Fc-binding fragment containing a CD137 antigen-binding site

Abstract

To solve the problem in which: clinical development of CD137 agonist molecules has been held back due to treatment being either associated with dose-limiting high-grade liver inflammation (urelumab) or low clinical efficacy (utomilumab).SOLUTION: The invention relates to specific binding members that bind CD137. The specific binding members comprise a CD137 antigen-binding site located in a constant domain of the specific binding member and find application in the treatment of cancer and infectious diseases, for example.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to specific binding members that bind to CD137. This specific binding member includes a CD137 antigen binding site located in the constant domain of the specific binding member, and is utilized, for example, in the treatment of cancer and infectious diseases.

Background Art

[0002] Background of the Invention Cell signaling is an essential part of the life of all organisms and usually requires cell surface receptors that interact with soluble ligands or ligands expressed on the surface. This interaction brings about changes in the receptor, ligand, or both. For example, ligand binding can induce a structural change in the receptor that clusters the receptors together into dimers or oligomers. This clustering effect then leads to the activation of intracellular signaling pathways. There are many such activated receptors, including members of the tumor necrosis factor receptor superfamily (TNFRSF), such as CD137.

[0003] CD137 (4-1BB; TNFRSF9) is a co-stimulatory molecule of the tumor necrosis factor receptor superfamily (TNFRSF). CD137 is widely known to be upregulated in CD8 + T cells after activation, and can also be expressed in activated CD4 + helper T cells, B cells, regulatory T cells, natural killer (NK) cells, natural killer T (NKT) cells, and dendritic cells (DC) (Bartkowiak & Curran, 2015). The main functional role of CD137 in promoting T cell cytotoxicity was first described in 1997 (Shuford et al., 1997), and soon after, anti-CD137 mAb was proposed as an anti-cancer therapeutic agent.

[0004] CD137 is a transmembrane protein that has four extracellular cysteine-rich domains called CRD1-4 and a cytoplasmic region involved in CD137 signaling. The ligand for CD137 is CD137L. Although the crystal structure does not exist for the CD137 / CD137L complex, it is predicted that CD137 forms a trimer / trimer complex with CD137L (Won et al., 2010). Engagement of CD137L results in the formation of receptor trimers and subsequent clustering of multiple receptor trimers, leading to the activation of the CD137 signaling cascade. This signaling cascade provides survival signals to T cells against activation-induced cell death (Hurtado et al., 1997), thereby playing an important role in maintaining an effective T cell immune response and generating immune memory (Bartkowiak & Curran, 2015).

[0005] The role of CD137 in leukocyte biology has a clear biological rationale and is generally well understood in contrast to its role in tumor immunology. CD137 is expressed by activated T cells and is used as a marker to identify antigen-specific CD4 + and CD8 + T cells. Typically, the expression of CD137 is higher in CD8 + T cells than in CD4 + T cells (Wen et al., 2002). In the case of CD8 + T cells, proliferation, survival, and cytotoxic effector functions via the production of interferon γ and interleukin 2 have been attributed to the clustering of CD137. Clustering of CD137 also contributes to the differentiation and maintenance of memory CD8 + T cells. In some subsets of CD4 + T cells, clustering of CD137 similarly leads to proliferation and activation, resulting in the release of cytokines such as interleukin 2 (Makkouk et al., 2016).

[0006] Natural killer (NK)-mediated antibody-dependent cell cytotoxicity (ADCC) via tumor-targeted mAbs has been demonstrated to be enhanced as a result of CD137 stimulation via agonistic anti-CD137 monoclonal antibodies in vitro and in vivo (Bartkowiak & Curran, 2015). NK cells bind to antibodies via their Fc receptors depending on the antibody isotype, which can lead to NK cell activation, causing cytotoxic granule release and lysis of target cells (Kohrt et al., 2012). Kohrt et al. demonstrated that an anti-CD137 agonist antibody enhanced the antitumor activity of the therapeutic antibodies rituximab, trastuzumab, and cetuximab by enhancing ADCC when co-administered (Kohrt et al., 2014; Kohrt et al., 2011). Furthermore, human NK cells upregulate the expression of CD137 after encountering cell-bound antibodies via their FcγRs. Subsequent stimulation of these NK cells by anti-CD137 antibodies has been shown to enhance their ADCC against tumor cells (Chester et al., 2015; Chester et al., 2016).

[0007] B lymphocytes also express CD137 upon activation. Binding of the CD137 ligand to CD137 promotes B cell proliferation, survival, and cytokine production. CD137 expression is also induced in normal and malignant human B cells after binding of CD40 to its ligand CD154 (CD40 ligand), resulting in promotion of B cell survival when CD137 is subsequently activated (Vinay and Kwon, 2011).

[0008] CD137 has also been demonstrated to be expressed in a tumor-reactive subset of tumor-infiltrating lymphocytes (TILs). CD137 monotherapy has been shown to be effective in several preclinical immunogenic tumor models such as MC38, CT26, and B cell lymphoma. Combinations of CD137 engagement with other anti-cancer agents such as chemotherapy, cytokines, and other checkpoint regulators have been demonstrated to promote a reduction in the growth of established tumors. Specifically, combinations of anti-CD137 antibodies with anti-CD20, anti-EGFR, and anti-HER-2 antibodies have been shown to have a synergistic effect on reducing tumor growth in various preclinical xenograft models (Kohrt et al., 2014; Kohrt et al., 2012; Kohrt et al., 2011).

[0009] Combining tumor-targeted monoclonal antibody therapy with treatment using an anti-CD137 agonist antibody has shown promising 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). Several tumor-targeted monoclonal antibodies, including the anti-CD20 mAb rituximab (NCT01307267, NCT02951156), the anti-EGFR mAb cetuximab (NCT02110082), and the anti-CS1 mAb elotuzumab (NCT02252263), have also been tested clinically in combination with a CD137 agonist antibody. However, clinical development has been delayed due to dose-limiting severe hepatitis 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 stopped after significant on-target dose-dependent hepatotoxicity was observed (Chester et al., 2018). More recently, clinical trials of urelumab in the treatment of solid cancers have been reopened, combining urelumab treatment with radiotherapy (NCT03431948) or other therapeutic antibodies, such as rituximab (NCT01775631), cetuximab (NCT02110082), the anti-PD-1 antibody nivolumab (NCT02253992, NCT02534506, NCT02845323), and a combination of nivolumab and the anti-LAG-3 antibody BMS986016 (NCT02658981). However, in order to reduce the hepatotoxicity associated with urelumab treatment, the administration of urelumab in these trials had to be restricted, and the results of efficacy were disappointing (Chester et al., 2018).

[0010] In a Phase I clinical trial of cancer, in the dose range of 0.03 mg / kg to 10 mg / kg of Pfizer's anti-CD137 antibody utomilumab (PF-05082566), a human IgG2 isotype antibody, dose-limiting toxicity was not observed (Chester et al. 2016; Segal et al., 2018). However, the overall objective response rate with this antibody was only 3.8% in patients with solid tumors, indicating that utomilumab may have a weaker potency and clinical efficacy than urelumab while having a more favorable safety profile (Chester et al., 2018; Segal et al., 2018). Utomilumab has been tested in combination with radiotherapy (NCT03217747) or chemotherapy, as well as 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 toxicity (DLT), maximum tolerated dose (MTD), and efficacy of various treatment combinations. These trials are currently ongoing, and the initial results show no DLT for doses up to 5 mg / kg and a response rate of 26% for the combination of utomilumab and pembrolizumab. A three-drug combination of utomilumab with avelumab and other cancer immunotherapies has also been tested (NCT02554812, NCT03217747).

[0011] Several bispecific molecules targeting CD137 are also in the early stages of development, and many of them are obtained from non-antibody-based scaffold proteins or fusion protein technologies. The development of bispecific molecules targeting CD137 and FAPα using DARPin scaffold protein-based technology has been reported (Link et al., 2018; Reichen et al., 2018). Tumor-targeted T cell activation by CD137 agonism using HER2-targeted and EphA2-targeted DART molecules has also been shown (Liu et al., 2017). CD137L fusion proteins targeting tumors via FAPα or CD19 in solid tumors and lymphomas have also been developed. The most clinically advanced CD137 bispecific molecule (and the only one containing a full-length antibody) is PRS-343, a CD137 / HER2 bispecific molecule. In this molecule, CD137 is linked via an artificial binding protein (anticalin) that binds to the Fc portion of the HER2-targeted antibody trastuzumab in the IgG4 format. PRS-343 has been reported to provide tumor-targeted dependent activation of CD137 on lymphocytes at sites where HER2 is overexpressed in a humanized mouse model, but no improvement in tumor growth inhibition beyond trastuzumab monotherapy was observed (Hinner et al., 2016 and International Publication No. WO 2016 / 177802 A1). PRS-343 has recently entered a Phase I clinical trial for the treatment of various solid tumors to evaluate its safety, tolerability, and efficacy (NCT03330561). Summary of the Invention Problems to be Solved by the Invention

[0012] Description of the Invention As described in the Background Art section above, the clinical development of CD137 agonist molecules has been delayed because the treatment is associated with either dose-limiting severe hepatitis (urelumab) or low clinical efficacy (utomilumab).

[0013] The inventors recognized that there is a need in the art for CD137 agonist molecules that exhibit high activity and are not associated with dose-limiting hepatitis. Such molecules can be administered to an individual at a dose that optimizes the potency of the molecule and thus its efficacy, and can be used as immunotherapeutic agents for the treatment of cancer, for example, or for the treatment of infectious diseases.

[0014] Without wishing to be bound by theory, it is believed that T cells present in the liver can be activated by anti-CD137 agonist molecules and have the potential to cause hepatitis. CD8+ T cells have been shown to promote hepatitis and apoptosis after sepsis / viral infection (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). Collectively, the results from these studies indicate that anti-CD137 agonist antibodies with high activity, such as urelumab, can cause infiltration of activated CD8+ T cells into the liver, thereby causing hepatitis. Alternatively, the dose-limiting hepatotoxicity observed with urelumab treatment may be due to a specific epitope bound by this antibody.

Means for Solving the Problems

[0015] The inventors conducted an extensive selection and affinity maturation program to isolate a series of antigen-binding Fc fragments (also referred to herein as "Fcab") that contain the CD137 binding site in the CH3 domain and bind to dimeric CD137 with higher affinity than monomeric CD137.

[0016] As used herein, "affinity" can refer to the strength of the binding interaction between an antibody molecule and its cognate antigen as measured by K D As will be readily apparent to those skilled in the art, when an antibody molecule is capable of forming multiple binding interactions with an antigen (e.g., when the antibody molecule is capable of binding to the antigen bivalently, optionally when the antigen is a dimer), KD The affinity measured by [reference] can also be affected by the binding force, whereby the binding force refers to the overall strength of the antibody-antigen complex.

[0017] The expression of CD137 by T cells is upregulated upon activation. Without wishing to be bound by theory, due to the high expression of CD137 in activated T cells, CD137 is thought to form dimers, trimers and higher order multimers on the surface of such cells. In contrast, naive immune cells such as naive T cells express low or very low levels of CD137 on their cell surface, so the CD137 present is likely to be in monomeric form. Thus, it is expected that an Fcab that binds to dimeric CD137 with higher affinity than monomeric CD137 will preferentially bind to activated immune cells such as activated T cells, as opposed to naive immune cells, for example.

[0018] The Fcab of the present invention was also able to bind to dimeric cynomolgus CD137. This is beneficial as it allows toxicology and efficacy studies to be carried out in cynomolgus monkeys during preclinical development. This is particularly advantageous for antibody molecules that bind to CD137, considering that hepatitis has been seen with some anti-CD137 antibodies. Two of the isolated Fcabs, FS22-053-014 and FS22-053-017, also bound to mouse CD137. This is advantageous as it allows the same Fcab to be tested in mice prior to administration to cynomolgus monkeys or humans. Under normal circumstances, an Fcab that binds to mouse CD137 is required for this purpose.

[0019] The inventors have unexpectedly found that all isolated anti-CD137 Fcab molecules that preferentially bind to the dimer over the monomer CD137 and are capable of affinity maturation contained the motif PPY, as well as a 5-amino acid insertion, in the AB loop of their CH3 domain. Another line of anti-CD137 Fcab isolated after the initial library screen did not have these features and could not be affinity matured, and thus was not pursued further. Without wishing to be bound by theory, it is thought that the presence of the PPY motif may promote the formation of an extended antigen-binding region by forming a more rigid or exposed loop structure as a result of the limited flexibility of the proline residues. Alternatively, since proline-rich sequences have been demonstrated to bind, for example, aromatic sequences in SH3 domain proteins, the PPY sequence may represent a specific conserved motif involved in binding to CD137. Furthermore, since the PPY conserved sequence has been independently selected in two separate lines of Fcab, it may be important for epitope binding in CD137. Additionally, the conserved LE or LD sequence is present in the EF loop of the CH3 domain of most of the isolated Fcab, suggesting that this amino acid sequence may also be important for CD137 binding.

[0020] As described in the Background section above, the initial ligation of the CD137 ligand to its receptor, CD137, initiates a series of events that result in trimerization of CD137, followed by receptor clustering, activation of the NFkB intracellular signaling pathway, and subsequent activation of immune cells. For a therapeutic agent to efficiently activate CD137, several CD137 monomers need to be crosslinked together in a way that mimics the trimeric ligand.

[0021] Utrumumab is an IgG2 molecule and depends on cross-linking by Fcγ receptors for its agonistic activity. Urelumab is an IgG4 molecule with constitutive activity and thus does not require cross-linking by Fcγ receptors for activity, although its agonistic activity is enhanced when cross-linked by some Fcγ receptors. Fcγ receptors are found throughout the human body. Therefore, the immunocyte activation activities of utrumumab and urelumab are not limited to specific sites in the body and can occur in the liver or other locations in the body.

[0022] The inventors have shown that the Fcab of the present invention clusters CD137 and requires cross-linking to activate it. However, it should be noted that this is not an inherent feature of Fcab that binds to CD137. Instead, many of the Fcabs isolated during the screening program bound to CD137 but did not require cross-linking for CD137 clustering and activation, or induced limited CD137 clustering and activation in the absence of cross-linking.

[0023] As described above, Fcγ receptor-mediated cross-linking has the drawback that since Fcγ receptors are found throughout the human body, the activation of CD137 is not limited to specific sites. Therefore, the inventors introduced mutations into the CH2 domain of Fcab to reduce or suppress Fcγ receptor binding. Therefore, in the absence of cross-linking by agents other than Fcγ receptors, the Fcab of the present invention does not exhibit CD137 agonistic activity and is not expected to induce hepatitis.

[0024] The inventors recognized that the anti-CD137 Fcab of the present invention can be used to prepare a multispecific, e.g., bispecific molecule that binds to a second antigen such as a tumor antigen in addition to CD137. Preferably, the multispecific molecule binds bivalently to the second antigen, but when the second antigen is a cell-binding tumor antigen, it is expected that monovalent binding of the antigen will be sufficient to crosslink the specific binding member / antibody molecule and induce clustering and activation of CD137. Specifically, the inventors prepared an antibody molecule comprising the anti-CD137 Fcab of the present invention that binds bivalently to a second antigen via the Fab region. The inventors showed that such a bispecific antibody molecule can conditionally activate CD137 in the presence of the second antigen without the need for, e.g., Fcγ receptor-mediated crosslinking required by conventional antibody molecules. Binding of the antibody molecule to the second antigen is thought to cause crosslinking of the antibody molecule at the site of the antigen, which in turn leads to clustering and activation of CD137 on the T cell surface. Thus, the agonist activity of the antibody molecule depends on the presence of both the second antigen and CD137. In other words, the agonist activity depends on the presence of both antigens. Furthermore, crosslinking of the antibody in the presence of the second antigen is thought to assist in clustering of CD137 bound via the constant domain antigen-binding site of the antibody molecule. Thus, when the second antigen is a disease antigen such as a tumor antigen, the antibody molecule is expected to be able to activate immune cells depending on the disease, e.g., in the tumor microenvironment. This targeted activation of immune cells is expected to be beneficial in avoiding hepatitis, for example, as seen with urelumab treatment.

[0025] The inventors have also shown that the bispecific antibody molecule comprising the anti-CD137 Fcab of the present invention is capable of suppressing tumor growth in vivo, wherein the second antigen bound by the antibody molecule was an immune cell antigen, a tumor antigen, or an antigen expressed in both tumor cells and immune cells. Furthermore, more effective suppression of tumor growth was observed with these bispecific antibody molecules compared to a combination of two monospecific antibody molecules, wherein one of the antibody molecules comprised the same constant domain as the bispecific molecule and the other antibody molecule comprised the same variable domain binding site as the bispecific molecule, indicating that enhanced clustering and signaling of CD137, and thus T cell activation and the corresponding anti-tumor effect, were seen when the two binding sites were present in the same molecule.

[0026] The antibody molecule comprising the anti-CD137 Fcab of the present invention and a Fab region specific for a second antigen preferably binds bivalently to both CD137 and the second antigen. This is advantageous because bivalent binding of both targets stabilizes the cross-bridge between T cells expressing CD137 and the second antigen, thereby prolonging the time during which the T cells can be localized to a specific site, such as the tumor microenvironment, and act on the disease, such as a tumor. This is different from most conventional bispecific antibody formats which are heterodimers and bind monovalently to each target antigen via one Fab arm. Such monovalent interactions are expected to be not only more unstable but also often insufficient to first induce clustering of TNF receptors such as CD137.

[0027] In another preferred embodiment, the antibody molecule comprises the anti-CD137 Fcab of the present invention capable of binding bivalently to CD137 and a monovalent binding site specific for a second antigen, such as a single Fab domain. The monovalent binding site can bind, for example, to a tumor-associated antigen. For such a molecule, monovalent binding of the second antigen is expected to allow for closer packing of the antibody molecules at the cell surface, resulting in enhanced clustering of CD137 and thus T cell activation.

[0028] A further feature of the antibody molecule comprising the anti-CD137 Fcab of the present invention is that both of the two antigen-binding sites for CD137 and the second antigen are contained within the antibody structure itself. In particular, the antibody molecule results in a molecule that binds bivalently to both of its targets, such that no other protein needs to be fused to the antibody molecule via a linker or other means. This has a number of advantages. Specifically, since the antibody molecule does not contain additional fused parts, it can be produced using methods similar to those used for the production of standard antibodies. This structure is also predicted to result in improved antibody stability, since the linker can degrade over time and result in a heterogeneous population of antibody molecules. Those antibodies in a population with only one protein fused may not be able to induce conditional agonism of TNF receptors such as CD137 as efficiently as antibodies with two proteins fused. Cleavage or degradation of the linker can occur before or after administration of the therapeutic agent to the patient (e.g., by enzymatic cleavage or by the patient's in vivo pH), thereby resulting in a decrease in its effectiveness in the patient's circulation. When there is no linker in the antibody molecule, the antibody molecule is expected to retain the same number of binding sites both before and after administration. Furthermore, the introduction of a fused protein or linker or both can induce immunogenicity when the antibody molecule is administered to a patient and result in a decrease in the effectiveness of the therapeutic agent, and thus the structure of this molecule is also preferred from the perspective of the immunogenicity of the molecule.

[0029] The inventors have further shown that the proximity resulting from the robust arrangement of the CD137 antigen-binding site and / or the robust structure of the Fcab molecule of the present invention is advantageous for inducing clustering of CD137 compared to the molecule, where the CD137 binding site is not integral with the antibody structure but is provided, for example, by a binding moiety attached to an antibody molecule, or a portion thereof, via a flexible linker.

[0030] Accordingly, the present invention provides the following: [1] A specific binding member that binds to CD137 and contains a CD137 antigen-binding site located in the CH3 domain of the specific binding member, wherein the CD137 antigen-binding site contains a first sequence located in the AB structural loop of the CH3 domain, and the sequence contains PPY (SEQ ID NO: 10), the specific binding member.

[0031] [2] The specific binding member according to [1], wherein the specific binding member contains an insertion in the AB structural loop.

[0032] [3] The specific binding member according to [2], wherein the insertion is 1 to 10 amino acids in length.

[0033] [4] The specific binding member according to [3], wherein the insertion is 4 to 6 amino acids in length.

[0034] [5] The specific binding member according to [4], wherein the insertion is 5 amino acids in length.

[0035] [6] The specific binding member according to 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, where the amino acid residue numbering follows the ImMunoGeneTics (IMGT) numbering scheme.

[0036] [7] The specific binding member according to [6], wherein the insertion is located between positions 14 and 17 of the CH3 domain of the specific binding member.

[0037] [8] The specific binding member according to [7], wherein the insertion is located between positions 16 and 17 of the CH3 domain of the specific binding member.

[0038] [9] The specific binding member according to any one of [2] to [8], wherein the insertion is located between positions 16.5 and 16.1 of the CH3 domain of the specific binding member, where the amino acid residue numbering follows the ImMunoGeneTics (IMGT) numbering scheme.

[0039]

[10] The PPY sequence is located between positions 15 and 17 of the CH3 domain, where the amino acid residue numbering follows the IMGT numbering scheme, and is a specific binding member described in any one of [1] to [9].

[0040]

[11] The PPY sequence is located between positions 16 and 17 of the CH3 domain, where the amino acid residue numbering follows the IMGT numbering scheme, and is a specific binding member described in any one of [1] to

[10] .

[0041]

[12] The PPY sequence is located at positions 16.3, 16.2 and 16.1 of the CH3 domain, and is a specific binding member described in

[11] .

[0042]

[13] The first sequence is a specific binding member: (i) FS22-172-003 described in SEQ ID NO: 138; (ii) FS22-172-002 described in SEQ ID NO: 129; (iii) FS22-172-004 described in SEQ ID NO: 147; (iv) FS22-172-001 described in SEQ ID NO: 120; (v) FS22-172-005 described in SEQ ID NO: 156; (vi) FS22-172-006 described in SEQ ID NO: 110; or (vii) FS22-172 described in SEQ ID NO: 110 and is the first sequence of a specific binding member described in any one of [1] to

[12] .

[0043]

[14] The first sequence is a specific binding member: (i) FS22-172-003 described in SEQ ID NO: 138; (ii) FS22-172-002 described in SEQ ID NO: 129; (iii) FS22-172-004 described in SEQ ID NO: 147; (iv) FS22-172-001 described in SEQ ID NO: 120; (v) FS22-172-005 described in SEQ ID NO: 156; or (vi) FS22-172-006 described in SEQ ID NO: 110 which is the first array of the specific binding member described in

[13] .

[0044]

[15] The first array is the specific binding member: (i) FS22-172-003 described in SEQ ID NO: 138; (ii) FS22-172-002 described in SEQ ID NO: 129; or (iii) FS22-172-004 described in SEQ ID NO: 147 which is the first array of the specific binding member described in

[14] .

[0045]

[16] The first array is the first array of the specific binding member FS22-172-003 described in SEQ ID NO: 138, which is the specific binding member described in

[15] .

[0046]

[17] The PPY array is located at positions 16, 16.5, and 16.4 of the CH3 domain, which is the specific binding member described in

[10] .

[0047]

[18] The first array is the first array 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 described in SEQ ID NO: 19, preferably the first array of the specific binding member FS22-053-008, which is the specific binding member described in any one of [1] to

[10] or

[17] .

[0048]

[19] The first array is located between positions 14 and 17 of the CH3 domain of the specific binding member, where the numbering of amino acid residues follows the IMGT numbering scheme, which is the specific binding member described in any one of

[13] to

[16] or

[18] .

[0049]

[20] The specific binding member according to

[19] , wherein the first array 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.

[0050]

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

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

[0051]

[22] The specific binding member according to

[21] , wherein the second array is the second array 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 described in SEQ ID NO: 111, preferably the specific binding member FS22-172-003.

[0052]

[23] The second array is the specific binding member: (i) FS22-053-008 described in SEQ ID NO: 20; (ii) FS22-053-009 described in SEQ ID NO: 29; (iii) FS22-053-011 described in SEQ ID NO: 47; (iv) FS22-053-017 described in SEQ ID NO: 101; (v) FS22-053-014 described in SEQ ID NO: 74; (vi) FS22-053-010 described in SEQ ID NO: 38; (vii) FS22-053-012 described in SEQ ID NO: 56; (viii) FS22-053-013 described in SEQ ID NO: 65; (ix) FS22-053-015 described in SEQ ID NO: 83; (x) FS22-053-016 described in SEQ ID NO: 92; or (xi) FS22-053 described in SEQ ID NO: 174 The specific binding member described in

[21] , which is the second array of

[0053]

[24] The second array is a specific binding member: (i) FS22-053-008 described in SEQ ID NO: 20; (ii) FS22-053-009 described in SEQ ID NO: 29; (iii) FS22-053-011 described in SEQ ID NO: 47; (iv) FS22-053-017 described in SEQ ID NO: 101; or (v) FS22-053-014 described in SEQ ID NO: 74 The specific binding member described in

[23] , which is the second array of

[0054]

[25] The second array is a specific binding member: (i) FS22-053-008 described in SEQ ID NO: 20; (ii) FS22-053-009 described in SEQ ID NO: 29; (iii) FS22-053-011 described in SEQ ID NO: 47; or (iv) FS22-053-017 described in SEQ ID NO: 101 The specific binding member described in

[24] , which is the second array of

[0055]

[26] The specific binding member described in

[25] , where the second array is the second array of the specific binding member FS22-053-008 described in SEQ ID NO: 20.

[0056]

[27] The specific binding member described in any one of

[21] to

[26] , where the second array is located at positions 92 to 98 of the CH3 domain of the specific binding member, and the numbering of amino acid residues follows the IMGT numbering scheme.

[0057]

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

[27] , where the specific binding member further includes a third array located in the CD structural loop of the CH3 domain.

[0058]

[29] The third array is located at positions 43 to 78 of the specific binding member, where the amino acid residue numbering follows the IMGT numbering scheme, the specific binding member described in

[28] .

[0059]

[30] The third array has the array described in SEQ ID NO: 2, the specific binding member described in any one of

[28] to

[29] .

[0060]

[31] The CH3 domain is a human IgG1 CH3 domain, the specific binding member described in any one of [1] to

[30] .

[0061]

[32] The specific binding member is the specific binding member: (i) FS22-172-003 described in SEQ ID NO: 139; (ii) FS22-172-002 described in SEQ ID NO: 130; (iii) FS22-172-004 described in SEQ ID NO: 148; (iv) FS22-172-001 described in SEQ ID NO: 121; (v) FS22-172-005 described in SEQ ID NO: 157; (vi) FS22-172-006 described in SEQ ID NO: 165; or (vii) FS22-172 described in SEQ ID NO: 112 The specific binding member comprising the CH3 domain sequence of, the specific binding member described in any one of [1] to

[16] ,

[19] to

[22] and

[27] to

[31] .

[0062]

[33] The specific binding member is the specific binding member: (i) FS22-172-003 described in SEQ ID NO: 139; (ii) FS22-172-002 described in SEQ ID NO: 130; (iii) FS22-172-004 described in SEQ ID NO: 148; (iv) FS22-172-001 described in SEQ ID NO: 121; (v) FS22-172-005 described in SEQ ID NO: 157; or (vi) FS22-172-006 described in SEQ ID NO: 165 The specific binding member described in

[32] , comprising the CH3 domain sequence thereof.

[0063]

[34] The specific binding member is the specific binding member: (i) FS22-172-003 described in SEQ ID NO: 139; (ii) FS22-172-002 described in SEQ ID NO: 130; or (iii) FS22-172-004 described in SEQ ID NO: 148 The specific binding member described in

[33] , comprising the CH3 domain sequence thereof.

[0064]

[35] The specific binding member is the specific binding member described in

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

[0065]

[36] The specific binding member is the specific binding member: (i) FS22-053-008 described in SEQ ID NO: 21; (ii) FS22-053-009 described in SEQ ID NO: 30; (iii) FS22-053-011 described in SEQ ID NO: 48; (iv) FS22-053-017 described in SEQ ID NO: 102; (v) FS22-053-014 described in SEQ ID NO: 75; (vi) FS22-053-010 described in SEQ ID NO: 39; (vii) FS22-053-012 described in SEQ ID NO: 57; (viii) FS22-053-013 described in SEQ ID NO: 66; (ix) FS22-053-015 described in SEQ ID NO: 84; (x) FS22-053-016 described in SEQ ID NO: 93; or (xi) FS22-053 described in SEQ ID NO: 175 A specific binding member according to any one of [1] to

[10] ,

[17] to

[18] ,

[19] to

[21] , and

[23] to

[31] , comprising the CH3 domain sequence of .

[0066]

[37] The specific binding member is a specific binding member: (i) FS22-053-008 described in SEQ ID NO: 21; (ii) FS22-053-009 described in SEQ ID NO: 30; (iii) FS22-053-011 described in SEQ ID NO: 48; (iv) FS22-053-017 described in SEQ ID NO: 102; or (v) FS22-053-014 described in SEQ ID NO: 75 A specific binding member according to

[36] , comprising the CH3 domain sequence of .

