating infectious diseases, the specific binding members described herein can be administered to an individual in combination with another therapeutic agent for treating an infectious disease, such as a therapeutic agent that has been shown to be suitable or expected to be suitable for treating the infectious disease. For example, a specific binding member can be administered to an individual in combination with an immunotherapeutic agent. The immunotherapeutic agent administered in combination with the antibody molecules described herein can be a therapeutic antibody molecule. For example, the therapeutic antibody molecule can bind to a receptor of the innate immune system. Examples of receptors of the innate immune system that the therapeutic antibody molecule can bind to include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-1-like receptors (e.g., RIG-1 and MDA-5) and STING.

[0548] When a specific binding member is used to prevent an infectious disease, the specific binding member can be administered in combination with a vaccine for the pathogen. Without wishing to be bound by theory, it is believed that the specific binding members described herein can act as an adjuvant in vaccination. Specifically, it is believed that the combination of a specific binding member and a vaccine administered to an individual will elicit a more potent immune response against the pathogen than using a separate vaccine.

[0549] Other aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure, including the following experimental examples.

[0550] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0551] As used herein, "and / or" should be taken as specific disclosure that each of the two specified features or components has or does not have the other. For example, "A and / or B" would be taken as specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually listed herein.

[0552] Unless the context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0553] Other aspects and embodiments of the invention provide the above-described aspects and embodiments wherein the term "consisting of" or "consisting essentially of" replaces the term "comprising," unless the context indicates otherwise.

[0554] Certain aspects and embodiments of the present invention will now be described, by way of example, and with reference to the above-identified drawings. Example

[0555] Example 1: Production, characterization and selection of human, mouse and cynomolgus monkey antigens

[0556] 1.1 Recombinant antigens

[0557] Tumor necrosis factor receptor superfamily (TNFRSF) members are known to tend to form multimers, clustering when bound to their cognate ligands (Croft, M. 2003). This tendency to aggregate due to their function makes it challenging to produce soluble recombinant proteins that do not aggregate in solution for in vitro selection, such as phage and yeast display, and for characterization of selected proteins.

[0558] Several commercially available recombinant antigens were tested, and most were found to be unsuitable for these options due to the levels of aggregates present. Of those tested, only biotinylated human secreted CD137, hFc fusion protein (BPS Biosciences, catalog number 71171), hereinafter referred to as "hCD137-hFc-Avi-BPS", had sufficiently low aggregation to be suitable for use in the experiments, although with limited success (see Example 2).

[0559] Since most commercially available antigens were deemed unsuitable, the following recombinant dimeric and monomeric CD137 antigens (see Table 1) were produced in-house for selection:

[0560] Table 1

[0561]

[0562] Monomeric antigens were prepared by cloning DNA encoding the extracellular domain of human (SEQ ID NO: 181) or mouse CD137 (SEQ ID NO: 185) along with the Avi sequence and six C-terminal histidine residues into a pFUSE vector (Invitrogen, catalog number pfuse-mg2afc2) modified with EcoRI-HF and BamHI-HF restriction enzymes. The vectors were transfected into HEK293-6E cells (National Research Council of Canada) and expressed using HisTrap. TM The expressed CD137 was purified using an excel nickel column (GE Life Sciences 29048586) and size exclusion chromatography (SEC) to ensure that the antigen was a single species and did not contain aggregates.

[0563] To generate dimeric antigens, DNA constructs encoding the extracellular domain of human, mouse, or cynomolgus monkey CD137 fused to the mIgG2a Fc domain with Avi sequences were cloned into a modified pFUSE vector and transfected into HEK293-6E cells. TM Recombinant CD137 was purified using a protein A column (GE Healthcare, 11003494) and size exclusion chromatography (SEC) to ensure that the antigen was a single species and did not contain aggregates.

[0564] Each dimeric and monomeric antigen was biotinylated using the BirA biotin-biotin protein ligase reaction kit (Avidity LLC, BirA500) to produce monomeric CD137 antigens labeled with a single biotin molecule and dimeric CD137 antigens labeled with two biotin molecules, one on each of the two monomers. 3 mg of antigen was mixed with 7.8 μl of BirA enzyme mix to give an enzyme-to-substrate molar ratio of 1:50. Additives (142 μl Biomix A, 142 μl Biomix B, 142 μl biotin) were then added according to the manufacturer's recommendations, and the reaction mixture was incubated at room temperature for 2 hours. The reaction mixture was immediately buffer-exchanged into DPBS (Life Technologies 14190-169) using an Amicon 30 μm filter (Merck Millipore UFC503096).

[0565] The protein was further purified by SEC to ensure the removal of the BirA enzyme and to produce a high-quality, monodisperse protein formulation free of high molecular weight aggregates. In more detail, materials from the same production batch were mixed together and analyzed for stability and purity by size exclusion high performance liquid chromatography (SE-HPLC), SDS polyacrylamide gel electrophoresis (SDS-PAGE), and size exclusion chromatography multi-angle light scattering (SEC-MALS). Complete biotinylation of the protein was confirmed in a streptavidin-migrated SDS-PAGE gel. Recombinant human and mouse antigens were confirmed to bind in vitro to anti-CD137 positive control antibodies (20H4.9 (U.S. Patent No. 7288638) and Lob12.3 (University of Southampton)) by surface plasmon resonance (SPR), and binding to DO11.10 cells expressing human and mouse CD137 ligands was confirmed by flow cytometry. Cells were incubated with CD137 antigen for 1 hour, and cell binding was detected using a fluorescently labeled anti-mouse Fc fragment antibody. As described above, binding of the recombinant cynomolgus monkey antigen to DO11.10 cells expressing the cynomolgus monkey CD137 ligand (National Jewish Health Center) was confirmed by flow cytometry. To ensure the highest possible purity of the material used in the selection protocol, the antigen was subjected to a thorough protein characterization to ensure that no more than 2% protein aggregates were present.

[0566] 1.2 Cell-expressed antigens

[0567] DO11.10 cells (National Jewish Health) expressing full-length mouse CD137 (SEQ ID NO: 184) or human CD137 (SEQ ID NO: 180), designated "DO11.10.mCD137" and "DO11.10.hCD137," respectively, were generated to present antigen in a membrane-bound conformation, most similar to its native form, for selection and further characterization of selected Fcabs, as listed in Table 2.

[0568] These DO11.10 cells overexpressing human or mouse CD137 receptors were generated using lentiviral transduction using the Lenti-X HTX packaging system (Takara, catalog number 631249). Lenti-X expression vector (pLVX) (Takara, catalog number 631253) containing cDNA encoding human CD137 (SEQ ID NO: 180) or mouse CD137 (SEQ ID NO: 184) was co-transfected with the Lenti-X HTX packaging mixture into the Lenti-X293T cell line (Takara, catalog number 632180) to produce virus. These lentiviral vectors were then used to transduce the DO11.10 cell line.

[0569] The expression of human CD137 or mouse CD137 on these cells was confirmed by flow cytometry by binding 20H4.9 and Lob12.3 anti-CD137 positive control antibodies to the cells, respectively. Cells were incubated with human or mouse positive control antibodies for 1 hour, and cell binding was detected using a fluorescently labeled anti-human Fc detection antibody (Stratech Technologies, Inc., catalog number 109-546-098-JIR).

[0570] DO11.10 cells expressing cynomolgus macaque CD137 were also generated using the same lentiviral transduction method, designated "DO11.10.cCD137", and used to test the cross-reactivity of anti-human CD137 Fcab with cynomolgus macaque CD137. As previously described, expression of cynomolgus macaque CD137 was confirmed by flow cytometry by binding an anti-CD137 positive control antibody (MOR_7480.1, US2012 / 0237498 A1) to the cells.

[0571] Table 2

[0572] type name species introduce cell DO11.10.hCD137 people Cell expression cell DO11.10.mCD137 mice Cell expression cell DO11.10.cCD137 cynomolgus macaque Cell expression

[0573] Example 2: Preliminary selection of anti-human CD137 antibodies

[0574] To find Fcabs that bind to human CD137 and to maximize the diversity of identified binders, yeast and phage display selection activities were employed. Since CD137 is expressed at low levels in many non-immune cell types, it was decided to select anti-human CD137 Fcabs that selectively target cells that express a large amount of CD137, such as activated T cells. Without wishing to be bound by theory, it is speculated that cells with very low or negligible CD137 expression levels are more likely to have CD137 in a monomeric state on their cell surface, in contrast to activated T cells, which have highly upregulated CD137 expression, where most of the protein is expected to be present on the cell surface in a dimer, trimer, or higher multimer state.

[0575] Cells overexpressing CD137 or recombinant dimeric human CD137 protein were used in Fcab selection to expose representative epitopes that promote binding interactions with cells that highly upregulate multimeric CD137. In addition, the use of dimeric antigens is thought to favor the selection of bivalent Fcabs that bind tightly to CD137 and remain bound to the target for a longer period of time, which in turn is thought to favor preferential binding to cells with upregulated CD137 expression levels. In this case, monomeric recombinant CD137 was not used to prevent the selection of very high-affinity monovalent Fcab binders that might bind CD137 too strongly.

[0576] The goal of this selection strategy is to obtain Fcabs that preferentially bind to activated T cells and that do not bind well to naive T cells that display only monomeric CD137 or other cells that express very low levels of CD 137. By selecting CD137 Fcabs that preferentially bind divalently to dimeric and multimeric antigens relative to monomeric antigens, it is believed that potential off-target T cell activation will be reduced, with a concomitant reduction in toxicity.

[0577] Phage display

[0578] Six primary phage libraries displaying the human IgG1 CH3 domain were used for selection via phage display. All six libraries contained a randomized AB loop (containing residues at positions 14-18 according to IMGT numbering) and a randomized EF loop (containing residues at positions 92-101 according to IMGT numbering). One library contained clones with two or four amino acids (encoded by two or four NNK codons) inserted at position 101 of the EF loop (residues inserted at positions 101.4–101.1 according to IMGT numbering).

[0579] A total of 12 selection campaigns were conducted to identify anti-human CD137 binders. Six phage libraries were used, and selections were performed using either in-house hCD137-mFc-Avi antigen or commercially available hCD137-hFc-Avi-BPS antigen and / or DO11.10.hCD137 antigen-expressing cells. Because only one functional Fcab sequence was identified from early selections using the hCD137-hFc-Avi-BPS recombinant antigen, in-house hCD137-mFc-Avi was used for the remainder of the selections with recombinant CD137. Initial rounds were performed using 100 nM biotinylated antigen, and deselection steps were performed using 500 nM unlabeled recombinant human Fc fragments produced in-house. Phage binders were captured using magnetic beads coated with streptavidin or neutravidin. For the selected outputs, non-specific binding to Fc fragments was detected by phage ELISA (A450-630nm above 0.4), and a further deselection step was introduced, whereby biotinylated Fc fragments (produced in-house) were incubated with the phage outputs. Non-Fc binders were separated from Fc binders by magnetic capture of the biotinylated Fc-binding phage.

[0580] To find binders to the membrane-bound native conformation of CD137, cell selection was performed as follows: phage output of the recombinant antigen selection was incubated with DO11.10 cells lacking human CD137 to remove unwanted binders, such as those that bind nonspecifically to cells. Next, phage were incubated with 1×10 7DO11.10.hCD137 cells were incubated, the conjugates were eluted by trypsinization, and then the next round of propagation was performed. The third round followed a similar process by reducing the number of DO11.10.hCD137 cells to 5 × 10 6 to increase selection pressure.

[0581] Phage ELISA was performed after each round of selection to determine the enrichment of antigen-specific phage. 96-well streptavidin plates were coated overnight with 1 μg / ml biotinylated antigen. After blocking the plates with 4% PBS, 50 μl of bacterial supernatant containing phage was added to each well and incubated with shaking at 450 rpm for 1 hour at room temperature. The phage solution was removed by inverting the plates and washing 4 times with PBS containing 0.1% Tween and 4 times with PBS. Anti-M13 phage-HRP conjugated antibody was then added to detect phage bound to the immobilized antigen. The phage solution was removed by inverting the plates and washing 4 times with PBS containing 0.1% Tween and 4 times with PBS. TMB microwell peroxidase solution was added to each well and color was developed over a period of 30 minutes. The reaction was stopped with 1 M sulfuric acid and the plate was counted in a microtiter plate reader at OD 450-630 Specific hits were defined as those that exhibited a signal intensity at least 4-fold higher than background (defined as negative control, i.e., PBS or negative non-binding phage, e.g., wild-type CH3) for recombinant CD137 and at least 10-fold higher than binding to biotinylated recombinant Fc fragments.

[0582] Phage fluorescence activated cell sorting (phage FACS) analysis was performed on the cell selection outputs of rounds 2 and 3. Briefly, 2×10 5 The cells were transferred to a round-bottom microtiter plate. Phage supernatant was added to the cells and incubated at 4°C for 1 hour. The cells were then washed twice in ice-cold 2% BSA in PBS buffer, resuspended in 100 μl of a solution of anti-M13 antibody and FITC-conjugated goat anti-mouse IgG F(ab') fragment and incubated with the cells for 1 hour. After washing three times in PBS, the cells were analyzed in a flow cytometer. In phage FACS, a specific hit was defined as a signal with a geometric mean fluorescence intensity (MFI) presented to CD137-positive cells that was at least 10 times higher than the binding signal of recombinant CD137 and at least 4 times higher than the background (as defined by the negative control (PBS)).

[0583] 3230 phage clones were screened for binding to dimeric recombinant hCD137 antigen by phage ELISA, and recombinant Fc was used as a negative control. 1140 phage clones were screened for specific binding to DO11.10.hCD137 cells by phage FACS. Individual hits were then sequenced, and the resulting 76 unique sequences were assigned Fcab clone identifiers and subcloned into the pTT5 expression vector (National Research Council of Canada) containing the HelD1.3 IgG1 heavy chain expression cassette for in vivo mAb expression. 2 The Fcab clones were in the form of (see Example 3.1).

