Cd137 binding molecules

Abstract

This invention relates to a specific binding object for binding CD137. The specific binding object includes a CD137 antigen binding address located in a constant domain of the specific binding object and, for example, has applications in the treatment of cancer and infectious diseases.

Description

Technical Field

[0001] Invention Field This invention relates to a specific binding object for CD137. The specific binding object includes a CD137 antigen binding address located in a constant domain of the specific binding object and, for example, has applications in the treatment of cancer and infectious diseases. Prior Technology

[0002] Background of the Invention Cellular signaling is an essential part of all life and typically involves the interaction of cell surface receptors with soluble or surface-expressed ligands. This interaction leads to changes in the receptor, ligand, or both. For example, ligand binding can induce conformational changes in receptors, causing them to cluster together as dimers or oligomers. This clustering effect subsequently activates intracellular signaling pathways. Many receptor systems are activated in this way, 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). It is well known that CD137 on CD8+ T cells is upregulated upon activation and is also expressed on activated CD4+ helper T cells, B cells, regulatory T cells, natural killer (NK) cells, natural killer T (NKT) cells, and dendritic cells (DCs) (Bartkowiak & Curran, 2015). The major functional role of CD137 in enhancing T cell cytotoxicity was first described in 1997 (Shuford et al., 1997), and shortly thereafter, anti-CD137 mAbs were proposed as an anticancer therapy.

[0004] CD137 is a transmembrane protein containing four extracellular cysteine-rich domains, called CRD1-4, and a cytoplasmic domain responsible for CD137 signaling. The ligand for CD137 is CD137L. Although the CD137 / CD137L complex does not exist in a crystalline form, it is predicted that CD137 will form a trimer / trimer complex with CD137L (Won et al., 2010). CD137L binding leads to receptor trimer formation and subsequent clustering of multiple receptor trimers, resulting in activation of the CD137 signaling cascade. This cascade provides survival signals to T cells to combat activation-induced cell death (Hurtado et al., 1997), thereby playing a crucial role in supporting effective T-cell immune responses and generating immune memory (Bartkowiak & Curran, 2015).

[0005] Because the role of CD137 in tumor immunology has a well-defined biological basis, its role in leukocyte biology is generally well understood. Activated T cells express CD137 and have been used as an indicator of antigen-specific CD4+ and CD8+ T cells. Typically, CD137 expression is higher on CD8+ T cells than on CD4+ T cells (Wen et al., 2002). For CD8+ T cells, proliferation, survival, and cytotoxic effector functions via the production of interferon-γ and interleukin-2 have been attributed to CD137 clustering. CD137 clustering also contributes to the differentiation and maintenance of memory CD8+ T cells. For some CD4+ T cell subsets, CD137 clustering also leads to proliferation and activation, resulting in the release of interleukins such as interleukin-2 (Makkouk et al., 2016).

[0006] CD137 stimulation induced in vitro and in vivo by agonist anti-CD137 monoclonal antibodies has been shown to enhance antibody-dependent cell cytotoxicity (ADCC) of natural killer (NK) cells mediated by tumor-targeting mAbs (Bartkowiak & Curran, 2015). NK cells bind to antibodies via their Fc receptors, and depending on antibody isotype, this can lead to NK cell activation, cytotoxic granule release, and target cell lysis (Kohrt et al., 2012). Kohrt et al. demonstrated that anti-CD137 agonist antibodies enhance the antitumor activity of therapeutic antibodies rituximab, trastuzumab, and cetuximab by increasing ADCC when used in combination (Kohrt et al., 2014; Kohrt et al., 2011). Furthermore, human NK cells upregulate CD137 expression via their FcγR after encountering cell-bound antibodies. Subsequently, stimulation of these NK cells with anti-CD137 antibodies has been shown to enhance their ADCC against tumor cells (Chester et al., 2015; Chester et al., 2016).

[0007] CD137 is also expressed when B lymphocytes are activated. Binding of CD137 ligands to CD137 enhances B cell proliferation, survival, and cytokine production. Normal or malignant human B cells also induce CD137 expression after CD40 binds to its ligand CD154 (CD40 ligand), and subsequent activation of CD137 may lead to increased B cell survival (Vinay and Kwon, 2011).

[0008] CD137 has also been shown to be expressed in tumor-reactive subsets of tumor-infiltrating lymphocytes (TILs). CD137 monotherapy has been shown to be effective in several preclinical immunotherapeutic tumor modalities, such as MC38, CT26, and B-cell lymphoma. Combinations of CD137 with other anticancer agents, such as chemotherapy, cytokines, and other checkpoint modulators, have been shown to enhance established tumor growth reduction. 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 tumor growth reduction in various preclinical xenograft modalities (Kohrt et al., 2014; Kohrt et al., 2012; Kohrt et al., 2011).