[0067]

[38] The specific binding member is a specific binding member: (i) FS22-053-008 described in SEQ ID NO: 21; (ii) FS22-053-009 described in SEQ ID NO: 30; (iii) FS22-053-011 described in SEQ ID NO: 48; or (iv) FS22-053-017 described in SEQ ID NO: 102 A specific binding member according to

[37] , comprising the CH3 domain sequence of .

[0068]

[39] A specific binding member according to

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

[0069]

[40] A specific binding member according to

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

[0070]

[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 set forth in SEQ ID NO: 75.

[0071]

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

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

[0072]

[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 containing a CH2 and a CH3 domain.

[0073]

[44] The specific binding member according to

[42] or

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

[0074]

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

[42] to

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

[0075]

[46] The specific binding member according to

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

[0076]

[47] The specific binding member according to

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

[0077]

[48] The specific binding member is the specific binding member described in any one of [1] to

[16] ,

[19] to

[22] ,

[27] to

[35] , and

[42] to

[47] , including the 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 described in SEQ ID NOs: 141, 132, 150, 123, 159, 167, and 114 respectively.

[0078]

[49] The specific binding member is the specific binding member described in

[48] , including the 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 described in SEQ ID NOs: 141, 132, 150, 123, 159, and 1672 respectively.

[0079]

[50] The specific binding member is the specific binding member described in

[49] , including the sequences of the specific binding members FS22-172-003, FS22-172-002, or FS22-172-004 described in SEQ ID NOs: 141, 132, and 150 respectively.

[0080]

[51] The specific binding member is the specific binding member described in

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

[0081]

[52] The specific binding member is the specific binding member described in each of SEQ ID NOs: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95, and 175, namely 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 the sequence of FS22-053, and is the specific binding member described in any one of [1] to

[10] ,

[17] to

[18] ,

[19] to

[21] ,

[23] to

[31] , and

[36] to

[47] .

[0082]

[53] The specific binding member is the specific binding member described in

[52] , and contains the sequence of the specific binding member described in each of SEQ ID NOs: 23, 32, 50, 104, and 77, namely FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, or FS22-053-014.

[0083]

[54] The specific binding member is the specific binding member described in

[53] , and contains the sequence of the specific binding member described in each of SEQ ID NOs: 23, 32, 50, and 103, namely FS22-053-008, FS22-053-009, FS22-053-011, or FS22-053-017.

[0084]

[55] The specific binding member is the specific binding member described in

[54] , and contains the sequence of the specific binding member FS22-053-008 described in SEQ ID NO: 23.

[0085]

[56] The specific binding member binds to human CD137, and is the specific binding member described in any one of [1] to

[55] .

[0086]

[57] Human CD137 has, contains, or consists of the sequence described in SEQ ID NO: 181, and is the specific binding member described in

[56] .

[0087]

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

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

[0088]

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

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

[0089]

[60] The cynomolgus CD137 according to

[59] , having, comprising, or consisting of the sequence set forth in SEQ ID NO: 183, or an antibody molecule.

[0090]

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

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

[0091]

[62] The specific binding member according to

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

[0092]

[63] The specific binding member according to

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

[0093]

[64] The specific binding member according to

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

[0094]

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

[61] to

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

[0095]

[66] The specific binding member according to

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

[0096]

[67] The second antigen-binding site is the specific binding member according to any one of

[61] to

[66] , which comprises a heavy-chain variable and a light-chain variable domain.

[0097]

[68] The specific binding member is an antibody molecule, which is the specific binding member according to any one of

[61] to

[67] .

[0098]

[69] The antibody molecule is a human IgG1 molecule, which is the antibody molecule according to

[68] .

[0099]

[70] The CDR-based antigen-binding site of the antibody molecule binds to a second antigen selected from the group consisting of immune cell antigens and disease antigens, which is the antibody molecule according to any one of

[65] to

[69] .

[0100]

[71] The disease antigen is a tumor antigen or a pathogenic antigen, which is the antibody molecule according to

[70] .

[0101]

[72] The immune cell antigen is an immune regulatory molecule such as PD-L1, which is the antibody molecule according to

[70] .

[0102]

[73] The immune cell antigen is a member of the tumor necrosis factor receptor superfamily (TNFRSF), which is the antibody molecule according to

[70] .

[0103]

[74] The tumor antigen is a tumor-associated antigen (TAA), which is the antibody molecule according to

[71] .

[0104]

[75] The tumor antigen is a cell surface antigen on cancer cells, which is the antibody molecule according to

[71] or

[74] .

[0105]

[76] The tumor antigen is a soluble multimer, which is the antibody molecule according to

[71] .

[0106]

[77] The soluble multimer is at least a dimer, which is the antibody molecule according to

[76] .

[0107]

[78] The antibody molecule according to

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

[0108]

[79] The antibody molecule according to

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

[0109]

[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.

[0110]

[81] The antibody molecule according to any one of

[70] to

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

[0111]

[82] The antibody molecule according to

[80] or

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

[0112]

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

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

[0113]

[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.

[0114]

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

[83] or

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

[0115]

[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 bioactive molecule.

[0116]

[87] A 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.

[0117]

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

[85] .

[0118]

[89] The nucleic acid molecule is (i) the nucleic acid 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 the CH3 domain of FS22-053, each described in SEQ ID NO: 22, 31, 49, 103, 76, 40, 58, 67, 85, 94, and 176; or (ii) the nucleic acid 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 the CH3 domain of FS22-172, each described in SEQ ID NO: 140, 131, 149, 122, 158, 166, and 113 The nucleic acid molecule according to

[88] , comprising.

[0119]

[90] The nucleic acid molecule is a specific binding member: (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, each described in SEQ ID NO: 24, 33, 51, 105, 78, 42, 60, 69, 87, 96, and 177; or (ii) The nucleic acid molecules described in

[88] or

[89] that contain the nucleic acid sequences of FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172, respectively, as described in SEQ ID NOs: 142, 133, 151, 124, 160, 168, and 115. The nucleic acid molecules described in

[88] or

[89] that contain the nucleic acid sequence of .

[0120]

[91] The nucleic acid molecule is a specific binding member: (i) FS22-172-003; or (ii) The CH3 domain nucleic acid sequence of FS22-053-008 The nucleic acid molecules described in

[89] or

[90] that contain the CH3 domain nucleic acid sequence of , or the nucleic acid sequence.

[0121]

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

[88] to

[91] .

[0122]

[93] A recombinant host cell that contains the nucleic acid described in any one of

[88] to

[91] , or the vector described in

[92] .

[0123]

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

[85] , which includes culturing the recombinant host cell described in

[93] under conditions for producing the specific binding member or the antibody molecule.

[0124]

[95] The method described in

[61] , which further includes isolating and / or purifying the specific binding member or the antibody molecule.

[0125]

[96] A pharmaceutical composition that contains the specific binding member or the antibody molecule described in any one of [1] to

[94] and a pharmaceutically acceptable excipient.

[0126]

[97] The specific binding member or the antibody molecule described in any one of [1] to

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

[0127]

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

[87] .

[0128]

[99] A specific binding member or antibody molecule for use as described in

[97] , or a method as described in

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

[0129]

[10] A specific binding member or antibody molecule for use as described in

[97] or

[99] , or a method as described in

[98] or

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

[0130] Brief Description of the Drawings

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Mode for Carrying Out the Invention

[0131] Detailed Description 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 members preferably bind to human CD137, more preferably to human and cynomolgus CD137, and even more preferably to dimeric human and cynomolgus CD137. The portion of CD137 bound by the specific binding member is preferably the CD137 extracellular domain. The extracellular domains of human and cynomolgus CD137 may comprise or consist of the sequences set forth in SEQ ID NOs: 181 and 183, respectively. The specific binding members are preferably capable of binding to CD137 expressed on the surface of cells. The cells are preferably immune cells, 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 (DC), or tumor infiltrating lymphocytes (TIL).

[0132] The specific binding member preferably binds specifically to CD137. The term "specific" can refer to a situation where the specific binding member does not show significant binding to molecules other than its specific binding partner, here CD137. The term "specific" also applies when the specific binding member is specific for a particular epitope, such as an epitope on CD137 carried by several antigens, in which case the specific binding member would be able to bind to various antigens carrying the epitope. The specific binding member preferably does not bind or shows no significant binding to CD40, OX40 and / or GITR.

[0133] As described in the background art section above, treatment of patients with the anti-CD137 antibody urelumab was associated with dose-limiting severe hepatitis. Without wishing to be bound by theory, it is thought that the hepatitis seen with urelumab treatment may have been 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 reduced or no hepatitis, the inventors selected Fcabs with high binding affinity for CD137. Specifically, the inventors selected Fcabs that bound to dimeric CD137 with higher affinity than monomeric CD137. Expression of CD137 by T cells is upregulated upon priming and activation. Due to the higher expression of CD137 in activated T cells, CD137 is thought to exist in the form of dimers, trimers and higher order multimers on the surface of such cells. In contrast, CD137 expression by inactive T cells is low or undetectable. Thus, CD137 is likely to be in monomeric form as long as it is expressed on the surface of such T cells. Therefore, Fcabs that bind to CD137 with high binding affinity preferentially bind to activated T cells as opposed to inactive T cells such as those present in the liver, and thus are thought to show reduced or no hepatitis.

[0134] The specific binding member preferably binds to dimeric human CD137 with an affinity (K D ) of 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, or 2 nM, or with a higher affinity.

[0135] In preferred embodiments, the specific binding member binds to dimeric CD137 with a higher affinity than monomeric CD137. In preferred embodiments, 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 higher than the affinity of the specific binding member for monomeric CD137.

[0136] Human CD137 can have, for example, the sequence set forth in SEQ ID NO: 183. The sequence is the same regardless of whether the antigen is monomeric or in dimeric form.

[0137] Specific binding members from the FS22-53 and FS22-172 lines have also been shown to bind to dimeric cynomolgus CD137. Binding to cynomolgus CD137 as well as human CD137 is beneficial to enable testing of the specific binding member in cynomolgus for efficacy and toxicity prior to administration to humans.

[0138] In preferred embodiments, the specific binding member can bind to dimeric cynomolgus CD137 with an affinity (K D ) 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, or with a higher affinity. Preferably, the specific binding member binds to cynomolgus CD137 with an affinity (K D ) of 2 nM, or with a higher affinity.

[0139] The specific binding member can bind to dimeric human CD137 and dimeric cynomolgus monkey CD137 with similar affinity. It is considered beneficial to conduct efficacy and toxicity tests using the specific binding member in cynomolgus monkeys, which can be predictive of the efficacy and toxicity of the specific binding member in humans.

[0140] Accordingly, in a preferred embodiment, the specific binding member binds to dimeric cynomolgus monkey CD137 with an affinity that is 10-fold or less, preferably 5-fold or less, lower or higher than the affinity with which the specific binding member binds to dimeric human CD137.

[0141] The specific binding member can bind to dimeric mouse CD137, dimeric cynomolgus monkey CD137, and dimeric human CD137. This is considered beneficial because the same specific binding member can be used to conduct initial efficacy tests in mice, toxicity tests in cynomolgus monkeys, and clinical trials in humans, and thus may simplify the path to the clinic. Specifically, the specific binding members FS22-053-014 and FS22-053-017 were unexpectedly found to bind to dimeric mouse, human, and cynomolgus monkey CD137.

[0142] The binding affinity of the specific binding member to an alloantigen such as human or cynomolgus monkey CD137 can be determined by surface plasmon resonance (SPR), such as Biacore.

[0143] The term "specific binding member" refers to an immunoglobulin, or a fragment thereof, that includes a constant domain containing a CD137 antigen-binding site. Thus, the term "specific binding member" as used herein includes an antigen-binding fragment when the antigen-binding fragment includes the CD137 antigen-binding site located in the constant domain of the specific binding member. The constant domain can be a CL, CH1, CH2, CH3, or CH4 domain, preferably the constant domain is a CH1, CH2, or CH3 domain, more preferably a CH2 or CH3 domain, and most preferably a CH3 domain. The specific binding member can be produced synthetically, in part or in whole.

[0144] Preferably, the specific binding member includes CH2 and CH3 domains, where the CH2 or CH3 domain, preferably the CH3 domain, includes the CD137 antigen-binding site. The specific binding member is preferably a dimer of two (identical) polypeptide chains each including CH2 and CH3 domains. In a preferred embodiment, the specific binding member further includes 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™. The hinge region can consist of or include the sequence set forth in SEQ ID NO: 179 or a fragment thereof. Preferably, the fragment is a C-terminal fragment of the sequence set forth in SEQ ID NO: 179. The fragment can be up to 20, up to 10, up to 8, or up to 6 amino acids in length. The fragment can be at least 3, at least 4, at least 5, or at least 6 amino acids in length. In a preferred embodiment, the hinge region has the sequence set forth in SEQ ID NO: 7.

[0145] 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 a human or humanized antibody. 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.

[0146] Since antibodies can be modified in several ways, the term "antibody molecule" should be interpreted to include antibody fragments, derivatives of antibodies, functional equivalents, and homologs, whether natural or wholly or partially synthetic. An example of an antibody fragment containing the CH3 domain is the Fc domain of an antibody. An example of an antibody fragment containing both the CDR sequence and the CH3 domain is a minibody containing an scFv linked to the CH3 domain (Hu et al. (1996), Cancer Res., 56(13):3055-61).

[0147] 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 in the CH3 domain. The CD137 antigen binding site comprises one or more modified structural loops in the constant domain of the specific binding member. Manipulation of antibody constant domain structural loops to form an antigen binding site for a target antigen is known in the art and is described, for example, in Wozniak-Knopp G et al., (2010), International Publication No. 2006 / 072620 and International Publication No. 2009 / 132876.

[0148] After a broad selection and affinity maturation program, the inventors isolated two panels of Fcabs that preferentially bound to the dimer rather than monomeric human CD137. Unexpectedly, all of the isolated Fcabs contained the sequence PPY, as well as a 5 - amino acid insertion, in their AB structural loops. The two panels of Fcabs were selected independently from different selection campaigns, each using a library that contained a 5 - amino acid insertion in the structural AB loop. Thus, the PPY sequence was selected independently twice, indicating the importance of this sequence for CD137 binding. Anti - CD137 Fcabs isolated from an Fcab library that did not contain a 5 - amino acid insertion in the AB structural loop could not be advanced further, for example because the selected Fcabs could not be affinity matured. This indicates that the amino acid insertions present in the AB structural loop can also be important for CD137 binding. Thus, the presence of the sequence PPY and the 5 - amino acid insertion in the AB structural loop, where the PPY sequence can optionally be fully or partially present within the 5 - amino acid insertion, can be important for CD137 binding.

[0149] Thus, the CD137 antigen - binding site of the specific binding member may comprise the first and / or second sequences, preferably, the first and second sequences, where the first and second sequences are each located in the AB and EF structural loops of the constant domain, preferably, the CH3 domain of the specific binding member.

[0150] 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 of these residues are located in the EF structural loop. Unless otherwise indicated, the positions of amino acid residues are numbered herein according to the ImMunoGeneTics (IMGT) numbering scheme, which is described in Lefranc et al., 2005.

[0151] The first array preferably includes the array PPY (SEQ ID NO: 10).

[0152] The PPY array can 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 array is located at positions 16, 16.5 and 16.4 of the CH3 domain. Alternatively, the PPY array can be located between positions 16 and 17 of the CH3 domain. In another preferred embodiment, the PPY array is located at positions 16.3, 16.2 and 16.1 of the CH3 domain. In the IMGT numbering scheme, the inserted residues are numbered according to the direction of the loop in which they are located. When the loop "goes up", the inserted residues take the number of the residue immediately before the insertion, and the numbers of the inserted residues in the sequence are indicated by ascending decimals, e.g., 16, 16.1, 16.2, 16.3, in which case there are three mutations after residue 16. When the loop "goes down", the inserted residues take the number of the residue immediately before the insertion, and the numbers of the inserted residues in the sequence are indicated by descending decimals, e.g., 16, 16.3, 16.2, 16.1, in which case there are three mutations after residue 16 (LeFranc et al., 2005, and LeFranc et al. 2015).

[0153] In a preferred embodiment, the AB structure loop contains an amino acid insertion. The insertion can be 1 to 10, 2 to 9, 3 to 7, 4 to 6 or 5 amino acids in length. Preferably, the insertion is 5 amino acids in length.

[0154] The insertion can be located between positions 10 and 19, preferably between positions 14 and 17, more preferably between positions 16 and 17, of the CH3 domain of the specific binding member. In a preferred embodiment, the insertion is located from position 16.5 to position 16.1 of the CH3 domain of the specific binding member as shown in Figure 1.

[0155] Most of the specific binding members identified after affinity maturation contained a leucine (L) residue at position 97 of the CH3 domain. Many of the specific binding members also contained an aspartic acid (D) residue or a glutamic acid (E) residue 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 suggest that one or both of these residues may be important for CD137 binding. Thus, the second sequence preferably contains the sequence LD or LE, where the LD or LE sequence is preferably located at positions 97 and 98 of the CH3 domain of the specific binding member.

[0156] The first and second sequences can be the first and second sequences of the CH3 domain 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 specific binding members FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, or FS22-172-006, more preferably the specific binding members FS22-172-003, FS22-172-002, or FS22-172-004, and even more preferably the specific binding member FS22-172-003.

[0157] Alternatively, the first and second arrays can be 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, or FS22-053-016, or FS22-053, preferably the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, or FS22-053-014, more preferably the specific binding members FS22-053-008, FS22-053-009, FS22-053-011, or FS22-053-017, even more preferably the first and second arrays of the CH3 domain of the specific binding member FS22-053-008. In another preferred embodiment, the first and second arrays are the specific binding members FS22-053-017 or FS22-053-014, more preferably the first and second arrays of the CH3 domain of the specific binding member FS22-053-017.

[0158] 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 set forth in SEQ ID NOs: 175, 21, 30, 39, 49, 58, 66, 75, 84, 93, 102, 112, 121, 130, 139, 148, 157, and 166, respectively.

[0159] 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 the sequences between positions 14 and 17 and between positions 91 and 99 of the respective CH3 domains 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.

[0160] 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 between positions 92 and 99 of the respective CH3 domains of the specific binding members FS22-053-008, FS22-053-010, FS22-053-011, FS22-053-012, and FS22-053-016.

[0161] The first and second sequences of the specific binding member FS22-053-015 can alternatively be the sequences between positions 14 and 17 and between positions 92 and 98 of the respective CH3 domain of the specific binding member FS22-053-015.

[0162] The CD loop array of the specific binding member is preferably unmodified, i.e., wild-type. Thus, the CD loop array preferably has the sequence set forth in SEQ ID NO: 2. The CD loop array is preferably located at positions 43 to 78 of the CH3 domain of the specific binding member.

[0163] The first and second sequences can be the complete AB and EF structural loop arrays 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. For example, determination of the positions of the AB, CD, and EF structural loops in the CH3 domain sequence, according to the IMGT, IMGT exon, EU, or Kabat numbering scheme, is within the ability of one of ordinary skill in the art and is described in Hasenhindl et al. (2013). In a preferred embodiment, the AB, CD, and EF structural loops according to the IMGT numbering scheme are located between positions 10 and 19, 42 and 79, and 91 and 102 of the CH3 domain of the specific binding member, respectively. In a preferred embodiment, thus, the first, second, and third sequences are the sequences between positions 10 and 19, 42 and 79, and 91 and 102 of the CH3 domain of each 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, FS22-053, FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172.

[0164] In a preferred embodiment, the specific binding member is the specific binding member: (i) FS22-172-003 set forth in SEQ ID NO: 141; (ii) FS22-172-002 set forth in SEQ ID NO: 132; (iii) FS22-172-004 set forth in SEQ ID NO: 150; (iv) FS22-172-001 set forth in SEQ ID NO: 123; (v) FS22-172-005 set forth in SEQ ID NO: 159; (vi) FS22-172-006 set forth in SEQ ID NO: 167; or (vii) FS22-172 set forth in SEQ ID NO: 114 comprising the first and / or second, preferably the first and second sequences; wherein the first and second sequences are each preferably located between positions 14 and 17, and between positions 91 and 99 of the CH3 domain of the specific binding member.

[0165] In a more preferred embodiment, the specific binding member is the specific binding member: (i) FS22-172-003 set forth in SEQ ID NO: 141; (ii) FS22-172-002 set forth in SEQ ID NO: 132; (iii) FS22-172-004 set forth in SEQ ID NO: 150; (iv) FS22-172-001 set forth in SEQ ID NO: 123; (v) FS22-172-005 set forth in SEQ ID NO: 159; or (vi) FS22-172-006 set forth in SEQ ID NO: 167 comprising the first and / or second, preferably the first and second sequences.

[0166] In an even more preferred embodiment, the specific binding member is the specific binding member: (i) FS22-172-003 set forth in SEQ ID NO: 141; (ii) FS22-172-002 set forth in SEQ ID NO: 132; or (iii) FS22-172-004 described in SEQ ID NO: 150 comprises the first and / or second, preferably the first and second sequences thereof.

[0167] In an even more preferred embodiment, the specific binding member comprises the first and / or second, preferably the first and second sequences of the specific binding member FS22-172-003 described in SEQ ID NO: 141.

[0168] In a preferred embodiment, the specific binding member, particularly the specific binding member comprising the first and / or second, preferably the first and second sequences of the specific binding member FS22-172-006, may comprise leucine (L) at position 19 of the CH3 domain of the specific binding member.

[0169] In another preferred embodiment, the specific binding member is the specific binding member: (i) FS22-053-008 described in SEQ ID NO: 23; (ii) FS22-053-009 described in SEQ ID NO: 32; (iii) FS22-053-011 described in SEQ ID NO: 50; (iv) FS22-053-017 described in SEQ ID NO: 104; (v) FS22-053-014 described in SEQ ID NO: 77; (vi) FS22-053-010 described in SEQ ID NO: 41; (vii) FS22-053-012 described in SEQ ID NO: 59; (viii) FS22-053-013 described in SEQ ID NO: 68; (ix) FS22-053-015 described in SEQ ID NO: 86; (x) FS22-053-016 described in SEQ ID NO: 95; or (xi) FS22-053 described in SEQ ID NO: 15 comprising the first and / or second, preferably, the first and second arrays; wherein the first and second arrays are each preferably located between positions 14 and 17 and between positions 91 and 99 of the CH3 domain of the specific binding member.

[0170] In a more preferred embodiment, the specific binding member is a specific binding member: (i) FS22-053-008 as set forth in SEQ ID NO: 23; (ii) FS22-053-009 as set forth in SEQ ID NO: 32; (iii) FS22-053-011 as set forth in SEQ ID NO: 50; (iv) FS22-053-017 as set forth in SEQ ID NO: 104; or (v) FS22-053-014 as set forth in SEQ ID NO: 77 comprising the first and / or second, preferably, the first and second arrays.

[0171] In an even more preferred embodiment, the specific binding member is a specific binding member: (i) FS22-053-008 as set forth in SEQ ID NO: 23; (ii) FS22-053-009 as set forth in SEQ ID NO: 32; (iii) FS22-053-011 as set forth in SEQ ID NO: 50; or (iv) FS22-053-017 as set forth in SEQ ID NO: 104 comprising the first and / or second, preferably, the first and second arrays of the CH3 domain.

[0172] In an even more preferred embodiment, the specific binding member comprises the first and / or second, preferably, the first and second arrays of the specific binding member FS22-053-008 as set forth in SEQ ID NO: 23.

[0173] As an alternative to IMGT numbering, the amino acid residue positions, including the amino acid sequences, substitutions, deletions, and positions of insertions described herein, can be numbered according to IMGT exon numbering (also referred to as consecutive numbering), EU numbering, or Kabat numbering. The correspondence between the residue positions of the CH3 domain for IMGT numbering, IMGT exon numbering, EU numbering, and Kabat numbering is shown in Figure 1. Thus, for example, as shown in Figure 1, if the present application refers to a first sequence located at positions 14-17 of the CH3 domain of a specific binding member (where the residue positions are numbered according to the IMGT numbering scheme), the first sequence is located at positions 18-21 of the CH3 domain (where the residue positions are numbered according to the IMGT exon numbering scheme). Alternatively, the positions of the amino acid residues in the CH3 domain, including the amino acid sequences, substitutions, deletions, and positions of insertions in the CH3 domain described herein, can be defined by reference to their positions in the wild-type CH3 domain sequence set forth in SEQ ID NO: 4. The correspondence between IMGT numbering and the wild-type CH3 domain sequence is also shown in Figure 1.

[0174] In a preferred embodiment, the specific binding member comprises, has, or consists of a CH3 domain comprising the CH3 domain sequence of 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 CH3 domain sequence of 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 CH3 domain sequence of specific binding member FS22-172-003, FS22-172-002, or FS22-172-004, and even more preferably the CH3 domain sequence of specific binding member FS22-172-003.

[0175] In another preferred embodiment, the specific binding member is 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 the CH3 domain sequence of FS22-053, 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 even more preferably comprises, has, or consists of the CH3 domain of the CH3 domain sequence of the specific binding member FS22-053-008.

[0176] The CH3 domain of the specific binding member may optionally contain an additional lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

[0177] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may require the introduction of CDRs, or variable regions, into different immunoglobulins. The introduction of the CDRs of one immunoglobulin into another immunoglobulin is described, for example, in European Patent Application Publication No. A-184187, British Patent Application Publication No. 2188638A, and European Patent Application Publication No. A-239400. Using similar techniques, the constant domain sequence constituting the CD137 antigen-binding site of the specific binding member according to the present invention can be introduced into the constant domain of another specific binding member, for example, the CH3 domain, thereby resulting in a specific binding member that contains the CD137 antigen-binding site in its constant domain. Alternatively, the entire constant domain sequence of the specific binding member can be replaced with the constant domain sequence of the specific binding member according to the present invention to prepare a specific binding member that contains the CD137 antigen-binding site in its constant domain. Similarly, a fragment of the constant domain sequence of the specific binding member can be replaced with the corresponding fragment of the constant domain sequence of the specific binding member according to the present invention that contains the CD137 antigen-binding site.

[0178] The CH2 domain of the specific binding member may contain one or more mutations in the CH2 domain that reduce or suppress binding to one or more Fcγ receptors such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII, and / or complement. The inventors hypothesize that reducing or suppressing binding to the Fcγ receptor reduces or abolishes ADCC mediated by the specific binding member. Similarly, reducing or suppressing binding to complement is expected to reduce or abolish CDC mediated by the specific binding member. Mutations in the CH2 domain that reduce or suppress binding to one or more Fcγ receptors and / or complement are known in the art (Wang et al., 2018). These mutations include the "LALA mutations" described in Bruhns et al., 2009 and Hezareh et al., 2001, which include substitution of leucine residues at IMGT positions 1.3 and 1.2 of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, production of an a-glycosyl antibody via mutation of the conserved N-linked glycosylation site by mutating asparagine (N) at IMGT position 84.4 of the CH2 domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q) is also known to reduce IgG1 effector function (Wang et al., 2018). As a further alternative, complement activation (C1q binding) and ADCC are known to be reduced by mutation of proline at IMGT position 114 of the CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations may also be combined to produce specific binding members with further reduced ADCC or CDC activity or no ADCC or CDC activity.

[0179] Accordingly, the specific binding member may comprise a CH2 domain, wherein the CH2 domain preferably (i) alanine residues at positions 1.3 and 1.2; and / or (ii) Alanine or glycine at position 114; and / or (iii) Alanine, glutamine or glycine at position 84.4 comprising; wherein the numbering of the amino acid residues follows the IMGT numbering scheme.

[0180] In a preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain preferably (i) Alanine residues at positions 1.3 and 1.2; and / or (ii) Alanine or glycine at position 114 comprising; wherein the numbering of the amino acid residues follows the IMGT numbering scheme.

[0181] In another preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain (i) Alanine residue at position 1.3; and (ii) Alanine residue at position 1.2 comprising; wherein the numbering of the amino acid residues follows the IMGT numbering scheme.

[0182] For example, the CH2 domain may have the sequence set forth in SEQ ID NO: 6. [LALA]

[0183] In another preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain (i) Alanine residue at position 1.3; (ii) Alanine residue at position 1.2; and (iii) Alanine at position 114 comprising; wherein the numbering of the amino acid residues follows the IMGT numbering scheme.

[0184] For example, the CH2 domain may have the sequence set forth in SEQ ID NO: 5. [LALA-PA]

[0185] In a preferred embodiment, the specific binding member is 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, more preferably the CH2 and CH3 domain sequences of the specific binding members FS22-172-003, FS22-172-002, or FS22-172-004, even more 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 start from amino acid 7 onwards and are shown in SEQ ID NOs: 141, 132, 150, 123, 159, 167, and 114 respectively.