[0584] Yeast display

[0585] Four original yeast libraries displaying the CH1 to CH3 domains of human IgG1 were used for selection by yeast display. All four libraries contained a randomized AB loop (containing residues at positions 14 to 18 according to IMGT numbering) and a randomized EF loop (containing residues at positions 92 to 101 according to IMGT numbering) in the CH3 domain. Two libraries also contained a five-amino acid residue insertion at position 16 in the AB loop of the CH3 domain (residues at positions 16.5 to 16.1 according to IMGT numbering).

[0586] In-house hCD137-mFc-Avi antigen or commercially available hCD137-hFc-Avi-BPS antigen was used for selection against four yeast libraries. Although similar to phage selection, commercially available antigen was not used after the early selection, but in-house antigen was used. For each library, magnetic cell separation (MACS) was used for the first round of selection. After a deselection step with 1.25 μM unlabeled human or mouse Fc, the library was grown, induced, and 1×10 10Cells were incubated with 250 or 300 nM biotinylated recombinant antigen. Yeast binders were isolated by adding streptavidin magnetic beads and using MACS LS columns (Miltenyi Biotech 130-042-401). Subsequent selection was performed by fluorescence-activated cell sorting (FACS) on a FACS-Aria II instrument (BD Bioscience), and bound antigen was detected by using fluorescently labeled antibodies (anti-biotin-APC (Miltenyi Biotech 130-090-856), streptavidin-APC (BD Bioscience 349024) or neutravidin-DyLight-488 (Thermo Fisher Scientific 22832), correctly folded IgG scaffolds (anti-human IgG CH2 domain-FITC (Biorad AbDSerotec (MCA647F) or expression of aga2-IgG constructs (anti-Xpress (Life Technologies R91025) and anti-mouse IgG-FITC (Sigma F2653-.5ML)). Whenever possible, antigen markers were used in conjunction with a structural marker to normalize the binding intensity signal by Fcab expression on the yeast surface. Unstained and control yeast populations were used to establish the following sorting gates: yeast cells in FSC-A and SSC-A plots, yeast in FSC-W and FSC-H plots to distinguish multiplying or budding yeast cells, and FITC-A and APC-A plots to detect bound Fcab double positives.

[0587] The antigen concentration used in each round was determined empirically by performing output quality control using the antigen concentration used in the previous round. If binding enrichment increased (>5-fold) in the previous round, the antigen concentration was decreased by 1:2 or 1:3, otherwise it was kept constant. This process was also followed to determine how many rounds of selection were needed and whether the selection branch was considered exhausted (if diversity decreased to only a few dominant sequences, or if there was no increase in antigen binding after two rounds).

[0588] 2784 yeast monoclones were identified from the library selection and screened as follows: After each round of selection, single yeast cells were spotted on SDCAA agar plates using a FACS-Aria II (BD Biosciences) instrument and screened in the following flow cytometry antigen binding assay: monoclonal colonies were grown until the colonies reached a diameter of 2 mm and then transferred to deep-well plates containing 600 μl SDCAA liquid medium. The cultures were grown overnight at 30°C with shaking at 1000 rpm. The expression of the aga2-IgG protein scaffold was induced by replacing the growth medium with an induction medium SGRCAA at an optical density of 1 (OD600=1). The cells were incubated with biotinylated recombinant dimeric human antigens or mouse Fc fragments to distinguish them from yeast clones that bound to the Fc portion of the recombinant hCD137 antigen. Yeast cells that bound to both proteins were labeled using streptavidin-APC. Anti-CH2-FITC antibody was also used as a structural IgG marker. To analyze the results of the screen, binding to the Fc fragment was plotted and any clones that bound to the biotinylated Fc were removed. Binding was defined as cells with greater than 0.2% APC fluorescence-positive cells, and a gate was placed using unstained and negative control samples.

[0589] Selection was repeated at different antigen concentrations and conditions, such as increasing the induction temperature, decreasing the selection stringency, or reducing the number of rounds, to increase the number of hits. Sequencing of the hits showed fairly low output diversity, with only nine Fcab clones identified with unique sequences: FS22-053, FS22-172, FS22-173, FS22-174, FS22-175, FS22-176, FS22-177, FS22-178, and FS22-179.

[0590] Example 3: Characterization of anti-human CD137 Fcabs from the initial selection

[0591] 3.1 Preparation of “mock” mAbs 2 Anti-human CD137 Fcab

[0592] Producing a "mimetic" mAb composed of IgG1 molecules 2 Antibodies, including 76 anti-human CD137 Fcab clones isolated from phage and 9 clones isolated from yeast selection, were expressed as mAb 2 Format characterization of Fcab. A mimetic mAb was prepared by replacing part of the CH3 domain of Fcab, including the AB, CD, and EF loops, with the corresponding region of the CH3 domain of the anti-hen egg lysozyme antibody HelD1.3. 2The generation of HelD1.3 antibody is described in Tello et al., 1993. The heavy and light chain sequences of antibody HelD1.3 are shown in SEQ ID 186 and 173, respectively. 2 The molecules were produced by transient expression in HEK293-6E cells. To assess the amount of protein produced, IgG protein content was quantified by biolayer interferometry using an Octall QKe platform from PALL with a Protein A quantitative biosensor (18-5021). The protein was purified by Protein A affinity chromatography using mAbSelectSure columns. 53 phage-derived CD137 mAbs 2 The protein detection results were below the detection threshold and therefore were considered unsuitable for further analysis. 32 mAbs were purified using mAb Select SuRe Protein A columns (GE Healthcare, 11003494). 2 : FS22-005, FS22-007, FS22-033, FS22-042, FS22-049, FS22-050, FS22-052, FS22-053 , FS22-054, FS22-167, FS22-169, FS22-170, FS22-171, FS22-172, FS22-173, FS22-174 , FS22-175, FS22-176, FS22-177, FS22-178, FS22-179, FS22-180, FS22-181, FS22-183 , FS22-184, FS22-186, FS22-187, FS22-191, FS22-192, FS22-193, FS22-194, FS22-195.

[0593] To compare expression levels, some early Fcabs were also subcloned and expressed as soluble Fcabs (containing a truncated hinge) in HEK293-6E cells and purified using a mAb Select SuRe Protein A column. 2 The results showed that the biophysical behavior and yield of the soluble Fcab were significantly better than those observed with the soluble Fcab. This was the case with the original clone FS22-053, which was produced in very low yield as a soluble Fcab but showed good yield when expressed as a mimetic mAb. 2 When expressed in the same manner, this clone was increased 25-fold, resulting in more clones available for characterization.

[0594] 3.2 Binding to recombinant antigens by BLI

[0595] In single-site binding experiments, 31 purified mock mAbs were tested by bio-layer interferometry using the Octet QKe platform. 2 Binding of molecules (excluding clone FS22-175) to human recombinant antigens. Biotinylated hCD137-mFc-Avi antigen was captured at 10 μg / ml in kinetic buffer (PALL) using a streptavidin BLI biosensor (PALL 18-5021). The sensor was then incubated with purified mAb diluted 1:1 in the same kinetic buffer. 2 The cells were immersed in wells containing 1x kinetic buffer for 240 seconds and then immersed in wells containing 1x kinetic buffer for 240 seconds. Binding hits were classified according to a simple Boolean yes / no criterion defined by the BLI response of the above buffer and the wild-type IgG1 control HelD1.3 mAb (G1 / HelD1.3): 12 mAbs 2 None bound, while 19 bound to CD137-coated sensors (FS22-007, FS22-033, FS22-042, FS22-049, FS22-050, FS22-052, FS22-053, FS22-054, FS22-169, FS22-172, FS22-173, FS22-174, FS22-179, FS22-180, FS22-181, FS22-183, FS22-187, FS22-194, FS22-195).

[0596] 3.3 Selected anti-CD137 mimetic mAbs 2 Activity in human NF-κB reporter gene assays

[0597] TNFR signaling requires multimerization and aggregation (Bitra et al., 2017). When CD137 interacts with its cognate ligand, CD137L, it aggregates and activates the NF-κB signaling pathway. Agonist molecules mimic the ligand in driving CD137 aggregation and activation, thereby activating the NF-κB signaling pathway. Some agonistic antibodies are known to inherently induce CD137 aggregation upon binding, such as utomilumab, while others require additional cross-linking of the antibody itself to induce CD137 aggregation, such as utomilumab (Fisher et al., 2012). Fcγ receptors on effector cells are known to induce this cross-linking in vivo, although this cross-linking is inefficient and may occur outside the therapeutic target site. Because dose-limiting toxicities have been associated with treatment with some anti-CD137 antibodies, it was decided to select anti-CD137 binding FcAbs that do not possess inherent agonistic capabilities, selecting only those that require additional cross-linking to induce CD137 aggregation. Therefore, an assay was developed that can detect activation of the NF-κB signaling pathway in cells when CD137 expressed on the cell surface is aggregated by a cross-linked antibody, but shows minimal activity when the antibody is not cross-linked. This method was then used to test a mimetic mAb 2 27 anti-CD137 Fcab clones and 6 anti-CD20 mAbs in the form of 2 The agonist functional activity of anti-CD137 Fcab clones was determined independently of whether the Fcab bound the recombinant antigen by BLI.

[0598] In the test, protein L was used as a cross-linker to drive the mimetic mAb 2 Cross-linking was performed via its Fab portion, and NF-κB activation was measured.

[0599] The cDNA encoding human CD137 (SEQ ID 180) was subcloned into a pMSCV-neomycin vector (Takara Clontech, catalog number (Cat. No.) 634401) using EcoRI-HF and XhoI restriction enzymes. RetroPack PT67 cell line (Clontech, catalog number (Cat. No.) 631510) was used to produce retroviral particles according to the manufacturer's protocol. The retrovirus was subsequently used to transduce the HEK.FRT.luc cells previously produced by Qiagen Cignal Lenti NFkBReporter (luc) (Qiagen, catalog number (Cat. No.) 336851) slow virus transduction Flp-In T-REx 293HEK cell line (Life Technologies, R780-07) containing the NF-κB sensitive promoter for controlling luciferase expression. These HEK.FRT.luc.hCD137 cells were used to screen for the simulation mAb containing CD137 binding substances identified in the selection.2 .

[0600] Each mock mAb was prepared in DPBS (Life Technologies, 14190169). 2 The 2 μM dilutions of the 500 μg / ml IgG antibodies were prepared and diluted 1:3 in reporter cell culture medium (DMEM (Gibco, catalog no. 61965-026); 10% FCS (Gibco, catalog no. 10270-106); 1x penicillin-streptomycin (Gibco, catalog no. 15140-122); elastin 15 μg / ml (Melford Laboratories Ltd., catalog no. B1105); puromycin 5 μg / ml (Life Technologies, catalog no. A11113803); bleomycin 100 μg / ml (InvivGen, catalog no. 11006-33-0); geneticin 500 μg / ml (Life Technologies, catalog no. 10131-027). Protein L (Life Technologies, 21189) was used as an artificial cross-linker and was covalently coupled to the mAb at a molar ratio of 1:4. 2 After 24 h of incubation, the cells were mixed with 100 μl of Promega Bio-Glo TM Cells were treated with luciferase assay reagent (Promega catalog number G7941) and luminescence was measured on a microplate reader using Gen5 software (BioTek) with an integration time of 0.5 seconds. Luminescence is a measure of luciferase activity, which is produced in response to cross-linked Fcab-induced CD137 aggregation and activation of the NF-κB signaling pathway. Luminescence values ​​were plotted against the logarithmic concentration of Fcab, and the resulting curves were fitted using the logarithmic (agonist) relative response equation in GraphPad Prism.

[0601] Hits were identified by demonstrating at least a 10-fold increase in luciferase signal upon cross-linking with Protein L compared to uncross-linked clones. These clones were determined to be capable of inducing CD137 aggregation and subsequent activation of downstream signaling pathways. Of all clones tested, two were able to induce a 10-fold increase in luciferase upon cross-linking, FS22-053 and FS22-172, although an EC50 could not be determined for either. Both were selected for further characterization in a DO11.10 T cell activation assay. Unexpectedly, despite binding to the CD137 target by BLI, no activity was observed for the remaining clones under cross-linking conditions, potentially indicating that they bind to an unrelated epitope of CD137 or that these clones do not bind CD137 with sufficient affinity to initiate the NF-κB signaling cascade.

[0602] Overall, although more than 30 Fcabs were tested, only two Fcabs (FS22-053 and FS22-172) were identified from the initial selection that exhibited the desired function in the NF-κB reporter gene assay when cross-linked but had little activity when not cross-linked.

[0603] 3.4 Selected anti-CD137 mimetic mAbs 2 Activity in DO11.10 T cell activation assay

[0604] CD137 aggregation by agonist molecules on activated T cells triggers T cell activation and downstream signaling, leading to, but not limited to, IL-2 production. Since FS22-053 and FS22-172 were identified as active in an NFKB reporter gene assay, their ability to activate CD137 was tested in a T cell activation assay. A DO11.10 T cell activation assay was developed using DO11.10 T cells engineered to overexpress human CD137, and T cell activation was assessed by measuring IL-2 release.