[0009] Tumor-targeted monoclonal antibody therapies combined with anti-CD137 agonist antibodies have shown promising results in preclinical models for lymphoma (Kohrt et al., 2011), head and neck cancer, colorectal cancer (Kohrt et al., 2014), and breast cancer (Kohrt et al., 2012). Several combinations of tumor-targeted monoclonal antibodies and CD137 agonist antibodies have also been tested clinically, including the anti-CD20 mAb rituximab (NCT01307267, NCT02951156), the anti-EGFR mAb cetuximab (NCT02110082), and the anti-CS1 mAb elotuzumab (NCT02252263). However, clinical development has shown dose-limited high hepatitis associated with CD137 agonist antibody therapy. Urelumab (BMS-663513), a nonligand-blocking human IgG4 isotype antibody (Chester et al., 2018), was the first anti-CD137 antibody to enter clinical trials, but these trials were discontinued after significant, dose-dependent hepatotoxicity was observed (Chester et al., 2018). Recently, clinical trials of urogenumab for solid tumors have been restarted, using urogenumab in combination with radiotherapy (NCT03431948) or other therapeutic antibodies, such as rituximab (NCT01775631), cetuximab (NCT02110082), the anti-PD-1 antibody nivolumab (NCT02253992, NCT02534506, NCT02845323), and the combination of nivolumab and the anti-LAG-3 antibody BMS986016 (NCT02658981). However, to reduce hepatotoxicity associated with urogenumab treatment, the dosage of urogenumab in these trials has been limited, and efficacy results have been disappointing (Chester et al., 2018).

[0010] Pfizer's anti-CD137 antibody, utomilumab (PF-05082566), a human IgG2 isotype antibody, showed no dose-limiting toxicities in a phase I clinical trial in advanced cancer at doses ranging from 0.03 mg / kg to 10 mg / kg (Chester et al., 2016; Segal et al., 2018). However, the overall objective response rate in patients with solid tumors was only 3.8%, possibly indicating that utomilumab has weaker efficacy and clinical efficacy than utomilumab, but with a more favorable safety profile (Chester et al., 2018; Segal et al., 2018). Utamicilab 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), has been tested to evaluate the safety, tolerability, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and efficacy of different treatment combinations. These trials are ongoing, and early results show that the utamicilab and pembrolizumab combination has no DLTs at doses up to 5 mg / kg and a patient response rate of 26%. Triple combinations of utamicilab with avelumab and other immuno-oncology therapies have also been tested (NCT02554812, NCT03217747).

[0011] Several dual-specific molecules targeting CD137 are in early stages of development, many of which are derived from non-antibody-based architectures or fusion protein technologies. The development of dual-specific molecules targeting CD137 and FAPalpha using DARPin architecture-based technologies has been reported (Link et al., 2018; Reichen et al., 2018). T-cell activation via CD137-induced tumor targeting using HER2- and EphA2-targeted DART molecules has also been demonstrated (Liu et al., 2017). The targeting of CD137L fusion proteins to tumors via FAPalpha or CD19 in solid tumors and lymphomas is also under development. The most advanced clinically available dual-specific CD137 molecule (containing only one full-length antibody) is PRS-343, a CD137 / HER2 dual-specific molecule. Regarding this molecule, CD137 binds to the Fc region of trastuzumab, a HER2-targeting antibody in IgG4 format, via an artificial binding protein (anticalin). PRS-343 has been reported to provide tumor-target-dependent CD137 activation on lymphocytes in a humanized mouse model of HER2 with overexpression, but no improvement in tumor growth inhibition was observed compared to trastuzumab monotherapy (Hinner et al., 2016 and WO 2016 / 177802 A1). PRS-343 has recently entered a series of Phase I clinical trials for the treatment of solid tumors to evaluate its safety, tolerability, and efficacy (NCT03330561). Summary of the Invention

[0012] Invention Summary As mentioned in the background section above, the clinical development of CD137 agonist molecules has been hampered because dose-limiting high hepatitis (uregula) or very low clinical efficacy (utromizumab) are both associated with treatment.

[0013] The inventors have recognized a continued need for CD137 agonist molecules that exhibit high activity but are not associated with dose-limiting hepatitis. These molecules can be administered to individuals at doses that optimize their efficacy and effectiveness, and, for example, can be used as immunotherapies to treat cancer or to treat infectious diseases.

[0014] While not wishing to be bound by theory, it is generally believed that T cells present in the liver may be activated by anti-CD137 agonist molecules, leading to liver inflammation. CD8+ T cells have been shown to promote liver inflammation and apoptosis following sepsis / viral infection (Wesche-Soldato et al., 2007). Anti-CD137 agonist antibody therapy in mice has been shown to induce CD137-dependent T cell infiltration into the liver (Dubrot J et al., 2010). These findings, taken together, suggest that highly active anti-CD137 agonist antibodies, such as urogenumab, may induce activated CD8+ T cell infiltration into the liver, thereby leading to liver inflammation. Alternatively, the dose-limiting hepatotoxicity observed with urogenumab treatment may be due to specific epitopes bound to this antibody.