[0186] In another preferred embodiment, the specific binding member is 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 the CH2 and CH3 domain sequences of FS22-053, preferably the CH2 and CH3 domain sequences of the specific binding member 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 member FS22-053-008, FS22-053-009, FS22-053-011, or FS22-053-017, even more preferably comprises, has, or consists of the CH2 and CH3 domain sequences of the specific binding member FS22-053-008, wherein 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 the CH2 and CH3 domain sequences of FS22-053 start from amino acid 7 and onwards, as shown in SEQ ID NOs: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95, and 15 respectively.

[0187] In another preferred embodiment, the specific binding member comprises, consists of, or has 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 the sequence of specific binding member FS22-053-008, FS22-053-009, FS22-053-011, FS22-053-017, or FS22-053-014, more preferably the sequence of specific binding member FS22-053-008, FS22-053-009, FS22-053-011, or FS22-053-017, even more preferably the sequence of specific binding member FS22-053-008, wherein the sequences of 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 set forth in SEQ ID NOs: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95, and 15, respectively.

[0188] In a preferred embodiment, the specific binding member may comprise one or more additional antigen binding sites that bind to one or more additional antigens in addition to the CD137 antigen binding site located in the constant domain of the specific binding member. The one or more additional antigen binding sites preferably specifically bind to their homologous antigens.

[0189] The one or more additional antigen binding sites may bind to CD137 or another antigen. Thus, the specific binding member can be a multispecific, e.g., bispecific, trispecific, or tetravalent molecule, preferably a bispecific molecule. In a preferred embodiment, the specific binding member is capable of binding to CD137 and one or more additional antigens simultaneously.

[0190] Antibody molecules are known to have a modular structure comprising individual domains that can be combined in a plurality of different ways to form multi-specific, for example bispecific, trispecific, or tetra-specific antibody formats. Exemplary multi-specific antibody formats are described, for example, in Spiess et al. (2015) and Kontermann (2012). The specific binding members of the present invention can be used in such multi-specific antibody formats. This has the further advantage of introducing additional antigen binding sites into such multi-specific antibody formats via the presence of the constant domain of the antigen binding site of the specific binding member, for example the CH3 domain.

[0191] For example, the specific binding member of the present invention can be a heterodimeric antibody molecule, for example, a heterodimeric full immunoglobulin molecule, or a fragment thereof. In this case, a part of the antibody molecule will have one or more of the sequences described herein. For example, if the specific binding member of the present invention is a bispecific heterodimeric antibody molecule, the specific binding member can include a heavy chain that is paired with a heavy chain comprising the CH3 domain described herein that binds to an antigen other than CD137. Techniques for preparing heterodimeric antibodies are known in the art and include the knobs-into-holes (KIH) technique, which involves engineering the CH3 domain of the antibody molecule to form either a "knob" or a "hole" to facilitate heterodimerization of the chains. Alternatively, the heterodimeric antibody can be prepared by introduction of a charge pair into the antibody molecule to avoid homodimerization of the CH3 domain by electrostatic repulsion and direct heterodimerization by electrostatic attraction. Examples of heterodimeric antibody formats include CrossMab, mAb-Fv, SEED-body, and KIH IgG.

[0192] Alternatively, the multispecific binding member of the present invention may comprise a full immunoglobulin molecule or a fragment thereof and one or more additional antigen-binding portions. The antigen-binding portion may be, for example, an Fv, scFv or single domain antibody and may be fused to the full immunoglobulin molecule or a fragment thereof. Examples of multispecific antibody molecules comprising an additional antigen-binding portion fused to a full 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 an additional antigen-binding portion fused to an immunoglobulin fragment comprising a CH3 domain include, for example, sc diabody-CH3, diabody-CH3, and scFv-CH3 KIH (Spiess et al., 2015; Figure 1).

[0193] Other suitable multispecific formats will be readily apparent to those skilled in the art.

[0194] In a preferred embodiment, the binding member comprises a second antigen-binding site that binds to a second antigen, and the second antigen-binding site is preferably a CDR-based antigen-binding site. A CDR-based antigen-binding site is an antigen-binding site in the antibody variable region. A CDR-based antigen-binding site is formed by six CDRs; three light chain variable domain (VL) CDRs and three heavy chain variable domain (VH) CDRs.

[0195] The preparation of antibody molecules against a given antigen and the determination of the CDR sequences of such antibody molecules are well established 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).

[0196] For example, the binding member is an mAb 2(Trademark) It can be a bispecific antibody. The mAb mentioned in this specification 2 A bispecific antibody is an IgG immunoglobulin that contains a CDR-based antigen-binding site in each of its variable regions and at least one antigen-binding site in the constant domain. When the specific binding member of the present invention is in the mAb 2 format, the specific binding member contains a CDR-based antigen-binding site in each of its variable regions in addition to the CD137 antigen-binding site in the constant domain of the specific binding member.

[0197] The three VH domain CDRs of the antigen-binding site may be located in the immunoglobulin VH domain, and the three VL domain CDRs may be located in the immunoglobulin VL domain. For example, the CDR-based antigen-binding site may be located in the antibody variable region.

[0198] The specific binding member may have one or preferably two or more, for example two, CDR-based antigen-binding sites for a second antigen. Thus, the specific binding member may contain one VH and one VL domain, but preferably contains two VH and two VL domains, i.e., two VH / VL domain pairs, as in the case of a natural IgG molecule.

[0199] In a preferred embodiment, the specific binding member can be an immunoglobulin containing two variable regions, and each variable region contains a CDR-based antigen-binding site for a second antigen.

[0200] Thus, in a preferred embodiment, the antibody molecule is an antibody that binds to CD137 and a second antigen, and the antibody molecule (i) two antigen-binding sites for CD137 located in the two CH3 domains of the antibody molecule; and (ii) two CDR-based antigen-binding sites for a second antigen formed by the immunoglobulin VH domain and the immunoglobulin VL domain, respectively including.

[0201] In a more preferred embodiment, the antibody is a complete immunoglobulin molecule, for example, a complete IgG1 molecule that binds to CD137 and a second antigen, and the antibody molecule is (i) two antigen-binding sites for CD137 located in the two CH3 domains of the antibody molecule; and (ii) two CDR-based antigen-binding sites for the second antigen, formed by the immunoglobulin VH domain and the immunoglobulin VL domain, respectively comprising; wherein the immunoglobulin molecule further comprises CH1, CH2 and CL domains.

[0202] Activation of CD137 requires clustering of CD137 on the surface of immune cells, such as the surface of T cells, which in turn stimulates intracellular signaling pathways and immune cell activation. Binding of a specific binding member to CD137 on the surface of immune cells in the absence of cross-linking of the specific binding member does not cause CD137 to form clusters and, as a result, does not lead to immune cell activation.

[0203] The inventors have shown that the specific binding members of the FS22-53 and FS22-172 lines do not effect T cell activation in the absence of cross-linking of the specific binding member (see Example 5).

[0204] As explained above, cross-linking of antibody molecules by binding to Fcγ receptors is inefficient and cannot be targeted to a specific location, such as the site of a disease, when Fcγ receptor-expressing cells are present throughout the human body. Therefore, the second antigen bound by the second antigen-binding site is preferably not an Fcγ receptor.

[0205] Accordingly, in a preferred embodiment, the specific binding member of the present invention comprises a second antigen-binding site that binds to a second antigen, wherein the second antigen can bind to and cross-link a plurality of specific binding members.

[0206] For example, the inventors have shown that when the second antigen is a multimeric molecule, binding of the specific binding member to the second antigen results in or promotes T cell activation. Thus, the second antigen is preferably a multimeric antigen, such as a dimer, trimer or higher order multimer, so that it is possible to crosslink several specific binding members.

[0207] The inventors have also shown that, using the CD137 / second antigen mAb 2 molecule, when the second antigen can be monomeric or multimeric and is a surface antigen, such as one present at high concentration on a surface, for example a cell surface, and / or a cell surface antigen that is clustered, binding of the antibody molecule to the second antigen results in or promotes T cell activation. Without wishing to be bound by theory, it is thought that binding of the antibody molecule to an abundant cell surface antigen results in, for example, a high concentration of antibody molecules bound to the cell surface that are arranged sufficiently closely that the antibody molecule can cause clustering of CD137 and activation of immune cells. Thus, in a preferred embodiment, the second antigen is a surface antigen that is expressed at high concentration on a surface, for example a cell surface.

[0208] A specific binding member that binds to the second antigen described herein and activates immune cells such as T cells only upon binding to the second antigen, or whose immune cell activating activity is promoted upon binding to the second antigen, is also referred to as a conditional agonist. This immune cell activating activity upon binding to the second antigen is independent of binding of the specific binding member to Fcγ receptors and / or an external crosslinking agent, such as protein A or G or a secondary antibody, and thus allows the conditional agonist activity of the specific binding member to be targeted to the site where the second antigen is present. For example, if the second antigen is a disease antigen, the specific binding member can selectively promote activation of immune cells at the site of the disease in an individual and not promote activation of T cells elsewhere.

[0209] Furthermore, specific binding members that activate immune cells such as T cells only upon binding to a second antigen preferably have increased immune cell activation activity compared to specific binding members that rely on cross-linking by other mechanisms such as external cross-linking agents or cross-linking by Fcγ receptor interactions. Since activation of CD137 is more efficient, immune cell activation can be achieved with a lower concentration of the specific binding members described herein compared to other specific binding members.

[0210] Accordingly, the specific binding members of the present invention preferably induce increased activation of immune cells such as T cells when the specific binding member is cross-linked, for example, by binding to a second antigen, compared to when the specific binding member is not cross-linked.

[0211] 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. Accordingly, a T cell activation assay can measure IL-2 release to determine the level of T cell activation induced by an antibody molecule or specific binding member.

[0212] 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 by T cells in a T cell activation assay when the specific binding member or antibody molecule is cross-linked. This is referred to as the EC 50 of the following antibody molecule or specific binding member. A lower EC 50 indicates that a lower concentration of the antibody molecule or specific binding member is required to achieve half-maximal release of IL-2 by T cells in a T cell activation assay and, thus, that the antibody molecule or specific binding member has higher T cell activation activity. The specific binding member or antibody molecule can be cross-linked, for example, using an anti-CH2 antibody.

[0213] In a preferred embodiment, the antibody molecule or specific binding member has an EC in a T cell activation assay that is within 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold of the EC of FS22-172-003 / HelD1.3 (including the LALA mutation) in the same assay 50 where FS22-172-003 / HelD1.3 (LALA) consists of or comprises the heavy chain set forth in SEQ ID NO: 145 and the light chain set forth in SEQ ID NO: 173. 50 In another preferred embodiment, the antibody molecule or specific binding member has an EC in a T cell activation assay that is within 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold of the EC of FS22-053-008 / HelD1.3 (including the LALA mutation) in the same assay

[0214] where FS22-053-008 / HelD1.3 (LALA) consists of or comprises the heavy chain set forth in SEQ ID NO: 27 and the light chain set forth in SEQ ID NO: 173. 50 For example, the antibody molecule or specific binding member can have an EC in a T cell activation assay of 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. 50 In addition to, or alternatively, the ability of the antibody molecule or specific binding member to activate T cells can be determined by measuring the highest 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, where the antibody molecule or specific binding member is crosslinked.

[0215] 50

[0216]

[0217] ​​​In a preferred embodiment, in the presence of crosslinking, in the presence of an antibody molecule or specific binding member, the maximum concentration of IL-2 released by T cells in a T cell activation assay is within 3-fold, 2-fold, or 1.5-fold of the maximum concentration of IL-2 released by T cells in the same assay in the presence of FS22-053-008 / HelD1.3 (including the LALA mutation) or FS22-172-003 / HelD1.3 (including the LALA mutation).

[0218] The T cell activation assay can be a T cell assay described herein, such as a CD8+ T cell assay, as described in the examples of the present invention. See Example 5.4.

[0219] For example, the T cell activation assay can be an IL-2 release assay based on CD8+ T cells isolated from human peripheral blood mononuclear cells (PBMC). For example, the T cell activation assay can include isolating human PBMC from a leukapheresis cone. Methods for isolating PBMC are known in the art and are described in the examples of the present invention. Next, CD8+ T cells can be isolated from PBMC. Methods for isolating CD8+ T cells from PBMC are known in the art and are described in the examples of the present invention.

[0220] Next, the CD8+ T cells can be added to a multiwell plate coated with anti-human CD3 antibody. A suitable dilution of each test antibody molecule or specific binding member can be prepared and added to the wells. Next, the T cells are incubated with the test antibody for 24 hours at 37°C, 5% CO 2 and can be incubated at. 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 solution are known in the art and are described in the examples of the present invention. The concentration of human IL-2 can be plotted against the log concentration of the antibody molecule or specific binding member. The resulting curve can be fitted using the log(agonist) vs. response equation.

[0221] 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. Examples of disease antigens include pathogenic antigens and tumor antigens.

[0222] The immune cell antigen bound by the specific binding member can be present on the same immune cell as CD137 or on a different immune cell.

[0223] The immune cell antigen can be a member of the tumor necrosis factor receptor superfamily (TNFRSF) other than CD137. TNFRSF receptors are membrane-bound cytokine receptors that contain an extracellular cysteine-rich domain that binds to one or more ligands of the tumor necrosis factor superfamily (TNFSF).

[0224] The TNFRSF receptor can be located on the surface of immune cells. Upon binding of the TNFRSF ligand, the TNFRSF receptor forms clusters on the surface of immune cells that activate the immune cells. For example, the ligand-bound TNFRSF receptor can form multimers such as trimers or clusters of multimers. The presence of a cluster of ligand-bound NFRSF receptors stimulates intracellular signaling pathways that activate immune cells.

[0225] Without wishing to be bound by theory, it is believed that by engaging CD137 and a second TNFRSF receptor on the surface of immune cells, the specific binding member clusters both CD137 and the second TNFRSF receptor and activates the immune cells. In other words, the specific binding member acts as a TNFRSF receptor agonist when both targets are bound.

[0226] Examples of TNFRSF receptors include CD27, CD40, EDA2R, EDAR, FAS, LTBR, RELT, TNFRSF1A, TNFRSF1B, TNFRSF4, TNFRSF6B, TNFRSF8, TNFRSF10A-10D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, and TNFRSF25.

[0227] CD27 (TNFRSF7: Gene ID 939) has the reference amino acid sequence of NP_001233.1 and can be encoded by the reference nucleotide sequence of NM_001242.4. CD40 (TNFRSF5: Gene ID 958) has the reference amino acid sequence of NP_001241.1 and can be encoded by the reference nucleotide sequence of NM_001250.5. EDA2R (TNFRSF27: Gene ID 60401) has the reference amino acid sequence of NP_001186616.1 and can be encoded by the reference nucleotide sequence of NM_001199687.2. EDAR (Gene ID 10913) has the reference amino acid sequence of NP_071731.1 and can be encoded by the reference nucleotide sequence of NM_022336.3. FAS (TNFRSF6: Gene ID 355) has the reference amino acid sequence of NP_000034.1 and can be encoded by the reference nucleotide sequence of NM_000043.5. LTBR (TNFRSF3: Gene ID 4055) has the reference amino acid sequence of NP_001257916.1 and can be encoded by the reference nucleotide sequence of NM_001270987.1. RELT (TNFRSF19L: Gene ID 84957) has the reference amino acid sequence of NP_116260.2 and can be encoded by the reference nucleotide sequence of NM_032871.3. TNFRSF1A (Gene ID 7132) has the reference amino acid sequence of NP_001056.1 and can be encoded by the reference nucleotide sequence of NM_001065.3. TNFRSF1B (Gene ID 7133) has the reference amino acid sequence of NP_001057.1 and can be encoded by the reference nucleotide sequence of NM_001066.2. TNFRSF4 (Gene ID 7293) has the reference amino acid sequence of NP_003318 and can be encoded by the reference nucleotide sequence of NM_003327. TNFRSF6B (Gene ID 8771) has the reference amino acid sequence of NP_003814.1 and can be encoded by the reference nucleotide sequence of NM_003823.3. TNFRSF8 (Gene ID 943) has the reference amino acid sequence of NP_001234.3 and can be encoded by the reference nucleotide sequence of NM_001243.4.TNFRSF10A (Gene ID 8797) has the reference amino acid sequence of NP_003835.3 and can be encoded by the reference nucleotide sequence of NM_003844.3. TNFRSF10B (Gene ID 8795) has the reference amino acid sequence of NP_003833.4 and can be encoded by the reference nucleotide sequence of NM_003842.4. TNFRSF10C (Gene ID 8794) has the reference amino acid sequence of NP_003832.2 and can be encoded by the reference nucleotide sequence of NM_003841.4. TNFRSF10D (Gene ID 8793) has the reference amino acid sequence of NP_003831.2 and can be encoded by the reference nucleotide sequence of NM_003840.4. TNFRSF11A (Gene ID 8792) has the reference amino acid sequence of XP_011524547.1 and can be encoded by the reference nucleotide sequence of XM_11526245.2. TNFRSF11B (Gene ID 4982) has the reference amino acid sequence of NP_002537.3 and can be encoded by the reference nucleotide sequence of NM_002546.3. TNFRSF12A (Gene ID 51330) has the reference amino acid sequence of NP_057723.1 and can be encoded by the reference nucleotide sequence of NM_016639.2. TNFRSF13B (Gene ID 23495) has the reference amino acid sequence of NP_0036584.1 and can be encoded by the reference nucleotide sequence of NM_012452.2. TNFRSF13C (Gene ID 115650) has the reference amino acid sequence of NP_443177.1 and can be encoded by the reference nucleotide sequence of NM_052945.3. TNFRSF14 (Gene ID 8764) has the reference amino acid sequence of NP_001284534.1 and can be encoded by the reference nucleotide sequence of NM_001297605.1. TNFRSF17 (Gene ID 608) has the reference amino acid sequence of NP_001183.2 and can be encoded by the reference nucleotide sequence of NM_001192.2. TNFRSF18 (Gene ID 8784) has the reference amino acid sequence of NP_004195.2 and can be encoded by the 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 the 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 the 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 the reference nucleotide sequence of NM_001039664.1.

[0228] Alternatively, the immune cell antigen bound by the second antigen-binding site can be a molecule having a regulatory function in the immune system other than a TNFRSF member, such as a costimulatory molecule or an inhibitory checkpoint molecule. Examples of such immune regulatory 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.

[0229] The immune cell to which the immune cell antigen is present may belong to any immune cell subset and can be a T cell, tumor-infiltrating leukocyte (TIL), myeloid lineage cell, such as an antigen-presenting cell (APC), NK cell, and / or B cell. When the immune cell antigen is a TNFRSF receptor, the immune cell in which the TNFRSF receptor is present is preferably a T cell.

[0230] Alternatively, the second antigen-binding site can bind to the disease antigen described above. Without wishing to be bound by theory, it is believed that the binding of the specific binding member to CD137 and the disease antigen results in activation of T cells near the disease. Next, the activated T cells can then initiate, promote, or participate in an immune response, for example, an immune response against a pathogen or cancer cells. An overview of the role played by the immune system in recognizing and eliminating cancer cells is provided by Chen and Mellman (2013).

[0231] In a preferred embodiment, the disease antigen can bind to a tumor antigen. A tumor antigen is an antigen that is mainly present in the tumor environment and not ubiquitously present elsewhere in the individual. For example, a tumor antigen can be present on the surface of tumor cells, or can be present in other stromal cells of the tumor microenvironment or in the body fluid near the tumor. Thus, a tumor antigen is a marker of the location of tumor cells in an individual.

[0232] In one embodiment, the tumor antigen can be an antigen located on the surface of cancer cells. Preferably, the tumor antigen is upregulated or overexpressed in tumor cells, while it is not highly expressed by corresponding normal somatic cells from the same tissue in the absence of the tumor.

[0233] In one embodiment, the tumor antigen is upregulated or overexpressed in stromal cells of the tumor microenvironment compared to stromal cells of the corresponding normal tissue in the absence of the tumor.

[0234] Preferred tumor antigens are present on the cell surface and are not rapidly internalized.

[0235] Tumor antigens suitable for targeting by specific binding members can be identified using methods known in the art. For example, specific binding members targeting the CD137 receptor and tumor antigens can be used in an assay in which CD137-expressing cells are co-cultured with tumor antigen-expressing cells, and activation of the CD137-expressing cells is measured, for example, by a T cell activation assay, a proliferation assay, or a cytotoxicity assay.

[0236] Cell surface tumor antigens can be tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).

[0237] Examples of tumor antigens expressed by cancer cells include, for example, cancer germline genes such as those encoded by 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-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, 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-I, SSX-I, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-I and XAGE, as well as 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).

[0238] Other cell surface tumor antigens include, for example, AFP, α v β 3 (vitronectin receptor), α v β 6, B cell maturation antigen (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-idiotype, carcinoembryonic antigen (CEA), for example carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), TAG-72, folate-binding protein, A33, G250, ferritin, glycolipids, for example, gangliosides, glycoproteins, for example, CA-125, IL-2 receptor, fibroblast activation protein (FAP), IGF1R, B7H3, B7H4, PD-L1, CD200, EphA2, and mesothelin or variants thereof. 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.

[0239] Other tumor antigens include out-of-frame peptide-MHC complexes generated by non-AUG translation initiation mechanisms used by "stressed" cancer cells (Malarkannan et al., 1999).

[0240] Other tumor antigens include peptide-MHC complexes on the surface of tumor cells or on the surface of cells in the tumor microenvironment, where the peptide-MHC complex contains a tumor-specific neoantigen peptide fragment of a mutated intracellular tumor antigen, and the peptide neoantigen has one or more tumor-specific mutations (Gubin et al., 2015). Other tumor antigens are well known in the art (see, for example, WO 00 / 20581; Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge). The sequences of these tumor antigens are readily available from public databases, but are also found in WO 92 / 020356 A1, WO 94 / 005304 A1, WO 94 / 023031 A1, WO 95 / 020974 A1, WO 95 / 023874 A1 and WO 96 / 026214 A1.

[0241] Preferred tumor antigens include HER2, FAP, EpCAM, CEACAM5, CD20, CD73, PSMA, mesothelin, EphA2, IGF1R, CD200, α v β 6 , BCMA, PD-L1, B7H3, B7H4 and EGFR.

[0242] In a more preferred embodiment, the tumor antigen is mesothelin (MSLN).

[0243] In an alternative more preferred embodiment, the tumor antigen is PD-L1.

[0244] HER2 (ERBB2; Gene ID 2064) may have the reference amino acid sequence of NP_001005862.1 and may be encoded by the reference nucleotide sequence of NM_001005862.2. FAP (Gene ID 2191) may have the reference amino acid sequence of NP_001278736.1 and may be encoded by the reference nucleotide sequence of NM_001291807.1. EpCAM (Gene ID 4072) may have the reference amino acid sequence of NP_002345.2 and may be encoded by the reference nucleotide sequence of NM_002354.2. CEACAM5 (Gene ID 1048) may have the reference amino acid sequence of NP_001278413.1 and may be encoded by the reference nucleotide sequence of NM_001291484.2. CD20 (MS4A1; Gene ID 931) may have the reference amino acid sequence of NP_068769.2 and may be encoded by the reference nucleotide sequence of NM_021950.3. CD73 (NT5E; Gene ID 4907) may have the reference amino acid sequence of NP_001191742.1 and may be encoded by the reference nucleotide sequence of NM_001204813.1. PSMA (FOLH1; Gene ID 2346) may have the reference amino acid sequence of NP_001014986.1 and may be encoded by the reference nucleotide sequence of NM_001014986.1. Mesothelin (MSLN; Gene ID 10232) may have the reference amino acid sequence of NP_001170826.1 and may be encoded by the reference nucleotide sequence of NM_001177355.2. EphA2 (Gene ID 1969) may have the reference amino acid sequence of NP_001316019.1 and may be encoded by the reference nucleotide sequence of NM_001329090.1. IGF1R (Gene ID 3480) may have the reference amino acid sequence of NP_000866.1 and may be encoded by the reference nucleotide sequence of NM_000875.4. CD200 (Gene ID 4345) may have the reference amino acid sequence of NP_001004196.2 and may be encoded by the reference nucleotide sequence of NM_001004196.3. Alpha v Beta 6is a heterodimer composed of integrin subunit αV and integrin subunit β6. Integrin subunit αV (ITGAV; Gene ID 3685) may have the reference amino acid sequence of NP_001138471.1 and may be encoded by the reference nucleotide sequence of NM_001144999.2. Integrin subunit β6 (ITGB6; Gene ID 3694) may have the reference amino acid sequence of NP_000879.2 and may be encoded by the reference nucleotide sequence of NM_000888.4. BCMA (TNFRSF17; Gene ID 608) may have the reference amino acid sequence of NP_001183.2 and may be encoded by the reference nucleotide sequence of NM_001192.2. PD-L1 (CD274; Gene ID 29126) may have the reference amino acid sequence of NP_001254635.1 and may be encoded by the reference nucleotide sequence of NM_001267706.1. B7H3 (CD276; Gene ID 80381) may have the reference amino acid sequence of NP_001019907.1 and may be encoded by the reference nucleotide sequence of NM_001024736.1. B7H4 (VTCN1; Gene ID 79679) may have the reference amino acid sequence of NP_001240778.1 and may be encoded by the reference nucleotide sequence of NM_001253849.1. EGFR (Gene ID 1956) may have the reference amino acid sequence of NP_001333826.1 and may be encoded by the reference nucleotide sequence of NM_001346897.1.

[0245] In other embodiments, the tumor antigen can be a soluble tumor antigen, such as a growth factor produced by or in response to cancer cells. The soluble factor can be upregulated or overexpressed in the body fluid near the tumor. The soluble tumor antigen can be a multimer, such as a dimer or trimer. The soluble tumor antigen can be present at a higher concentration in the tumor site or tumor microenvironment than in other parts of the individual's body. The tumor microenvironment and related soluble tumor antigens are described in more detail by Bhome et al. (2015).

[0246] 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.

[0247] VEGF (VEGFA; Gene ID 7422) has the reference amino acid sequence of NP_001020537.2 and can be encoded by the reference nucleotide sequence of NM_001025366.2. HGF (Gene ID 3082) has the reference amino acid sequence of NP_000592.3 and can be encoded by the reference nucleotide sequence of NM_000601.5. SDF1 (CXCL12; Gene ID 6387) has the reference amino acid sequence of NP_000600.1 and can be encoded by the reference nucleotide sequence of NM_000609.6. TGF-β-1 (TGFB1; Gene ID 7040) may have the reference amino acid sequence of NP_000651.3 and can be encoded by the reference nucleotide sequence of NM_000660.6. TGF-β-2 (TGFB2; Gene ID 7042) may have the reference amino acid sequence of NP_001129071.1 and can be encoded by the reference nucleotide sequence of NM_001135599.3. TGF-β-3 (TGFB3; Gene ID 7043) may have the reference amino acid sequence of NP_001316867.1 and can be encoded by the reference nucleotide sequence of NM_001329938.1. TGF-β-4 (LEFTY2; Gene ID 7044) may have the reference amino acid sequence of NP_001165896.1 and can be encoded by the reference nucleotide sequence of NM_001172425.2.

[0248] In another preferred embodiment, the disease antigen is a pathogenic antigen.

[0249] Activation of immune cells such as T cells and / or macrophages by specific binding members near the site of infection is expected to be useful for the treatment of infections. The infection can be an acute or persistent infection, but is preferably a persistent infection.

[0250] A pathogenic antigen is preferably an antigen expressed by a human pathogen, such as a viral, bacterial, fungal or parasitic antigen (e.g., protozoan antigen), preferably a viral or bacterial antigen. A pathogenic antigen is an antigen that is mainly present near the pathogen or the site of infection and is not ubiquitously present elsewhere in the individual.

[0251] For example, a pathogenic antigen can be an antigen present on the surface of a virus, bacterium, fungus or parasite, or a soluble antigen expressed by a virus, bacterium, fungus or parasite. The virus, bacterium, fungus, or parasite can be the virus, bacterium, fungus, or parasite referred to elsewhere in this specification.

[0252] When the pathogenic antigen is a soluble antigen, the antigen can be upregulated or overexpressed in the body fluid near the site of infection. For example, the soluble pathogenic antigen can be present at a higher concentration at or near the site of infection than elsewhere in the body of the individual. The soluble pathogenic antigen can be a multimer, such as a dimer or trimer.

[0253] Pathogenic antigens suitable for targeting by specific binding members can be identified using methods known in the art. For example, a specific binding member targeting CD137 and a pathogenic antigen can be used in an assay in which CD137-expressing cells are co-cultured with a pathogen or a pathogenic antigen, and the activation of OX40-expressing cells is measured, for example, by a T cell activation assay, a proliferation assay or a cytotoxicity assay.

[0254] Many pathogenic antigens suitable for targeting by specific binding members are better known in the art and can be selected by one of ordinary skill in the art depending on the infectious disease to be treated. Examples of viral antigens include the proteins p24, gp120, and gp41 expressed by the human immunodeficiency virus (HIV), the hepatitis B surface antigen (HBsAg) expressed by the hepatitis B virus (HBV), and the hemagglutinin and neuraminidase expressed by the influenza virus. Examples of bacterial antigens include Rv1733, Rv2389, and Rv2435n expressed by Mycobacterium tuberculosis.