[0605] DO11.10 T cells (National Jewish Health) were transduced with lentiviral vectors designed to overexpress mouse or human CD137 as described previously. As well as FS22-053 and FS22-172, the following clones were tested in this DO11.10 T cell activation assay: FS22-007, FS22-033, FS22-042, FS22-049, FS22-050, FS22-052, FS22-054 (all with “mock” HelD1.3 mAb) 2 Preparation of mAbs with or without recombinant protein L (Life Technologies, 21189) cross-linker 2 Or dilution of 20H4.9 positive control mAb and add it to DO11.10.hCD137 cells in 96-well round-bottom plates coated with 0.1 μg / ml anti-CD3 antibody (clone 17A2, BioLegend, 100208) overnight. After incubation for 18 hours, supernatant was collected according to the manufacturer's instructions and analyzed with mouse IL-2 ELISA kit (eBioscience, 88-7024-86). Plates were read at 450nm using a microplate reader with Gens software (BioTek). The absorbance at 570nm was subtracted (corrected) from the absorbance at 450nm. The standard curve for calculating cytokine concentrations was based on four-parameter logarithmic curve fitting (Gens software, BioTek). The concentration of mlL-2 was plotted relative to mAb. 2 The logarithmic concentrations of agonist or benchmark mAb were plotted and the resulting curves were fitted using the log (agonist) relative response equation in GraphPad Prism.

[0606] When cross-linked with protein L in this assay, clones FS22-053 and FS22-172 showed significantly enhanced activity. The activity of FS22-053 when not cross-linked was 126 nM and the activity when cross-linked was 21 nM (a 6-fold increase), while the activity of FS22-172 when not cross-linked was 950 nM and the activity when cross-linked was 44 nM (a 22-fold increase). As a result, both clones were selected for affinity maturation. In addition, although clone FS22-033 had no activity in this assay, it was also selected for affinity maturation because it may bind to a different epitope than the recombinant antigen, as it did not compete with clones FS22-053 and FS22-172 for binding in the BLI binding assay (data not shown). It is believed that the improvement in the binding affinity of the clones to CD137 may also lead to an improvement in functional activity (see Example 4.1).

[0607] 3.5 CD137 / CD20 mAb 2 Preparation of anti-human CD137 Fcab

[0608] A panel of anti-CD137 Fcabs (FS22-053, FS22-175, FS22-176, FS22-177, FS22-178, FS22-179) was prepared to identify mAbs with higher biological relevance. 2 Format Characterization Fcab. mAb was prepared by replacing the partial CH3 domain Fcab containing the AB, CD, and EF loops with the corresponding region of the CH3 domain of the anti-CD20 2F2 clone (from US 8,529,902 B2). 2 , to produce mAbs that bind to both CD137 and CD20 2 These CD137 / CD20 mAbs 2 It was produced by transient expression in HEK293-6E cells and purified using a mAb Select SuRe Protein A column.

[0609] 3.6 Anti-CD137 / CD20 mAb 2 Activity in DO11.10 T cell activation assay

[0610] Example 3.4 describes a T cell activation assay in which protein L is used to mimic the anti-CD137 mAb 2 Cross-linking. This setup provides a reliable and reproducible way to screen a large number of molecules, although when antibodies and mAbs 2 When higher order structures are formed, cross-linking is suboptimal and not representative of the physiological environment. A more biologically relevant setting is one in which mAbs 2A setup where molecules are cross-linked via their Fab arms to bind to targets present in biological systems. This cell-based in vitro system allows for the optimization of antibodies and mAbs via Fab binding to cell membranes. 2 presentation, thereby driving antibody aggregation and subsequently increasing the affinity of the CD137 Fcab arm to its target on T cells.

[0611] CD20 + Daudi cells (ATCC CCL-213) were seeded in a 96-well round-bottom plate at a 1:1 ratio with the DO11.10.hCD137 cells used in Example 3.4. The six CD137 / CD20 mAbs generated in Example 3.5 were tested in this DO11.10 T cell activation assay. 2 Preparation of mAbs with or without recombinant protein L (Life Technologies, 21189) cross-linker 2 Dilutions of the positive control mAb were added to DO11.10.hCD137 and Daudi cells in 96-well round-bottom plates coated overnight with 0.1 μg / ml anti-CD3 antibody (clone 17A2, BioLegend, 100208). After 18 hours of incubation, the supernatant was collected according to the manufacturer's instructions and analyzed using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86). The plate was read at 450 nm using a microplate reader with Gens software (BioTek). The absorbance at 570 nm was subtracted from the absorbance at 450 nm (correction). The standard curve for calculating cytokine concentrations was based on a four-parameter logarithmic curve fit (Gens software, BioTek). The concentration of mlL-2 was plotted relative to mAb. 2 The logarithmic concentrations of the agonist or benchmark mAb were plotted and the resulting curves were fitted in GraphPad Prism using the logarithmic (agonist) relative response equation. Of all clones tested, FS22-053 was the only clone that showed significant IL-2 production in this CD20 cell-based cross-linking system, with an EC50 of 0.3 nM (126 nM in the absence of CD20+ cells).

[0612] Examples 3.4 and 3.6 show that when the tested mAb is bound by protein L or by Fab 2 Upon cell-based cross-linking of another target in the form of HeID1.3 and CD20, FS22-053 can drive the aggregation and activation of CD137 on the surface of DO11.10 T cells.

[0613] Example 4: Affinity maturation of anti-human Fcab

[0614] As previously described, three clones were selected for affinity maturation based on their functional properties in NF-κB reporter assays, DO11.10 T cell activation assays (FS22-053 and FS22-172), or thought to bind to different regions of CD137 (FS22-033).

[0615] 4.1 Affinity Maturation of FS22-033

[0616] Two yeast and two phage display libraries were constructed from clone FS22-033: one by randomizing five residues in the AB loop of the CH3 domain and the other by randomizing five residues in the EF loop of the CH3 domain using ELLA primers. ELLA primers specify the codon used for each amino acid and its relative abundance in the mixture. Only cysteine ​​was excluded from the mixture, and no other amino acids were biased.

[0617] For the phage FS22-033 AB and FS22-033 EF libraries, two rounds of selection were performed for affinity-matured clones using 200 nM hCD137-mFc-Avi in ​​round 1 and 10 nM hCD137-mFc-Avi in ​​round 2. 96 clones from each selection were screened by phage ELISA. This screen identified 24 unique clones (FS22-033-001 to FS22-033-024), all of which were expressed with a HelD1.3-containing mock mAb. 2 24 affinity-matured clones performed better than the parental FS22-033 clone in this assay and were therefore not studied. For the yeast FS22-033 AB and FS22-033 EF libraries, three rounds of selection were performed as follows: 1st and 2nd rounds of selection were performed with 300 nM hCD137-mFc-Avi, followed by a third round of selection with 300 nM hCD137-mFc-Avi, 300 nM cynomolgus monkey CD137-mFc-Avi, or 300 nM hCD137-Avi-his. 1056 clones from rounds 2 and 3 output were sequenced and all identified unique clones were screened in antigen binding flow cytometry analysis using 30 nM human dimer recombinant antigen for improved binding to antigen compared to the parental FS22-033 clone and ranked by the percentage of APC+ cells that correlated with the strength of binding. The best five clones (FS22-033-025, FS22-033-026, FS22-033-027, FS22-033-028, FS22-033-029) were then used as HelD1.3 mAbs 2FS22-033 clones were produced (as described in Example 3.1) and tested for binding to human dimeric recombinant antigen by BLI as described previously, but none of the clones showed improved kinetics relative to the parental FS22-033 clone and therefore these clones were not studied further.

[0618] 4.2 Affinity Maturation of FS22-053 and FS22-172

[0619] Four yeast display libraries were constructed from the FS22-053 and FS22-172 Fcab clones. Libraries FS22-053 AB and FS22-172 AB were generated by randomizing seven residues (positions 15-16.1 according to IMGT) in the AB loop of the CH3 domain of each clone using ELLA primers with the same trinucleotide distribution as described in Example 4.1. Five residues (positions 92-94 and 97-98 according to IMGT) were randomized in the EF loop of the CH3 domain using ELLA primers to generate libraries FS22-053EF and FS22-172 EF.

[0620] For libraries FS22-053 AB and FS22-053 EF, as well as FS22-172 AB and FS22-172 EF, three or four rounds of selection were performed in yeast using either dimeric hCD137-mFc-Avi or monomeric hCD137-Avi-His antigens to affinity mature clones. Monomeric and dimeric antigens were used alternately to ensure that clones retained affinity for the antigen and did not bind exclusively through affinity. The use of monomeric or dimeric antigens, as well as the concentrations used, were determined empirically in each round by flow cytometry, based on whether enrichment for the monomeric or dimeric antigens was observed in the previous round. Whenever possible, a higher sorting gate than the parental molecule was used to isolate affinity-matured clones. Selection pressure was increased to 1 nM for the dimeric antigen. Individual clones were spotted on agar plates during each selection round to assess selection progress. Each clone was grown and induced individually, and its binding and structural parameters were determined by flow cytometry using biotinylated dimeric antigen and anti-CH2 structural markers as described previously. This screening cascade was followed to allow determination of selection success based on a sample of clones from the selection output and to allow individual clones from early screening to be subsequently produced as soluble protein.

[0621] As previously described, a total of 1,152 yeast monoclonal clones were screened for binding to biotinylated recombinant antigen by antigen-binding flow cytometry. Selection of the FS22-053 EF library resulted in the enrichment of 138 unique loop sequences. Similarly, 30 unique loop sequences were isolated from the FS22-172 AB library. Libraries FS22-053 AB and FS22-172 EF did not contain any clones that showed any improvement in binding compared to the parental clones. Sequence analysis of the best-binding clones from the FS22-053 EF and FS22-172 AB libraries revealed a conserved PPY sequence pattern in the AB loop. Because this sequence is conserved after affinity maturation and was independently selected in two separate FcAb lineages, it is likely important for epitope binding on CD137. Furthermore, a conserved LE or LD sequence pattern was located in the EF loop of the CH3 domain of clones of the FS22-053 and FS22-172 lineages, suggesting that this amino acid motif in the EF loop is essential for improved binding.

[0622] To assess the progress of the selection and whether it was necessary to recombine the mutated AB and EF loops between affinity-matured clones, the top five unique clones of the FS22-053 EF library (FS22-053-008, FS22-053-009, FS22-053-010, FS22-053-011, FS22-053-012) were ranked by demonstrating specific binding to 10 nM dimeric human antigen (above 30% APC-positive cells in a flow cytometric binding assay), FS22-172 The first six unique clones of the AB library (FS22-172-001, FS22-172-002, FS22-172-003, FS22-172-004, FS22-172-005, FS22-172-006, all showed greater than 10% APC-positive cells when screened with 10 nM dimeric human antigen in the same experiment) were used to simulate mAb 2 (HelD1.3) and mimetic mAbs 2 (PD-L1)mAb 2 generated to evaluate the functional and kinetic improvements of randomized rings.

[0623] Example 5: "Mock" mAb 2 Construction, expression, and characterization of an affinity-matured anti-human CD137 Fcab

[0624] 5.1 “Simulated” and Model mAbs 2 Construction of anti-human CD137 Fcab

[0625] Sixteen affinity mature clones derived from the parental FS22-053 clone (FS22-053-001 to FS22-053-016) and six clones derived from the parental FS22-172 clone (FS22-172-001 to FS22-172-006) were used as mAbs. 2 Clones FS22-053-001 to FS22-053-007 were not studied further because they did not function as mAbs. 2 The remaining clones were found to have at least 95% sequence identity in their CH3 domains compared to the CH3 sequence derived from the parental clone. A percent sequence similarity matrix was generated by comparing each amino acid position to the reference sequence (parental clone FS22-053 or FS22-172). Figure 1D Shown are the percentage sequence identities of the CH3 domains of clones FS22-053-008 to FS22-053-016 and FS22-053-017 (see Example 10.1) compared to the parental FS22-053 CH3 domain. Figure 1E Shown are the percentage sequence identities of the CH3 domains of clones FS22-172-001 to FS22-172-006 compared to the parental FS22-053 CH3 domain.

[0626] A "mock" mAb containing anti-human CD137 Fcab in HelD1.3 was prepared 2 Antibodies to further characterize mAbs 2 These mAbs were prepared as described in Example 3.1. 2 .

[0627] mAb was also produced 2 Models comprising anti-human CD137 Fcab and PD-L1 binding Fab region (clone YW243.55.S70 from US 8,217,149B2). They were prepared similarly to the method described in Example 3.1 by replacing a portion of the CH3 of the anti-PD-L1 binding antibody containing the AB, CD and EF loops with the corresponding region of the Fcab. These PD-L1 type mAbs 2 The LALA mutation is included in the CH2 domain (AA). Introduction of the LALA mutation into the CH2 domain of human IgG1 is known to reduce Fcγ receptor binding (Bruhns, P. et al., (2009) and Hezareh M. et al., (2001)).

[0628] CD137 / HelD1.3 and CD137-AA / PD-L1 mAbs were produced by transient expression in HEK293-6E cells 2 and purified using a mAb Select SuRe Protein A column.

[0629] 5.2 Mimicking mAbs in human NF-κB reporter cell assays 2 Human Fcab activity

[0630] The mock mAbs listed in Table 3 were tested in the same NF-κB luciferase assay as described in Example 3.3. 2 Functional activity of the affinity-matured anti-human CD137 Fcab (HelD1.3) was measured using Gen5 software (BioTek) in a microplate reader with an integration time of 0.5 seconds. The results of this assay are shown in Table 3 and Figure 2 As expected, no Fcab showed activity without Protein L crosslinking (-XL). All affinity-matured CD137 Fcabs showed substantial improvement compared to the parental CD137 Fcab, however, although positive in this assay, EC could not be calculated. 50 When cross-linked with Protein L (+XL), FS22-053-008 and FS22-172-003 showed the best activity, with EC values ​​of each family 50 lowest.

[0631] Table 3

[0632]

[0633]

[0634] N / A - Not applicable, EC50 cannot be determined due to low signal

[0635] 5.3 Affinity maturation of mimetic mAbs in human DO11.10 T cell activation assays 2 Activity of human Fcab

[0636] The affinity matured analog mAbs listed in Table 4 2 The functional activity of a human Fcab (PD-L1 LALA) in the form of FLAG-ELISA was tested in a DO11.10 T cell activation assay, similar to the assay described in Example 3.6.