[0015] The inventors conducted a large-scale selection and affinity maturation program to isolate a set of antigen-binding Fc fragments (also referred to herein as "Fcabs") containing a CD137 binding site located in its CH3 domain and having a higher affinity for the dimer CD137 than for the monomer CD137.

[0016] As described herein, 'affinity' refers to the strength of the binding interaction between an antibody molecule and its homologous antigen, as measured by KD. In cases where an antibody molecule can form multiple binding interactions with an antigen (e.g., where the antibody molecule can bind divalently to the antigen and selectively, the antigen is a dimer), affinity measured by KD may also be affected by total binding capacity, which will be apparent to those skilled in the art. Therefore, total binding capacity refers to the overall strength of the antibody-antigen complex.

[0017] T cells upregulate CD137 expression upon activation. While not wishing to be bound by theory, it is believed that due to the high expression of CD137 on activated T cells, CD137 would exist in dimer, trimer, and higher-order multimeric forms on the surface of these cells. Conversely, naive immune cells, such as naive T cells, exhibit low or negligible CD137 locating on their cell surfaces, and any CD137 present would likely be in monomeric form. Therefore, Fcabs, which have a higher affinity for dimer CD137 than monomeric CD137, are expected to preferentially bind to activated immune cells, such as activated T cells, rather than, for example, naive immune cells.

[0018] The Fcabs of this invention can also bind to the dimer CD137 of cynomolgus monkeys. This is advantageous because it allows for toxicity and efficacy testing in cynomolgus monkeys during preclinical development. Given that some anti-CD137 antibodies have been associated with hepatitis, this is particularly advantageous in cases involving binding to CD137 antibody molecules. Two of the isolated Fcabs, FS22-053-014 and FS22-053-017, also bind to mouse CD137. This is advantageous because it allows the same Fcabs to be tested in mice before being administered to cynomolgus monkeys or humans. For this purpose, an Fcab that binds to mouse CD137 under normal conditions is required.

[0019] The inventors unexpectedly discovered that all isolated anti-CD137 Fcab molecules that preferentially bind to the dimer rather than the monomeric CD137 and mature with affinity contain a motif of PPY and five amino acid insertions within the AB ring of its CH3 domain. Anti-CD137 Fcabs isolated from another lineage after initial library screening did not possess these characteristics and could not mature with affinity, thus not proceeding further. While not wishing to be bound by theory, it is thought that the presence of the PPY motif may promote the formation of extended antigen-binding regions by forming more rigid or exposed ring structures due to the flexibility of proline residue restriction. Alternatively, the PPY sequence may represent a specific conserved portion involved in CD137 binding, as proline-rich sequences have been shown to bind, for example, aromatic sequences of SH3 domain proteins. Moreover, since two separate Fcab lineages have independently selected conserved PPY sequences, these may be important for CD137 epitope binding. Furthermore, most isolated Fcabs contain conserved LE or LD sequences within the EF ring of the CH3 domain, suggesting that this amino acid sequence may also be important for CD137 binding.

[0020] As described in the background section above, the initiation of the connection between the CD137 ligand and its receptor, CD137, triggers a series of events leading to CD137 trimerization, followed by receptor clustering, activation of the intracellular NF-κB signaling pathway, and subsequent immune cell activation. For therapeutic agents to efficiently activate CD137, several CD137 monomers must be bridged together in a trimer-mimicking manner.

[0021] Utamide is an IgG2 molecule, and its agonist activity depends on Fcγ receptor cross-linking. Urelumab is a persistently active IgG4 molecule, and therefore its activity does not require Fcγ receptor cross-linking, although some Fcγ receptor cross-linking can enhance its agonist activity. Fcγ receptors have been found to be distributed throughout the human body. Therefore, the immune cell activation activities of utamide and urelumab are not limited to specific sites in the body and can occur in the liver or other parts of the body.

[0022] The inventors have demonstrated that the Fcabs of this invention require cross-linking to cluster and activate CD137. However, it should be noted that this is not an inherent characteristic of Fcabs that bind to CD137. Conversely, many isolated Fcabs during the screening program bind to CD137 but do not require cross-linking for CD137 clustering and activation, or induce limited CD137 clustering and activation in the absence of cross-linking.

[0023] As mentioned above, Fcγ receptors are found to be distributed throughout the human body, and therefore CD137 activation is not limited to specific sites, which is a disadvantage for Fcγ receptor-mediated cross-linking. Therefore, the inventors have introduced a mutation into the CH2 domain of Fcabs to reduce or eliminate Fcγ receptor binding. Thus, in the absence of cross-linking via agents other than Fcγ receptors, the Fcabs of this invention will not exhibit CD137 agonist activity and are therefore not expected to induce hepatitis.