[0255] Specific binding members may also include variants of the first or second sequences, AB, CD, or EF structural loop sequences, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain, and / or heavy chain sequences disclosed herein. Suitable variants can be obtained by methods of sequence variation, or mutagenesis, and screening. In a preferred embodiment, a specific binding member comprising one or more variant sequences retains one or more of the functional properties of the parent specific binding member, such as the binding specificity and / or binding affinity for CD137. For example, a specific binding member comprising one or more variant sequences preferably binds to CD137 with the same or higher affinity as the (parent) specific binding member. A parent specific binding member is a specific binding member that does not include amino acid substitutions, deletions, and / or insertions incorporated into the variant specific binding member.

[0256] For example, the specific binding member 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 first, second, or third array, AB, CD, or EF structural loop array, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain, or heavy chain array disclosed herein, and may include the first, second, or third array, AB, CD, or EF structural loop array, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain, and / or heavy chain array.

[0257] In a preferred embodiment, the specific binding member 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 set forth in SEQ ID NOs: 21, 30, 48, 102, 75, 39, 57, 66, 84, 93, 175, 139, 130, 148, 121, 157, 165, or 112, and has or comprises a CH3 domain sequence having such sequence identity.

[0258] In a further preferred embodiment, the specific binding member has or comprises a CH2 domain sequence having 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 set forth in SEQ ID NO: 5 or 6.

[0259] In another preferred embodiment, the specific binding member has, comprises, or consists of a sequence having 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 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.

[0260] Sequence identity is generally defined in relation to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences to maximize the number of matches and minimize the number of gaps. Generally, default parameters with a gap creation penalty equal to 12 and a gap extension penalty equal to 4 are used. Although the use of GAP may be preferred, other algorithms may be used, such as BLAST (using the method of Altschul et al. (1990)), FASTA (using the method of Pearson and Lipman (1988)), or the Smith-Waterman algorithm (Smith and Waterman (1981)), or the TBLASTN program of Altschul et al. (1990) described above, generally using default parameters. In particular, the psi-Blast algorithm may be used.

[0261] Specific binding members may include a first, second, or third sequence, an AB, CD, or EF structural loop sequence, a CH3 domain, a CH2 domain, an Fcab, a CDR, a VH domain, a VL domain, a light chain, or a heavy chain sequence having one or more amino acid sequence variations (additions, deletions, substitutions, and / or insertions of amino acid residues), preferably 20 or fewer variations, 15 or fewer variations, 10 or fewer variations, 5 or fewer variations, 4 or fewer variations, 3 or fewer variations, 2 or fewer variations, or 1 variation, as 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.

[0262] In a preferred embodiment, the specific binding member may comprise a CH3 domain sequence having one or more amino acid sequence variations (addition, deletion, substitution and / or insertion of amino acid residues), preferably 20 or fewer variations, 15 or fewer variations, 10 or fewer variations, 5 or fewer variations, 4 or fewer variations, 3 or fewer variations, 2 or fewer variations, or 1 variation, compared to the CH3 domain sequences 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.

[0263] In a further preferred embodiment, the specific binding member comprises a CH2 domain sequence having one or more amino acid sequence variations (addition, deletion, substitution and / or insertion of amino acid residues), preferably 20 or fewer variations, 15 or fewer variations, 10 or fewer variations, 5 or fewer variations, 4 or fewer variations, 3 or fewer variations, 2 or fewer variations, or 1 variation, compared to the CH2 domain sequences set forth in SEQ ID NO: 5 or 6.

[0264] In a further preferred embodiment, the specific binding member comprises or consists of a sequence having one or more amino acid sequence variations (addition, deletion, substitution and / or insertion of amino acid residues), preferably 40 or fewer variations, 30 or fewer variations, 20 or fewer variations, 15 or fewer variations, 10 or fewer variations, 5 or fewer variations, 4 or fewer variations, 3 or fewer variations, 2 or fewer variations, or 1 variation, compared to the Fcab sequences 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.

[0265] When the specific binding member comprises a variant of the first array, AB structure loop array, CH3 domain, Fcab, or heavy chain array 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. Further, 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 a further preferred embodiment, the specific binding member preferably retains a sequence at positions 97 and 98 of the CH3 domain of the specific binding member.

[0266] In particular, the specific binding member can be a variant of the specific binding member FS22 - 053 (or the antibody molecule can comprise it), where the variant is: (i) comprises one or more amino acid sequence variations (additions, deletions, substitutions and / or insertions of amino acid residues) compared to the sequence of the specific binding member FS22 - 053 disclosed herein, preferably 20 or fewer variations, 15 or fewer variations, 10 or fewer variations, 5 or fewer variations, 4 or fewer variations, 3 or fewer variations, 2 or fewer variations, or 1 variation; or (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; wherein the specific binding member or antibody molecule comprises the sequence PPY between positions 15 and 17 of the CH3 domain of the specific binding member or antibody molecule, and optionally comprises a 5 - amino acid insertion between positions 16 and 17; wherein residue numbering follows the IMGT residue numbering scheme.

[0267] Furthermore, or, when the specific binding member comprises a variant of the CH3 domain, CH2 and CH3 domains, Fcab, light chain or heavy chain sequences disclosed herein, the variant preferably does not contain amino acid mutations in the first, second and third sequences located in the AB, CD and EF structural loops of the CH3 domain of the specific binding member. For example, the variant does not contain amino acid mutations in the AB, CD and EF structural loops of the CH3 domain of the specific binding member.

[0268] In a preferred embodiment where one or more amino acids are substituted with another amino acid, the substitution can be a conservative substitution represented, for example, in the following table. In certain embodiments, amino acids in the same category in the middle column are substituted with each other, i.e., nonpolar amino acids are substituted with, for example, another nonpolar amino acid. In certain embodiments, amino acids in the same row in the rightmost column are substituted with each other.

[0269]

Table 1

[0270] In certain embodiments, the substitution can be functionally conservative. That is, in certain embodiments, the substitution does not (or does not substantially) affect one or more functional properties (e.g., binding affinity) of the specific binding member containing the substitution as compared to the equivalent unsubstituted specific binding member.

[0271] Constant domains of the specific binding member, preferably including the CD137 antigen-binding site located in the CH3 domain, and competing with the specific binding member of the present invention for binding to CD137, or binding to the same epitope on CD137 as the specific binding member of the present invention are also contemplated. Methods for determining competition for an antigen by two specific binding members are known in the art. For example, competition for binding to an antigen by two specific binding members can be determined using surface plasmon resonance such as Biacore. Methods for mapping the epitope bound by a specific binding member are similarly known in the art.

[0272] In certain embodiments, the specific binding member does not include a CDR-based antigen-binding site.

[0273] In particular, the specific binding member does not include a CDR-based antigen-binding site that binds to PD-L1.

[0274] In addition or alternatively, the specific binding member does not include a CDR-based antigen-binding site that binds to mesothelin (MSLN).

[0275] For example, the specific binding member does not include a CDR-based antigen-binding site that binds to PD-L1 or MSLN, where the specific binding member includes 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.

[0276] In particular, the specific binding member does not include the CDR, VH and / or VL domains, and / or the heavy and / or light chain sequences of FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 described below.

[0277] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VH domain CDRs HCDR1(IMGT) GYPFTSYG HCDR1(Kabat) SYGIS HCDR2(IMGT) ISAYSGGT HCDR2(Kabat) WISAYSGGTNYAQKLQG HCDR3(IMGT) ARDLFPTIFGVSYYYY HCDR3(Kabat) DLFPTIFGVSYYYY

[0278] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VH domains

Chemical formula

[0279] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VL domain CDRs LCDR1(IMGT) QSIGNR LCDR1(Kabat) RASQSIGNRLA LCDR2(IMGT) EAS LCDR2(Kabat) EASTSET LCDR3(IMGT) QQSYSTPYT LCDR3(Kabat) QQSYSTPYT

[0280] FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 VL domains

Chemical formula

[0281] Heavy chain FS22-172-003-AA / E12v2 [Chem.]

[0282] Light chain FS22-172-003-AA / E12v2 [Chem.]

[0283] Heavy chain FS22-053-008-AA / E12v2 [Chem.]

[0284] Light chain FS22-053-008-AA / E12v2 [Chem.]

[0285] In addition to, or alternatively, the specific binding member does not include the CDR and / or VH and / or VL domains of the anti-MSLN antibody FS28-256-271 described below.

[0286] FS28-256-271 VH domain CDR HCDR1(AA)(IMGT) GFTFTHTY HCDR1(AA)(Kabat) HTYMS HCDR2(AA)(IMGT) ISPTYSTT HCDR2(AA)Kabat) AISPTYSTTNYADSVKG HCDR3(AA)(IMGT) ARYNAYHAALDY HCDR3(AA)(Kabat) YNAYHAALDY

[0287] FS28 - 256 - 271 VH domain

Chem.

[0288] FS28 - 256 - 271 VL domain CDR LCDR1(AA)(IMGT) QSVSSSY LCDR1(AA)(Kabat) RASQSVSSSYLA LCDR2(AA)(IMGT) GAS LCDR2(AA)(Kabat) GASSRAT LCDR3(AA)(IMGT) QQTVPYPYT LCDR3(AA)(Kabat) QQTVPYPYT

[0289] FS28 - 256 - 271 VL domain

Chem.

[0290] The specific binding member can be conjugated to a bioactive molecule or a detectable label. In this case, the specific binding member can be referred to as a conjugate. Such conjugates are utilized in the treatment of the diseases described herein.

[0291] For example, the bioactive molecule can be an immunomodulatory agent 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 for conjugation to the specific binding member include IL - 2, IL - 10, IL - 12, IL - 15, IL - 21, GM - CSF, and IFN - γ.

[0292] Alternatively, the bioactive molecule can be a ligand trap of a cytokine, such as a ligand trap of TGF - β or IL - 6.

[0293] Suitable detectable labels that can be conjugated to the specific binding member are known in the art and include radioisotopes such as iodine-125, iodine-131, yttrium-90, indium-111, and technetium-99; fluorescent dyes such as fluorescein, rhodamine, phycoerythrin, Texas Red, and cyanine dye derivatives such as Cy7 and Alexa750; chromogenic dyes such as diaminobenzidine; latex beads; enzyme labels such as horseradish peroxidase; fluorescent or laser dyes having spectrally separated absorption or emission characteristics; and specific homologous detectable moieties such as chemical moieties like biotin that can be detected via binding to a labeled avidin.

[0294] The specific binding member can be conjugated to the bioactive molecule or detectable label by any suitable covalent or non-covalent bond such as a disulfide or peptide bond. If the bioactive molecule is a cytokine, the cytokine can be bound 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.

[0295] In certain embodiments, the bioactive molecule can be conjugated to the specific binding member by a cleavable linker. The linker can allow for the release of the bioactive molecule from the specific binding member at the site of treatment. The linker can include an amide bond (such as a peptide linker), a disulfide bond, or a hydrazone. The peptide linker can be cleaved, for example, by a site-specific protease, the disulfide bond can be cleaved by the reducing environment of the cytosol, and the hydrazone can be cleaved by acid-mediated hydrolysis.

[0296] The conjugate can be a fusion protein comprising a specific binding member and a bioactive molecule. In this case, the bioactive molecule can be conjugated to the specific binding member by a peptide linker or a peptide bond. When the specific binding member is a multimeric molecule, for example, when the specific binding member is or comprises or is an Fcab or mAb 2 is, the bioactive molecule can be conjugated to one or more chains of the specific binding member. For example, the bioactive molecule can be conjugated to one or both of the heavy chains of the mAb 2 molecule. The fusion protein has the advantage of being easy to produce and purify, facilitating the manufacture of clinical grade materials.

[0297] The present invention also provides one or more isolated nucleic acid molecules encoding the specific binding members of the present invention. Those skilled in the art will have no difficulty in preparing such nucleic acid molecules using methods well known in the art.

[0298] In a preferred embodiment, the nucleic acid molecule encodes the CH3 domain 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 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.

[0299] In another preferred embodiment, the nucleic acid molecule encodes the CH3 domain of 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 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.

[0300] The CH3 domain sequences of these specific binding members are described herein.

[0301] For example, a nucleic acid molecule encoding the CH3 domain of a specific binding member: (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, each described in SEQ ID NOs: 22, 31, 49, 103, 76, 40, 58, 67, 85, 94, and 176; or (ii) FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172, each described in SEQ ID NOs: 140, 131, 149, 122, 158, 166, and 113.

[0302] In a preferred embodiment, the nucleic acid molecule encodes a 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.

[0303] In another preferred embodiment, the nucleic acid molecule encodes a 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 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.

[0304] For example, a nucleic acid molecule encoding a specific binding member: (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 each of SEQ ID NOs: 24, 33, 51, 105, 78, 42, 60, 69, 87, 96, and 177; or (ii) FS22-172-003, FS22-172-002, FS22-172-004, FS22-172-001, FS22-172-005, FS22-172-006, or FS22-172 as described in each of SEQ ID NOs: 142, 133, 151, 124, 160, 168, and 115.

[0305] The isolated nucleic acid molecule can be used to express the specific binding member of the present invention. The nucleic acid is generally provided in the form of a recombinant vector for expression. Accordingly, another aspect of the present invention provides a vector containing the nucleic acid described above. If necessary, a suitable vector containing appropriate control sequences including a promoter sequence, a terminator fragment, a polyadenylation sequence, an enhancer sequence, a marker gene, and other sequences can be selected or constructed. Preferably, the vector contains appropriate control sequences for causing the expression of the nucleic acid in a host cell. The vector can be, if necessary, a plasmid, a virus, such as a phage, or a phagemid.

[0306] The nucleic acid molecule or vector described herein can be introduced into a host cell. Techniques for introducing the nucleic acid or vector into a host cell are well established in the art and any suitable technique can be used. Various host cells suitable for the production of the recombinant specific binding member are known in the art and include bacterial, yeast, insect, or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NS0, or HEK cells, such as HEK293 cells. Most preferred host cells are CHO cells.

[0307] Another aspect of the present invention is a method for producing a specific binding member of the present invention, which comprises expressing a nucleic acid encoding the specific binding member in a host cell and optionally isolating and / or purifying the specifically produced binding member thus produced. Methods for culturing host cells are well known in the art. The method may further comprise isolating and / or purifying the specific binding member. Techniques for purifying recombinant specific binding members are well known in the art and include, for example, HPLC, FPLC or affinity chromatography using Protein A or Protein L. In certain embodiments, purification can be performed using an affinity tag on the specific binding member. The method may optionally also include formulating the specific binding member into a pharmaceutical composition using a pharmaceutically acceptable excipient or other substances described hereinafter.

[0308] As described above, CD137 is expressed in 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). In particular, activation of CD137 has been shown to play a role in promoting the proliferation, survival and cytotoxic effector functions of CD8 + T cells, as well as the differentiation of CD8 + T cells and the maintenance of memory CD8 + T cells. CD137 is expressed at lower levels in CD4 + T cells than in CD8 + T cells, but has also been shown to be involved in inducing the proliferation and activation of some subsets of CD4 + T cells. Activation of CD137 has also been demonstrated to promote NK cell-mediated ADCC, as well as B cell proliferation, survival and cytokine production.

[0309] In view of the immune response promoting the activity of CD137, CD137 agonist molecules have been investigated for cancer treatment and for the treatment of chronic infections.

[0310] Therefore, the specific binding members described herein may be useful for therapeutic applications, particularly for the treatment of cancer. Furthermore, the specific binding members are expected to be useful for the treatment of infectious diseases such as persistent infections.

[0311] The specific binding members described herein can be used in methods for treating the human or animal body. Related aspects of the invention provide: (i) the specific binding members described herein for use as a medicament, (ii) the specific binding members described herein for use in a method for treating a disease or disorder, (iii) use of the specific binding members described herein in the manufacture of a medicament for use in treating a disease or disorder; and (iv) a method of treating a disease or disorder in an individual, the method comprising administering to the individual a therapeutically effective amount of the specific binding members described herein.

[0312] The individual can be a patient, preferably a human patient.

[0313] Treatment can be any treatment or therapy by which a desired therapeutic effect is achieved, for example, inhibition or delay of the progression of a disease state, reduction in the rate of progression, arrest of the rate of progression, improvement of the disease state, cure or alleviation (whether partial or total) of the disease state, prevention, improvement, delay, reduction or arrest of one or more symptoms and / or signs of the disease state, or extension of the lifespan of the individual or patient beyond that expected in the absence of treatment.

[0314] Treatment also includes treatment as a prophylactic measure (i.e., prevention). For example, individuals who are prone to or at risk of developing or recurring of a disease such as cancer can be treated as described herein. Such treatment can prevent or delay the onset or recurrence of the disease in the individual.

[0315] The methods of treatment described herein may include administering to the subject at least one additional treatment in addition to the specific binding member. Thus, the specific binding members described herein may be administered to a subject alone or in combination with one or more other treatments. When the specific binding member is administered to the subject in combination with another treatment, the additional treatment may be administered to the subject simultaneously with, consecutively with, or separately from the administration of the specific binding member. When the additional treatment is administered simultaneously with the specific binding member, the specific binding member and the additional treatment may be administered to the subject as a combined formulation. For example, the additional treatment may be a known method of treatment or therapeutic agent for the disease being treated.

[0316] The specific binding member may be administered alone, but will generally be administered in the form of a pharmaceutical composition that may include at least one component in addition to the specific binding member. Thus, another aspect of the invention provides a pharmaceutical composition comprising a specific binding member described herein. Also provided is a method comprising formulating a specific binding member into a pharmaceutical composition.

[0317] The pharmaceutical composition may include, in addition to the specific binding member, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those of skill in the art. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human) within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable risk / benefit ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The exact nature of the carrier or other material will depend on the route of administration, which, as described below, may be by infusion, injection, or any other suitable route.

[0318] For example, for parenteral, such as subcutaneous or intravenous administration by injection, a pharmaceutical composition containing a specific binding member is pyrogen-free and in the form of an aqueous solution that is parenterally acceptable and has a suitable pH, isotonicity, and stability. One skilled in the art can adequately prepare a suitable solution, for example, using an isotonic vehicle such as sodium chloride injection solution, Ringer's injection solution, or lactated Ringer's injection solution. Buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol, etc.); low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG) can be used as preservatives, stabilizers, buffers, antioxidants, and / or other additives as needed.

[0319] In certain embodiments, the specific binding member can be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized specific binding member can be reconstituted with sterile water and mixed with physiological saline prior to administration to an individual.

[0320] Administration can be at a "therapeutically effective amount", which is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site to which the composition is being delivered, the type of specific binding member, the method of administration, the scheduling of administration and other factors known to the physician. Prescription of treatment, e.g., decisions regarding dosage, etc., is the responsibility of the general practitioner and other physicians and may depend on the severity of the symptoms and / or the progression of the disease being treated. Appropriate dosages of immunoglobulins are well known in the art (Ledermann et al. (1991) Int. J. Cancer 47:659-664; and Bagshawe et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922). Specific dosages shown herein or, where appropriate, in the Physician’s Desk Reference (2003) for the antibody molecules being administered may be used. For antibody molecules, a therapeutically effective amount or a suitable dosage 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 test animals to humans are known. The exact dosage will depend on several factors including the size and location of the site being treated and the exact nature of the specific binding member.

[0321] Typical immunoglobulin dosages range from 100 μg to 1 g for systemic application and from 1 μg to 1 mg for local application. Initial higher loading doses, followed by one or more lower doses, may be administered. This is the dosage for a single treatment of an adult individual and may be proportionally adjusted for pediatric and infant patients and also for other specific binding member formats in proportion to the molecular weight.

[0322] Treatment can be repeated daily, twice a week, at one-week or one-month intervals, at the discretion of the physician. The treatment schedule for an individual can depend on the pharmacokinetic and pharmacodynamic properties of the specific binding member composition, the route of administration, and the nature of the condition being treated.

[0323] Treatment can be periodic, and the period between administrations can be about two weeks or more, such as about three weeks or more, about four weeks or more, about one month or more, about five weeks or more, or about six weeks or more. 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.

[0324] In a preferred embodiment, the specific binding members described herein can be for use in a method of treating cancer.

[0325] Cancer can be characterized by the abnormal proliferation of malignant cancer cells. When a specific type of cancer, such as breast cancer, is mentioned, this refers to the abnormal proliferation of malignant cells in related tissues such as breast tissue. A secondary cancer that is located in the breast but is the result of the abnormal proliferation of malignant cells in another tissue, such as ovarian tissue, is, when mentioned herein, ovarian cancer, not breast cancer.

[0326] Cancer can be primary or secondary. Thus, the specific binding members described herein can be for use in a method of treating cancer in an individual, where the cancer is a primary tumor and / or tumor metastasis.

[0327] Tumors of cancer treated with the specific binding members described herein can contain, for example, TILs that express CD137 on their cell surface. In one embodiment, the tumor may have been determined to contain TILs that express CD137. Methods for determining the expression of an antigen on the cell surface are known in the art and include, for example, flow cytometry.

[0328] For example, cancers treated using the specific binding members described herein include leukemia, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma; and solid cancers, such as sarcoma (e.g., soft tissue sarcoma), skin cancer (e.g., Merkel cell carcinoma), melanoma, bladder cancer (e.g., urothelial carcinoma), brain tumor (e.g., glioblastoma multiforme), breast cancer, uterine / 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 cancer, stomach cancer, testicular cancer, gallbladder and biliary tract cancer (e.g., cholangiocarcinoma), thyroid cancer, thymic cancer, osteosarcoma, and brain tumor, and can be selected from the group consisting of.

[0329] 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 that can be selected from the group consisting of sarcoma, melanoma, bladder cancer, brain tumor, breast cancer, uterine cancer / endometrial cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer, and stomach cancer.

[0330] Regarding cancer, treatment can include inhibiting cancer growth, and / or inhibiting cancer metastasis, and / or inhibiting cancer recurrence, including complete remission of cancer. Cancer growth generally refers to any one of several indicators that show changes within the cancer to a more developed form. Thus, indicators for measuring inhibition of cancer growth include decreased cancer cell survival, decreased tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasound, or other imaging methods), delayed tumor growth, disruption of the tumor vasculature, improved results in delayed hypersensitivity skin tests, increased activity of anti-cancer immune cells or other anti-cancer immune responses, and decreased levels of tumor-specific antigens. Activating or promoting an immune response against a cancerous tumor in an individual can improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the tendency for cancer growth in the individual.

[0331] Regarding cancer treatment, the specific binding members described herein can be administered to an individual in combination with another anti-cancer treatment method or treatment agent, such as an anti-cancer treatment method or treatment agent that has been shown to be suitable or is expected to be suitable for the treatment of the cancer. For example, the specific binding member can be administered to an individual in combination with a chemotherapeutic agent, radiation therapy, immunotherapeutic agent, anti-tumor vaccine, oncolytic virus, adoptive cell transfer (ACT) therapy (such as adoptive NK cell therapy or a treatment method using chimeric antigen receptor (CAR) T cells, autologous tumor-infiltrating lymphocytes (TIL), or γ / δ T cells), or an agent for hormone therapy.

[0332] Without wishing to be bound by theory, the specific binding members described herein are thought to be able to act as adjuvants in anti-cancer treatment methods. Specifically, administration of the specific binding member to an individual in combination with chemotherapy and / or radiation therapy, or in combination with an anti-tumor vaccine, is thought to cause a greater immune response against cancer, for example, than can be achieved with chemotherapy and / or radiation therapy alone, or with the anti-tumor vaccine alone.

[0333] One or more chemotherapeutic agents for administration in combination with the specific binding members described herein may 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, antitumor chemotherapeutic agents, and others. Taxanes include docetaxel, paclitaxel, and nab-paclitaxel; cytotoxic antibiotics include actinomycin, bleomycin, and anthracyclines such as doxorubicin, mitoxantrone, and valrubicin; tyrosine kinase inhibitors include erlotinib, gefitinib, axitinib, PLX3397, imatinib, cobemitinib, and trametinib; PARP inhibitors include niraparib; 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 defactinib, enzastaurin, eribulin, irinotecan, and vinblastine.

[0334] Preferred therapeutic agents for administration together with the antibody molecules described herein are doxorubicin, mitoxantrone, cyclophosphamide, cisplatin, and oxaliplatin.

[0335] Radiation therapy for administration in combination with the specific binding members described herein can be external beam radiation therapy or brachytherapy.

[0336] Immunotherapeutic agents for administration in combination with the specific binding members described herein can be therapeutic antibody molecules, nucleic acid cytokines, or cytokine-based therapies. For example, therapeutic antibody molecules can bind to immunomodulatory molecules, such as inhibitory checkpoint molecules or costimulatory molecules, or tumor antigens, such as cell surface tumor antigens or soluble tumor antigens. Examples of immunomodulatory molecules to which therapeutic antibody molecules can bind include CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, PD-L1, PD-1, CD47, CD73, CSF-1R, KIR, CD40, HVEM, IL-10, and CSF-1. Examples of receptors of the innate immune system to which therapeutic antibody molecules can bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (e.g., RIG-I and MDA-5), and STING. Examples of tumor antigens to which therapeutic antibody molecules can bind include HER2, EGFR, CD20, and TGF-β.

[0337] The nucleic acid for administration in combination with the specific binding members described herein can be siRNA.

[0338] Cytokines or cytokine-based therapies can be selected from the group consisting of IL-2, a prodrug of conjugated IL-2, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21, and type I interferons.

[0339] Antitumor vaccines for the treatment of cancer have been realized clinically and are described in detail in the scientific literature (such as Rosenberg, S. 2000, etc.). This mainly requires a technique for promoting the immune system to respond to various cell markers expressed by those cells by using autologous or allogeneic cancer cells as a vaccination method, with or without granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF, when used by the said technique, causes a strong response in antigen presentation and functions particularly well.

[0340] Agents for chemotherapy, radiotherapy, immunotherapy, anti-tumor vaccines, oncolytic viruses, ACT therapy, or hormonal therapy are preferably agents for chemotherapy, radiotherapy, immunotherapy, anti-tumor vaccines, oncolytic viruses, ACT therapy, or hormonal therapy for the cancer, i.e., agents for chemotherapy, radiotherapy, immunotherapy, anti-tumor vaccines, oncolytic viruses, ACT therapy, or hormonal therapy that have been shown to be effective in treating the cancer. The selection of suitable agents for chemotherapy, radiotherapy, immunotherapy, anti-tumor vaccines, oncolytic viruses, ACT therapy, or hormonal therapy that have been shown to be effective for the cancer is well within the ability of those skilled in the art.

[0341] Considering the immune response that promotes the activity of CD137, CD137 agonist molecules are expected to be utilized in the treatment of infectious diseases. Thus, in another preferred embodiment, the antibody molecules described herein can be for use in a method of treating an infectious disease such as an acute or persistent infectious disease.

[0342] Without wishing to be bound by theory, CD137 agonist molecules may induce rapid infiltration and activation of innate immune cells such as neutrophils and monocytes, thereby promoting the removal of pathogens causing acute infectious diseases, and thus may promote the immune response against acute infectious diseases caused by pathogens. Thus, in a further embodiment, the antibody molecules described herein can be for use in a method of treating an acute infectious disease such as an acute bacterial disease. In a preferred embodiment, the acute infectious disease is an acute bacterial disease caused by an infection with a Gram-positive bacterium such as a bacterium of the genus Listeria, Streptococcus pneumoniae, or Staphylococcus aureus.

[0343] Infections are usually eliminated by the immune system, but some infections persist over long periods, such as months or years, and cannot be effectively halted by the immune system. Such infections are also called persistent or chronic infections.

[0344] Preferably, the antibody molecules described herein are used to treat persistent infections, such as persistent viral, bacterial, fungal or parasitic infections, preferably persistent viral or bacterial infections.

[0345] In a preferred embodiment, the persistent viral infections treated with the antibody molecules described herein are persistent infections of human immunodeficiency virus (HIV), Epstein - Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, or varicella - zoster virus.

[0346] In a preferred embodiment, the persistent bacterial infections treated with the antibody molecules described herein are persistent infections of Staphylococcus aureus, Hemophilus influenza, Mycobacterium tuberculosis, Mycobacterium leprae, Salmonella typhi, Helicobacter pylori, Treponema pallidum, or Streptococcus pneumoniae.

[0347] CD137 agonism has been described as beneficial for the treatment of infections caused by Gram-positive bacteria. Thus, in a preferred embodiment, the persistent bacterial infections treated with the antibody molecules described herein are persistent infections caused by Gram-positive bacteria. In a more preferred embodiment, the persistent bacterial infection is a persistent infection caused by a Gram-positive bacterium selected from the group consisting of Staphylococcus aureus, Mycobacterium leprae, and Streptococcus pneumoniae.