[0637] HEK.mPD-L1 cells were generated by subcloning the cDNA encoding mouse PD-L1 (SEQ ID NO: 188) into the pcDNA5FRT vector (Life Technologies) using KpnI and NotI restriction sites, and then transforming the vector into the Flp-In T-REx293 cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). The cells were grown in DMEM containing 10% FBS, 100 μg / ml hygromycin B (Melford Laboratories, Ltd., Z2475), and 15 μg / ml blasticidin (Melford Laboratories, Ltd., B1105) for 3-4 weeks until stably transformed cell colonies were formed. These colonies were expanded in the presence of 1 μg / ml doxycycline (Sigma-Aldrich, D9891) and tested for PD-L1 expression using PE-conjugated anti-mouse PD-L1 (MIH5) antibody (BD Biosciences, 558091).

[0638] Detach the cells using cell dissociation buffer, wash once with PBS, and plate 2x10 5 Cells were plated in 96-well plates and incubated with antibodies diluted 1:20 in PBS for 1 hour at 4°C. Cells were washed once in PBS and then measured on an Accuri C6 cytometer (BD Biosciences), with data analyzed using FlowJoX. Mouse PD-L1 expression was confirmed.

[0639] The CD137 / PD-L1 mAb described in Example 5.1 was tested in this DO11.10 T cell activation assay. 2 15 clones were generated. Preparation of mAb 2 Dilutions of the positive control mAb or mAb were added to DO11.10.hCD137 (7.5×10 per well) in a 96-well flat-bottom plate that had been coated overnight with 0.1 μg / ml anti-CD3 antibody (clone 17A2, BioLegend, 100208). 3 cells) and HEK.mPD-L1 cells (2x10 4 cells) or DO11.10.hCD137 (7.5x10 3 HEK cells that were not transduced to express mPD-L1 (2 x 10 cells per well) 4After 18 hours of incubation, the supernatant was collected according to the manufacturer's instructions and analyzed using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86). The plate was read at 450 nm using a microplate reader with Gens software (BioTek). The absorbance at 570 nm was subtracted from the absorbance at 450 nm (correction). The standard curve for calculating cytokine concentrations was based on a four-parameter logarithmic curve fit (Gens software, BioTek). The concentration of mIL-2 was plotted relative to the mAb. 2 The logarithmic concentrations of the agonist or benchmark mAb were plotted and the resulting curves were fitted in GraphPad Prism using the logarithmic (agonist) relative response equation. T cell activation was detected by measuring IL-2 release.

[0640] The results of this determination are shown in Table 4 and Figure 3 No T cell activity was observed without crosslinking by binding to cells expressing PD-L1 (no cell-based XL column). After crosslinking, high levels of IL-2 and subnanomolar EC 50 As can be seen from the values, all mAbs 2 All had potent T cell activity. The positive control anti-human CD137 mAb (G1-AA / 20H4.9) showed increased mIL-2 release at increasing concentrations, but the maximal release was significantly lower than that of the anti-human CD137 Fcab. All clones except FS22-053-009 and FS22-172-005 had EC50s less than 0.3 nM, thus performing as well as, if not better than, the positive control. The lowest observed Emax was 7758 pg / ml, higher than the positive control and a measure of maximal T cell activation, which may be associated with greater T cell anti-tumor activity in vivo.

[0641] Table 4

[0642]

[0643] N / A - Not applicable, EC50 cannot be determined due to low signal

[0644] 5.4 Primary human CD8+ T cell activation assay

[0645] The activity of Fcabs in activating CD137 on HEK cells that overexpress CD137 is shown in Example 5.3. To test the activity of Fcabs on cells that have not been engineered to overexpress CD137, a primary human T cell assay is required. +T cells are responsible for directly killing cancer cells and express CD137 on their cell surface (Ye et al., 2014). It is known that the aggregation of CD137 is crucial for inducing downstream signaling and further CD8+ T cell activation. Therefore, the CD8+ T cell activation assay is used to evaluate the efficacy of Fcab (in the form of mAb) 2 The ability of CD8+ T cells to drive aggregation and subsequent CD137 downstream signaling is determined by the release of IL-2.

[0646] In order to isolate T cells, peripheral blood mononuclear cells (PBMC) were isolated from leucocyte depletion cones, a byproduct of platelet donation. In brief, the leucocyte cone contents were rinsed with PBS and covered on a Ficoll (Sigma-Aldrich, 1440-02) gradient. PBMC were separated by centrifugation and cells that did not pass through the Ficoll gradient were recovered. PBMC were further washed with PBS and the remaining red blood cells were lysed by adding 10 ml of 1X red blood cell lysis buffer (eBioscience, 00-4300-54) according to the manufacturer's instructions. CD8 + CD8 T cell isolation kit II (Miltenyi Biotech, 130-096-495) was used to isolate CD8 T cells from PBMCs in the eluate. + T cells.

[0647] Incubation with anti-CD3 antibody serves as the first signal to drive initial T cell activation. 96-well flat-bottom tissue culture plates were coated with 8 μg / ml anti-CD3 antibody (clone UCHT1, R&D Systems, MAB100-SP) in PBS overnight at 4°C. The plates were then washed three times with 200 μl of PBS.

[0648] For affinity-matured PD-L1 mimetic mAbs 2 HEK293 cells overexpressing hPD-L1 (HEK.hPD-L1) were produced essentially as described in Example 5.3, but by subcloning the cDNA encoding human PD-L1 (SEQ ID NO: 187) instead of mouse PD-L1. HEK.hPD-L1 cells were plated at 2 x 10 cells per well. 5Cells were seeded in 100 μl of T cell culture medium (RPMI medium (Life Technologies, 61870-044) containing 10% FBS (Life Technologies), 1X penicillin-streptomycin (Life Technologies, 15140122), 1 mM sodium pyruvate (Gibco, 11360-070), 10 mM Hepes (Sigma-Aldrich, H0887), 2 mM L-glutamine (Sigma-Aldrich, G7513) and 50 μM 2-mercaptoethanol (Gibco, M6250)) in 96-well flat-bottom plates coated with anti-CD3 antibody (8 μg / ml). After incubation for 4 hours, once HEK.hPD-L1 cells or HEK cells not transduced to express hPD-L1 have adhered, all T cell culture medium was removed and cells were replaced with 5.0 x 10 5 cells / ml of T cells were replaced with 100 μl of T cell culture medium to yield 5.0 x 10 4 cells / well.

[0649] mAb 2 Dilute 100 μl of mAb in T cell culture medium at 2X final concentration starting from 500 nM and titrate 1:3. 2 The titration solution was added to the cells so that the total assay volume was 200 μl and the antibody concentration was 1X.

[0650] Positive control anti-CD137 antibody (G1-AA / 20H4.9) and negative control isotype IgG antibody (G1-AA / HelD1.3) were each diluted in T cell culture medium at 2X final concentration, starting at 500 nM (with 500 nM cross-linker (anti-human CH2, clone MK1A6 (Jefferis et al., 1985 and Jefferis et al., 1992), produced in-house) and titrated 1:3. 100 μl of the diluted positive control antibody / cross-linker mixture or negative control IgG antibody / cross-linker mixture was added to the cells to achieve a total assay volume of 200 μl and an antibody concentration of 1X.

[0651] The assay was incubated at 37°C, 5% CO2 for 72 hours. Supernatants were collected and assayed using human IL-2 ELISA Ready-SET-Go! Kit (eBioscience, catalog number 88-7025-88) according to the manufacturer's instructions. Plates were read at 450 nm using a plate reader with Gen5 software (BioTek). The absorbance at 630 nm was subtracted (corrected) from the absorbance at 450 nm. The standard curve for calculating cytokine concentration was based on a four-parameter logarithmic curve fit (Gen5 software, BioTek). The logarithmic concentration of the relative antibody concentration of human IL-2 (hIL-2) was plotted, and the resulting curve was fitted using the logarithmic (agonist) relative response equation in GraphPad Prism.

[0652] Table 5 shows the PD-L1 mimetic mAbs tested in cell-based cross-linking assays. 2 In the presence of affinity-matured Fcab clones, ECs were observed in T cell activation assays. 50 The positive control anti-human CD137 mAb (20H4.9) showed increased hIL-2 release when cross-linked with anti-hCH2 antibody with an EC50 of 0.5 nM. All clones were active in the assay, with most clones showing good potency with sub-nanomolar EC50. mAbs including Fcabs FS22-053-007, FS22-053-008, FS22-053-010, FS22-053-011, FS22-053-012, FS22-172-003, FS22-172-004, FS22-172-005 2 The maximal T cell response was elicited with the lowest EC50 (ranging from 0.19 to 0.49 nM). mAbs were also tested 2 As expected, a subset of Fcabs (including FS22-053-008, FS22-053-011, FS22-053-014, FS22-173-003, and FS22-172-004) had no activity in this assay when not cross-linked by PD-L1 expressed on HEK cells. This confirms the activity seen in the NF-kB assay and the DO11.10 T cell activation assay. Figure 4 The results for FS22-053-007-AA / PD-L1, FS22-053-008-AA / PD-L1 and FS22-172-002-AA / PD-L1, FS22-172-003-AA / PD-L1, FS22-172-004-AA / PD-L1 mAbs are shown. 2 Representative graphs of IL-2 release from T cell activation assays.

[0653] Table 5

[0654]

[0655]

[0656] 5.5 Determination of the specificity of anti-human CD137 Fcab by surface plasmon resonance (SPR)

[0657] The specificity of the anti-human CD137 Fcab for human CD137 was tested compared with other related TNFSFR family members. 2 Eight Fcabs were tested in the (HelD1.3) format and measured by SPR in a Biacore T200 (GE Healthcare) by testing binding to other human TNFRSF receptors: CD40, OX40, and GITR. Human CD40, GITR, and OX40 were coated to approximately 1000 RU on a Biacore CM5 chip (GE Healthcare, catalog number 29149603) using an amine coupling reagent (amine coupling kit, GE Healthcare, BR-1000-50). 2 Anti-human CD137 Fcab (FS22-053-008 / HelD1.3, FS22-053-009 / HelD1.3, FS22-053-010 / HelD1.3, FS22-053-011 / HelD1.3, FS22-053-012 / HelD1.3, FS22-053-014 / HelD1.3, FS22-172-003 / HelD1.3, FS22-172-004 / HelD1.3) dilutions were prepared starting at 1 μM in HBS-EP+ buffer (BR100669), injected at 30 μl / min for 3 minutes, and then dissociated in buffer for 4 minutes. The chip was regenerated by injecting 10 mM glycine, pH 2.5, at 30 μl / min for 12 seconds. Specific antibodies against different TNFRSF members were used as positive controls to validate Biacore chip coating. Data were analyzed using double-reference subtraction using BIAevaluation 3.2 software. Fcabs did not bind to any of the tested TNFRSF receptors, demonstrating their specificity for CD137. The results suggest that Fcabs do not cause off-target binding.

[0658] 5.6 Simulated mAb testing by SPR 2 Binding affinity of anti-human CD137 Fcab to human, cynomolgus monkey, and mouse CD137

[0659] The affinity of anti-human CD137 Fcabs (FS22-053-008, FS22-053-011, FS22-053-014, FS22-172-004, FS22-172-004) for human, cynomolgus monkey (cyno) and mouse CD137 was determined by SPR to determine whether Fcabs can be used for detection in animal studies. Anti-human Fab capture antibodies were immobilized on all four flow cells of a CM5 Series S chip (GE Healthcare #BR-1005-30) with an average surface density of 6000 RU according to the manufacturer's recommendations (GE Healthcare, Human Fab Capture Kit, #28958325). 3 μg / ml mAb diluted in HBS-EP+ buffer (GE Healthcare #BR1006-69) was injected at a rate of 30 μl / min. 2 solution, each mAb 2 Captured to approximately 150 RU. Different concentrations of human, cynomolgus monkey or mouse CD137 antigen (non-biotinylated human, cynomolgus monkey or mouse CD137-mFc-Avi or human CD137-Avi-His) in HBS-EP+ buffer were then flowed through the chip at a flow rate of 60 μl / min for 3 minutes and then allowed to dissociate for 10 minutes. After each antigen concentration, the chip was regenerated by injecting 10 mM glycine at pH 2.1 for 30 seconds at a flow rate of 30 μl / min. Buffer HBS-EP+ was injected before the highest concentration of antigen and after the lowest concentration of antigen for reference subtraction, and one of the concentrations was randomly repeated twice. The binding kinetics were fitted with a 1:1 Langmuir model to generate the equilibrium binding constant (K) for each sample. D Data analysis was performed using BiaEvaluation software version 3.2. The results are shown in Table 6.

[0660] The results show that all affinity-matured clones have improved binding to human and cynomolgus monkey CD137 compared to each parent molecule. The binding affinity to monomeric human CD137 antigen is weaker (at least 100 times) than to dimer and cynomolgus monkey Fc fusion antigen. As described in Example 2, the selected Fcab preferentially binds to dimeric CD137 rather than monomeric CD137, and the data confirm that the selection strategy is successful. This kinetic behavior makes it unlikely that they will bind to monomeric CD137 expressed at the lowest level on unstimulated T cells, thereby reducing the risk of liver or systemic toxicity associated with certain anti-CD137 monoclonal antibody therapies.

[0661] This data also demonstrates that anti-human CD137 Fcab binds to cynomolgus monkey dimeric CD137 with an affinity comparable to human dimeric CD137.

[0662] The ability of Fcabs to bind to mouse dimeric CD137 was also tested. D Except for 24 nM, other clones did not show strong binding to mouse antigen (as shown in Table 6, where N / A indicates that K cannot be calculated). D This was unexpected because mouse CD137 and human CD137 share less than 57% sequence homology.