[0024] The inventors have identified that the anti-CD137 Fcabs of the present invention can be used to prepare multispecific molecules, such as bispecific molecules, that bind to a second antigen other than CD137, such as a tumor antigen. Multispecific molecules preferably bind bivalently to the second antigen, although it is generally expected that in the case where the second antigen is a cell-binding tumor antigen, monovalent binding would be sufficient to crosslink the specific binding agent / antibody molecule and induce CD137 clustering and activation. Specifically, the inventors have prepared antibody molecules comprising the anti-CD137 Fcabs of the present invention, which bind bivalently to the second antigen via their Fab regions. The inventors have demonstrated that such bispecific antibody molecules can conditionally activate CD137 in the presence of the second antigen without requiring, for example, the crosslinking action mediated by the Fcγ receptor required by conventional antibody molecules. It is generally believed that the binding of the antibody molecule to the second antigen causes crosslinking of the antibody molecule at the site of the antigen, which in turn leads to CD137 clustering and activation on the surface of T cells. The agonist activity of the antibody molecule therefore depends on the presence of both the second antigen and CD137. In other words, the agonist activity requires the presence of both antigens. Furthermore, it is believed that in the presence of a second antigen, the cross-linked antibody assists in the aggregation of the bound CD137 clusters via the constant domain antigen-binding site of the antibody molecule. When the second antigen is a disease antigen, such as a tumor antigen, the antibody molecule is therefore expected to activate immune cells in a disease-dependent manner, for example, within the tumor microenvironment. This targeted immune cell activation is expected to be beneficial in avoiding, for example, the hepatitis seen with ureriganumab treatment.

[0025] The inventors have also demonstrated that the bispecific molecule containing the anti-CD137 Fcabs of this invention can inhibit tumor growth in vivo when the second antigen bound by the antibody molecule is an immune cell antigen, a tumor antigen, or an antigen expressed by both tumor cells and immune cells. Furthermore, compared to a combination of two monospecific antibody molecules, this bispecific antibody molecule, in which one antibody molecule contains the same constant domain as the bispecific molecule and the other antibody molecule contains the same variable domain binding address as the bispecific molecule, has been observed to exhibit more effective tumor growth inhibition. This demonstrates that the presence of two binding addresses within the same molecule enhances CD137 clustering and signal transduction, thereby increasing T cell activation and the corresponding anti-tumor effect.

[0026] Antibody molecules comprising the anti-CD137 Fcabs of the present invention and a Fab moiety specific to a second antigen are preferably divalently bound to both CD137 and the second antigen. This is advantageous because the divalent binding of the two targets is expected to stabilize the bridging between immune cells expressing CD137 and the second antigen, thereby prolonging the time T cells are localized to specific sites, such as the tumor microenvironment, and to act on diseases, such as tumors. This differs from most conventional bispecific antibody formats, which are heterodimers and monovalently bound to each target antigen via a Fab arm. This monovalent interaction is expected to be not only less stable but also, in many cases, insufficient to induce clustering of TNF receptors such as CD137 from the outset.

[0027] In a preferred embodiment, the antibody molecule comprises an anti-CD137 Fcabs that binds bivalently to CD137 and a monovalent binding address specific to a second antigen, such as a single Fab arm. The monovalent binding address could bind, for example, a tumor-associated antigen. In the case of such molecules, the monovalent binding to the second antigen is expected to allow the antibody molecule to be more tightly packed on the cell surface, leading to CD137 clustering and increased T cell activation.

[0028] The present invention includes an antibody molecule against CD137 Fcab, characterized in that the antibody structure itself contains two antigen-binding sites for both CD137 and a second antigen. Specifically, the antibody molecule does not require the fusion of other proteins with the antibody molecule via linkers or other components to produce a molecule that binds to two targets bivalently. This has several advantages. Specifically, the antibody molecule can be produced using methods similar to those used to produce standard antibodies, as they do not contain any additional fusion portions. This structure is also expected to lead to improved antibody stability, as linkers may degrade over time, resulting in a heterogeneous population of antibody molecules. Antibodies with only one fused protein in the population may not induce conditional toxicity of TNF receptors such as CD137 as efficiently as those with two fused proteins. Cleavage / degradation of the linker can occur before or after administration of the therapeutic agent to the patient (e.g., via enzymatic cleavage or the patient's in vivo pH), thereby reducing its efficacy during circulation in the patient. Because antibody molecules lack linkers, it is expected that the same number of binding sites will be retained before and after administration. Furthermore, the structure of antibody molecules is also preferred from a molecular immunogenicity perspective, as the introduction of fusion proteins or linkers, or both, into patients during administration may induce immunogenicity, leading to reduced therapeutic efficacy.