[0348] In a preferred embodiment, the persistent fungal infections treated with the specific binding members described herein are persistent infections of Candida (e.g., Candida albicans), Cryptococcus (e.g., Cryptococcus gattii or Cryptococcus neoformans), Talaromyces (Penicillium) (e.g., Talaromyces marneffe), Microsporum (e.g., Microsporum audouinii), or Trichophyton tonsurans.

[0349] In a preferred embodiment, the persistent parasitic infections treated with the specific binding members described herein are persistent infections of Plasmodium, e.g., Plasmodium falciparum, or Leishmania, e.g., Leishmania donovani.

[0350] Regarding the treatment of persistent infections, the treatment can include removing the infection, reducing the individual pathogenic load, and preventing recurrence of the infection. For example, the treatment can include preventing, ameliorating, delaying, suppressing, or halting one or more symptoms and / or signs of the persistent infection. Alternatively, the treatment can include preventing the infection.

[0351] Regarding the treatment of an infection, the specific binding members described herein can be administered to an individual in combination with another therapeutic agent for the treatment of the infection, such as a therapeutic agent that has been shown to be suitable or is expected to be suitable for the treatment of the infection. For example, the specific binding member can be administered to an individual in combination with an immunotherapeutic agent. The immunotherapeutic agent for administration 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 to which the therapeutic antibody molecule can bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (such as RIG-I and MDA-5), and STING.

[0352] When the specific binding member is used to prevent an infection, the specific binding member can be administered in combination with a vaccine against the pathogen. Without wishing to be bound by theory, it is contemplated that the specific binding members described herein can act as adjuvants in vaccination. Specifically, administration of the specific binding member to an individual in combination with a vaccine is thought to elicit a greater immune response against the pathogen than can be achieved with the vaccine alone.

[0353] Considering that the present disclosure includes the exemplification of the following experiments, further aspects and embodiments of the invention will be apparent to those skilled in the art.

[0354] All documents mentioned herein are hereby incorporated by reference in their entirety.

[0355] As used herein, "and / or" shall be construed as specific disclosure of each of two specified features or components, with or without the other. For example, "A and / or B" shall be construed as (i) A, (ii) B, and (iii) specific disclosure of each of A and B, as if each were individually described herein.

[0356] Unless the context requires otherwise, the descriptions and definitions of the features described above are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0357] Other aspects and embodiments of the invention provide the aspects and embodiments described above using the term "comprising" replaced by the terms "consisting of" or "consisting essentially of", unless the context requires otherwise.

[0358] Particular aspects and embodiments of the invention are illustrated by way of example and with reference to the figures described above.

Examples

[0359] Examples Example 1: Production, Characterization, and Selection of Human, Mouse, and Cynomolgus Monkey Antigens 1.1 Recombinant Antigens Members of the tumor necrosis factor receptor superfamily (TNFRSF) are known to form multimers that cluster together when bound to their cognate ligands (Croft, M. 2003). This tendency to assemble for their functionality makes it difficult to produce soluble recombinant proteins that do not assemble in solution for use in in vitro selections such as phage and yeast display, as well as for characterization of the selected proteins.

[0360] Several commercially available recombinant antigens were tested and most of them were found to be unsuitable for use in these selections 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 aggregates low enough to be suitable for selection and was used in the selection, with limited success (see Example 2).

[0361] Since most of the commercially available antigens were considered inappropriate, the following recombinant dimeric and monomeric CD137 antigens (see Table 1) were produced in-house for use in the selection.

[0362]

Table 2

[0363] The monomeric antigen was produced by cloning DNA encoding the extracellular domain of human (SEQ ID NO: 181) or mouse CD137 (SEQ ID NO: 185) together with the Avi sequence and six C-terminal histidine residues into a modified pFUSE vector (Invivogen catalog number pfuse-mg2afc2) using EcoRI-HF and BamHI-HF restriction enzymes. The vector was transfected into HEK293-6E cells (National Research Council of Canada) and the expressed CD137 was purified using a HisTrap™ Excel nickel column (GE LifeSciences 29048586) and size exclusion chromatography (SEC) to ensure that the antigen was a single species and did not contain aggregates.

[0364] To produce the dimeric antigen, a DNA construct encoding the extracellular domain of human, mouse or cynomolgus CD137 fused with the mIgG2a Fc domain together with the Avi sequence was cloned into a modified pFUSE vector and transfected into HEK293-6E cells. Recombinant CD137 was purified using a MabSelect SuRe™ 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.

[0365] Each of the dimeric and monomeric antigens was biotinylated using a BirA biotin-biotin protein ligase reaction kit (Avidity LLC, BirA500) to produce monomeric CD137 antigen labeled with a single biotin molecule and dimeric CD137 antigen labeled with two biotin molecules, one on each of the two monomers. 3 mg of the antigen was mixed with 7.8 μl of BirA enzyme mix until a 1:50 enzyme-to-substrate molar ratio was reached. Next, additives were added according to the manufacturer's recommendations (142 μl of Biomix A, 142 μl of Biomix B, 142 μl of biotin), and the reaction mix was incubated at room temperature for 2 hours. The reaction mix was immediately buffer-exchanged into DPBS (Life Technologies 14190-169) using an Amicon 30 μm filter (Merck Millipore UFC503096).

[0366] The protein was further purified by SEC to ensure removal of the BirA enzyme and production of a final high-quality monodisperse protein formulation free of high molecular weight aggregates. More specifically, materials from the same production lot 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 on a streptavidin-shift 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. 7,288,638) and Lob12.3 (University of Southampton), respectively) by surface plasmon resonance (SPR), and to bind to DO11.10 cells expressing human and mouse CD137 ligand by flow cytometry. Cells were incubated with the CD137 antigen for 1 hour and then cell binding was detected using a fluorescently labeled anti-mouse Fc fragment antibody. The recombinant cynomolgus antigen was confirmed to bind by flow cytometry, as described above, to DO11.10 cells (National Jewish Health) expressing cynomolgus CD137 ligand. To ensure the highest possible purity of the materials used in the selection protocol, thorough protein characterization of the antigen was performed to ensure that the presence of protein aggregates did not exceed 2% assuredly.

[0367] 1.2 Antigens Expressed in Cells DO11.10 cells (National Jewish Health) expressing full-length mouse CD137 (SEQ ID NO: 184) or human CD137 (SEQ ID NO: 180), designated as "DO11.10.mCD137" and "DO11.10.hCD137", respectively, were produced to represent the antigen in its membrane-bound structure most similar to its native form for the selection and further characterization of the selected Fcabs, as listed in Table 2.

[0368] Lentiviral transduction was used to generate these DO11.10 cells that overexpress human or mouse CD137 receptor using the Lenti-X HTX Packaging System (Takara, catalog number 631249). A Lenti-X expression vector (pLVX) (Takara, catalog number 631253) containing the cDNA encoding human CD137 (SEQ ID NO: 180) or mouse CD137 (SEQ ID NO: 184) was co-transfected into Lenti-X 293T cell line (Takara, catalog number 632180) together with the Lenti-X HTX Packaging Mix to generate the virus. Next, these lentiviral vectors were transduced into the DO11.10 cell line.

[0369] The expression of human CD137 or mouse CD137 in these cells was confirmed by flow cytometry by the binding of the 20H4.9 and Lob12.3 anti-CD137 positive control antibodies to the cells, respectively. The cells were incubated with the human or mouse positive control antibody for 1 hour, and then cell binding was detected using a fluorescently labeled anti-human Fc detection antibody (Stratech Scientific Ltd, catalog number 109-546-098-JIR).

[0370] DO11.10 cells expressing cynomolgus CD137 (designated as "DO11.10.cCD137") were also generated using the same lentiviral transduction method and used to test the cross-reactivity between anti-human CD137 Fcab and cynomolgus CD137. The expression of cynomolgus CD137 was confirmed by flow cytometry by the binding of the anti-CD137 positive control antibody (MOR_7480.1, US Patent Application Publication No. 2012 / 0237498 A1) to the cells as described above.

[0371]

Table 3

[0372] Example 2: Naive Selection of Anti-Human CD137 Fcab To discover Fcabs that bind to human CD137 and maximize the diversity of the identified binders, both yeast and phage display selection procedures were used. Since CD137 is expressed at low levels in some non-immune cell types, it was determined to select anti-human CD137 Fcabs that selectively target cells that express large amounts of CD137, such as activated T cells. Without wishing to be bound by theory, it is hypothesized that cells with very low or very little CD137 expression are more likely to have monomeric CD137 on their cell surface, in which case most of the protein is expected to be present in a dimeric, trimeric, or higher-order multimeric state on the cell surface.

[0373] Cells overexpressing CD137, or recombinant dimeric human CD137 protein, were used for Fcab selection to expose their representative epitopes that promote binding interactions with cells using highly upregulated multimeric CD137. Furthermore, using a dimeric antigen was considered beneficial for selecting bivalent Fcabs that were hypothesized to bind strongly to CD137, stay more stably engaged with the target, and thus promote preferential binding to cells using upregulated expression levels of CD137. In this regard, monomeric recombinant CD137 was not used to prevent the selection of very high-affinity monovalent Fcab binders that could bind too strongly to CD137.

[0374] The purpose of this selection approach was to obtain Fcabs that preferentially bind to activated T cells and do not bind well to naive T cells that show only monomeric CD137 or other cells that express very low levels of CD137. By selecting CD137 Fcabs that bind divalently and preferentially to dimeric and multimeric antigens compared to monomeric antigens, it was thought that potential off-target T cell activation could be reduced, along with the associated reduced toxicity.

[0375] Phage display Six naive phage libraries representing the CH3 domain of human IgG1 were used for selection by phage display. All six libraries contained a randomized AB loop (residues 14–18 according to IMGT numbering) and a randomized EF loop (residues 92–101 according to IMGT numbering). One of the libraries contained clones with insertions of two or four amino acids (encoded by two or four NNK codons) at position 101 of the EF loop (the inserted residues are at positions 101.4–101.1 according to IMGT numbering).

[0376] A total of 12 selection operations were performed to identify anti-human CD137 binding agents. In-house hCD137-mFc-Avi antigen or commercially supplied hCD137-hFc-Avi-BPS antigen and / or DO11.10.hCD137 antigen-expressing cells were used for selection with the six phage libraries. Since only one functional Fcab sequence was identified from the initial selection using the hCD137-hFc-Avi-BPS recombinant antigen, in-house hCD137-mFc-Avi was used for the remaining rounds of selection on recombinant CD137. The first round was performed using 100 nM biotinylated antigen, which was accompanied by a deselection step using 500 nM unlabeled recombinant human Fc fragment produced in-house. Phage binders were captured by electromagnetic beads coated with either streptavidin or neutravidin. A further deselection step was introduced due to phage ELISA-detected non-specific binding to the Fc fragment (A450-630nm higher than 0.4) of the phage selection output, by incubating the biotinylated Fc fragment (produced in-house) with the phage output. Non-Fc binders were separated from Fc binders by magnetic capture of biotinylated Fc-binding phage.

[0377] To discover binders to the membrane-bound native conformation of CD137, cell selection was performed as follows: Phage output from recombinant antigen selection was incubated with DO11.10 cells lacking human CD137 to discard unwanted binders such as those that bind non-specifically to the cells. Next, the phages were incubated with 1×10 7 DO11.10.hCD137 cells, the binders were eluted by trypsin digestion, and then propagated for the next round. For the third round, the selection pressure was increased by reducing the number of DO11.10.hCD137 cells to 5×10 6 cells and following the same process.

[0378] Phage ELISA was performed after each round of selection to determine the enrichment of antigen-specific phages. A 96-well streptavidin plate was coated overnight with 1 μg / ml biotinylated antigen. After blocking the plate with 4% marvel PBS, 50 μl of the phage-containing bacterial supernatant was added to each well and incubated at room temperature for 1 hour with shaking at 450 rpm. The phage solution was discarded by inverting the plate and washing 4 times with 0.1% Tween in PBS and 4 times with PBS. Next, anti-M13 phage-HRP conjugate antibody was added to detect the phages bound to the immobilized antigen. The phage solution was discarded by inverting the plate and washing 4 times with 0.1% Tween in PBS and 4 times with PBS. TMB microwell peroxidase solution was added to each well and allowed to develop color for up to 30 minutes. The reaction was stopped using 1 M sulfuric acid, and the plate was read at OD 450~630 in a microtiter plate reader. Specific hits were defined as those showing signal intensity to recombinant CD137 that was at least 4-fold higher than the background defined by the negative control, i.e., a negative non-binding phage such as PBS or wild-type CH3, and at least 10-fold higher than the binding strength to the biotinylated recombinant Fc fragment.

[0379] Phage fluorescence-activated cell sorting (phage FACS) assays were performed on the cell selection outputs from the second and third rounds. Briefly, 2 × 10 5 cells were transferred to round-bottom microtiter plates. Phage supernatant was added to the cells and incubated at 4 °C for 1 h. The cells were then washed twice in ice-cold PBS 2% BSA buffer and resuspended in a solution of 100 μl of anti-M13 antibody conjugated to FITC and goat anti-mouse IgG F(ab’) fragments and incubated with the cells for 1 h on ice. The cells were analyzed on a flow cytometer after three washes in PBS. In phage FACS, specific hits were defined as those showing a geometric mean fluorescence intensity (MFI) to CD137-positive cells that was at least 10-fold higher than the binding signal to recombinant CD137 and at least 4-fold higher than the background defined by the negative control (PBS).

[0380] 3230 phage clones were screened by phage ELISA for binding to the dimeric recombinant hCD137 antigen, using recombinant Fc as a negative control. 1140 phage clones were screened by phage FACS for specific binding to DO11.10.hCD137 cells. The individual hits were then sequenced, and Fcab clone identifiers were assigned to the 76 unique sequences obtained, and Fcab clones in the mAb 2 format were expressed by subcloning into the pTT5 expression vector (National Research Council of Canada) containing the HelD1.3 IgG1 heavy chain expression cassette (see Example 3.1).

[0381] Yeast display Four naive yeast libraries representing the CH1 through CH3 domains of human IgG1 were used for selection by yeast display. All four libraries contained a randomized AB loop (residues 14 through 18 according to IMGT numbering) and a randomized EF loop (residues 92 through 101 according to IMGT numbering) in the CH3 domain. Two of the libraries further contained an insertion of five amino acid residues (residues at positions 16.5 through 16.1 according to IMGT numbering) at position 16 in the AB loop of the CH3 domain.

[0382] The in-house hCD137-mFc-Avi antigen or the commercially supplied hCD137-hFc-Avi-BPS antigen was used for selection with the four yeast libraries, but as in phage selection, the commercially supplied antigen was not used after the initial round of selection and the in-house antigen was used instead. For each library, the first round of selection was performed using magnetic cell separation (MACS). The libraries were grown, induced, and 1×10 10Individual cells were incubated with 250 or 300 nM of biotinylated recombinant antigen after an exclusion step using 1.25 μM of unlabeled human or mouse Fc. Yeast binders were separated by adding streptavidin electromagnetic beads and using a MACS LS column (Miltenyi Biotech 130-042-401). Subsequent rounds of selection were performed by fluorescence-activated cell sorting (FACS) on a FACS-Aria II instrument (BD Bioscience) using fluorescently labeled antibodies to detect the expression of bound antigen (anti-biotin-APC (Miltenyi Biotech 130-090-856), streptavidin-APC (BD Biosciences 349024), or NeutrAvidin-DyLight-488 (Thermo Fisher 22832), correctly folded IgG scaffold (anti-human IgG CH2 domain-FITC (Biorad AbD Serotec (MCA647F))), or aga2-IgG construct (anti-Xpress (Life Technologies R91025) and anti-mouse IgG-FITC (Sigma F2653-.5ML)). Whenever possible, antigen markers were used together with one of the structural markers to normalize the binding strength signal by Fcab expression on the yeast surface. Sorting gates were set as follows using unstained and control yeast populations: yeast cells in FSC-A and SSC-A plots to distinguish between multi-budded or budding yeast cells and single-budded cells, FSC-W and FSC-H plots, and FITC-A and APC-A plots to detect bound Fcab double positivity.

[0383] The antigen concentration used for each round was determined empirically by performing output quality control using the antigen concentration used in the previous round. When the binding enhancement increased (>5-fold) compared to the previous round, the antigen concentration was decreased to 1:2 or 1:3, or it was kept constant. Also, according to this process, it was determined how many selections were required and whether the options were considered exhausted (whether the diversity dropped to just a few major sequences, or the antigen binding did not increase after two rounds).

[0384] A total of 2,784 yeast single clones identified from library selections were individually screened as follows: After each round of selection, single yeast cells were spotted onto SDCAA agar plates using a FACS-Aria II (BD Biosciences) instrument and screened in the following flow cytometry antigen binding assay: Single clone colonies were grown until the colony reached a diameter of 2 mm and then transferred onto a deep well plate containing 600 μl of SDCAA liquid medium. The cultures were grown at 30 °C with shaking at 1000 rpm overnight. Expression of the aga2-IgG protein scaffold was induced by replacing the growth medium with the induction medium SGRCAA at an optical density of 1 (OD600 = 1). Cells were incubated with either biotinylated recombinant dimeric human antigen or mouse Fc fragment to discriminate yeast clones that bind to the Fc portion of the recombinant hCD137 antigen. Yeast cells that bound to either protein were labeled with streptavidin-APC. Also, an anti-CH2-FITC antibody was used as a structural IgG marker. To analyze the screening results, binding to the Fc fragment was plotted and clones that bound to biotinylated Fc were discarded. Binding was defined as cells that were positive for APC fluorescence above 0.2% as set by the gates established using non-stained and negative control samples.

[0385] Selections were repeated using various antigen concentrations and conditions, such as increasing the induction temperature, decreasing the stringency of selection, or decreasing the number of rounds to increase the number of hits. Hit sequencing showed a fairly low output diversity, and only 9 Fcab clones with unique sequences were identified: FS22-053, FS22-172, FS22-173, FS22-174, FS22-175, FS22-176, FS22-177, FS22-178, and FS22-179.

[0386] Example 3: Characterization of anti-human CD137 Fcab from naive selection 3.1 "Mock" mAb 2 Preparation of anti-human CD137 Fcab in the format A "mock" mAb consisting of IgG1 molecules containing 76 anti-human CD137 Fcab clones isolated from phage and 9 clones isolated from yeast selection 2 The antibody was produced as an mAb 2 To enable characterization of Fcab in the format, the mock mAb was prepared by replacing the portion of the CH3 domain Fcab containing the AB, CD, and EF loops with the corresponding region of the CH3 domain of the anti-chicken egg lysozyme antibody HelD1.3. 2 The generation of the HelD1.3 antibody is described in Tello et al. 1993. The heavy and light chain sequences of the antibody HelD1.3 are shown in SEQ ID NOs: 186 and 173, respectively. The mock mAb 2 molecules were produced by transient expression in HEK293-6E cells. To evaluate the amount of protein produced, the IgG protein content was quantified by biolayer interferometry using an Octet QKe platform with a Protein A quantification biosensor manufactured by PALL (18-5021). The protein was purified by protein A affinity chromatography using an mAb SelectSure column. The CD137 mAb 2 protein driven by 53 phages showed measurements below the detection threshold and was thus found to be unsuitable for further analysis. 32 mAbs2 were purified using an mAb Select SuRe Protein A column (GE Healthcare, 11003494): 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.

[0387] To compare the expression levels, several of the previous Fcabs were also subcloned and expressed as soluble Fcabs (including the truncated hinge) in HEK293-6E cells and purified using an mAb Select SuRe Protein A column. Interestingly, several of the Fcabs were found to have significantly better biophysical behavior and production yields in the mock mAb 2 format. This was also true for the naive clone FS22-053, which was produced at a very low yield as a soluble Fcab, but which was 25-fold improved when expressed in the mock mAb 2 format, making more clones available for characterization.

[0388] 3.2 Binding to the Recombinant Antigen by BLI The purified mock mAb 2Thirty-one of the molecules (excluding clone FS22-175) were tested for binding to the human recombinant antigen in a single-point binding experiment by biolayer interferometry using the Octet QKe platform. The streptavidin BLI biosensor (PALL 18-5021) was used to capture the biotinylated hCD137-mFc-Avi antigen at 10 μg / ml in kinetic buffer (PALL). Next, the sensor was immersed in wells containing the purified mAb 2 diluted 1:1 in the same kinetic buffer for 240 seconds, and then immersed in wells containing 1× kinetic buffer for 240 seconds. Binding hits were classified by a simple Boolean yes / no criterion defined by a BLI response exceeding that of the buffer and the wild-type IgG1 control HelD1.3 mAb (G1 / HelD1.3): twelve mAbs 2 did not bind and 19 bound to the CD137-coated sensor (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).

[0389] 3.3 Activity of selected anti-CD137 mock mAbs in the human NF-κB reporter assay 2 of Multimerization and clustering are required for TNFR signaling (Bitra et al, 2017). CD137 clusters and activates the NF-κB signaling pathway when it interacts with its cognate ligand, CD137L. Agonist molecules mimic the ligand by causing clustering and activation of CD137, thereby activating the NF-κB signaling pathway. Some agonist antibodies (e.g., urelumab) are known to originally cause clustering of CD137 upon binding, while others such as utomilumab require further cross-linking of the antibody itself to induce clustering of CD137 (Fisher et al, 2012). Fcγ receptors on effector cells are known to induce such cross-linking in vivo, but this is inefficient and can occur away from the site of treatment. Since dose-limiting toxicity has been associated with treatment with some anti-CD137 antibodies, it was decided to select anti-CD137 binding Fcabs that do not have the ability to agonise intrinsically, but only those that require further cross-linking to induce clustering of CD137. Thus, an assay was developed that could detect activation of the intracellular NF-κB signaling pathway upon clustering of CD137 expressed on the cell surface by cross-linked antibodies, but showed little activity when the antibody was not cross-linked. Next, using this assay, mock mAb 2 in the format of 27 anti-CD137 Fcab clones, and anti-CD20 mAb 2 The agonist functional activity of 6 anti-CD137 Fcab clones in the format was tested.

[0390] Protein L was used as a cross-linking agent to cause cross-linking of mock mAb via their Fab portions in the assay 2 and NF-κB activation was measured.

[0391] The cDNA encoding human CD137 (SEQ ID NO: 180) was subcloned into the pMSCV-neomycin vector (Takara Clontech, Cat. 634401) using EcoRI-HF and XhoI restriction enzymes. Retroviral particles were produced using the RetroPack PT67 cell line (Clontech, Cat. 631510) according to the manufacturer's protocol. Subsequently, the HEK.FRT.luc cells generated previously were transduced with this retrovirus by transducing the Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) with the Qiagen Cignal Lenti NFkB Reporter (luc) (Qiagen, catalog number 336851) lentivirus containing an NF-κB-responsive promoter that controls the expression of luciferase. Using these HEK.FRT.luc.hCD137 cells, a mock mAb 2 containing the CD137 binder identified in the selection was screened.

[0392] Each mock mAb 2 at a 2 μM dilution was prepared in DPBS (Life Technologies, 14190169) and further diluted 1:3 in reporter cell medium (DMEM (Gibco, Cat. 61965-026); 10% FCS (Gibco, Cat. 10270-106); 1× PennStrep (Gibco, Cat. 15140-122); blasticidin 15 μg / ml (Melford Laboratories Ltd., Cat. B1105); puromycin 5 μg / ml (Life Technologies, Cat. A11113803); Zeocin 100 μg / ml (InvivGen, Cat. 11006-33-0); Geneticin 500 μg / ml (Life Technologies, Cat. 10131-027)). Protein L (Life Technologies, 21189) was used as an artificial cross-linking agent and the mAb 2It was mixed with the molecule. After 24 hours of incubation, the cells were treated with 100 μl of Promega Bio-Glo™ Luciferase Assay Reagent (Promega catalog number G7941) according to the manufacturer's instructions, and luminescence was measured in a plate reader using Gen5 Software, BioTek with an integration time of 0.5 seconds. The luminescence value is a measure of luciferase produced in response to activation of the NF-κB signaling pathway by clustering of CD137 induced by cross-linked Fcab. The luminescence values were plotted against the log concentration of Fcab, and the resulting curve was fitted in GraphPad Prism using the log(agonist) vs response equation.

[0393] Hit was identified by having at least a 10-fold increase in luciferase signal when cross-linked with Protein L compared to when not cross-linked. These clones were found to be able to induce clustering of CD137 and subsequent activation of downstream signaling pathways. Of all the clones tested, two, FS22-053 and FS22-172, were able to induce this 10-fold increase in luciferase upon cross-linking, but the EC50 could not be determined for either. Both were selected for further characterization in the DO11.10 T cell activation assay. Surprisingly, despite binding to the CD137 target by BLI, activity was not observed for clones that remained under cross-linking conditions, which probably indicates that they were binding at an irrelevant epitope in CD137 or that the affinity of such clones was not strong enough to bind to CD137 sufficiently to initiate the NF-κB signaling cascade. Overall, more than 30 Fcabs were tested, but only two Fcabs (FS22-053 and FS22-172) were identified from naïve selection, which showed the desired function in the NF-κB reporter assay when cross-linked and had little activity when not cross-linked.

[0394] 3.4 Activity of selected anti-CD137 mock mAbs in the DO11.10 T cell activation assay 2 activity Clustering of CD137 via agonist molecules in activated T cells causes T cell activation and downstream signaling, resulting in, but not limited to, IL-2 production. Since FS22-053 and FS22-172 were confirmed to have activity in the NFKB reporter assay, their ability to activate CD137 was tested in the T cell activation assay. A DO11.10 T cell activation assay was created using DO11.10 T cells engineered to overexpress human CD137, and T cell activation was evaluated by measuring IL-2 release.

[0395] DO11.10 T cells (National Jewish Health) were transduced with a lentiviral vector designed to overexpress mouse or human CD137 as described above. Not only FS22-053 and FS22-172, but also 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 "mock" HelD1.3 mAbs 2 in the format). mAbs with or without the recombinant protein L (Life Technologies, 21189) crosslinker 2Alternatively, dilutions of 20H4.9 positive control mAb were prepared and added to DO11.10.hCD137 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 supernatants were collected and assayed using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86) according to the manufacturer's instructions. The plates were read at 450 nm using a plate reader with Gens Software, BioTek. The absorbance value at 570 nm was subtracted from the absorbance value at 450 nm (corrected). The standard curve for cytokine concentration calculation was based on a 4-parameter logistic curve fit (Gens Software, BioTek). The concentration of mlL-2 was plotted against the log concentration of mAb 2 or the benchmark mAb, and the resulting curve was fitted in GraphPad Prism using the log(agonist) vs. response equation.

[0396] Clones FS22-053 and FS22-172 showed significantly enhanced activity when cross-linked with Protein L in this assay. FS22-053 had an activity of 126 nM when not cross-linked and 21 nM when cross-linked (a 6-fold improvement), while FS22-172 had an activity of 950 nM when not cross-linked and 44 nM when cross-linked (a 22-fold improvement). As a result, both clones were selected for affinity maturation. Additionally, clone FS22-033 did not show activity in this assay, but since it did not compete with clones FS22-053 and FS22-172 for binding in the BLI binding assay (data not shown) and is likely to bind to the recombinant antigen at a different epitope, it was also selected for affinity maturation. Improvement in the binding affinity of the clones to CD137 can also lead to improved functional activity (see Example 4.1).

[0397] 3.5 CD137 / CD20 mAb2 Preparation of anti-human CD137 Fcab in a format A series of anti-CD137 Fcab (FS22-053, FS22-175, FS22-176, FS22-177, FS22-178, FS22-179) were produced to enable characterization of the Fcab in a format. The portion of the CH3 domain Fcab containing the AB, CD, and EF loops was replaced with the corresponding region of the CH3 domain of an anti-CD20 2F2 clone (from US Patent No. 8,529,902 B2) to produce an mAb 2 that binds to both CD137 and CD20. By producing an mAb 2 in this way, an mAb 2 was prepared. These CD137 / CD20 mAbs 2 were produced by transient expression in HEK293-6E cells and purified using an mAb Select SuRe protein A column.

[0398] 3.6 Activity of anti-CD137 / CD20 mAbs in the DO11.10 T cell activation assay 2 of the mAbs Example 3.4 describes a T cell activation assay in which an anti-CD137 mock mAb 2 was cross-linked using protein L. The setting provided a reliable and reproducible method for screening a significant number of molecules, but the higher-order structures formed by the antibody and mAb 2 when cross-linked were sub-optimal and not representative of the physiological environment. A more biologically relevant setting is one in which the mAb 2 molecule is cross-linked by the binding of its Fab arms to the target present in the biological system. This cell-based in vitro system optimizes the presentation of the antibody and mAb 2 via the Fab that binds to the cell membrane, causing clustering of the antibody, which in turn increases the affinity of the CD137 Fcab arm for its target in T cells.