[0663] Table 6

[0664]

[0665] N / A – Not applicable because K cannot be determined due to low signal D

[0666] 5.7 Determination of Fcab Binding Valency Using Heterodimeric and Homodimeric Fcabs

[0667] Fcab typically contains two homodimeric Fc chains with an antigen binding site in the CH3 domain. Since these antigen binding sites in the two CH3 domains are very close, the binding valence of Fcab was tested to determine whether the CH3 domains can bind to CD137 independently of each other. Using the mortar mutation (mortar: T22W, mortar: T22S L24A Y66V) (AtwellS et al., 1997), a heterodimeric Fcab containing a single antigen binding CH3 domain was constructed by combining one chain of FS22-172-003 with one chain of wild-type Fc. Thus, each heterodimer contains CH3 from FS22-172-003 (SEQ ID NO: 139) in one chain and wild-type CH3 (SEQ ID NO: 4) in the other chain. With mAb 2 Fcab was prepared in this manner.

[0668] Heterodimeric Fcabs were compared to FS22-172-003 homodimeric molecules (containing two antigen-binding CH3 domains) by SPR binding analysis. For this experiment, monomeric human CD137-mFc-Avi was preferred over dimeric CD137 antigen. To compensate for the weaker binding to monomeric antigen described in Example 5.6, human CD137-mFc-Avi was immobilized on a CM5 chip at a higher density of 450 RU. Heterodimeric or homodimeric Fcabs were injected and flowed over the immobilized antigen. Figure 14The results in Example 2 demonstrate that even under these suboptimal conditions, heterodimeric Fcabs containing only one CD137-binding CH3 domain are able to bind antigen. The observed dissociation rate of the heterodimeric Fcab is significantly faster than that of the homodimeric Fcab. These results confirm that the FS22-172-003 Fcab is able to bind to CD137 via either or both CH3 domains and demonstrate that the selection strategy described in Example 2 was successful, as the Fcab is able to bind its target bivalently.

[0669] 5.8 Binding of anti-CD137 Fcab to cells with different CD137 expression levels

[0670] As described in Example 2, Fcabs were selected to bind CD137 such that they preferentially bind to cells with elevated CD137 expression levels. The bivalency and thus affinity binding of FS22-172-003 was confirmed by SPR in Example 5.6: FS22-172-003 exhibited high affinity and stronger binding to dimeric CD137 than to monomeric CD137.

[0671] As described in Example 3.5, a series of DO11.10 cells expressing different levels of CD137 were generated. To determine the relative expression of CD137 in each cell line, the cells were cloned according to the manufacturer's protocol (Quantum TM Antibody binding capacity (ABC) was determined by Bangs Labs (#816). After background subtraction, each cell line was ranked in order of CD137 expression level: hCD137 high (ABC: 1206283), hCD137 medium (ABC: 404597), hCD137 medium / low (ABC: 143065), hCD137 low (ABC: 14208), and hCD137 negative (ABC: 0).

[0672] Simulated mAb 2 The binding of anti-human CD137 Fcab (FS22-172-003-AA / HelD1.3), positive control antibody (G1-AA / 20H4.9) or isotype control (G1-AA / HelD1.3) to each of the above cells was tested as follows: DO11.10 cells were harvested from T175 cell culture flasks and centrifuged at 1200 rpm for 3 minutes, and then 2×10 6Cells were resuspended in ice-cold FACS buffer (DPBS (Life Technologies, 14190169) and 1% BSA (Sigma-Aldrich, A7906) at 50 μl per well in a 96-well V-bottom plate (Costar, 3894). All tested antibodies were diluted in 120 μl of FACS buffer. DO11.10 cells were then centrifuged, the supernatant removed, and the cells were resuspended in 100 μl of each antibody dilution and incubated at 4°C for 45 minutes. The cells were washed twice by centrifugation with 150 μl of FACS buffer and resuspended in 100 μl of a 1:1000 dilution of goat anti-human IgG (γ chain specific) F(ab')2 fragment-R-phycoerythrin antibody (Sigma, P8047) in FACS buffer and incubated at 4°C for 45 minutes. The cells were washed once with 150 μl FACS buffer, then washed with 150 μl DPBS, resuspended in 150 μl DPBS containing DAPI (Biotium, 40043) at a dilution ratio of 1:10.000, and read on a BD Canto II or iQue (Intellicyt). Data were analyzed using FlowJo v10 to determine the geometric mean of the PE signal of live cells in each well.

[0673] like Figure 15 As shown, FS22-172-003-AA / HelD1.3 bound more strongly to cells with higher CD137 expression compared to the positive control G1-AA / 20H4.9 ( Figure 15 D: CD137 medium and Figure 15 E: CD137 high) and does not bind to cells expressing very low levels of CD137 ( Figure 15 B: CD137 low and Figure 15 C: CD137 medium / low). In contrast, the positive control G1-AA / 20H4.9 bound better to both dimer and monomer antigens, with a K of 0. D Within 10-fold of each other, unlike FS22-172-003-AA / HelD1.3, which binds to the monomeric CD137 antigen at least 200-fold weaker than the dimeric antigen, as described in Example 5.6.

[0674] Example 6: Initial selection of anti-mouse CD137 antibodies

[0675] To test the activity of anti-CD137 Fcabs in an in vivo mouse model, an Fcab that specifically binds to mouse CD137 was generated and characterized.

[0676] Phage display

[0677] Six naive phage libraries displaying human IgG1 CH3 domains, previously used to select Fcabs binding to human CD137, were used to select Fcabs binding to mouse CD137, where recombinant mouse dimeric CD137 or cells expressing full-length mouse CD137 were used as antigens.

[0678] In-house mCD137-mFc-Avi antigen and DO11.10 cells expressing mCD137 (DO11.10.mCD137) were used in the screening of six phage pools. A simple selection protocol was followed in which 100 nM biotinylated antigen was used for all selection rounds (three rounds in total) and 500 nM unlabeled recombinant human Fc fragment was used for the deselection step. Bounds were captured by magnetic beads coated with streptavidin or neutravidin. In addition, the first round output bound to the recombinant antigen was also used in the selection on DO11.10.mCD137 cells expressing mouse CD137. Briefly, the phage output was incubated with DO11.10 cells lacking mouse CD137 to remove unwanted binders, such as those that bind nonspecifically to the cells. Next, the phage was incubated with 1 × 10 7 DO11.10.mCD137 cells were incubated. The binders were then eluted by trypsinization and then subjected to a second round of selection. The third round followed a similar process by reducing the number of DO11.10.mCD137 cells to 5 × 10 6 to increase selection pressure.

[0679] All round 3 recombinant antigen outputs (576 clones) and all round 3 cell selection outputs (576 clones) were screened by phage ELISA (as described above) and used for cell binding to DO11.10.mCD137 cells. For ELISA, most clones showed high antigen binding signal intensity (OD450>1). Therefore, clones that showed less than a 10-fold increase in antigen binding compared to binding to mouse-Fc were removed. For cell binding, FITC MFI was higher than 5×10 5 The performance of the three phage libraries was significantly poorer, with many clones showing nonspecific binding to DO11.10.mCD137 cells and recombinant antigens. As described in Example 3.1, 34 Fcab clone hits were subcloned and generated as HeID1.3 mAbs. 2 .

[0680] Yeast display

[0681] Four natural yeast libraries displaying the CH1 to CH3 domains of human IgG1, which were previously used to select Fcabs that bind to human CD137, were used to select Fcabs that bind to mouse CD137.

[0682] A total of 53 independent selections were performed to identify anti-mouse CD137 binders. In-house produced recombinant dimer biotinylated mouse CD137 (mCD137-mFc-Avi) antigen was used to select binders from a natural yeast library. In brief, the first round of binders was selected by incubating the natural library with 300nM recombinant antigen and deselecting with 2.5μM unlabeled mouse IgG2aFc fragment. The output was separated using MACS and streptavidin magnetic beads. As described in Example 2 above, three rounds of FACS selection and deselection were performed using 300nM recombinant antigen and 1.5μM mouse Fc, respectively.

[0683] Individual clones from rounds 2, 3, and 4 were spotted onto agar plates. To determine output diversity, at least 96 clones from each selection output were sequenced. 126 unique clones (one library of 50 clones from round 3, another library of 48 clones from round 3, and the remaining library of 18 clones from round 4) were screened for binding to recombinant antigen by flow cytometry. Clones that showed greater than 10% positive cells in the APC fluorescence channel when incubated with recombinant antigen and less than 0.2% when incubated with recombinant mFc were considered hits.

[0684] Example 7: Characterization of anti-mouse CD137 Fcabs from the initial selection

[0685] 7.1 Determining the specificity of anti-mouse CD137 Fcab by BLI

[0686] Anti-mouse CD137 Fcab specific for mouse CD137 is used as a HelD1.3 "mimic" mAb 2 The binding of Fcabs to other mouse TNFRSF receptors (CD40, OX40, GITR) was measured by BLI in the Octali QKe system. Streptavidin biosensors (PALL ForteBio18-5021) were coated with 10 ng / μl mouse CD40, GITR, and OX40 receptors (all obtained from R&D Systems and biotinylated using the EZ-Link Sulfo-NHS-SS-Biotin Kit from Thermo Fisher Scientific #21328). 2 Anti-mouse CD137 Fcab in the form of mAb was diluted 1:1 in kinetic buffer (PALL18-1092) to a final concentration of at least 1 μM. 2The cells were incubated in 1x kinetic buffer for 180 seconds, followed by immersion in 1x kinetic buffer for 180 seconds. Antibodies to each TNFRSF receptor were used as positive controls. Fcab clones FS22m-055, FS22m-063, FS22m-066, FS22m-075, FS22m-135, FS22m-055, FS22m-063, and FS22m-066 did not bind to any of the TNFRSF receptors tested, demonstrating their specificity for mouse CD137.

[0687] 7.2 Mimicking mAbs in Mouse NF-κB Reporter Cell Assays 2 Activity of mouse Fcab

[0688] HEK.FRT.luc cells expressing mouse CD137 sequence (SEQ ID NO: 184) were prepared in the same manner as in Example 3.3. This cell line, HEK.FRT.luc.mCD137, was used to screen mAbs containing previously selected anti-mouse CD137 Fcabs according to the method described in Example 3.3. 2 56 mAbs tested 2 , 29 of which tested positive for NF-kB activity. Lob12.3 containing a human IgG1 Fc with the LALA mutation (G1AA / Lob12.3) was used as a positive control anti-mouse CD137 mAb and showed increased luminescence, confirming the validity of the assay. HelD1.3 (also containing a human IgG1 Fc with the LALA mutation) was used as a negative control human IgG isotype to exclude interference from human IgG mimetic Fabs in this assay. EC50 was calculated where possible for mAbs whose activity had not reached steady state. 2 are ignored in favor of mAbs that display classic S-type activity kinetics. 2 mAb 2 They were ranked in order of EC50 and fold change in activity after cross-linking with protein L. FS22m-063 was selected based on its best cross-linking EC50 (1.44 nM) and highest fold change in cross-linking activity (27-fold). Figure 5 HelD1.3 mimic mAb was shown to be effective in HEKmCD137 NF-kB reporter assays. 2 The anti-mouse CD137 Fcab FS22m-063, when cross-linked to Protein L, drives CD137 aggregation and NF-kB signaling.

[0689] Example 8: FS22m-063Fcab (FS22m-063-AA / PD-L1 mAb 2 ) in vivo antitumor activity

[0690] FS22m-063Fcab has been shown to drive CD137 recruitment and activation in vitro, so their ability to activate CD137 in vivo needs to be tested.

[0691] 8.1 Preparation of mAbs 2 FS22m-063Fcab in a form for in vivo testing in mice

[0692] Mock mAbs were generated using a method similar to that described in Example 7.1. 2 A mAb containing anti-mouse CD137 Fcab (FS22m-063) and PD-L1-specific Fab region was prepared by 2 , and tested its in vivo antitumor activity in the MC38 syngeneic mouse tumor model.

[0693] Control: G1-AA / Lob12.3, G1-AA / S70, G1-AA / 4420.

[0694] The control antibody for in vivo experiments was generated by linking the heavy chain variable region of the anti-PD-L1 antibody S70 (clone YW243.55.S70 from US 8,217,149 B2) to a human IgG1 (G1m17) constant region containing the LALA mutation, and the light chain variable region from the S70 antibody was linked to a human constant region (Lm1) via a human kappa J region. mAbs were generated by replacing the CH3 domain of the recombinant construct with FS22m-063. 2 and named it "FS22m-063-AA / S70".

[0695] 8.2FS22m-063-AA / S70 mAb 2 Activity in the MC38 syngeneic tumor model

[0696] Syngeneic mouse models are considered suitable murine systems for testing the anti-tumor effects of inhibiting therapeutic targets and have been widely used to validate the development of human therapeutics. The MC38 syngeneic tumor model was used in this experiment because MC38 tumors are known to be highly immunogenic and responsive to anti-CD137 antibody monotherapy (Kocak et al., 2006) and express PD-L1 (Juneja et al., 2017).

[0697] Before the start of the study, 9-10 week old C57BL / 6 female mice (Jackson Laboratory) weighing 17.92 to 23.89 g each were rested for one week. All animals were implanted with microchips and given unique identifiers. There were 12 mice in each cohort. The MC38 colon cancer cell line (National Cancer Institute) was first expanded and stored, and then pre-screened for pathogens, which showed that they were pathogen-free. Each animal received a subcutaneous injection on the right side of 1x 10 in 100 μl of serum-free medium (Dulbecco's modified Eagle medium). 6 Seven days after tumor cell inoculation, mice that were tumor-free at this time point were removed from the study.