[0029] The inventors have further demonstrated that the fixed position and / or very close CD137 antigen binding address due to the rigid structure of the Fcab molecule of the present invention are advantageous for inducing CD137 clustering, compared to cases where the CD137 binding address is provided for antibody structures that are incomplete but are provided, for example, by attaching to, for example, the binding portion of an antibody molecule or a portion thereof via a flexible linker.

[0030] Therefore, the present invention provides:

[0031] [1] A specific binding object that binds to CD137 and includes a CD137 antigen binding address located in a CH3 domain of the specific binding object, wherein the CD137 binding address includes an AB structure loop with a first sequence located in the CH3 domain, wherein the sequence includes the sequence PPY (sequence identification number: 10).

[0032] [2] A specific bonding object as in [1], wherein the specific bonding object includes an insertion into the AB structure ring.

[0033] [3] As in [2] specific binding objects, wherein the length of the insertion is between 1 and 10 amino acids.

[0034] [4] Such as [3] specific binding objects, wherein the length of the insertion is between 4 and 6 amino acids.

[0035] [5] Such as [4] specific binding objects, wherein the length of the insertion is 5 amino acids.

[0036] [6] A specific binding object as in any of [2] to [5], wherein the insertion is located between position 10 and 19 of the CH3 domain of the specific binding object, wherein the amino acid residue is numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0037] [7] A specific binding object as in [6], wherein the insertion is located between position 14 and 17 of the CH3 domain of the specific binding object.

[0038] [8] A specific binding object as in [7], wherein the insertion is located between position 16 and 17 of the CH3 domain of the specific binding object.

[0039] [9] A specific binding object as in any of [2] to [8], wherein the insertion is located at position 16.5 to 16.1 of the CH3 domain of the specific binding object, wherein the amino acid residue is numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0040]

[10] A specific binding object as in any of [1] to [9], wherein the PPY sequence is located between position 15 and 17 of the CH3 domain, and wherein the amino acid residue is numbered according to the IMGT numbering scheme.

[0041]

[11] A specific binding object as in any of [1] to

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

[0042]

[12] As in

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

[0043]

[13] A specific combination of any of [1] to

[12] , wherein the first sequence is a first sequence of the following specific combination of objects: (i) The first sequence of [FS22-172-003], listed in the sequence identification number:

[0138] Inside; (ii) The first sequence of [FS22-172-002] is listed in the sequence identification number:

[0129] Inside; (iii) The first sequence of [FS22-172-004], listed in the sequence identification number:

[0147] Inside; (iv) The first sequence of [FS22-172-001], listed in the sequence identification number:

[0120] Inside; (v) The first sequence of [FS22-172-005], listed in the sequence identification number:

[0156] Inside; (vi) The first sequence of [FS22-172-006], listed in the sequence identification number:

[0110] Inside; or (vii) The first sequence of [FS22-172], listed in the sequence identification number:

[0110] Inside.

[0044]

[14] A specific combination of objects as in

[13] , wherein the first sequence is a first sequence of the following specific combination objects: (i) The first sequence of [FS22-172-003] is listed in sequence identification number: 138; (ii) The first sequence of [FS22-172-002] is listed in sequence identification number: 129; (iii) The first sequence of [FS22-172-004] is listed in sequence identification number: 147; (iv) The first sequence of [FS22-172-001] is listed in sequence identification number: 120; (v) The first sequence of [FS22-172-005] is listed in sequence identification number: 156; or (vi) The first sequence of [FS22-172-006] is listed in sequence identification number: 110.

[0045]

[15] A specific combination of objects as in

[14] , wherein the first sequence is a first sequence of the following specific combination objects: (i) The first sequence of [FS22-172-003] is listed in sequence identification number: 138; (ii) The first sequence of [FS22-172-002] is listed in sequence identification number: 129; or (iii) The first sequence of [FS22-172-004] is listed in sequence identification number: 147.

[0046]

[16] As in

[15] , a specific combination object, wherein the first sequence is a specific combination object. The first sequence of [FS22-172-003] is listed in sequence identification number: 138.

[0047]

[17] As in

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

[0048]

[18] A specific combination object as in any of [1] to

[10] or

[17] , wherein the first sequence is a specific combination object. [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 a specific bonding object) The first sequence of [FS22-053-008] is listed in sequence identification number: 19.

[0049]

[19] A specific binding object of any of

[13] to

[16] or

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

[0050]

[20] A specific binding object as in

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

[0051]

[21] A specific binding object as in any of [1] to

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

[0052]

[22] As in

[21] , a specific combination object, wherein the second sequence is a specific combination object. [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] [Or FS22-172 () is preferred as a specific type of object] The second sequence of [FS22-172-003] is listed in sequence identification number: 111.