[0399] CD20 +Daudi cells (ATCC CCL-213) were seeded at a 1:1 ratio with the DO11.10.hCD137 cells used in Example 3.4 in 96-well round-bottom plates. CD137 / CD20 mAb 2 Six clones produced in Example 3.5 in 2 were tested in this DO11.10 T cell activation assay. mAb 2 dilutions with or without the recombinant Protein L (Life Technologies, 21189) crosslinker or positive control mAb were prepared and 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 supernatants were collected and assayed using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86) according to the manufacturer's instructions. Plates were read at 450 nm using a plate reader with Gens Software, BioTek. The absorbance value at 570 nm was subtracted from the absorbance value at 450 nm (corrected). The standard curve for cytokine concentration calculation was based on a 4-parameter l logistic curve fit (Gens Software, BioTek). The concentration of mlL-2 was plotted against the log concentration of mAb 2 or benchmark mAb, and the resulting curve was fitted in GraphPad Prism using the log(agonist) vs response equation. Of all the clones tested, FS22-053 was the only one that showed significant IL-2 production in this CD20 cell-based crosslinking setting with an EC50 of 0.3 nM (and 126 nM in the absence of CD20+ cells).

[0400] Examples 3.4 and 3.6 show that FS22-053 was crosslinked by Protein L or the mAb being tested 2When cross-linked by cell-based cross-linking via Fab that binds to another target in the format (HelD1.3 and CD20), it is shown that it can cause clustering and activation of CD137 on the surface of DO11.10 T cells.

[0401] Example 4: Affinity Maturation of Anti-Human Fcab As described above, three clones were selected for affinity maturation based on their functional properties in the NF-κB reporter assay, DO11.10 T cell activation assays (FS22-053 and FS22-172), or were thought to bind to different regions of CD137 (FS22-033).

[0402] 4.1 Affinity Maturation of FS22-033 Two yeast and two phage-displayed libraries were constructed from the FS22-033 clone, 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 the ELLA primer. The ELLA primer defines the codons used for each amino acid and their relative abundance within the mixture. Only cysteine was excluded from the mixture and there was no bias towards any other amino acid.

[0403] For the phage FS22-033 AB and FS22-033 EF libraries, two rounds of selection of affinity-matured clones were performed, first using 200 nM hCD137-mFc-Avi and second using 10 nM hCD137-mFc-Avi. 96 clones from each selection output were screened by phage ELISA. This screening identified 24 unique clones (from FS22-033-001 to FS22-033-024), all of which were mock mAbs containing HelD1.3 2It was generated as such and then tested for the binding rate improved by BLI as described in Example 3.2. None of these 24 affinity matured clones functioned better than the parental FS22-033 clone in this assay, so it could not be advanced. For the yeast FS22-033 AB and FS22-033 EF libraries, three selections were performed as follows: the first and second times with 300 nM hCD137-mFc-Avi, and then the third time with 300 nM hCD137-mFc-Avi, 300 nM cynomolgus CD137-mFc-Avi, or 300 nM hCD137-Avi-his. 1056 clones from the second and third outputs were sequenced, and all the identified unique clones were screened in an antigen binding flow cytometry assay for improved binding to the antigen compared to the parental FS22-033 clone using 30 nM human dimeric recombinant antigen, and the clones were ranked by the percentage of APC+ cells correlated with the binding strength. Next, the top 5 clones (FS22-033-025, FS22-033-026, FS22-033-027, FS22-033-028, FS22-033-029) were produced as HelD1.3 mAb 2 (as described in Example 3.1) and the binding to human dimeric recombinant antigen was tested by BLI as described above, but none of the clones showed an improved kinetic profile compared to the parental FS22-033 clone. These clones could not be advanced further.

[0404] 4.2 Affinity Maturation of FS22-053 and FS22-172 Four yeast-displayed libraries were constructed from the FS22-053 and FS22-172 Fcab clones. Seven residues (at positions 15 - 16.1 according to IMGT) were randomized using the ELLA primer in the AB loop of the CH3 domain of each clone with the same trinucleotide distribution as described in Example 4.1 to create libraries FS22-053 AB and FS22-172 AB. Five residues (at positions 92 - 94 and 97 - 98 according to IMGT) were randomized using the ELLA primer in the EF loop of the CH3 domain to obtain libraries FS22-053 EF and FS22-172 EF.

[0405] For libraries FS22-053 AB and FS22-053 EF, and FS22-172 AB and FS22-172 EF, three or four rounds of selection were performed in the yeast library to select clones affinity matured using either the dimeric hCD137-mFc-Avi antigen or the monomeric hCD137-Avi-His antigen. The monomeric antigen was used alternately with the dimeric antigen to ensure that the clones retained affinity for the antigen and did not bind by avidity alone. The use of the monomeric or dimeric antigen, and the concentration used, were determined empirically between each round by flow cytometry, which was determined by whether enrichment for the monomeric or dimeric antigen was observed in the previous round. Where possible, sorting gates beyond the parent were used to isolate affinity matured clones compared to the parental molecule. The selection pressure was increased up to 1 nM of the dimeric antigen. Between each round of selection, individual clones were spotted onto agar plates to assess the progress of selection. Each clone was grown, induced individually, and then its binding and structural parameters were determined by flow cytometry using the biotinylated dimeric antigen and the anti-CH2 structural marker as described above. This screening cascade enabled the determination of the success of selection based on samples of clones from the selection output and enabled the initial screening of individual clones that could then be produced as soluble proteins.

[0406] A total of 1152 yeast single clones were screened for binding to biotinylated recombinant antigen in antigen - binding flow cytometry as described above. Selection in 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. The libraries FS22 - 053 AB and FS22 - 172 EF did not contain clones and showed improved binding over the parental clones. Sequence analysis across the best - binding clones from the FS22 - 053 EF and FS22 - 172 AB libraries showed a conserved PPY sequence pattern in the AB loop. This sequence was conserved after affinity maturation and was independently selected in two separate lineages of Fcab, so it may be important for epitope binding to CD137. Furthermore, a conserved LE or LD sequence pattern was shown in the EF loop of the CH3 domain of clones from both the FS22 - 053 and FS22 - 172 lineages, suggesting that this amino acid motif in the EF loop is required for improved binding.

[0407] To evaluate the selection process and whether it may be necessary to recombine the mutated AB and EF loops between affinity - matured clones, the top 5 unique clones from the FS22 - 053 EF library (FS22 - 053 - 008, FS22 - 053 - 009, FS22 - 053 - 010, FS22 - 053 - 011, FS22 - 053 - 012), and the top 6 unique clones from the FS22 - 172 AB library (FS22 - 172 - 001, FS22 - 172 - 002, FS22 - 172 - 003, FS22 - 172 - 004, FS22 - 172 - 005, FS22 - 172 - 006, all of which show more than 10% APC - positive cells when screened with 10 nM dimeric human antigen in the same assay), ranked by showing specific binding (more than 30% APC - positive cells in a flow cytometry binding assay) to 10 nM dimeric human antigen, were used as mock mAb 2(HelD1.3) and model mAb 2 (PD-L1) mAb 2 were produced to evaluate the improvement of the function and dynamics of the randomized loop.

[0408] Example 5: "Mock" mAb 2 Construction, expression, and characterization of affinity-matured anti-human CD137 Fcab in a format 5.1 "Mock" and model mAb 2 Construction of anti-human CD137 Fcab in a format Sixteen affinity-matured clones (FS22-053-001 to FS22-053-016) obtained from the parental FS22-053 clone, and six clones (FS22-172-001 to FS22-172-006) obtained from the parental FS22-172 clone were prepared in the mAb 2 format. Clones FS22-053-001 to FS22-053-007 did not express the mAb 2 format at a level that would allow downstream purification for further testing and characterization, and thus were not pursued further. The remaining clones were found to have at least 95% sequence identity in their CH3 domains when compared to the CH3 sequence of the parental clone from which they were obtained. A sequence similarity matrix percentage was generated by comparing each amino acid position to that of the reference sequence (parental clone FS22-053 or FS22-172). (Figure 1D shows the sequence identity percentage 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 shows the sequence identity percentage of the CH3 domains of clones FS22-172-001 to FS22-172-006 compared to the parental FS22-053 CH3 domain.

[0409] "Mock" mAb containing anti-human CD137 Fcab in HelD1.3 2 The antibody was in the mAb 2Prepared for further characterization of affinity matured Fcab in the format. These mAbs 2 were prepared as described in Example 3.1.

[0410] Model mAbs containing anti-human CD137 Fcab and further a PD-L1 binding Fab region (clone YW243.55.S70 from US Patent No. 8,217,149 B2) 2 were also produced. They were prepared in the same manner as the method described in Example 3.1 by substitution of the corresponding region of Fcab in the CH3 portion of the anti-PD-L1 binding antibody containing the AB, CD and EF loops. These PD-L1 model mAbs 2 contain the LALA mutation in the CH2 domain (AA). Introduction of the LALA mutation in the CH2 domain of human IgG1 is known to reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Hezareh M., et al. (2001)).

[0411] CD137 / HelD1.3 and CD137-AA / PD-L1 mAbs 2 were produced by transient expression in HEK293-6E cells and purified using an mAb Select SuRe protein A column.

[0412] 5.2 Mock mAbs in the human NF-κB reporter cell assay 2 Activity of human Fcab in the format Mock mAbs listed in Table 3 2(HelD1.3) format, the functional activity of affinity matured anti-human CD137 Fcab was tested in the same NF-κB luciferase assay described in Example 3.3. Luminescence was measured in a plate reader using Gen5 Software, BioTek with an integration time of 0.5 seconds. The results of this assay are shown in Table 3 and Figure 2. As expected, none of the Fcabs showed activity without Protein L cross-linking (-XL). All affinity matured CD137 Fcabs showed a significant improvement over the parental CD137 Fcab, which was positive in this assay while the EC 50 value could not be calculated (see Example 3.3). FS22-053-008 and FS22-172-003 showed the best activity from each family with the lowest EC 50 when cross-linked with Protein L (+XL).

[0413]

Table 4

[0414] 5.3 Activity of Model mAbs in Human DO11.10 T Cell Activation Assay 2 Activity of Affinity Matured Human Fcabs in (PD-L1 LALA) Format Model mAbs listed in Table 4 2 (PD-L1 LALA) format, the functional activity of affinity matured human Fcabs was tested in a DO11.10 T cell activation assay similar to the assay described in Example 3.6.

[0415] Using KpnI and NotI restriction sites, the cDNA encoding mouse PD-L1 (SEQ ID NO: 188) was subcloned into the pcDNA5FRT vector (Life Technologies), and the vector was transformed into Flp-In T-REx 293 cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019) to generate HEK.mPD-L1 cells. 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 colonies of stably transformed cells were formed. These colonies were amplified in the presence of 1 μg / ml doxycycline (Sigma Aldrich, D9891) and tested for PD-L1 expression using the PE-conjugated anti-mouse PD-L1 (MIH5) antibody (BD Biosciences, 558091).

[0416] The cells were separated using cell dissociation buffer, washed once with PBS, and 2×10 5 cells were plated in the wells of a 96-well plate and then incubated with the antibody diluted 1:20 in PBS for 1 hour at 4°C. The cells were washed once in PBS and then measured on an Accuri C6 cytometer (BD Biosciences), and the data were analyzed using FlowJoX. The expression of mouse PD-L1 was confirmed again.

[0417] CD137 / PD-L1 mAb 2 Fifteen clones produced in Example 5.1 in CD137 / PD-L1 mAb were tested in this DO11.10 T cell activation assay. mAb 2Prepare dilutions of the mAb or positive control mAb and add them to either DO11.10.hCD137 (7.5×10 3 cells per well) and HEK.mPD-L1 cells (2×10 4 cells per well) or DO11.10.hCD137 (7.5×10 3 cells per well) and non-transduced HEK cells expressing mPD-L1 (2×10 4 cells per well) in 96-well flat-bottom plates coated overnight with 0.1 μg / ml anti-CD3 antibody (clone 17A2, BioLegend, 100208). After 18 hours of incubation, collect the supernatants and assay them using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86) according to the manufacturer's instructions. Read the plates at 450 nm using a plate reader with Gens Software, BioTek. Subtract the absorbance value at 570 nm from the absorbance value at 450 nm (correction). The standard curve for cytokine concentration calculation was based on a four-parameter logistic curve fit (Gens Software, BioTek). Plot the concentration of mlL-2 against the log concentration of the mAb 2 or benchmark mAb and fit the resulting curve using the log(agonist) vs response equation in GraphPad Prism. Detect T cell activation by measuring the release of IL-2.

[0418] This assay is shown in Table 4 and Figure 3. T cell activation was not observed without cross-linking by binding to PD-L1-expressing cells (column without cell-based XL). Upon cross-linking, all mAbs 2 showed high levels of IL-2 release and sub-nanomolar EC 50As can be seen by the values, it had strong T cell activity. At increasing concentrations, the positive control anti-human CD137 mAb, G1-AA / 20H4.9, showed an increase in mIL-2 release, but the maximum release was significantly lower than that of anti-human CD137 Fcab. All clones except FS22-053-009 and FS22-172-005 had an EC50 of less than 0.3 nM and were thus comparable to being no better than the positive control. The lowest Emax observed, which is a measure of maximum T cell activation and may be related to higher T cell anti-tumor activity in vivo, was 7758 pg / ml, which was higher than the positive control.

[0419]

Table 5

[0420] 5.4 Primary human CD8+ T cell activation assay The activity of Fcab that activates CD137 in HEK cells overexpressing CD137 was shown in Example 5.3. A primary human T cell assay was required to test the activity of Fcab in cells not engineered to overexpress CD137. Activated cytotoxic CD8 + T cells are involved in directly killing cancer cells and express CD137 on their cell surface (Ye et al, 2014). Clustering of CD137 is known to be essential for inducing downstream signaling and further CD8+ T cell activation. Therefore, a CD8+ T cell activation assay was used to evaluate the ability of Fcab (mAb in the format detailed below) to cause clustering of CD137 and subsequent downstream signaling. CD8+ T cell activation was determined by the release of IL-2. 2 in the format).

[0421] To isolate T cells, peripheral blood mononuclear cells (PBMCs) were isolated from leukoreduction cone cells, which are a byproduct of platelet component donation. Briefly, the contents of the leukoreduction cone cells were flushed with PBS and overlaid on a Ficoll (Sigma-Aldrich, 1440 - 02) gradient. PBMCs were isolated by centrifugation, and the cells that did not pass through the Ficoll gradient were collected. The PBMCs were further washed with PBS, and the remaining red blood cells were lysed by the addition of 10 ml of 1× red blood cell lysis buffer (eBioscience, 00 - 4300 - 54) according to the manufacturer's instructions. CD8 + T cells were isolated from the PBMCs present in the eluate using the CD8 + T cell isolation kit II (Miltenyi Biotec Ltd, 130 - 096 - 495) according to the manufacturer's instructions.

[0422] Incubation with anti-CD3 antibody was used as the first signal to trigger the initial activation of T cells. A 96-well flat-bottom tissue culture plate was coated overnight at 4°C with 8 μg / ml of anti-CD3 antibody (Clone UCHT1, R&D Systems, MAB100 - SP) in PBS. Next, the plate was washed three times with 200 μl of PBS.

[0423] PD-L1 model mAb 2For cell-based crosslinking of affinity matured human CD137 Fcab in the format, HEK293 cells overexpressing hPD-L1 (HEK.hPD-L1) were produced essentially as described in Example 5.3, except that the cDNA encoding human PD-L1 (SEQ ID NO: 187) was subcloned instead of mouse PD-L1.HEK.hPD-L1 cells, in 100 μl of T cell culture medium containing 10% FBS (Life Technologies), 1× 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)) (RPMI medium (Life Technologies, 61870-044)), plated at 2×10 5 cells per well on 96-well flat-bottom plates coated with anti-CD3 antibody (8 μg / ml). If non-transduced HEK.hPD-L1 cells or HEK cells expressing hPD-L1 had attached after 4 hours of incubation, all T cell culture medium was removed and replaced with 100 μl of T cell culture medium containing T cells at a concentration of 5.0×10 5 cells / ml to obtain 5.0×10 4 cells / well.

[0424] mAb 2 was diluted in T cell medium starting at 500 nM and in 2× final concentrations and a 1:3 titration was performed. 100 μl of the titration of mAb 2 was added to the cells for a total assay volume of 200 μl and a 1-fold concentration of the antibody.

[0425] The positive control anti-CD137 antibody (G1-AA / 20H4.9) and the negative control isotype IgG antibody (G1-AA / HelD1.3) were each diluted in T cell medium starting at 500 nM with a crosslinker (anti-human CH2 produced in-house, clone MK1A6 (Jefferis et al., 1985 and Jefferis et al., 1992)) containing 500 nM and diluted at 2× final concentration, and a 1:3 titration was performed. 100 μl of the diluted positive control antibody / crosslinker mixture or negative control IgG antibody / crosslinker mixture was added to the cells for a total assay volume of 200 μl and a 1-fold concentration of antibody.

[0426] The assay was incubated at 37 °C, 5% CO 2 for 72 hours. The supernatant was collected and assayed using the human IL-2 ELISA Ready-SET-Go! kit (eBioscience, Cat. 88-7025-88) according to the manufacturer's instructions. The plate was read at 450 nm using a plate reader with Gen5 Software, BioTek. The absorbance value at 630 nm was subtracted from the absorbance value at 450 nm (corrected). The standard curve for the calculation of cytokine concentration was based on a 4-parameter logistic curve fit (Gen5 Software, BioTek). The concentration of human IL-2 (hIL-2) was plotted against the log concentration of the antibody, and the resulting curve was fitted in GraphPad Prism using the log(agonist) vs response equation.

[0427] Table 5 shows the PD-L1 model mAb tested using cell-based crosslinking 2 EC observed in the T cell activation assay in the presence of affinity matured Fcab clones in the format 50Shows the values and the maximum response of IL-2 release. The positive control anti-human CD137 mAb, 20H4.9, shows an increase in hIL-2 release with an EC50 of 0.5 nM when cross-linked with an anti-hCH2 antibody. All clones are active in the assay, and most show good potency with sub-nanomolar EC50s. mAbs containing Fcab 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 induced the largest T cell response with the lowest EC50 in the range of 0.19 - 0.49 nM. A series of mAbs 2 (containing Fcab FS22-053-008, FS22-053-011, FS22-053-014, FS22-173-003 and FS22-172-004) were also tested without cross-linking by PD-L1 expressed in HEK cells and, as expected, showed no activity in this assay. This confirms the activity seen in the NF-kB assay and the DO11.10 T cell activation assay. Figure 4 shows representative plots of IL-2 release for the T cell activation assay 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 2

[0428]

Table 6

[0429] 5.5 Determination of the specificity of anti-human CD137 Fcab by surface plasmon resonance (SPR) The specificity of anti-human CD137 Fcab for human CD137 was tested in comparison to other related TNFSFR family members. Eight of the Fcabs were tested against mock mAbs 2 ​(HelD1.3) format was tested and tested for binding to other human TNFRSF receptors: CD40, OX40 and GITR, and measured by SPR in a Biacore T200 (GE Healthcare). Using amine coupling (amine coupling kit, GE Healthcare, BR-1000-50), human CD40, GITR and OX40 were coated in a Biacore CM5 chip (GE Healthcare, catalog number 29149603) to approximately 1000 RU. Mock mAb starting from 1 μM 2 Dilutions of anti-human CD137 Fcab in formats (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) were prepared 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 injection of 10 mM glycine pH 2.5 at 30 μl / min for 12 seconds. Biacore chip coating was confirmed using antibodies specific for various TNFRSF members as positive controls. Data were double reference subtracted and analyzed using BIAevaluation 3.2 software. Fcab did not bind to any of the TNFRSF receptors tested, demonstrating their specificity for CD137. As a result, it is not expected that Fcab will cause off-target binding.

[0430] 5.6 Mock mAbs against human, cynomolgus monkey and mouse CD137 by SPR 2 Binding affinity of anti-human CD137 Fcab in format The affinity of anti-human CD137 Fcab (FS22-053-008, FS22-053-011, FS22-053-014, FS22-172-004, FS22-172-004) for human, cynomolgus (cynomolgus (cyno)), and mouse CD137 was measured by SPR to determine whether the Fcab could be useful for testing in animal studies. Anti-human Fab capture antibody was immobilized on all four flow cells of a CM5 series S chip (GE Healthcare #BR-1005-30) to an average surface density of 6000 RU according to the manufacturer's recommendations (GE Healthcare, human Fab capture Kit, #28958325). A 3 μg / ml solution of mAb 2 was injected at 30 μl / min for 60 seconds to capture each mAb 2 to approximately 150 RU. Next, various concentrations of human, cynomolgus, or mouse CD137 antigen (non-biotinylated human, cynomolgus, or mouse CD137-mFc-Avi or human CD137-Avi-His) in HBS-EP+ buffer were flowed over the chip at 60 μl / min for 3 minutes and then dissociated for 10 minutes. After each antigen concentration, the chip was regenerated by infecting it with 10 mM glycine pH 2.1 at a flow rate of 30 μl / min for 30 seconds. For reference subtraction, HBS-EP+ buffer was injected before the highest concentration of antigen and after the lowest antigen, and one of the concentrations was randomly repeated twice. The association rates were fit to a 1:1 Langmuir model to generate the equilibrium binding constant (K D ) for each sample. Data analysis was performed using BiaEvaluation software version 3.2. The results are shown in Table 6.

[0431] Analysis of the results showed improved binding to both human and cynomolgus CD137 by all affinity matured clones as compared to their respective parental molecules. The binding affinity to monomeric human CD137 antigen was (at least 100-fold) weaker than to dimeric human and cynomolgus Fc fusion antigens. As described in Example 2, Fcabs that preferentially bind dimeric CD137 over the monomeric form of CD137 were selected, and this data confirms that the selection approach was successful. By this kinetic behavior, they are less likely to bind to monomeric CD137, which is expressed at minimal levels in unstimulated T cells, reducing the risk of liver or systemic toxicity associated with some anti-CD137 monoclonal antibody therapies.

[0432] The data also shows that the anti-human CD137 Fcab bound to cynomolgus dimeric CD137 with equivalent affinity to human dimeric CD137.

[0433] The ability of Fcabs to bind to mouse dimeric CD137 was also tested. With the exception of clone FS22-053-014, which was unexpectedly found to have a K D of 24 nM for the mouse antigen, none of the clones showed strong binding to the mouse antigen (as shown in Table 6 (where N / A indicates that the K D could not be calculated)). This was unexpected since mouse CD137 and human CD137 share less than 57% sequence homology.

[0434]

Table 7

[0435] 5.7 Determination of Fcab binding valency using heterodimeric and homodimeric Fcabs Fcab typically contains two homodimeric Fc chains together with antigen-binding sites in the CH3 domains. Since these antigen-binding sites in the two CH3 domains are in close proximity, the valency of Fcab binding was tested to determine whether the CH3 domains could bind to CD137 independently of each other. Using the knob-into-hole mutation (knob: T22W, hole: T22S L24A Y66V) (Atwell S 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. As a result, each heterodimer contained CH3 from FS22-172-003 (SEQ ID NO: 139) in one chain and wild-type CH3 (SEQ ID NO: 4) in the other chain. Fcab was prepared in the mAb 2 format.

[0436] The heterodimeric Fcab was compared to the FS22-172-003 homodimeric molecule (containing two antigen-binding CH3 domains) by SPR binding analysis. For this experiment, monomeric human CD137-mFc-Avi was preferred over the dimeric CD137 antigen. To compensate for the weaker binding to the monomeric antigen described in Example 5.6, human CD137-mFc-Avi was immobilized on a CM5 chip to a higher density of 450 RU. The heterodimeric or homodimeric Fcab was injected and flowed over the immobilized antigen. The results in Figure 14 show that the heterodimeric Fcab containing only one CD137-binding CH3 domain was able to bind to the antigen even under these sub-optimal conditions. The off-rate observed for the heterodimeric Fcab was significantly faster than that of the homodimeric Fcab. These results confirm that FS22-172-003 Fcab was able to bind to CD137 via either or both of the CH3 domains, and that the selection procedure described in Example 2 was successful since the Fcab was able to bind bivalently to its target.

[0437] 5.8 Binding of anti-CD137 Fcab to cells at various CD137 expression levels As described in Example 2, Fcabs that bind to CD137 were selected to preferentially bind to cells at high CD137 expression levels. The bivalent binding of FS22-172-003, and thus strong binding, was confirmed by SPR in Example 5.6: FS22-172-003 showed high binding affinity and stronger binding to dimeric CD137 compared to monomeric CD137.

[0438] A series of DO11.10 cells expressing various levels of CD137 were generated as described in Example 3.5. To determine the relative expression of CD137 in each cell line, antibody binding capacity (ABC) was determined according to the manufacturer's protocol (Quantum™ Simply Cellular® #816 Bangs Labs). After subtracting the background, each cell line was ranked in order of CD137 expression level: hCD137 high (ABC: 1,206,283), hCD137 intermediate (ABC: 404,597), hCD137 intermediate / low (ABC: 143,065), hCD137 low (ABC: 14,208), hCD137 negative (ABC: 0).

[0439] Mock mAb to each of the cells described above 2 The binding of anti-human CD137 Fcab, positive control antibody (G1-AA / 20H4.9), or isotype control (G1-AA / HelD1.3) in the format (FS22-172-003-AA / HelD1.3) was tested as follows: DO11.10 cells were collected from a T175 cell culture flask, centrifuged at 1200 rpm for 3 minutes, and 2×10 6cells / ml and resuspended in ice-cold FACS buffer consisting of DPBS (Life Technologies, 14190169) and 1% BSA (Sigma-Aldrich, A7906). 50 μl per well was seeded into a 96-well V-bottom plate (Costar, 3894). All antibodies to be tested were diluted in 120 μl of FACS buffer. Next, DO11.10 cells were centrifuged, the supernatant was removed, 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, resuspended in 100 μl containing goat anti-human IgG (γ-chain specific) F(ab’)2 fragment-R-phycoerythrin antibody (Sigma, P8047) diluted 1:1000 in FACS buffer, and incubated at 4°C for 45 minutes. The cells were washed once with 150 μl of FACS buffer and then once with 150 μl of DPBS, resuspended in 150 μl of DPBS containing DAPI (Biotium, 40043) at 1:10,000, and read on a BD CantoII or iQue (Intellicyt). The data was analyzed using FlowJo v10 to determine the geometric mean of the PE signal for live cells in each well.

[0440] As shown in Figure 15, 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 15D: CD137 intermediate and Figure 15E: CD137 high), and did not bind to cells expressing very low levels of CD137 (Figure 15B: CD137 low and Figure 15C: CD137 intermediate / low). In contrast, as described in Example 5.6, unlike FS22-172-003-AA / HelD1.3 which had at least 200-fold weaker binding to monomeric CD137 antigen than to the dimeric antigen, the positive control G1-AA / 20H4.9 had a K D for both dimeric and monomeric antigens, and bound well to both.

[0441] Example 6: Naive selection of anti-mouse CD137 Fcab To test the activity of anti-CD137 Fcab in an in vivo mouse model, Fcabs that specifically bind to mouse CD137 were generated and characterized.

[0442] Phage display Six naive phage libraries showing the CH3 domain of human IgG1 previously used for the selection of Fcabs that bind to human CD137 were used for the selection of Fcabs that bind to mouse CD137, and cells expressing recombinant mouse dimer CD137 or full-length mouse CD137 were used as antigens.

[0443] The in-house mCD137-mFc-Avi antigen and DO11.10 cells expressing mCD137 (DO11.10.mCD137) were used for the selection using six phage libraries. All selection rounds (a total of 3 rounds) were performed using 100 nM biotinylated antigen, following a simple selection scheme with an exclusion step using 500 nM unlabeled recombinant human Fc fragment. The binder was captured by electromagnetic beads coated with either streptavidin or neutravidin. Furthermore, the first output that bound to the recombinant antigen was also used for selection in DO11.10.mCD137 cells expressing mouse CD137. Briefly, the phage output was incubated with DO11.10 cells lacking mouse CD137 to discard unwanted binders such as those that bind nonspecifically to the cells. Next, the phage was incubated with 1×10 7 cells of DO11.10.mCD137. Next, the binder was eluted by trypsin digestion and then propagated to the second selection. For the third time, the selection pressure was increased by reducing the number of DO11.10.mCD137 cells to 5×10 6 cells, following a similar process.

[0444] All three rounds of recombinant antigen output (576 clones) and all three rounds of cell selection output (576 clones) were screened by phage ELISA (as described above) and for cell binding to DO11.10.mCD137 cells. For ELISA, most clones showed high signal intensity for antigen binding (OD450 > 1). Thus, clones showing less than a 10-fold increase in antigen binding compared to binding to mouse-Fc were discarded. For cell binding, 5×10 5 Higher FITC MFI was considered positive. Three of the phage libraries functioned significantly worse, with many clones showing non-specific binding to DO11.10.mCD137 cells and recombinant antigens. 34 Fcab clone hits were subcloned and produced as HelD1.3 mAb 2 as described in Example 3.1.