[0698] FS22m-063-AA / S70 mAb 2 and control antibodies (G1-AA / Lob12.3 (CD137 positive control), G1-AA / S70 (positive control PD-L1), G1-AA / 4420 (isotype control)) were injected intraperitoneally into mice at a fixed concentration of 20 μg per dose in DPBS+1 mM arginine+0.05 Tween 80. On days 7, 9, and 11 after tumor inoculation, each mouse received mAb by 200 μl intraperitoneal (IP) injection 2 Molecules or control antibodies. Accurate tumor measurements were performed on the days indicated for any drug dose administration, and mice were closely observed for the remainder of the study. Tumor volume measurements were performed using calipers to determine the longest and shortest axes of the tumor. The following formula was used to calculate tumor volume:

[0699] LX(S 2 ) / 2

[0700] Where L = longest axis; S = shortest axis

[0701] like Figure 6 As shown, FS22m-063-AA / S70 mAb increased the expression of FS22m-063-AA / S70 mAb compared to mice treated with any control antibody. 2 The results showed significant tumor growth inhibition. All groups were compared using mixed model analysis, and the statistical significance of the growth rate over the entire study period was shown in pairs. As shown in Table 7, unexpectedly, at the end of the study, the FS22m-063-AA / S70mAb 2 All treated mice were tumor-free, compared to only 4 of 12 mice treated with a combination of anti-PD-L1 and anti-CD137 antibodies (G1-AA / S70+G1-AA / Lob12.3) or PD-L1 or CD137 antibodies alone.

[0702] Table 7: Tumor-free mice in each treatment group in the MC38 syngeneic tumor model

[0703]

[0704]

[0705] The study showed that in mice with a fully functional immune system, CD137 agonism, possibly through PD-L1 crosslinking, led to reduced tumor growth, possibly due to increased cytotoxicity of CD8+ T cells in the tumor.

[0706] Example 9: FS22m-063Fcab (FS22m-063-AA / PD-1 mAb 2 ) in vivo antitumor activity

[0707] Having demonstrated that FS22m-063Fcab is able to drive CD137 recruitment and activation in vitro, it is desirable to test their ability to activate CD137 via another Fab target (in this case only one target found on immune cells in vivo, PD-1).

[0708] 9.1 Preparation of mAbs 2 FS22m-063Fcab in a form for in vivo testing in mice

[0709] The same mock mAb produced in Example 7.1 was used 2 A similar approach was used to generate mAbs containing the Fab region of anti-mouse CD137 Fcab, FS22m-063, and specific for PD-1. 2 , and tested its in vivo antitumor activity in the MC38 syngeneic mouse tumor model.

[0710] The control antibody for in vivo experiments was generated by linking the heavy chain variable region of the anti-PD-L1 antibody F2 (clone PD1-F2 from WO 2004 / 056875 A1) to a human IgG1 (G1m17) constant region containing the LALA mutation, and the light chain variable region from the F2 antibody was linked to a human constant region (Lm1) via a human kappa J region. mAbs were generated by replacing the CH3 domain of the recombinant construct with FS22m-063. 2 and named it "FS22m-063-AA / F2".

[0711] 9.2FS22m-063-AA / F2 mAb 2 Activity in the MC38 syngeneic tumor model

[0712] The MC38 syngeneic tumor model was used for this experiment as described in Example 8.2, with the following deviations:

[0713] 9-11 week old female C57BL / 6 mice (Charles River) were microchipped and given a unique identifier. Each cohort consisted of 12 mice. Each animal received a subcutaneous injection in the right flank of 1 x 10 6 MC38 colorectal cancer cells.

[0714] FS22m-063-AA / F2 mAb 2 and control antibodies (G1-AA / Lob12.3 (CD137 positive control), G1-AA / F2 (positive control PD-1), G1-AA / 4420 (isotype control)) were injected intraperitoneally into mice at a fixed concentration of 20 μg per dose in DPBS + 1 mM arginine + 0.05 Tween 80. Once the tumor volume reached 50-60 mm 3 On day 0 and on days 2 and 4 after the first dose, each mouse received mAb via 200 μl intraperitoneal (IP) injection 2 Molecules or control antibodies. Tumors were measured as described in Example 8.2.

[0715] like Figure 7 As shown in A and B, FS22m-063-AA / F2 mAb was significantly inhibited in mice treated with an isotype control antibody and a positive control PD-1 antibody. 2 As shown in Table 8, at the end of the study, the FS22m-063-AA / F2 mAb 2 Twelve of the treated mice were tumor-free, compared to only seven of the 12 mice treated with the combination of anti-PD-L1 and anti-CD137 antibodies (G1-AA / S70+G1-AA / Lob12.3).

[0716] Table 8

[0717] Group % tumor-free mice Number of tumor-free mice G1 / Lob12.3 50％ 6 / 12 G1-AA / 4420 0％ 0 / 12 G1-AA / F2 16％ 2 / 12 G1-AA / F2+G1-AA / Lob12.3 50％ 6 / 12 FS22m-063-AA / F2 83％ 10 / 12

[0718] The study showed that in mice with fully functional immune systems, CD137 agonism, likely through cross-linking of PD-1 engagement, and additional PD-1 blockade led to reduced tumor growth, possibly due to increased cytotoxicity of CD8+ T cells in the tumor.

[0719] It was also shown that when CD137 Fcab was used as mAb 2 In the form of Fab arms, cross-linking can occur through the binding of Fab arms to immune cell targets, leading to the aggregation and activation of CD137.

[0720] Example 10: mCD137 / MSLN mAb 2 In vivo antitumor activity

[0721] mAbs containing FS22m-063Fcab and PD-L1 Fab have been shown to 2 Efficacy (Example 8), it is hoped to test mAb 2 The CD137 Fcab format results in mAb binding to a tumor-specific antigen (TAA), in this case mesothelin (MSLN), through its Fab arm. 2 This tumor-targeted approach is expected to be beneficial in localizing T cell activation to the tumor microenvironment, as mAb activation occurs only where MSLN is expressed. 2 Cross-linking and CD137 agonism.

[0722] A syngeneic mouse tumor model expressing mouse MSLN was constructed. CT26 colon cancer cells (ATCC, CRL-2638) expressing full-length mouse mesothelin (SEQ ID NO: 189) were prepared by lipofectamine transfection (Lipofectamine 3000, Thermo Fisher Scientific, Catalog No. L3000008) using the pcDNA3.1 vector (+) (Thermo Fisher Scientific, Catalog No. V79020). CT26 cells were transfected with the pcDNA3.1 vector containing the mouse MSLN cDNA according to the manufacturer's protocol. Stable transfection was achieved using Geneticin as the selective antibiotic (600 μg / ml) in complete culture medium (RPMI, 10% FBS).

[0723] Expression of mouse MSLN on CT26 cells was confirmed by flow cytometry using the positive control antibody MOR6626 (WO 2009 / 068204 A1). Specifically, cells were incubated with the positive control antibody for 1 hour, and then cell binding was detected using a fluorescently labeled anti-human IgG detection antibody (Stratech Technologies, Inc., catalog number 109-546-098-JIR). The clone population was expanded and then analyzed using the same flow cytometric method to determine its relative expression level. One clone was then selected and named CT26.G10.

[0724] CT26.G10 tumor growth was confirmed in vivo. 8-10 week old Balb / c female mice (Charles River) were microchipped and given a unique identifier. Each group had 17 mice, and each animal received a subcutaneous injection in the right flank of 1 x 10 mice in 100 μl of serum-free medium. 5 Tumor volume measurements were performed three times per week using calipers as described in Example 8. The study was performed in accordance with UK Home Office regulations as described in Example 8.

[0725] Tissues were collected at the end of the study and the expression of membrane-bound mesothelin was confirmed by immunohistochemical staining in formalin-fixed paraffin-embedded (FFPE) tumor tissue as follows: 4 μm FFPE tissue sections were deparaffinized and antigen retrieval was performed at 97°C using low pH 6.1 (Dako PT Link), followed by peroxidase blocking and protein blocking, and then incubated with anti-mesothelin primary antibody at a concentration of 1 μg / ml (Life Cycle Biosciences, catalog number LS-C407883). The anti-mesothelin antibody was detected using a polymer-HRP anti-rabbit secondary reagent and a DAB (3,3'-diaminobenzidine) chromogenic end point (Dako EnVision+ system).

[0726] To evaluate the efficacy of Fcab FS22m-063, the following molecules or combinations were tested in vivo: anti-MSLN FS28m-228-010 antibody of human IgG1 isotype with LALA mutation (G1-AA / FS28m-228-010), two mock mAbs 2 Format Fcab (FS22m-063-AA / HelD1.3 and FS22m-063-AA / 4420), FS28m-228-010 antibody and mimetic CD137 mAb with LALA mutation 2 (G1-AA / FS28m-228-010+FS22m-063-AA / HelD1.3), human isotype control antibody (G1-AA / HelD1.3), and finally CD137 / MSLN mAb with LALA mutation 2 (FS22m-063-AA / FS28m-228-010).

[0727] Prior to the start of the study, female Balb / c mice (Charles River), 8-10 weeks old and weighing 20-25 g each, were rested for 1 week. All animals were microchipped and uniquely identified. Each cohort consisted of 20 mice, except for FS22m-063-AA / 4420 (n=10 mice). The CT26.G10 colon cancer cell line was expanded and a cell bank was generated. Each animal received a subcutaneous injection of 1 x 10 cells in 100 μl of serum-free medium in the left flank. 5 Mice without tumors 12 days after tumor cell inoculation were removed from the study.

[0728] A 200 μg dose of each antibody (~10 mg / kg) was prepared and injected intraperitoneally (IP) into mice. In addition, for the combination group, FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 were prepared at 200 μg per dose (~10 mg / kg). On days 12, 14, and 16 after tumor inoculation, mice were administered a 200 μl dose (q2dx3). Tumor volume measurements were taken three times a week using a caliper, and the mice were closely monitored. The study endpoints were determined by humane endpoints based on the tumor volume and condition of the mice.

[0729] like Figure 9 As shown, FS22m-063-AA / FS28m-228-010 mAb was significantly more effective than G1-AA / HelD1.3 isotype control. 2 Tumor growth was significantly inhibited. Table 9 shows pairwise comparisons of tumor growth rates for all treatment groups using mixed model analysis throughout the study, comparing all groups to the G1-AA / HelD1.3 isotype control.

[0730] Table 9: Results of pairwise comparisons of tumor growth using mixed model analysis, comparing all groups to the G1-AA / HelD1.3 isotype control.

[0731]

[0732] NS p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 At the end of the study, all tumors were equal to or less than 62.5 mm 3 All animals with ≥50% response were considered complete responders (see Table 10). 2 Thirty-five percent of treated animals had a complete response to treatment compared to 0% in the G1-AA / HelD1.3 isotype control, FS22m-063-AA / HelD1.3, FS22m-063-AA / 4420, and the FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 combination groups.

[0733] Table 10: Tumor-free mice (tumors ≤ 62 mm) in the CT26.G10 syngeneic tumor model by the end of the study. 3 ) in terms of quantity and percentage.

[0734]

[0735] Survival analysis ( Figure 10and Table 11) show that FS22m-063-AA / FS28m-228-010mAb 2 induced a significant survival advantage, whereas the components (G1-AA / FS28m-228-010, FS22m-063-AA / HelD1.3) or G1-AA / FS28m-228-010+FS22m-063-AA / 4420 did not show a survival advantage. In addition, FS22m-063-AA / FS28m-228-010 mAb was significantly superior to G1-AA / HelD1.3 (29 days), FS22m-063-AA / HelD1.3 (30 days), FS22m-063-AA / 4420 (29 days), G1-AA / FS28m-228-010 (30 days), and the combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 mAb (29 days). 2 The median survival time was extended to 42.5 days.

[0736] Table 11: Median survival time of animals treated with each compound and results of pairwise statistical analysis (log-rank) in the CT26.G10 syngeneic tumor model.

[0737]

[0738] NS p≥0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 These data indicate that mAb 2 The ability to drive CD137 activation in tumors through MSLN cross-linking was superior to targeting CD137 and / or MSLN alone (or even in combination), significantly improving the survival of tumor-bearing mice. 2 The Fcab forms (FS22m-063-AA / HelD1.3 or FS22m-063-AA / 4420) did not show any intrinsic activity in this study.

[0739] Example 11: Selection to obtain Fcabs capable of binding to mouse and human CD137

[0740] Since it was unexpectedly found that some Fcabs that bind to human CD137 also bind to mouse CD137 (see Example 5.5 for specificity of binding to human, mouse and cynomolgus monkey CD137), it was decided to see if these clones could be improved.

[0741] 11.1: Site-directed modification to eliminate potential sequence liability in FS22-053-014 clone

[0742] Mouse-human cross-reactivity clone FS22-053-014 is selected because it is shown that it can be combined with mouse dimer CD137 antigen by SPR (referring to embodiment 5.5). However, after further sequence analysis, it has potential sequence responsibility, and this may be due to the Q98D mutation and the wild-type G99 position producing DG motif causing the result of aspartic acid isomerization after translation in its CH3 domain EF ring. Other affinity-matured clones in FS22-053 pedigree (referring to Table 12) comprise Q98E modification at the same position of EF ring. According to the manufacturer's advice, Q98D was mutated to Q98E using QuickChange II mutagenesis kit (Agilent, catalog number (Cat. No.) 200523) by site-directed mutagenesis, resulting in clone FS22-053-017. Table 12 below shows the frequently occurring LE motifs in the EF loop of the CH3 domains of clones FS22-053-008, FS22-172-003, and FS22-172-004, and the DG motif in the EF loop of the CH3 domain of clone FS22-053-014.

[0743] Table 12

[0744]

[0745] Example 12: Characterization of mutant FS22-053-017 Fcab clones

[0746] 12.1 Simulating mAbs in the Human CD137 DO11.10 T Cell Activation Assay 2 Activity of FS22-053-017Fcab

[0747] Fcab clone FS22-053-017 was subcloned and expressed as a HeID1.3 “mimetic” mAb 2 , and then in the human CD137 DO11.10 T cell activation assay as described in Example 3.4, compared with the same HelD1.3 mAb 2 The FS22-053-014 clone was compared with the FS22-053-014 clone. G1-AA / 20H4.9 was used as an anti-CD137 positive control and G1-AA / D1.3 was used as an IgG control. 2 It can be cross-linked with protein L or with protein L at a ratio of 1:4.