[0053]

[23] As in

[21] , the specific combination object, wherein the second sequence is a second sequence of the following specific combination objects: (i) The second sequence of [FS22-053-008], listed in the sequence identification number:

[20] Inside; (ii) The second sequence of [FS22-053-009] is listed in sequence identification number 29; (iii) The second sequence of [FS22-053-011] is listed in sequence identification number: 47; (iv) The second sequence of [FS22-053-017] is listed in sequence identification number: 101; (v) The second sequence of [FS22-053-014] is listed in sequence identification number: 74; (vi) The second sequence of [FS22-053-010] is listed in sequence identification number 38; (vii) The second sequence of [FS22-053-012] is listed in sequence identification number: 56; (viii) The second sequence of [FS22-053-013] is listed in sequence identification number: 65; (ix) The second sequence of [FS22-053-015] is listed in sequence identification number: 83; (x) The second sequence of [FS22-053-016] is listed in sequence identification number: 92; [or] [] (xi) The second sequence of [FS22-053] is listed in sequence identification number: 174.

[0054]

[24] As in

[23] , the specific combination object, wherein the second sequence is a second sequence of the following specific combination objects: (i) The second sequence of [FS22-053-008], listed in the sequence identification number:

[20] Inside; (ii) The second sequence of [FS22-053-009] is listed in sequence identification number 29; (iii) The second sequence of [FS22-053-011] is listed in sequence identification number: 47; (iv) The second sequence of [FS22-053-017] is listed in sequence identification number: 101; or (v) The second sequence of [FS22-053-014] is listed in sequence identification number: 74.

[0055]

[25] As in

[24] , the specific combination object, wherein the second sequence is a second sequence of the following specific combination objects: (i) The second sequence of [FS22-053-008], listed in the sequence identification number:

[20] Inside; (ii) The second sequence of [FS22-053-009] is listed in sequence identification number 29; (iii) The second sequence of [FS22-053-011] is listed in sequence identification number: 47; or (iv) The second sequence of [FS22-053-017] is listed in sequence identification number: 101. [。]

[0056]

[26] As in

[25] , a specific combination object, wherein the second sequence is a specific combination object. The second sequence of [FS22-053-008] is listed in sequence identification number: 20.

[0057]

[27] A specific binding compound of any of

[21] to

[26] , wherein the second sequence is located at position 92 to 98 of the CH3 domain of the specific binding compound, wherein the amino acid residue is numbered according to the IMGT numbering scheme.

[0058]

[28] A specific binding object as in any of [1] to

[27] , wherein the specific binding object further comprises a third sequence CD structure ring located in the CH3 domain.

[0059]

[29] A specific binding compound as in

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

[0060]

[30] A specific combination of objects as in any of

[28] to

[29] , wherein the third sequence has a sequence listed in sequence identification number: 2.

[0061]

[31] A specific binding object of any of [1] to

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

[0062]

[32] A specific binding object as described in any of [1] to

[16] ,

[19] to

[22] and

[27] to

[31] , wherein the specific binding object comprises the CH3 domain sequence of the following specific binding objects: (i) The CH3 domain sequence of [FS22-172-003] is listed in the sequence identification number:

[0139] Inside; (ii) The CH3 domain sequence of [FS22-172-002] is listed in sequence identification number 130; (iii) The CH3 domain sequence of [FS22-172-004] is listed in sequence identification number 148; (iv) The CH3 domain sequence of [FS22-172-001] is listed in sequence identification number 121; (v) The CH3 domain sequence of [FS22-172-005] is listed in sequence identification number 157; (vi) The CH3 domain sequence of [FS22-172-006] is listed in sequence identification number 165; or (vii) The CH3 domain sequence of [FS22-172] is listed in sequence identification number: 112.

[0063]

[33] A specific binding object as in

[32] , wherein the specific binding object comprises the following CH3 domain sequence of the specific binding object: (i) The CH3 domain sequence of [FS22-172-003] is listed in sequence identification number 139; (ii) The CH3 domain sequence of [FS22-172-002] is listed in sequence identification number 130; (iii) The CH3 domain sequence of [FS22-172-004] is listed in sequence identification number 148; (iv) The CH3 domain sequence of [FS22-172-001] is listed in sequence identification number 121; (v) The CH3 domain sequence of [FS22-172-005] is listed in sequence identification number 157; or (vi) The CH3 domain sequence of [FS22-172-006] is listed in sequence identification number 165.

[0064]

[34] A specific binding object as in

[33] , wherein the specific binding object comprises the following CH3 domain sequence of the specific binding object: (i) The CH3 domain sequence of [FS22-172-003] is listed in sequence identification number 139; (ii) The CH3 domain sequence of [FS22-172-002] is listed in sequence identification number 130; or (iii) The CH3 domain sequence of [FS22-172-004] is listed in sequence identification number 148.

[0065]

[35] A specific combination object as in

[34] , wherein the specific combination object comprises a specific combination object. The CH3 domain sequence of [FS22-172-003] is listed in sequence identification number 139.