[0445] Yeast display Four naive yeast libraries showing the CH1 to CH3 domains of human IgG1 previously used for the selection of Fcab binding to human CD137 were used for the selection of Fcab binding to mouse CD137.

[0446] A total of 53 separate rounds of selection were performed to identify anti-mouse CD137 binders. A recombinant dimeric biotinylated mouse CD137 (mCD137-mFc-Avi) antigen produced in-house was used to select binders from the yeast naive library. Briefly, the first round of binders was selected by incubating the naive library with 300 nM recombinant antigen and excluding with 2.5 μM unlabeled mouse IgG2a Fc fragment. The output was separated using MACS and streptavidin magnetic beads. As previously described in Example 2, three rounds of FACS selection were performed using 300 nM recombinant antigen and 1.5 μM mouse Fc used for selection and exclusion, respectively.

[0447] Single clones from each of the 2nd, 3rd, and 4th rounds were spotted onto agar plates. To determine the diversity of the output, at least 96 clones from each selected output were sequenced. 126 unique clones (50 clones from the 3rd round for one library, 48 clones from the 3rd round for another library, and 18 clones from the 4th round for the remaining library) were screened by flow cytometry for binding to the recombinant antigen. Clones that showed >10% positive cells in the APC fluorescence channel when incubated with the recombinant antigen and <0.2% when incubated with the recombinant mFc were considered hits.

[0448] Example 7: Characterization of anti-mouse CD137 Fcab from naive selection 7.1 Determination of anti-mouse CD137 Fcab specificity by BLI The specificity of the anti-mouse CD137 Fcab for mouse CD137 was tested in the HelD1.3 “mock” mAb 2 format and measured by BLI in an Octet QKe system by testing the binding of the Fcab to other mouse TNFRSF receptors (CD40, OX40, GITR). Streptavidin biosensors (PALL ForteBio 18-5021) were coated with 10 ng / μl of mouse CD40, GITR, OX40 receptors (all obtained from R&D Systems and biotinylated using the EZ-Link sulfo-NHS-SS-biotin kit from Thermoscientific #21328). The mock mAb 2 format of the anti-mouse CD137 Fcab was diluted 1:1 in kinetic buffer (PALL 18-1092) to a final concentration of at least 1 μM. The antigen-coated sensors were incubated with the mAb for 180 seconds 2It was then immersed in 1X kinetic buffer for 180 seconds. Antibodies against each of the TNFRSF receptors were used as positive controls. Fcab clones FS22m-055, FS22m-063, FS22m-066, FS22m-075, FS22m-135, FS22m-055, FS22m-063, FS22m-066 did not bind to any of the TNFRSF receptors tested and thus demonstrated their specificity for murine CD137.

[0449] 7.2 Mock mAbs in the mouse NF-κB reporter cell assay 2 Activity of mouse Fcab in the format HEK.FRT.luc cells expressing the murine CD137 sequence (SEQ ID NO: 184) were produced according to the same method as described above in Example 3.3. mAbs containing the previously selected anti-mouse CD137 Fcab 2 were screened using this cell line, HEK.FRT.luc.mCD137, according to the method described in Example 3.3. Fifty-six mAbs 2 were tested and 29 of them were positive for NF-κB activity. Lob12.3 containing the human IgG1 Fc with the LALA mutation (G1AA / Lob12.3) was used as a positive control anti-mouse CD137 mAb, showing an increase in luminescence and confirming the validity of the assay. HelD1.3 containing the human IgG1 Fc with the LALA mutation was also used as a negative control human IgG isotype to exclude interference from human IgG mock Fab in this assay. EC50 was calculated if possible, and mAbs 2 showing classical sigmoidal activity kinetics were prioritized over mAbs 2 that did not reach a plateau of activity. mAbs 2They were ranked in the order of EC50 and fold change in activity upon cross-linking of Protein L. FS22m-063 was selected based on its having the best EC50 (1.44 nM) upon cross-linking and the highest fold change in activity (27-fold) upon cross-linking. Figure 5 shows the anti-mouse CD137 Fcab FS22m-063 in the 2 format causes CD137 clustering and NF-κB signaling when cross-linked with Protein L in the HEK mCD137 NF-κB reporter assay.

[0450] Example 8: In vivo anti-tumor activity of FS22m-063 Fcab (in FS22m-063-AA / PD-L1 mAb 2 ) Since FS22m-063 Fcab was shown to be able to cause CD137 clustering and activation in vitro, it was desirable to test their ability to activate CD137 in vivo.

[0451] 8.1 mAb for in vivo testing in mice 2 Preparation of FS22m-063 Fcab in the format The anti-mouse CD137 Fcab, FS22m-063, and mAb containing the Fab region specific for PD-L1 2 were prepared using the same method as the model mAb produced in Example 7.1 2 and tested for in vivo anti-tumor activity in the MC38 syngeneic mouse tumor model.

[0452] Controls: G1-AA / Lob12.3, G1-AA / S70, G1-AA / 4420.

[0453] The variable heavy chain region of the anti-PD-L1 antibody S70 (clone YW243.55.S70 from U.S. Patent No. 8,217,149 B2) was conjugated to the human IgG1 (G1m17) constant region containing the LALA mutation to produce a control antibody for in vivo experiments, and the variable light chain region from the S70 antibody was conjugated to the human constant region (Lm1) via the human κ J-region. By replacing the CH3 domain of the reformatted construct described above with FS22m-063, mAb 2 was generated and designated "FS22m-063-AA / S70".

[0454] 8.2 Activity of FS22m-063-AA / S70 mAb in the MC38 syngeneic tumor model 2 of Allogeneic mouse models are recognized as suitable mouse systems for testing the antitumor effects of inhibiting therapeutic targets and are widely used to facilitate the development of human therapeutics. The MC38 syngeneic tumor model was used in this experiment because the MC38 tumor is known to be highly immunogenic and responsive to anti-CD137 antibody monotherapy (Kocak et al, 2006) and to express PD-L1 (Juneja et al, 2017).

[0455] C57BL / 6 female mice (The Jackson Laboratory) at 9 - 10 weeks of age and weighing 17.92 to 23.89 g each were rested for 1 week prior to the start of the study. All animals were implanted with a microchip to give a unique identifier. Each cohort had 12 mice. The MC38 colon cancer cell line (National Cancer Institute, USA) was first grown, stored, and then pre-screened for pathogens and shown to be pathogen-free. Each animal was given 1 × 10 6 cells subcutaneously in the right flank in 100 μl of serum-free culture medium (Dulbecco's Modified Eagle Medium). Seven days after tumor cell inoculation, mice that did not have a tumor at this time were removed from the study.

[0456] 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 intraperitoneally injected into mice at a constant concentration of 20 μg per administration in DPBS + 1 mM arginine + 0.05 Tween 80. Each mouse was given the mAb 2 molecule or control antibody by 200 μl intraperitoneal (IP) injection at 7, 9, and 11 days after tumor inoculation. Accurate measurements of the tumors were taken, drugs were administered up to the corresponding days, and the mice were closely observed for the remainder of the study. Tumor volume measurements were taken using calipers to determine the longest and shortest axes of the tumors. The tumor volume was calculated using the following formula: L×(S 2 ) / 2 (where L = longest axis; S = shortest axis).

[0457] As shown in Figure 6, FS22m-063-AA / S70 mAb 2 showed significant tumor growth inhibition compared to mice treated with any of the control antibodies. Statistical significance was shown pairwise for the growth rate over the entire time of the study using a mixed model analysis comparing all groups. As shown in Table 7, unexpectedly, all mice treated with FS22m-063-AA / S70 mAb 2 had no tumors at the end of the study compared to only 4 out of 12 mice treated with the combination of anti-PD-L1 and anti-CD137 antibodies (G1-AA / S70 + G1-AA / Lob12.3) or with PD-L1 or CD137 antibody alone.

[0458]

Table 8

[0459] The experiment shows that in mice with a fully functional immune system, CD137 agonism, presumably as a result of crosslinking via PD-L1, leads to a decrease in tumor growth, presumably due to the increased cytotoxic activity of CD8+ T cells in the tumor.

[0460] Example 9: In vivo antitumor activity of FS22m-063 Fcab (in FS22m-063-AA / PD-1 mAb 2 ) Since FS22m-063 Fcab has been shown to be able to cause clustering and activation of CD137 in vitro, it was desirable to test the ability of these, another Fab target, in this case targeting PD-1, a target found only on immune cells in vivo, to activate CD137 via it.

[0461] 9.1 mAb for in vivo testing in mice 2 Preparation of FS22m-063 Fcab in format mAb containing an anti-mouse CD137 Fcab, FS22m-063, and a Fab region specific for PD-1 2 was prepared using the same method as the model mAb 2 produced in Example 7.1 and tested for in vivo antitumor activity in an MC38 syngeneic mouse tumor model.

[0462] Control antibodies (G1 / Lob12.3, G1-AA / F2, G1-AA / 4420) for in vivo experiments were produced by binding the variable heavy chain region of the anti-PD-1 antibody F2 (clone PD1-F2 from WO 2004 / 056875 A1) to a human IgG1 (G1m17) constant region containing the LALA mutation, and binding the variable light chain region from the F2 antibody to a human constant region (Lm1) via the human κ J-region. mAb 2 was generated by replacing the CH3 domain of the reformatted construct described above with FS22m-063 and designated "FS22m-063-AA / F2".

[0463] 9.2 Activity of FS22m-063-AA / F2 mAb in MC38 syngeneic tumor model 2 was used The MC38 syngeneic tumor model was used in this experiment with the following differences as described in Example 8.2: C57BL / 6 female mice (Charles River) aged 9 - 11 weeks were implanted with microchips to give unique identifiers. Each cohort had 12 mice. Each animal was given 1 × 10 6 MC38 colon cancer cells subcutaneously injected into the right flank of the back in 100 μl of serum-free medium.

[0464] 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 intraperitoneally injected into mice at a constant concentration of 20 μg per administration in DPBS + 1 mM arginine + 0.05 Tween 80. After the tumor volume reached 50 - 60 mm 3 (day 0) and 2 days and 4 days after the first administration, each mouse was given the mAb 2 molecule or control antibody by 200 μl intraperitoneal (IP) injection. Tumors were measured as described in Example 8.2.

[0465] As shown in Figures 7A and B, FS22m-063-AA / F2 mAb 2 showed very significant tumor growth inhibition compared to mice treated with the isotype control antibody and the positive control PD-1 antibody. As shown in Table 8, unexpectedly, compared to only 7 out of 12 mice treated with the combination of anti-PD-1 and anti-CD137 antibodies (G1-AA / F2 + G1-AA / Lob12.3), 10 out of 12 mice treated with FS22m-063-AA / F2 mAb 2 had no tumors at the end of the study.

[0466]

Table 9

[0467] The study shows that in mice with a fully functional immune system, CD137 agonism, presumably as a result of cross-linking by PD-1 engagement, possibly with the addition of PD-1 inhibition, leads to a reduction in tumor growth, presumably due to the increased cytotoxic activity of CD8+ T cells in the tumor.

[0468] The study also shows that CD137 Fcab can be cross-linked when in the mAb 2 format by binding the Fab arms to immune cell targets, resulting in clustering and activation of CD137.

[0469] Example 10: In vivo antitumor activity of mCD137 / MSLN mAb 2 Since the efficacy of an mAb containing FS22m-063 Fcab and PD-L1 Fab (Example 8) was shown, it was desirable to test the ability of CD137 Fcab in the mAb 2 format to activate CD137 by cross-linking mAb2 through the binding of its Fab arms to a tumor-specific antigen (TAA), in this case mesothelin (MSLN). 2 Such a tumor-targeting approach is expected to be beneficial in restricting T cell activation to the tumor microenvironment as cross-linking of the mAb 2 and thus CD137 agonism will occur only when MSLN is expressed.

[0470] An autologous 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 produced by lipofection (Lipofectamine 3000, Thermo Fisher Scientific, catalog number L3000008) using the pcDNA3.1 vector (+) (Thermo Fisher Scientific, catalog number V79020). According to the manufacturer's protocol, CT26 cells were transfected with the pcDNA3.1 vector containing mouse MSLN cDNA. Stable transfection was performed using geneticin as a selection antibiotic (at 600 μg / ml) in complete medium (RPMI, 10% FBS).

[0471] Expression of mouse MSLN in CT26 cells was confirmed by flow cytometry by using the positive control antibody MOR6626 (International Publication No. 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 Scientific Ltd, catalog number 109-546-098-JIR). Clonal populations were expanded and then analyzed to determine relative expression levels using the same flow cytometry procedure, and then one clone was selected and named CT26.G10.

[0472] CT26.G10 tumor growth was confirmed in vivo. Microchips were implanted into 8- to 10-week-old Balb / c female mice (Charles River) to give unique identifiers. Each cohort had 17 mice, and each animal was given 1 × 10 5 cells subcutaneously injected into the left flank of the back in 100 μl of serum-free medium. Tumor volume measurements were performed three times a week using calipers as described in Example 8. The study was conducted in accordance with UK Home Office regulation as described in Example 8.

[0473] Tissues were harvested at the end of the test, and the expression of membrane-bound mesothelin was confirmed in formalin-fixed paraffin-embedded (FFPE) tumor tissues by immunohistochemical staining as follows: 4-μm FFPE tissue sections were deparaffinized, and antigens were retrieved using low pH 6.1 at 97 °C (Dako PT Link). Subsequently, peroxidase blocking and protein blocking were performed, followed by incubation with the primary anti-mesothelin antibody (LifeSpan Biosciences, catalog number LS-C407883) at a concentration of 1 μg / ml. The anti-mesothelin antibody was detected using a labeled polymer-HRP anti-rabbit secondary reagent and DAB (3,3'-diaminobenzidine) chromogenic endpoint (Dako EnVision+ System).

[0474] To evaluate the efficacy of Fcab FS22m-063, the following molecules or combinations were tested in vivo: the anti-MSLN FS28m-228-010 antibody (G1-AA / FS28m-228-010) in the human IgG1 isotype with the LALA mutation, two "mock" mAbs 2 Fcab in the format (FS22m-063-AA / HelD1.3 and FS22m-063-AA / 4420), the FS28m-228-010 antibody, and a mock CD137 mAb with the LALA mutation 2 in combination (G1-AA / FS28m-228-010 + FS22m-063-AA / HelD1.3), the human isotype control antibody (G1-AA / HelD1.3), and finally the CD137 / MSLN mAb with the LALA mutation 2 (FS22m-063-AA / FS28m-228-010).

[0475] Balb / c female mice (Charles River) at 8 - 10 weeks of age and weighing 20 - 25 g each were acclimatized for 1 week prior to the start of the test. All animals were implanted with a microchip to give a unique identifier. Except for FS22m - 063 - AA / 4420 (n = 10 mice), each cohort consisted of 20 mice. The CT26.G10 colon cancer cell line was propagated to generate a cell bank. Each animal was given 1 × 10 5 cells subcutaneously in the left flank in 100 μl of serum - free medium. Mice that did not have a tumor 12 days after tumor cell inoculation were excluded from the test.

[0476] Each antibody at a dose of 200 μg (about 10 mg / kg) was prepared and injected intraperitoneally (IP) into the mice. Additionally, both FS22m - 063 - AA / HelD1.3 and G1 - AA / FS28m - 228 - 010 were each prepared at 200 μg (about 10 mg / kg) per administration for the combination group. A 200 - μl dose was administered to the mice on days 12, 14, and 16 after tumor inoculation (q2d × 3). Tumor volume measurements were taken three times a week using calipers, and the mice were closely monitored. The test endpoint was determined by human endpoints based on the tumor volume and condition of the mice.

[0477] As shown in Figure 9, the FS22m - 063 - AA / FS28m - 228 - 010 mAb 2 significantly inhibited tumor growth compared to the G1 - AA / HelD1.3 isotype control. Table 9 shows the pairwise comparison of tumor growth rates for all treatment groups throughout the test using a mixed - model analysis comparing all groups to the G1 - AA / HelD1.3 isotype control.

[0478]

Table 10

[0479] At the end of the test, 62.5 mm 3All animals with the following tumors were counted as animals in which the treatment was fully effective (see Table 10). Anti-CD137 / MSLN mAb 2 35% of the animals treated with 2 showed complete response to the treatment at the end of the study, compared to 0% in the G1-AA / HelD1.3 isotype control, FS22m-063-AA / HelD1.3, FS22m-063-AA / 4420, and the combination group of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010.

[0480]

Table 11

[0481] Survival analysis (Figure 10 and Table 11) showed that FS22m-063-AA / FS28m-228-010 mAb 2 induced a significant survival benefit compared to the G1-AA / HelD1.3 antibody, while 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 benefit. Furthermore, FS22m-063-AA / FS28m-228-010 mAb 2 resulted in an improvement in the median survival time of 42.5 days compared 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 (29 days).

[0482]

Table 12

[0483] These data are for mAb via MSLN 2Crosslinking can cause a more potent bispecific antibody effect targeting CD137 agonism in tumors; CD137 and / or MSLN alone (and even in combination), suggesting a significantly improved survival of tumor-bearing mice. Mock mAb without MSLN-targeted Fab 2 The Fcab in format FS22m-063-AA / HelD1.3 or FS22m-063-AA / 4420 did not show intrinsic activity in this assay.

[0484] Example 11: Selection to obtain Fcab capable of binding to mouse and human CD137 Since some of the Fcab that bind to human CD137 were unexpectedly found to also bind to mouse CD137 (see Example 5.5 for the specificity of binding to human, mouse, and cynomolgus monkey CD137), it was decided to investigate whether these clones could be improved.

[0485] 11.1: Site-directed modification to remove potential sequence liability in clone FS22-053-014 The mouse-human cross-reactive clone FS22-053-014 was selected because it was shown to be able to bind to the mouse dimer CD137 antigen by SPR (see Example 5.5). However, during further sequence analysis, it had potential sequence liability that could result in post-translational aspartate isomerization in the EF loop of its CH3 domain as a result of the Q98D mutation from the wild-type G99 position that gives rise to the DG motif. Other affinity-matured clones in the FS22-053 line (see Table 12) instead contained the Q98E modification at the same position in the EF loop. Q98D was mutated to Q98E by site-directed mutagenesis using the QuickChange II mutagenesis kit (Agilent, catalog number 200523) according to the manufacturer's recommendations, thereby obtaining clone FS22-053-017. The following Table 12 shows the LE motif that is present at a high frequency in the EF loop of the CH3 domain of clones FS22-053-008, FS22-172-003, and FS22-172-004, as well as the DG motif in the EF loop of the CH3 domain of clone FS22-053-014.

[0486]

Table 13

[0487] Example 12: Characterization of the Mutant FS22-053-017 Fcab Clone 12.1 Mock mAb in the Human CD137 DO11.10 T Cell Activation Assay 2 Activity of FS22-053-017 Fcab in the Format The Fcab clone FS22-053-017 was subcloned and expressed as the HelD1.3 “mock” mAb 2 and then, in the human CD137 DO11.10 T cell activation assay as described in Example 3.4, similarly to the HelD1.3 mAb 2It was compared with the FS22-053-014 clone in the format. G1-AA / 20H4.9 was used as an anti-CD137 positive control, and G1-AA / D1.3 was used as an IgG control. The mAb 2 was tested without protein L crosslinking or crosslinked with protein L at a ratio of 1:4.

[0488]

Table 14

[0489] The results in Table 13 and Figure 12 show that the mock mAb 2 FS22-053-17 Fcab in the format and the mock mAb 2 FS22-053-014 Fcab in the format both had equivalent activity when crosslinked by protein L in the same DO11.10 T cell activation assay. Thus, the mutagenesis performed did not adversely affect the functional activity. The mock mAb 2 Both clones in the format had no activity without crosslinking.

[0490] As expected, the positive control anti-CD137 had activity only when crosslinked, and the IgG control had no activity regardless of crosslinking.

[0491] 12.2 Activity of the mock mAb in the mouse CD137 DO11.10 T cell activation assay 2 Activity of the mutant FS22-053-017 Fcab in the format Also, the FS22-053-017 clone (HelD1.3 mock mAb 2 in the format) was tested in the mouse CD137 DO11.10 T cell activation assay as described in Example 3.4, together with the mouse CD137-binding Fcab clone FS22m-063 (similarly HelD1.3 mock mAb 2 in the format), and the parental FS22-053-014 clone (HelD1.3 mock mAb 2compared to (in the format of) mAb 2 The molecule was crosslinked with Protein L at a molar ratio of 4:1 (mAb 2 :Protein L).

[0492] As expected, all of the molecules tested showed activity when crosslinked by Protein L as measured by IL-2 release, but had no activity when not crosslinked. FS22m-063, which was selected to bind to mouse CD137, had the best activity in the assay with an EC50 of 0.39 nM when crosslinked. Both FS22-053-14 and FS22-053-017 had activity in the assay, indicating that the function was not lost by mutagenesis. However, FS22-053-017 had a slightly lower activity with an EC50 that was approximately 8-fold worse than FS22-053-14 when crosslinked by Protein L. Figure 12 shows the HelD1.3 mock mAb 2 In the format of, affinity matured human and mouse cross-reactive CD137 Fcab FS22-053-014 and FS22-053-017, and anti-mouse CD137 Fcab FS22m-063 activate CD137 and result in the release of mIL-2 when crosslinked with Protein L in the DO11.10 T cell activation assay.

[0493]

Table 15

[0494] 12.3 Association rate of FS22-053-017 The equilibrium dissociation constant (K D ) of the Fcab clone FS22-053-017 was determined by SPR in a Biacore T200 system, compared to the equilibrium dissociation constant (K D) was compared with. For the hCD137-mFc-Avi antigen, the following method was used: Anti-human Fab molecules were immobilized on a CM5 chip until a surface density of 9,000 - 11,000 response units (RU) was achieved. The antibody was diluted to 4 μg / ml in HBS-EP buffer and captured by the anti-Fab molecules at a flow rate of 30 μl / sec. Eight different concentrations of hCD137-mFc antigen were used: 200 nM; 66.67 nM; 22.22 nM (included twice), 7.41 nM; 2.47 nM; 0.82 nM, 0.27 nM; 0.091 (diluted in HBS-EP+ buffer) were flowed over the captured mAb 2 onto it.

[0495] For the determination of the mCD137-mFc-Avi binding rate, different methods were used. Mouse PD-L1-mFc-Avi was immobilized on a CM5 chip until a surface density of 200 RU was achieved. mAb containing FS22-053-014 and FS22-053-017 Fcab in combination with anti-mouse PD-L1 Fab (S70) 2 was diluted to 7.5 μg / ml in HBS-EP buffer and captured by the immobilized mPD-L1 protein at a flow rate of 60 μl / sec. 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 flowed over the captured mAb 2 onto it. Data analysis was performed using Biacore T200 evaluation software. The curve was subtracted against the blank flow cell, and RI was set to 0 as a constant and R max was set locally, and fitted using a 1:1 Langmuir binding model using mass transfer. The results are summarized in Table 15, showing very similar kinetic profiles for both Fcab clones against the human antigen, thereby demonstrating that mutating Q98D to Q98E did not adversely affect binding to the human antigen. A two-fold decrease in binding strength to the mouse antigen also agrees with the functional data shown in Example 11.2.

[0496]

Table 16

[0497] Example 13: Involvement of PPY in antigen binding (alanine scanning of the conserved PPY motif) As described in Example 5, the PPY sequence motif was identified in two independently selected AB loops of Fcab, and this motif was conserved in all affinity matured clones analyzed. Therefore, it was desirable to understand the involvement of this motif in the binding to CD137 and the overall protein structure. Alanine scanning is a common biological technique used to determine the importance of specific residues in protein-protein interactions. Briefly, each of the three amino acids was substituted, in turn and then together, with alanine residues. Alanine is considered the most chemically inert and non-bulky residue and is therefore thought to be less likely to assist in binding.

[0498] 13.1 Generation of mutant clones for alanine scanning Mutant clones were generated by site-directed mutagenesis in 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 with their respective parents. As described in Example 3, next, the mAb 2 The mutants were transiently expressed in HEK293-6E cells and purified using an mAb Select SuRe protein A column. The expression yields of the mutants were similar to those of the parent, thus suggesting that the alanine substitutions did not affect protein production.

[0499]

Table 17

[0500] 13.2 Binding rate of mutant clones The binding rates of two parental clones and eight mutant clones were compared using the Octet QK e system from ForteBio. Dimeric biotinylated hCD137-mFc-Avi was captured on streptavidin sensors at 10 μg / ml, and then the interaction with each mAb 2 at a concentration of 200 nM was analyzed. The binding of mAb 2 to the dimeric antigen was reduced for all FS22-172-003 mutant clones compared to the parental clones. Clones FS22-172-003_AAA and FS22-172-003_APY had lost all binding, and clones FS22-172-003_PPA and FS22-172-003_PAY retained some binding with significantly decreased responses (5.5- and 4.4-fold lower response units) compared to the parent. The binding of FS22-053-008 was also affected. Mutants PAY and AAA had lost all binding, and mutants APY and PPA showed decreased binding to the antigen and a deceleration of the association profile when compared to the parental FS22-053-008 clone. This data suggests that the PPY motif is important for the binding of both mAbs 2 to the CD137 antigen.

[0501] 13.3 Homology modeling Considering the involvement of the PPY motif in the binding, evaluating the protein structure prediction in the CH3 domain was beneficial for in silico modeling of Fcab. Structure homology modeling and subsequent conformational searches were performed using the 2019.0101 version of the MOE software suite (Chemical Computing Group ULC). The Fc region in the Protein Data Bank [PDB] structure 5JII was used as a structural template for both Fcab regions of FS22-172-003 and FS22-053-008. Since the insertions were present in the AB and EF loop regions with respect to the selected structural template, de novo loop search using residue side-chain optimization was applied to generate the AB and EF loop structures. The obtained homology models were energy minimized and scored according to geometric criteria including backbone bond lengths, angles, dihedral angles, and chirality. The structure homology models with scores passing the criteria were used as a basis for conformational sampling using the LowModeMD simulation method as implemented within MOE.

[0502] The homology models in Figure 13 show that the AB and EF loops of both Fcabs adopt different conformations compared to the template 5JII, and the PPY motif plays a role in the AB loop protrusion from the core of the CH3 domain. The PPY motif also mediates interactions that result in AB loop conformational stabilization through both interactions within the loop and between the loop and the rest of the structure. The conformational search performed using LowModeMD simulation also highlighted that 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, presumably stabilizing the AB loop. When 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.

[0503] Sequence Listing Amino Acid Sequence of the WT Fcab CH3 Domain Structure Loop WT Fcab AB loop - RDELTKNQ (SEQ ID NO: 1) WT Fcab CD loop - SNGQPENNY (SEQ ID NO: 2) WT Fcab EF loop - DKSRWQQGNV (SEQ ID NO: 3)

[0504] Amino acid sequence of the CH3 domain of WT Fcab (SEQ ID NO: 4) The AB, CD, and EF loops are underlined

Chemical Structure

[0505] Amino acid sequence of the Fcab CH2 domain with the LALA - PA mutation (SEQ ID NO: 5) The LALA - PA mutation is underlined

Chemical Structure

[0506] Amino acid sequence of the Fcab CH2 domain with the LALA mutation (SEQ ID NO: 6) The LALA mutation is underlined

Chemical Structure

[0507] Amino acid sequence of the cleaved Fcab hinge region (SEQ ID NO: 7) TCPPCP

[0508] Amino acid sequence of WT Fcab with the LALA mutation (SEQ ID NO: 8) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chemical Structure

[0509] Amino acid sequence of WT Fcab without LALA mutation (SEQ ID NO: 9) Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

Chem.

[0510] Amino acid sequence of the PPY motif (SEQ ID NO: 10) PPY

[0511] Amino acid sequence of Fcab FS22-033 with LALA mutation (SEQ ID NO: 11) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0512] Amino acid sequence of Fcab FS22-033 without LALA mutation (SEQ ID NO: 12) Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

Chem.

[0513] FS22-033 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 13) VH domain (underlined)

Chem.

[0514] FS22-033 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 14) VH domain (underlined)

Chem.

[0515] Amino acid sequence of Fcab FS22-053 having LALA mutation (SEQ ID NO: 15) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0516] Amino acid sequence of Fcab FS22-053 without LALA mutation (SEQ ID NO: 16) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chem.

[0517] Heavy chain amino acid sequence of FS22-053 / HelD1.3 mock mAb having LALA mutation 2 (SEQ ID NO: 17) VH domain (underlined)

Chem.

[0518] Heavy chain amino acid sequence of FS22-053 / HelD1.3 mock mAb without LALA mutation 2 (SEQ ID NO: 18) VH domain (underlined)

Chem.