[0748] Table 13

[0749]

[0750] N / A - Not applicable, EC50 cannot be determined due to low signal

[0751] Table 13 and Figure 12The results showed that the simulated mAb 2 FS22-053-17Fcab and mimetic mAb formats 2 The FS22-053-014 Fcab version was comparable in activity when cross-linked with protein L in the same DO11.10 T cell activation assay. Therefore, mutagenesis did not negatively impact functional activity. 2 Both clones were inactive in the absence of cross-linking.

[0752] As expected, the positive control anti-CD137 was active only when cross-linked, whereas the IgG control was inactive regardless of cross-linking.

[0753] 12.2 Mimicking mAbs in Mouse CD137 DO11.10 T Cell Activation Assays 2 Activity of mutant forms of FS22-053-017Fcab

[0754] FS22-053-017 clone (HelD1.3 mimic mAb) was also 2 The Fcab clone FS22m-063 (also a HeID1.3 mimetic mAb 2 format) that binds to mouse CD137 and the parental clone FS22-053-014 (a HeID1.3 mimetic mAb 2 The mAb was compared in the mouse CD137 DO11.10 T cell activation assay as described in Example 3.4. 2 The molecule and protein L were in a 4:1 molar ratio (mAb 2 : Protein L) cross-linking.

[0755] As expected, all tested molecules showed activity, as measured by IL-2 release, when cross-linked by protein L, but showed no activity when not cross-linked. FS22m-063, selected for its binding to mouse CD137, exhibited the best activity in the assay, with an EC50 of 0.39 nM upon cross-linking. Both FS22-053-14 and FS22-053-017 were active in the assay, indicating no loss of function due to mutagenesis, although FS22-053-017 showed a slight decrease in activity, with an EC50 approximately 8-fold lower compared to FS22-053-14 when cross-linked with protein L. Figure 12 Display of HelD1.3 mimetic mAb 2 The affinity-matured human and mouse cross-reactive CD137 Fcabs FS22-053-014 and FS22-053-017, as well as the anti-mouse CD137 Fcab FS22m-063, activated CD137 when cross-linked with protein L, leading to the release of mIL-2 in a DO11.10 T cell activation assay.

[0756] Table 14

[0757]

[0758] N / A - Not applicable, EC50 cannot be determined due to low signal

[0759] 12.3 Binding Kinetics of FS22-053-017

[0760] The equilibrium dissociation constant (K) of Fcab clone FS22-053-017 was analyzed by SPR on the Biacore T200 system. D ) were compared with the equilibrium dissociation constant of FS22-053-014. For the hCD137-mFc-Avi antigen, the following method was used: anti-human Fab molecules were immobilized on a CM5 chip with a surface density between 9,000 and 11,000 response units (RU). The antibodies were diluted to 4 μg / ml in HBS-EP buffer and captured by the anti-Fab molecules at a flow rate of 30 μl / s. Eight different concentrations of hCD137-mFc antigen were used: 200nM; 66.67nM; 22.22nM (including twice), 7.41nM; 2.47nM; 0.82nM, 0.27nM: 0.091 (diluted with HBS-EP+ buffer) flowing through the captured mAb 2 .

[0761] For the mCD137-mFc-Avi binding kinetics assay, a different approach was used. Mouse PD-L1-mFc-Avi was immobilized on a CM5 chip at a surface density of 200 RUs. A mAb containing FS22-053-014 and FS22-053-017 Fcab was combined with anti-mouse PD-L1 Fab (S70). 2 Diluted to 7.5 μg / ml in HBS-EP buffer and captured by immobilized mPD-L1 protein at a flow rate of 60 μl / s. Eight different concentrations of mCD137-mFc antigen were used: 600 nM; 200 nM; 66.67 nM (included twice), 22.2 nM; 7.1 nM; 2.47 nM; 0.82 nM (diluted in HBS-EP+ buffer) were passed over the captured mAb. 2 .

[0762] Data analysis was performed using Biacore T200 evaluation software. A blank flow cell was subtracted and the curves were fitted using a 1:1 Langmuir binding model with mass transfer, RI set to a constant of zero, and R maxThe results are summarized in Table 15 and show that the kinetic properties of the two Fcab clones for human antigens are very similar, demonstrating that the Q98D to Q98E mutation has no negative impact on binding to human antigens. The 2-fold reduction in binding strength to mouse antigens is also consistent with the functional data presented in Example 11.2.

[0763] Table 15

[0764] molecular <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (nM)]]> FS22-053-014 / HelD1.3 <![CDATA[8.304×10 4 ]]> <![CDATA[3.656×10 -4 ]]> 4.403 FS22-053-017 / HelD1.3 <![CDATA[8.853×10 4 ]]> <![CDATA[4.062×10 -4 ]]> 4.588 FS22-053-014 / mPD-L1 <![CDATA[1.509×10 5 ]]> <![CDATA[4.025×- 3 ]]> 27 FS22-053-017 / mPD-L1 <![CDATA[1.064×10 5 ]]> <![CDATA[5.186×- 3 ]]> 49

[0765] Example 13: Involvement of PPY in Antigen Binding (Alanine Scanning of the Conserved PPY Motif)

[0766] As described in Example 5, a PPY sequence motif was identified in the AB loop of two independently selected Fcabs, and this motif was conserved in all affinity-matured clones analyzed. Therefore, it is necessary to understand the relationship between this motif and the binding of CD137 and the overall protein structure. Alanine scanning is a commonly used biological technique for determining the importance of specific residues in protein-protein interactions. Briefly, each of the three amino acids is sequentially replaced with an alanine residue. Alanine is considered to be the most chemically inert, non-bulky residue and is therefore considered unlikely to contribute to binding.

[0767] 13.1 Generation of Mutant Clones for Alanine Scanning

[0768] Mutant clones were generated by site-directed mutagenesis on the parental clones (FS22-172-003 and FS22-053-008). The positions of the amino acid substitutions within the AB loop are summarized in Table 16, and all other residues were conserved from their respective parents. mAbs were then transiently expressed in HEK293-6E cells as described in Example 3. 2 The mutants were expressed in yields similar to those of the parent, thus indicating that the alanine substitution had no effect on protein production.

[0769] Table 16

[0770]

[0771] 13.2 Binding Kinetics of Mutant Clones

[0772] Using Octet QK from ForteBio e The binding kinetics of two parental clones and eight mutant clones were systematically compared. Dimeric biotinylated hCD137-mFc-Avi was captured on a streptavidin sensor at 10 μg / ml and then analyzed for each mAb at a concentration of 200 nM. 2Compared with the parental clone, the mAbs of all FS22-172-003 mutant clones 2 Binding to the dimeric antigen was disrupted, with clones FS22-172-003_AAA and FS22-172-003_APY losing all binding, while clones FS22-172-003_PPA and FS22-172-003_PAY maintained some binding, although significantly reduced compared to the parent (response units decreased by 5.5 and 4.4 fold). Binding of FS22-053-008 was also affected compared to the parental FS22-053-008 clone, with variants PAY and AAA losing all binding, and variants APY and PPA showing reduced binding to the antigen and slower association properties. This data suggests that the PPY motif is essential for both mAbs. 2 Binding to the CD137 antigen is important.

[0773] 13.3 Homology Modeling

[0774] Given the involvement of the PPY motif in binding, it is beneficial to simulate Fcab in silico to assess protein structure predictions in the CH3 domain. Structural homology modeling and subsequent conformational exploration were performed using the MOE software suite (Chemical Computing Group, ULC), version 2019.0101. The Fc region from the Protein Data Bank [PDB] structure 5JII was used as a structural template for the Fcab regions of FS22-172-003 and FS22-053-008. Due to insertions in the AB and EF loop regions relative to the selected structural template, a de novo loop search with residue side-chain optimization was applied to generate the AB and EF loop structures. The resulting homology models were energy minimized and scored according to geometric criteria including backbone bond lengths, angles, dihedral angles, and chirality. Scores were used as the basis for conformational sampling using the LowModeMD simulation method, implemented within MOE, using standard structural homology models.

[0775] Figure 13Homology models in [ 13.1.1.2 ] show that the AB and EF loops of both Fcabs adopt different conformations compared to template 5JII, with the PPY motif playing a role in the AB loop extending from the CH3 domain core. The PPY motif also mediates interactions that lead to conformational stabilization of the AB loop, both through interactions within the loop and between the loop and the rest of the structure. Conformational searches using LowModeMD simulations also highlighted that the tyrosine residues in the PPY motif interact with Q3 (IMGT) and D12 (IMGT) in the CH3 domain of the same chain in the CH3 domain, potentially stabilizing the AB loop. Combined with the results shown in Example 13.2, this suggests that the PPY motif is important for the binding of both FS22-172-003 and FS22-053-008 to CD137.

[0776] Sequence Listing

[0777] Amino acid sequence of the wild-type (WT) Fcab CH3 domain structural loop

[0778] WT Fcab AB ring–RDELTKNQ (SEQ ID NO: 1)

[0779] WT Fcab CD ring–SNGQPENNY (SEQ ID NO: 2)

[0780] WT Fcab EF loop–DKSRWQQGNV (SEQ ID NO: 3)

[0781] Amino acid sequence of WT Fcab CH3 domain (SEQ ID NO: 4)

[0782] The underlined circles are AB, CD, and EF.

[0783] GQPREPQVYTLPPS RDELTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNY KTTPPVLDSDGS FFLYSKLTV DKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG

[0784] Amino acid sequence of Fcab CH2 domain with LALA-PA mutation (SEQ ID NO: 5)

[0785] The underlined LALA-PA mutation

[0786] APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL A APIEKTISKAK

[0787] Amino acid sequence of Fcab CH2 domain with LALA mutation (SEQ ID NO: 6)

[0788] The underlined LALA mutation

[0789] APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0790] Amino acid sequence of the truncated Fcab hinge region (SEQ ID NO: 7)

[0791] TCPPCP

[0792] Amino acid sequence of WT Fcab with LALA mutation (SEQ ID NO: 8)

[0793] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0794]

[0795] Amino acid sequence of WT Fcab without LALA mutation (SEQ ID NO: 9)

[0796] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0797]

[0798] Amino acid sequence of PPY motif (SEQ ID NO: 10)

[0799] PPY

[0800] Amino acid sequence of Fcab FS22-033 with LALA mutation (SEQ ID NO: 11)

[0801] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0802]

[0803] Amino acid sequence of Fcab FS22-033 without the LALA mutation (SEQ ID NO: 12)

[0804] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0805]

[0806] FS22-033 / HelD1.3 mimetic mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 13)

[0807] VH domain (underlined)

[0808] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYFEQEVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVARHRWQLGNVFSCSVMHEALHNHYTQKSLSLSPG

[0809] FS22-033 / HelD1.3 mimetic mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 14)

[0810] VH domain (underlined)

[0811] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYFEQEVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVARHRWQLGNVFSCSVMHEALHNHYTQKSLSLSPG

[0812] Amino acid sequence of Fcab FS22-053 with LALA mutation (SEQ ID NO: 15)

[0813] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0814]

[0815] Amino acid sequence of Fcab FS22-053 without the LALA mutation (SEQ ID NO: 16)

[0816] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0817]

[0818] FS22-053 / HelD1.3 mimetic mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 17)

[0819] VH domain (underlined)

[0820] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVYYNRWQDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0821] FS22-053 / HelD1.3 mimetic mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 18)

[0822] VH domain (underlined)

[0823] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVYYNRWQDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0824] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-008

[0825] FS22-053-008 First Sequence – NPPYLFS (SEQ ID NO: 19)

[0826] FS22-053-008 Second Sequence – DYWRWLE (SEQ ID NO: 20)

[0827] Amino acid sequence of Fcab FS22-053-008 CH3 domain (SEQ ID NO: 21)

[0828] The underlined parts are the first and second sequences

[0829] GQPREPQVYTLPPSRDEL NPPYLFS NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DYWRWLE GNVFSCSVMHEALHNHYTQKSLSLSPG

[0830] Nucleic acid sequence of Fcab FS22-053-008 CH3 domain (SEQ ID NO: 22)

[0831] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTACTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGCTGGAAGGGAACGTCTTCTCATGTCCCGTGATGCATGAGGCGCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGT

[0832] Amino acid sequence of Fcab FS22-053-008 with LALA mutation (SEQ ID NO: 23)

[0833] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0834]

[0835] Nucleic acid sequence of Fcab FS22-053-008 with LALA mutation (SEQ ID NO: 24)

[0836] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0837] Amino acid sequence of Fcab FS22-053-008 without the LALA mutation (SEQ ID NO: 25)

[0838] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0839]

[0840] Nucleic acid sequence of Fcab FS22-053-008 without the LALA mutation (SEQ ID NO: 26)

[0841] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0842] FS22-053-008 / HelD1.3 mimetic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 27)

[0843] VH domain (underlined)

[0844] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG

[0845] FS22-053-008 / HelD1.3 mimetic mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 28)

[0846] VH domain (underlined)

[0847] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG

[0848] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-009

[0849] FS22-053-009 First Sequence – NPPYLFS (SEQ ID NO: 19)

[0850] FS22-053-009 Second Sequence – EHTRWLD (SEQ ID NO: 29)

[0851] Amino acid sequence of Fcab FS22-053-009 CH3 domain (SEQ ID NO: 30)

[0852] Underline the first and second sequences

[0853] GQPREPQVYTLPPSRDEL NPPYLFS NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV EHTRWLD GNVFSCSVMHEALHNHYTQKSLSLSPG

[0854] Nucleic acid sequence of Fcab FS22-053-009 CH3 domain (SEQ ID NO: 31)

[0855] GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAACATACTAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0856] Amino acid sequence of Fcab FS22-053-009 with LALA mutation (SEQ ID NO: 32)