[0066]

[36] A specific binding object such as any one of [1] to

[10] ,

[17] to

[18] ,

[19] to

[21] and

[23] to

[31] , wherein the specific binding object comprises the CH3 domain sequence of the following specific binding objects: (i) The CH3 domain sequence of [FS22-053-008] is listed in sequence identification number 21; (ii) The CH3 domain sequence of [FS22-053-009] is listed in sequence identification number 30; (iii) The CH3 domain sequence of [FS22-053-011] is listed in sequence identification number 48; (iv) The CH3 domain sequence of [FS22-053-017] is listed in sequence identification number 102; (v) The CH3 domain sequence of [FS22-053-014] is listed in sequence identification number 75; (vi) The CH3 domain sequence of [FS22-053-010] is listed in sequence identification number 39; (vii) The CH3 domain sequence of [FS22-053-012] is listed in sequence identification number 57; (viii) The CH3 domain sequence of [FS22-053-013] is listed in sequence identification number 66; (ix) The CH3 domain sequence of [FS22-053-015] is listed in sequence identification number 84; (x) The CH3 domain sequence of [FS22-053-016] is listed in sequence identification number 93; or (xi) The CH3 domain sequence of [FS22-053] is listed in sequence identification number 175.

[0067]

[37] A specific binding object as in

[36] , wherein the specific binding object comprises the following CH3 domain sequence of the specific binding object: (i) The CH3 domain sequence of [FS22-053-008] is listed in sequence identification number 21; (ii) The CH3 domain sequence of [FS22-053-009] is listed in sequence identification number 30; (iii) The CH3 domain sequence of [FS22-053-011] is listed in sequence identification number 48; (iv) The CH3 domain sequence of [FS22-053-017] is listed in sequence identification number 102; or (v) The CH3 domain sequence of [FS22-053-014] is listed in sequence identification number 75.

[0068]

[38] A specific binding object as in

[37] , wherein the specific binding object comprises the following CH3 domain sequence of the specific binding object: (i) The CH3 domain sequence of [FS22-053-008] is listed in sequence identification number 21; (ii) The CH3 domain sequence of [FS22-053-009] is listed in sequence identification number 30; (iii) The CH3 domain sequence of [FS22-053-011] is listed in sequence identification number 48; or (iv) The CH3 domain sequence of [FS22-053-017] is listed in sequence identification number: 102.

[0069]

[39] A specific combination object as in

[38] , wherein the specific combination object comprises a specific combination object. The CH3 domain sequence of [FS22-053-008] is listed in sequence identification number 21.

[0070]

[40] A specific combination object as in

[37] , wherein the specific combination object comprises a specific combination object. The CH3 domain sequence of [FS22-053-017] is listed in sequence identification number: 102.

[0071]

[41] A specific combination object as in

[37] , wherein the specific combination object comprises a specific combination object. The CH3 domain sequence of [FS22-053-014] is listed in sequence identification number 75.

[0072]

[42] A specific binding object of any of [1] to

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

[0073]

[43] A specific binding object as in any of [1] to

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

[0074]

[44] Such as

[42] or

[43] specific binding objects, wherein the CH2 domain has a sequence listed in sequence identification number: 6 or 5.

[0075]

[45] The specific binding object of any of

[42] to

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

[0076]

[46] As in

[45] , a specific joint object, wherein the hinge region has a serial identification number listed below:

[0179] The sequence or fragment thereof within.

[0077]

[47] A specific combination object as in

[46] , wherein the hinge area has a sequence listed in sequence identification number: 7.

[0078]

[48] ​​A specific combination object as described in any of [1] to

[16] ,

[19] to

[22] ,

[27] to

[35] and

[42] to

[47] , wherein the specific combination object comprises a specific combination object [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] The sequences [or FS22-172] are listed in sequence identification numbers: 141, 132, 150, 123, 159, 167 and 114 respectively.

[0079]

[49] A specific combination object as in

[48] , wherein the specific combination object comprises a specific combination object. [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] The sequence [or FS22-172-006] is listed in sequence identification numbers: 141, 132, 150, 123, 159 and 167 respectively.

[0080]

[50] A specific combination object as in

[49] , wherein the specific combination object comprises a specific combination object. [FS22-172-003] [FS22-172-002] The sequence [or FS22-172-004] is listed in sequence identification numbers: 141, 132 and 150 respectively.

[0081]

[51] A specific combination object as in

[50] , wherein the specific combination object comprises a specific combination object. The sequence [FS22-172-003] is listed in sequence identification number: 141.

[0082]

[52] A specific combination object as described in any of [1] to

[10] ,

[17] to

[18] ,

[19] to

[21] ,

[23] to

[31] and

[36] to

[47] , wherein the specific combination object comprises a specific combination object [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] The sequences [or FS22-053] are listed in sequence identification numbers: 23, 32, 50, 104, 77, 41, 59, 68, 86, 95 and 175 respectively.