[0519] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-008 FS22-053-008 first sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-008 second sequence - DYWRWLE (SEQ ID NO: 20)

[0520] Amino acid sequence of the Fcab FS22-053-008 CH3 domain (SEQ ID NO: 21) The first and second sequences are underlined

Chemical Structure

[0521] Nucleic acid sequence of the Fcab FS22-053-008 CH3 domain (SEQ ID NO: 22)

Chemical Structure

[0522] Amino acid sequence of Fcab FS22-053-008 with the LALA mutation (SEQ ID NO: 23) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chemical Structure

[0523] Nucleic acid sequence of Fcab FS22-053-008 with the LALA mutation (SEQ ID NO: 24)

Chemical Structure

[0524] Amino acid sequence of Fcab FS22-053-008 without the LALA mutation (SEQ ID NO: 25) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chemical Structure

[0525] Nucleic acid sequence of Fcab FS22-053-008 without the LALA mutation (SEQ ID NO: 26)

Chemical Structure

[0526] FS22-053-008 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 27) VH domain (underlined)

Chemical Structure

[0527] FS22-053-008 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 28) VH domain (underlined)

Chemical Structure

[0528] Amino acid sequence of the loop region of the CH3 domain of Fcab FS22-053-009 FS22-053-009 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-009 Second sequence - EHTRWLD (SEQ ID NO: 29)

[0529] Amino acid sequence of the CH3 domain of Fcab FS22-053-009 (SEQ ID NO: 30) The first and second sequences are underlined

Chemical Structure

[0530] Nucleic acid sequence of the CH3 domain of Fcab FS22-053-009 (SEQ ID NO: 31)

Chemical Structure

[0531] Amino acid sequence of Fcab FS22-053-009 with LALA mutation (SEQ ID NO: 32) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0532] Nucleic acid sequence of Fcab FS22-053-009 with LALA mutation (SEQ ID NO: 33)

Chem.

[0533] Amino acid sequence of Fcab FS22-053-009 without LALA mutation (SEQ ID NO: 34) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chem.

[0534] Nucleic acid sequence of Fcab FS22-053-009 without LALA mutation (SEQ ID NO: 35)

Chem.

[0535] Heavy chain amino acid sequence of FS22-053-009 / HelD1.3 mock mAb with LALA mutation 2 (SEQ ID NO: 36) VH domain (underlined)

Chem.

[0536] Heavy chain amino acid sequence of FS22-053-009 / HelD1.3 mock mAb without LALA mutation 2 (SEQ ID NO: 37) VH domain (underlined)

Chem.

[0537] Amino acid sequence of the loop array of the Fcab FS22-053-010 CH3 domain FS22-053-010 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-010 Second sequence - DYMRWLD (SEQ ID NO: 38)

[0538] Amino acid sequence of the Fcab FS22-053-010 CH3 domain (SEQ ID NO: 39) The first and second sequences are underlined

Chem.

[0539] Nucleic acid sequence of the Fcab FS22-053-010 CH3 domain (SEQ ID NO: 40)

Chem.

[0540] Amino acid sequence of Fcab FS22-053-010 with the LALA mutation (SEQ ID NO: 41) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0541] Nucleic acid sequence of Fcab FS22-053-010 with the LALA mutation (SEQ ID NO: 42)

Chem.

[0542] Amino acid sequence of Fcab FS22-053-010 without LALA mutation (SEQ ID NO: 43) Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

Chem.

[0543] Nucleic acid sequence of Fcab FS22-053-010 without LALA mutation (SEQ ID NO: 44)

Chem.

[0544] FS22-053-010 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 45) VH domain (underlined)

Chem.

[0545] FS22-053-010 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 46) VH domain (underlined)

Chem.

[0546] Amino acid sequence of the CH3 domain loop sequence of Fcab FS22-053-011 FS22-053-011 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-011 Second sequence - DYWRWTD (SEQ ID NO: 47)

[0547] Amino acid sequence of the CH3 domain of Fcab FS22-053-011 (SEQ ID NO: 48) The first and second sequences are underlined [Chemistry]

[0548] Nucleic acid sequence of Fcab FS22-053-011 with CH3 domain (SEQ ID NO: 49) [Chemistry]

[0549] Amino acid sequence of Fcab FS22-053-011 with LALA mutation (SEQ ID NO: 50) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined) [Chemistry]

[0550] Nucleic acid sequence of Fcab FS22-053-011 with LALA mutation (SEQ ID NO: 51) [Chemistry]

[0551] Amino acid sequence of Fcab FS22-053-011 without LALA mutation (SEQ ID NO: 52) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic) [Chemistry]

[0552] Nucleic acid sequence of Fcab FS22-053-011 without LALA mutation (SEQ ID NO: 53) [Chemistry]

[0553] FS22-053-011 / HelD1.3 mock mAb with LALA mutation2 Amino acid sequence of the heavy chain (SEQ ID NO: 54) VH domain (underlined)

Chem.

[0554] FS22-053-011 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 55) VH domain (underlined)

Chem.

[0555] Amino acid sequence of the loop region of the Fcab FS22-053-012 CH3 domain FS22-053-012 first sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-012 second sequence - DHMRWLE (SEQ ID NO: 56)

[0556] Amino acid sequence of the Fcab FS22-053-012 CH3 domain (SEQ ID NO: 57) The first and second sequences are underlined

Chem.

[0557] Nucleic acid sequence of the Fcab FS22-053-012 CH3 domain (SEQ ID NO: 58)

Chem.

[0558] Amino acid sequence of Fcab FS22-053-012 with LALA mutation (SEQ ID NO: 59) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0559] Nucleic acid sequence of Fcab FS22-053-012 having the LALA mutation (SEQ ID NO: 60)

Chem.

[0560] Amino acid sequence of Fcab FS22-053-012 without the LALA mutation (SEQ ID NO: 61) Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

Chem.

[0561] Nucleic acid sequence of Fcab FS22-053-012 without the LALA mutation (SEQ ID NO: 62)

Chem.

[0562] FS22-053-012 / HelD1.3 mock mAb having the LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 63) VH domain (underlined)

Chem.

[0563] FS22-053-012 / HelD1.3 mock mAb without the LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 64) VH domain (underlined)

Chem.

[0564] Amino acid sequence of the CH3 domain loop array of Fcab FS22-053-013 FS22-053-013 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-013 Second sequence - GYERWLE (SEQ ID NO: 65)

[0565] Amino acid sequence of the CH3 domain of Fcab FS22-053-013 (SEQ ID NO: 66) The first and second sequences are underlined

Chem.

[0566] Nucleic acid sequence of the CH3 domain of Fcab FS22-053-013 (SEQ ID NO: 67)

Chem.

[0567] Amino acid sequence of Fcab FS22-053-013 having the LALA mutation (SEQ ID NO: 68) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0568] Nucleic acid sequence of Fcab FS22-053-013 having the LALA mutation (SEQ ID NO: 69)

Chem.

[0569] Amino acid sequence of Fcab FS22-053-013 without the LALA mutation (SEQ ID NO: 70) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chem.

[0570] Nucleic acid sequence of Fcab FS22-053-013 without LALA mutation (SEQ ID NO: 71)

Chemical formula

[0571] FS22-053-013 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 72) VH domain (underlined)

Chemical formula

[0572] FS22-053-013 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 73) VH domain (underlined)

Chemical formula

[0573] Amino acid sequence of the CH3 domain loop sequence of Fcab FS22-053-014 FS22-053-014 first sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-014 second sequence - YHWRWLD (SEQ ID NO: 74)

[0574] Amino acid sequence of the CH3 domain of Fcab FS22-053-014 (SEQ ID NO: 75) The first and second sequences are underlined

Chemical formula

[0575] Nucleic acid sequence of the CH3 domain of Fcab FS22-053-014 (SEQ ID NO: 76) [Chemistry]

[0576] Amino acid sequence of Fcab FS22-053-014 having the LALA mutation (SEQ ID NO: 77) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined) [Chemistry]

[0577] Nucleic acid sequence of Fcab FS22-053-014 having the LALA mutation (SEQ ID NO: 78) [Chemistry]

[0578] Amino acid sequence of Fcab FS22-053-014 without the LALA mutation (SEQ ID NO: 79) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic) [Chemistry]

[0579] Nucleic acid sequence of Fcab FS22-053-014 without the LALA mutation (SEQ ID NO: 80) [Chemistry]

[0580] FS22-053-014 / HelD1.3 mock mAb having the LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 81) VH domain (underlined) [Chemistry]

[0581] FS22-053-014 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 82) VH domain (underlined)

Chemical Structure

[0582] Amino acid sequence of the loop region of the CH3 domain of Fcab FS22-053-015 FS22-053-015 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-015 Second sequence - DHWRWLQ (SEQ ID NO: 83)

[0583] Amino acid sequence of the CH3 domain of Fcab FS22-053-015 (SEQ ID NO: 84) The first and second sequences are underlined

Chemical Structure

[0584] Nucleic acid sequence of the CH3 domain of Fcab FS22-053-015 (SEQ ID NO: 85)

[0585] Amino acid sequence of Fcab FS22-053-015 with LALA mutation (SEQ ID NO: 86) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chemical Structure

[0586] Nucleic acid sequence of Fcab FS22-053-015 with LALA mutation (SEQ ID NO: 87)

Chemical Structure

[0587] Amino acid sequence of Fcab FS22-053-015 without LALA mutation (SEQ ID NO: 88) Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

Chem.

[0588] Nucleic acid sequence of Fcab FS22-053-015 without LALA mutation (SEQ ID NO: 89)

Chem.

[0589] FS22-053-015 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 90) VH domain (underlined)

Chem.

[0590] FS22-053-015 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 91) VH domain (underlined)

Chem.

[0591] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-053-016 FS22-053-016 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-016 Second sequence - DYIRWLN (SEQ ID NO: 92)

[0592] Amino acid sequence of the CH3 domain of Fcab FS22-053-016 (SEQ ID NO: 93) Underlined in the 1st and 2nd arrays [Chemical formula]

[0593] Nucleic acid sequence of the Fcab FS22-053-016 CH3 domain (SEQ ID NO: 94) [Chemical formula]

[0594] Amino acid sequence of Fcab FS22-053-016 with the LALA mutation (SEQ ID NO: 95) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined) [Chemical formula]

[0595] Nucleic acid sequence of Fcab FS22-053-016 with the LALA mutation (SEQ ID NO: 96) [Chemical formula]

[0596] Amino acid sequence of Fcab FS22-053-016 without the LALA mutation (SEQ ID NO: 97) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic) [Chemical formula]

[0597] Nucleic acid sequence of Fcab FS22-053-016 without the LALA mutation (SEQ ID NO: 98) [Chemical formula]

[0598] FS22-053-016 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 99) VH domain (underlined)

Chem.

[0599] FS22-053-016 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 100) VH domain (underlined)

Chem.

[0600] Amino acid sequence of the loop region of the Fcab FS22-053-017 CH3 domain FS22-053-017 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053-017 Second sequence - YHWRWLE (SEQ ID NO: 101)

[0601] Amino acid sequence of the Fcab FS22-053-017 CH3 domain (SEQ ID NO: 102) The first and second sequences are underlined

Chem.

[0602] Nucleic acid sequence of the Fcab FS22-053-017 CH3 domain (SEQ ID NO: 103)

Chem.

[0603] Amino acid sequence of the Fcab FS22-053-017 with LALA mutation (SEQ ID NO: 104) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0604] Nucleic acid sequence of Fcab FS22-053-017 with LALA mutation (SEQ ID NO: 105)

Chem.

[0605] Amino acid sequence of Fcab FS22-053-017 without LALA mutation (SEQ ID NO: 106) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chem.

[0606] Nucleic acid sequence of Fcab FS22-053-017 without LALA mutation (SEQ ID NO: 107)

Chem.

[0607] Heavy chain amino acid sequence of FS22-053-017 / HelD1.3 mock mAb with LALA mutation (SEQ ID NO: 108) 2 VH domain (underlined)

Chem.

[0608] Heavy chain amino acid sequence of FS22-053-017 / HelD1.3 mock mAb without LALA mutation (SEQ ID NO: 109) 2 VH domain (underlined)

Chem.

[0609] Amino acid sequence of the loop array of the Fcab FS22-172 CH3 domain FS22-172 first sequence - RKYYPPY (SEQ ID NO: 110) FS22-172 second sequence - GADRWLE (SEQ ID NO: 111)

[0610] Amino acid sequence of the Fcab FS22-172 CH3 domain (SEQ ID NO: 112) The first and second sequences are underlined

Chem.

[0611] Nucleic acid sequence of the Fcab FS22-172 CH3 domain (SEQ ID NO: 113)

Chem.

[0612] Amino acid sequence of Fcab FS22-172 with the LALA mutation (SEQ ID NO: 114) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0613] Nucleic acid sequence of Fcab FS22-172 with the LALA mutation (SEQ ID NO: 115)

Chem.

[0614] Amino acid sequence of Fcab FS22-172 without the LALA mutation (SEQ ID NO: 116) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic) [Chemistry]

[0615] Nucleic acid sequence of Fcab FS22-172 without LALA mutation (SEQ ID NO: 117) [Chemistry]

[0616] FS22-172 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 118) VH domain (underlined) [Chemistry]

[0617] FS22-172 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 119) VH domain (underlined) [Chemistry]

[0618] Amino acid sequence of the loop region of the CH3 domain of Fcab FS22-172-001 FS22-172-001 First sequence - PFVMPPY (SEQ ID NO: 120) FS22-172-001 Second sequence - GADRWLE (SEQ ID NO: 111)

[0619] Amino acid sequence of the CH3 domain of Fcab FS22-172-001 (SEQ ID NO: 121) The first and second sequences are underlined [Chemistry]

[0620] Nucleic acid sequence of Fcab FS22-172-001 in the CH3 domain (SEQ ID NO: 122)

Chem.

[0621] Amino acid sequence of Fcab FS22-172-001 with the LALA mutation (SEQ ID NO: 123) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0622] Nucleic acid sequence of Fcab FS22-172-001 with the LALA mutation (SEQ ID NO: 124)

Chem.

[0623] Amino acid sequence of Fcab FS22-172-001 without the LALA mutation (SEQ ID NO: 125) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chem.

[0624] Nucleic acid sequence of Fcab FS22-172-001 without the LALA mutation (SEQ ID NO: 126)

Chem.

[0625] FS22-172-001 / HelD1.3 mock mAb with the LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 127) VH domain (underlined)

Chem.

[0626] FS22-172-001 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 128) VH domain (underlined)

Chemical Structure

[0627] Amino acid sequence of the loop region of the CH3 domain of Fcab FS22-172-002 FS22-172-002 First sequence - PFQMPPY (SEQ ID NO: 129) FS22-172-002 Second sequence - GADRWLE (SEQ ID NO: 111)

[0628] Amino acid sequence of the CH3 domain of Fcab FS22-172-002 (SEQ ID NO: 130) The first and second sequences are underlined

Chemical Structure

[0629] Nucleic acid sequence of the CH3 domain of Fcab FS22-172-002 (SEQ ID NO: 131)

Chemical Structure

[0630] Amino acid sequence of Fcab FS22-172-002 with LALA mutation (SEQ ID NO: 132) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chemical Structure

[0631] Nucleic acid sequence of Fcab FS22-172-002 having the LALA mutation (SEQ ID NO: 133)

Chem.

[0632] Amino acid sequence of Fcab FS22-172-002 without the LALA mutation (SEQ ID NO: 134) Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

Chem.

[0633] Nucleic acid sequence of Fcab FS22-172-002 without the LALA mutation (SEQ ID NO: 135)

Chem.

[0634] Heavy chain amino acid sequence of FS22-172-002 / HelD1.3 mock mAb having the LALA mutation 2 (SEQ ID NO: 136) VH domain (underlined)

Chem.

[0635] Heavy chain amino acid sequence of FS22-172-002 / HelD1.3 mock mAb without the LALA mutation 2 (SEQ ID NO: 137) VH domain (underlined)

Chem.

[0636] Amino acid sequence of the CH3 domain structural loop sequence of Fcab FS22-172-003 FS22-172-003 First sequence - PYIIPPY (SEQ ID NO: 138) FS22-172-003 Second Array - GADRWLE (SEQ ID NO: 111)

[0637] Amino acid sequence of the Fcab FS22-172-003 CH3 domain (SEQ ID NO: 139) The first and second arrays are underlined [Chemical formula]

[0638] Nucleic acid sequence of the Fcab FS22-172-003 CH3 domain (SEQ ID NO: 140) [Chemical formula]

[0639] Amino acid sequence of Fcab FS22-172-003 with LALA mutation (SEQ ID NO: 141) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined) [Chemical formula]

[0640] Nucleic acid sequence of Fcab FS22-172-003 with LALA mutation (SEQ ID NO: 142) [Chemical formula]

[0641] Amino acid sequence of Fcab FS22-172-003 without LALA mutation (SEQ ID NO: 143) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic) [Chemical formula]

[0642] Nucleic acid sequence of Fcab FS22-172-003 without LALA mutation (SEQ ID NO: 144)

Chem.

[0643] Amino acid sequence of the heavy chain of FS22-172-003 / HelD1.3 mock mAb with LALA mutation (SEQ ID NO: 145) 2 VH domain (underlined)

Chem.

[0644] Amino acid sequence of the heavy chain of FS22-172-003 / HelD1.3 mock mAb without LALA mutation (SEQ ID NO: 146) 2 VH domain (underlined)

Chem.

[0645] Amino acid sequence of the loop region of the CH3 domain of Fcab FS22-172-004 FS22-172-004 first sequence - NYIYPPY (SEQ ID NO: 147) FS22-172-004 second sequence - GADRWLE (SEQ ID NO: 111)

[0646] Amino acid sequence of the CH3 domain of Fcab FS22-172-004 (SEQ ID NO: 148) The first and second sequences are underlined

Chem.

[0647] Nucleic acid sequence of the CH3 domain of Fcab FS22-172-004 (SEQ ID NO: 149)

Chem.

[0648] The amino acid sequence of Fcab FS22-172-004 having the LALA mutation (SEQ ID NO: 150) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chem.

[0649] The nucleic acid sequence of Fcab FS22-172-004 having the LALA mutation (SEQ ID NO: 151)

Chem.

[0650] The amino acid sequence of Fcab FS22-172-004 without the LALA mutation (SEQ ID NO: 152) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chem.

[0651] The nucleic acid sequence of Fcab FS22-172-004 without the LALA mutation (SEQ ID NO: 153)

Chem.

[0652] The heavy chain amino acid sequence of the FS22-172-004 / HelD1.3 mock mAb having the LALA mutation 2 (SEQ ID NO: 154) VH domain (underlined)

Chem.

[0653] FS22-172-004 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 155) VH domain (underlined)

Chemical formula

[0654] Amino acid sequence of the loop region of the CH3 domain of Fcab FS22-172-005 FS22-172-005 First sequence - QQVYPPY (SEQ ID NO: 156) FS22-172-005 Second sequence - GADRWLE (SEQ ID NO: 111)

[0655] Amino acid sequence of the CH3 domain of Fcab FS22-172-005 (SEQ ID NO: 157) The first and second sequences are underlined

Chemical formula

[0656] Nucleic acid sequence of the CH3 domain of Fcab FS22-172-005 (SEQ ID NO: 158)

Chemical formula

[0657] Amino acid sequence of Fcab FS22-172-005 with LALA mutation (SEQ ID NO: 159) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chemical formula

[0658] Nucleic acid sequence of Fcab FS22-172-005 with LALA mutation (SEQ ID NO: 160)

Chemical formula

[0659] Amino acid sequence of Fcab FS22-172-005 without LALA mutation (SEQ ID NO: 161) Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

Chem.

[0660] Nucleic acid sequence of Fcab FS22-172-005 without LALA mutation (SEQ ID NO: 162)

Chem.

[0661] FS22-172-005 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 163) VH domain (underlined)

Chem.

[0662] FS22-172-005 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 164) VH domain (underlined)

Chem.

[0663] Amino acid sequence of the CH3 domain loop sequence of Fcab FS22-172-006 FS22-172-006 first sequence - RKYYPPY (SEQ ID NO: 110) FS22-172-006 second sequence - GADRWLE (SEQ ID NO: 111)

[0664] Amino acid sequence of the Fcab FS22-172-006 CH3 domain (SEQ ID NO: 165) The first and second sequences are underlined

Chemical Structure

[0665] Nucleic acid sequence of the Fcab FS22-172-006 CH3 domain (SEQ ID NO: 166)

Chemical Structure

[0666] Amino acid sequence of Fcab FS22-172-006 with the LALA mutation (SEQ ID NO: 167) Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

Chemical Structure

[0667] Nucleic acid sequence of Fcab FS22-172-006 with the LALA mutation (SEQ ID NO: 168)

Chemical Structure

[0668] Amino acid sequence of Fcab FS22-172-006 without the LALA mutation (SEQ ID NO: 169) Hinge region (underlined), CH2 domain (bold) and CH3 domain (italic)

Chemical Structure

[0669] Nucleic acid sequence of Fcab FS22-172-006 without the LALA mutation (SEQ ID NO: 170)

Chemical Structure

[0670] FS22-172-006 / HelD1.3 mock mAb with LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 171) VH domain (underlined)

Chemical Structure

[0671] FS22-172-006 / HelD1.3 mock mAb without LALA mutation 2 Amino acid sequence of the heavy chain (SEQ ID NO: 172) VH domain (underlined)

Chemical Structure

[0672] HelD1.3 mock mAb 2 Amino acid sequence of the light chain (SEQ ID NO: 173) VL domain (underlined)

Chemical Structure

[0673] Amino acid sequence of the loop region of the Fcab FS22-053 CH3 domain FS22-053 First sequence - NPPYLFS (SEQ ID NO: 19) FS22-053 Second sequence - YYNRWQD (SEQ ID NO: 174)

[0674] Amino acid sequence of the Fcab FS22-053 CH3 domain (SEQ ID NO: 175) (The first and second sequences are underlined.)

Chemical Structure

[0675] Nucleic acid sequence of the Fcab FS22-053 CH3 domain (SEQ ID NO: 176)

Chem.

[0676] Nucleic acid sequence of Fcab FS22 - 053 Fcab having LALA (SEQ ID NO: 177)

Chem.

[0677] Nucleic acid sequence of Fcab FS22 - 053 Fcab without LALA (SEQ ID NO: 178)

Chem.

[0678] Amino acid sequence of the full - length immunoglobulin hinge region (SEQ ID NO: 179) EPKSCDKTHTCPPCP

[0679] Amino acid sequence of human CD137 (SEQ ID NO: 180) Extracellular domain (italicized); transmembrane and intracellular domains (bold)

Chem.

[0680] Amino acid sequence of the extracellular domain of human CD137 (SEQ ID NO: 181)

Chem.

[0681] Amino acid sequence of cynomolgus monkey CD137 (SEQ ID NO: 182) Extracellular domain (italicized); transmembrane and intracellular domains (bold)

Chem.

[0682] Amino acid sequence of cynomolgus monkey CD137 extracellular domain (SEQ ID NO: 183)

Chem.

[0683] Amino acid sequence of mouse CD137 (SEQ ID NO: 184) Extracellular domain (italicized); transmembrane and intracellular domains (bold)

Chem.

[0684] Amino acid sequence of mouse CD137 extracellular domain (SEQ ID NO: 185)

Chem.

[0685] Amino acid sequence of the heavy chain of G1 / HelD1.3 mAb (SEQ ID NO: 186)

Chem.

[0686] Amino acid sequence of human PD-L1 (SEQ ID NO: 187)

Chem.

[0687] Amino acid sequence of mouse PD-L1 (SEQ ID NO: 188)

Chem.

[0688] Amino acid sequence of mouse mesothelin (SEQ ID NO: 189)

Chem.

[0689] FS22m-063-AA / FS28m-228 mAb 2 Amino acid sequence of the heavy chain (SEQ ID NO: 190)

Chem.

[0690] FS22m-063-AA / FS28m-228 mAb 2 Amino acid sequence of the light chain (SEQ ID NO: 191)

Chem.

[0691] Amino acid sequence of the heavy chain of G1AA / HelD1.3 mAb (with LALA) (SEQ ID NO: 192)

Chem.

[0692] Amino acid sequence of the heavy chain of FS22-172-004-AA / S70 (with LALA) (SEQ ID NO: 193)

Chem.

[0693] Amino acid sequence of the heavy chain of FS22-172-004-AA / S70 (without LALA) (SEQ ID NO: 194)

Chem.

[0694] Amino acid sequence of the light chain of FS22-172-004-AA / S70 (SEQ ID NO: 194)

Chem.

[0695] Amino acid sequence of the heavy chain of FS22-172-003-...

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, wherein the CD137 antigen-binding site comprises a first sequence and a second sequence respectively located in the AB structural loop and the EF structural loop of the CH3 domain, wherein the first sequence comprises a sequence consisting of PPY, and (a) the first sequence is (i) SEQ ID NO: 138; (ii) SEQ ID NO: 129; (iii) SEQ ID NO: 147; (iv) SEQ ID NO: 120; (v) SEQ ID NO: 156; or (vi) SEQ ID NO: 110 as described in the sequence, and the second sequence is the sequence described in SEQ ID NO: 111, or (b) the first sequence is the sequence described in SEQ ID NO: 19, and the second sequence is (i) SEQ ID NO: 20; (ii) SEQ ID NO: 29; (iii) SEQ ID NO: 47; (iv) SEQ ID NO: 101; (v) SEQ ID NO: 74; (vi) SEQ ID NO: 38; (vii) SEQ ID NO: 56; (viii) SEQ ID NO: 65; (ix) SEQ ID NO: 83; (x) SEQ ID NO: 92; or (xi) SEQ ID NO: 174 as described in the sequence, the first sequence and the second sequence are respectively located between positions 14 and 17, and between positions 91 and 99 of the CH3 domain, the AB structural loop and the EF structural loop are respectively located between positions 10 and 19, and between positions 91 and 102 of the CH3 domain, the positions of the amino acid residues of the CH3 domain are numbered according to the ImMunoGeneTics (IMGT) numbering scheme, a specific binding member, further comprising a CH2 domain, a specific binding member.

2. The specific binding member according to claim 1, wherein the CH2 domain comprises one or more mutations that reduce or suppress the binding of the specific binding member to one or more Fcγ receptors.

3. The specific binding member according to claim 1 or 2, wherein the CH2 domain has the sequence described in SEQ ID NO: 6 or 5.

4. The specific binding member is (i) SEQ ID NO: 139; (ii) SEQ ID NO: 130; (iii) SEQ ID NO: 148; (iv) SEQ ID NO: 121; (v) SEQ ID NO: 157; (vi) SEQ ID NO: 165; (vii) SEQ ID NO: 112; (viii) SEQ ID NO: 21; (ix) SEQ ID NO: 30; (x) SEQ ID NO: 48; (xi) SEQ ID NO: 102; (xii) SEQ ID NO: 75; (xiii) SEQ ID NO: 39; (xiv) SEQ ID NO: 57; (xv) SEQ ID NO: 66; (xvi) SEQ ID NO: 84; (xvii) SEQ ID NO: 93; or (xviii) SEQ ID NO: 175 The specific binding member according to claim 2 or 3, comprising the CH3 domain sequence described in any one of the above.

5. The specific binding member, respectively comprising the first sequence and the second sequence described in SEQ ID NOs: 138 and 111, the CH3 domain sequence described in SEQ ID NO: 139, the first sequence and the second sequence described in SEQ ID NOs: 19 and 20, respectively, the CH3 domain sequence described in SEQ ID NO: 21, the first sequence and the second sequence described in SEQ ID NOs: 19 and 101, respectively, or the CH3 domain sequence described in SEQ ID NO: 102, the specific binding member according to any one of claims 1 to 4.

6. The specific binding member, respectively comprising the first sequence and the second sequence described in SEQ ID NOs: 138 and 111, or the CH3 domain sequence described in SEQ ID NO: 139, the specific binding member according to claim 5.

7. The specific binding member according to any one of claims 1 to 6, further comprising an immunoglobulin hinge region, or a portion thereof, at the N-terminus of the CH2 domain.

8. The specific binding member according to claim 7, wherein the hinge region has the sequence described in SEQ ID NO: 179 or a fragment thereof.

9. The specific binding member according to any one of claims 1 to 8, wherein the CH3 domain further comprises an additional lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

10. The specific binding member according to any one of claims 1 to 9, further comprising a CDR-based antigen-binding site.

11. The specific binding member according to claim 10, wherein the specific binding member is an antibody molecule.

12. The specific binding member according to claim 10 or 11, wherein the CDR-based antigen-binding site binds to a second antigen selected from the group consisting of immune cell antigens, tumor antigens, and pathogenic antigens.

13. One or more nucleic acid molecules encoding the specific binding member according to any one of claims 1 to 12.

14. A recombinant host cell comprising the one or more nucleic acid molecules according to claim 13.

15. A method for producing a specific binding member according to any one of claims 1 to 12, the method comprising culturing a recombinant host cell according to claim 14 under conditions for the production of the specific binding member. **Claim 16** A pharmaceutical composition for the treatment of cancer or an infectious disease in an individual, comprising a specific binding member according to any one of claims 1 to 12.

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