[0857] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0858]

[0859] Nucleic acid sequence of Fcab FS22-053-009 with LALA mutation (SEQ ID NO: 33)

[0860] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAACATACTAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0861] Amino acid sequence of Fcab FS22-053-009 without the LALA mutation (SEQ ID NO: 34)

[0862] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0863]

[0864] Nucleic acid sequence of Fcab FS22-053-009 without the LALA mutation (SEQ ID NO: 35)

[0865] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAACATACTAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0866] FS22-053-009 / HelD1.3 mimetic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 36)

[0867] VH domain (underlined)

[0868] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVEHTRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0869] FS22-053-009 / HelD1.3 mimetic mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 37)

[0870] VH domain (underlined)

[0871] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVEHTRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0872] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-010

[0873] FS22 - 053 - 010 First Sequence – NPPYLFS (SEQ ID NO: 19)

[0874] FS22 - 053 - 010 Second Sequence – DYMRWLD (SEQ ID NO: 38)

[0875] Amino acid sequence of Fcab FS22-053-010 CH3 domain (SEQ ID NO: 39)

[0876] Underline the first and second sequences

[0877] GQPREPQVYTLPPSRDEL NPPYLFS NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DYMRWLD GNVFSCSVMHEALHNHYTQKSLSLSPG

[0878] Nucleic acid sequence of Fcab FS22-053-010 CH3 domain (SEQ ID NO: 40)

[0879] GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACATGAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0880] Amino acid sequence of Fcab FS22-053-010 with LALA mutation (SEQ ID NO: 41)

[0881] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0882] TCPPCP APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYMRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0883] Nucleic acid sequence of Fcab FS22-053-010 with LALA mutation (SEQ ID NO: 42)

[0884] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACATGAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0885] Amino acid sequence of Fcab FS22-053-010 without the LALA mutation (SEQ ID NO: 43)

[0886] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0887] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYMRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0888] Nucleic acid sequence of Fcab FS22-053-010 without the LALA mutation (SEQ ID NO: 44)

[0889] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACATGAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0890] FS22-053-010 / HelD1.3 mimetic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 45)

[0891] VH domain (underlined)

[0892] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYMRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0893] FS22-053-010 / HelD1.3 mimetic mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 46)

[0894] VH domain (underlined)

[0895] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYMRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0896] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-011

[0897] FS22-053-011 first sequence—NPPYLFS (SEQ ID NO: 19)

[0898] FS22-053-011 second sequence—DYWRWTD (SEQ ID NO: 47)

[0899] Amino acid sequence of Fcab FS22-053-011 CH3 domain (SEQ ID NO: 48)

[0900] Underline the first and second sequences

[0901] GQPREPQVYTLPPSRDEL NPPYLFS NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DYWRWTD GNVFSCSVMHEALHNHYTQKSLSLSPG

[0902] Nucleic acid sequence of Fcab FS22-053-011 CH3 domain (SEQ ID NO: 49)

[0903] GGACAGCCTCGAGAGCCTCAAGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGACTGATGGGAACGTCTTCTCATGTCCCGTGATGCATGAGGCGCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0904] Amino acid sequence of Fcab FS22-053-011 with LALA mutation (SEQ ID NO: 50)

[0905] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0906]

[0907] Nucleic acid sequence of Fcab FS22-053-011 with LALA mutation (SEQ ID NO: 51)

[0908] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGACTGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0909] Amino acid sequence of Fcab FS22-053-011 without the LALA mutation (SEQ ID NO: 52)

[0910] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0911]

[0912] Nucleic acid sequence of Fcab FS22-053-011 without the LALA mutation (SEQ ID NO: 53)

[0913] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGACTGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0914] FS22-053-011 / HelD1.3 mimetic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 54)

[0915] VH domain (underlined)

[0916] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWTDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0917] FS22-053-011 / HelD1.3 mimetic mAb without the LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 55)

[0918] VH domain (underlined)

[0919] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWTDGNVFSCSVMHEALHNHYTQKSLSLSPG

[0920] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-012

[0921] FS22 - 053 - 012 First Sequence – NPPYLFS (SEQ ID NO: 19)

[0922] FS22 - 053 - 012 Second Sequence – DHMRWLE (SEQ ID NO: 56)

[0923] Amino acid sequence of Fcab FS22-053-012 CH3 domain (SEQ ID NO: 57)

[0924] Underline the first and second sequences

[0925] GQPREPQVYTLPPSRDEL NPPYLFS NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DHMRWLE GNVFSCSVMHEALHNHYTQKSLSLSPG

[0926] Nucleic acid sequence of Fcab FS22-053-012 CH3 domain (SEQ ID NO: 58)

[0927] GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCATATGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0928] Amino acid sequence of Fcab FS22-053-012 with LALA mutation (SEQ ID NO: 59)

[0929] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0930]

[0931] Nucleic acid sequence of Fcab FS22-053-012 with LALA mutation (SEQ ID NO: 60)

[0932] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCATATGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0933] Amino acid sequence of Fcab FS22-053-012 without the LALA mutation (SEQ ID NO: 61)

[0934] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0935]

[0936] Nucleic acid sequence of Fcab FS22-053-012 without the LALA mutation (SEQ ID NO: 62)

[0937] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCATATGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0938] FS22-053-012 / HelD1.3 mimetic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 63)

[0939] VH domain (underlined)

[0940] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDHMRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG

[0941] FS22-053-012 / HelD1.3 mimetic mAb without the LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 64)

[0942] VH domain (underlined)

[0943] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDHMRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG

[0944] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-013

[0945] FS22 - 053 - 013 First Sequence – NPPYLFS (SEQ ID NO: 19)

[0946] FS22 - 053 - 013 Second Sequence – GYERWLE (SEQ ID NO: 65)

[0947] Amino acid sequence of Fcab FS22-053-013 CH3 domain (SEQ ID NO: 66)

[0948] Underline the first and second sequences

[0949] GQPREPQVYTLPPSRDEL NPPYLFS NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV GYERWLE GNVFSCSVMHEALHNHYTQKSLSLSPG

[0950] Nucleic acid sequence of Fcab FS22-053-013 CH3 domain (SEQ ID NO: 67)

[0951] GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGGTTACGAAAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0952] Amino acid sequence of Fcab FS22-053-013 with LALA mutation (SEQ ID NO: 68)

[0953] Hinge region (underlined), CH2 domain (bold), CH3 domain (italics), LALA mutation (bold and underlined)

[0954]

[0955] Nucleic acid sequence of Fcab FS22-053-013 with LALA mutation (SEQ ID NO: 69)

[0956] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGGTTACGAAAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0957] Amino acid sequence of Fcab FS22-053-013 without the LALA mutation (SEQ ID NO: 70)

[0958] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic)

[0959]

[0960] Nucleic acid sequence of Fcab FS22-053-013 without the LALA mutation (SEQ ID NO: 71)

[0961] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGGTTACGAAAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0962] FS22-053-013 / HelD1.3 mimetic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 72)

[0963] VH domain (underlined)

[0964] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVGYERWLEGNVFSCSVMHEALHNHYTQKSLSLSPG

[0965] FS22-053-013 / HelD1.3 mimetic mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO: 73)

[0966] VH domain (underlined)

[0967] QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTM LVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVGYERWLEGNVFSCSVMHEALHNHYTQKSLSLSPG

[0968] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-014

[0969] FS22 - 053 - 014 First Sequence – NPPYLFS (SEQ ID NO: 19)

[0970] FS22 - 053 - 014 First Sequence – YHWRWLD (SEQ ID NO: 74)

[0971] Amino acid sequence of Fcab FS22-053-014 CH3 domain (SEQ ID NO: 75)

[0972] Underline the first and second sequences

[0973] GQPREPQVYTLPPSRDEL NPPYLFS NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV YHWRWLD GNVFSCSVMHEALHNHYTQKSLSLSPG

[0974] Nucleic acid sequence of Fcab FS22-053-014 CH3 domain (SEQ ID NO: 76)

[0975] GGACAGCCTCGAGAGCCTCAAGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGTACCATTGGAGGTGGCTGGATGGGAACGTCTTCTCATGTCCCGTGATGCATGAGGCGCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA...

Claims

1. A specific binding member that binds to CD137 and comprises a CD137 antigen binding site located in the CH3 domain of the specific binding member, the CD137 antigen binding site comprising a first sequence located in the AB structural loop of the CH3 domain, wherein the first sequence comprises the sequence PPY (SEQ ID NO: 10).

2. The specific binding member of claim 1, wherein the specific binding member comprises an insertion in the AB structural loop.

3. A specific binding member as claimed in claim 2, wherein the insertion length is 5 amino acids.

4. The specific binding member of any one of claims 1 to 3, wherein the first sequence is the first sequence of the following specific binding member: (i) FS22-172-003 shown in SEQ ID NO: 138; (ii) FS22-172-002 shown in SEQ ID NO: 129; (iii) FS22-172-004 shown in SEQ ID NO: 147; (iv) FS22-172-001 shown in SEQ ID NO: 120; (v) FS22-172-005 shown in SEQ ID NO: 156; (vi) FS22-172-006 shown in SEQ ID NO: 110; or (vii) FS22-172 shown in SEQ ID NO:

110.

5. The specific binding member of any one of claims 1 to 4, wherein the specific binding member further comprises a second sequence located in the EF structural loop of the CH3 domain, and wherein the second sequence is the second sequence of the specific binding member FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172 shown in SEQ ID NO:

111.

6. The specific binding member of any one of claims 1 to 5, wherein The specific binding member comprises the CH3 domain sequence of the following specific binding member: (i) FS22-172-003 shown in SEQ ID NO: 139; (ii) FS22-172-002 shown in SEQ ID NO: 130; (iii) FS22-172-004 shown in SEQ ID NO: 148; (iv) FS22-172-001 shown in SEQ ID NO: 121; (v) FS22-172-005 shown in SEQ ID NO: 157; (vi) FS22-172-006 shown in SEQ ID NO: 165; or (vii) FS22-172 shown in SEQ ID NO:

112.

7. A specific binding member as described in any one of claims 1 to 6, wherein the specific binding member comprises the sequence of the specific binding members FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006 or FS22-172 as shown in SEQ ID NO: 141, 132, 150, 123, 159, 167 and 114, respectively.

8. The specific binding member of any one of claims 1 to 3, wherein the first sequence is the first sequence of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 and / or FS22-053 as shown in SEQ ID NO:

19.

9. The specific binding member of any one of claims 1 to 3 or 8, wherein the specific binding member further comprises a second sequence located in the EF structural loop of the CH3 domain, and wherein the second sequence is the second sequence of the following specific binding member: (i) FS22-053-008 shown in SEQ ID NO: 20; (ii) FS22-053-009 shown in SEQ ID NO: 29; (iii) FS22-053-011 shown in SEQ ID NO: 47; (iv) FS22-053-017 shown in SEQ ID NO: 101; (v) FS22-053-014 shown in SEQ ID NO: 74; (vi) FS22-053-010 shown in SEQ ID NO: 38; (vii) FS22-053-012 shown in SEQ ID NO: 56; (viii) FS22-053-013 shown in SEQ ID NO: 65; (ix) FS22-053-015 shown in SEQ ID NO: 83; (x) FS22-053-016 shown in SEQ ID NO:92; or (xi) FS22-053 shown in SEQ ID NO:

174.

10. The specific binding member of any one of claims 1 to 3 and 8 to 9, wherein the specific binding member comprises the CH3 domain sequence of the following specific binding member: (i) FS22-053-008 shown in SEQ ID NO: 21; (ii) FS22-053-009 shown in SEQ ID NO: 30; (iii) FS22-053-011 shown in SEQ ID NO: 48; (iv) FS22-053-017 shown in SEQ ID NO: 102; (v) FS22-053-014 shown in SEQ ID NO: 75; (vi) FS22-053-010 shown in SEQ ID NO: 39; (vii) FS22-053-012 shown in SEQ ID NO: 57; (viii) FS22-053-013 shown in SEQ ID NO: 66; (ix) FS22-053-015 shown in SEQ ID NO: 84; (x) FS22-053-016 shown in SEQ ID NO:93; or (xi) FS22-053 shown in SEQ ID NO:

175.

11. A specific binding member as described in any one of claims 1 to 3 and 8 to 10, wherein the specific binding member comprises the sequence of the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 as shown in SEQ ID NO: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95 and 15, respectively.

12. The specific binding member of any one of claims 1 to 11, wherein the specific binding member comprises the specific binding member FS22-172-003 or FS22-053-008, preferably the first sequence, the first and second sequence, the CH3 domain sequence of FS22-172-003, or a sequence thereof.

13. The specific binding member of any one of claims 1 to 12, wherein the specific binding member further comprises a CDR-based antigen binding site.

14. The specific binding member of claim 13, wherein the specific binding member is an antibody molecule.

15. The antibody molecule of claim 15, wherein the CDR-based antigen binding site binds a second antigen selected from the group consisting of an immune cell antigen, a tumor antigen, and a pathogenic antigen.

16. The specific binding member or antibody molecule of any one of claims 1 to 15, wherein the specific binding member or antibody molecule has been modified to reduce or eliminate binding of the specific binding member or antibody molecule to one or more Fcγ receptors.

17. A nucleic acid molecule encoding the specific binding member or antibody molecule of any one of claims 1 to 16.

18. A recombinant host cell comprising the nucleic acid of claim 17.

19. A method for producing the specific binding member or antibody molecule of any one of claims 1 to 16, comprising culturing the recombinant host cell of claim 18 under conditions where the specific binding member or antibody molecule is produced.

20. A specific binding member or antibody molecule as claimed in any one of claims 1 to 16 for use in a method of treatment of the human or animal body by therapy.

21. A specific binding member or antibody molecule for use as claimed in claim 20, wherein the treatment is treatment of cancer or an infectious disease in an individual.

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