[0083]

[53] A specific combination object as in

[52] , wherein the specific combination object comprises a specific combination object. [FS22-053-008] [FS22-053-009] [FS22-053-011] [、FS22-053-017] or The sequence [FS22-053-014] is listed in sequence identification numbers 23, 32, 50, 104 and 77 respectively.

[0084]

[54] A specific combination object as in

[53] , wherein the specific combination object comprises a specific combination object. [FS22-053-008] [FS22-053-009] [、FS22-053-011] or The sequence [FS22-053-017] is listed in sequence identification numbers 23, 32, 50 and 103 respectively.

[0085]

[55] A specific combination object as in

[54] , wherein the specific combination object comprises a specific combination object. The sequence [FS22-053-008] is listed in sequence identification number: 23.

[0086]

[56] A specific binding object as in any of [1] to

[55] , wherein the specific binding object is bound to human CD137.

[0087]

[57] As in

[56] , a specific combination of objects, wherein the human CD137 has, contains, or consists of the following: listed in the sequence identification number:

[0181] sequence within.

[0088]

[58] A specific binding object as described in any of [1] to

[55] , wherein the specific binding object has a higher affinity for the dimer CD137 than for the monomer CD137.

[0089]

[59] A specific binding object as in any of [1] to

[58] , wherein the specific binding object is bound to the cynomolgus macaque CD137.

[0090]

[60] Specific binding compounds or antibody molecules as in

[59] , wherein the cynomolgus macaque CD137 has, contains, or is composed of the following: listed in the sequence identification number:

[0183] sequence within.

[0091]

[61] A specific binding object as in any of [1] to

[60] , wherein the specific binding object further comprises a second antigen binding address.

[0092]

[62] Such as

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

[0093]

[63] Such as

[62] specific binding objects, wherein the specific binding object is a bis-specific, tri-specific or tetra-specific molecule.

[0094]

[64] Such as

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

[0095]

[65] A specific binding object as in any of

[61] to

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

[0096]

[66] A specific binding object as in

[65] , wherein the second antigen binding address includes a heavy chain variable domain CDR1, CDR2 and CDR3, and a light chain variable domain CDR1, CDR2 and CDR3.

[0097]

[67] A specific binding object as in any of

[61] to

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

[0098]

[68] A specific binding object as in any of

[61] to

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

[0099]

[69] The antibody molecule in

[68] is a human IgG1 molecule.

[0100]

[70] An antibody molecule such as any one of

[65] to

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

[0101]

[71] Such as the antibody molecule in

[70] , wherein the disease antigen is a tumor antigen and / or a pathogenic antigen.

[0102]

[72] Antibody molecules such as

[70] , wherein the disease antigen is an immunomodulatory molecule, such as PD-L1.

[0103]

[73] Antibody molecules such as

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

[0104]

[74] Such as the antibody molecule in

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

[0105]

[75] An antibody molecule such as any of

[71] to

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

[0106]

[76] Such as the antibody molecule in

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

[0107]

[77] The antibody molecule as in

[76] , wherein the soluble polymer is at least one dimer.

[0108]

[78] The antibody molecule as in

[77] , wherein the soluble polymer is at least one trimer.

[0109]

[79] Such as the antibody molecule in

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

[0110]

[80] An antibody molecule of any of

[70] to

[79] , wherein the antibody molecule, in the presence of the second antigen, can activate CD137 present on immune cells.

[0111]

[81] An antibody molecule such as any one of

[70] to

[80] , wherein the antibody molecule binds to CD137 and the second antigen, resulting in CD137 clusters on immune cells.

[0112]

[82] Such as the antibody molecule in

[80] or

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

[0113]

[83] A specific binding compound or antibody molecule as described in any of [1] to

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

[0114]

[84] A specific binding compound or antibody molecule as described in any of [1] to

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

[0115]

[85] A specific binding compound or antibody molecule as in

[83] or

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

[0116]

[86] A specific binding compound or antibody molecule as described in any of [1] to

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

[0117]

[87] A specific binding compound or antibody molecule as described in any of [1] to

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

[0118]

[88] A nucleic acid molecule that encodes a specific binding compound or antibody molecule as described in any of [1] to

[85] .

[0119]

[89] A nucleic acid molecule as in

[88] , wherein the nucleic acid molecule comprises: (i) Specific combination objects [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] The CH3 domain nucleic acid sequence of [or FS22-053] is listed in the sequence identification number: [twenty two] [、]

[31] [、]

[49] [、]

[0103] [、]

[76] [、]

[40] [、]

[58] [、]

[67] [、]

[85] [、]

[94] [and]

[0176] Inside; or (ii) Specific combination objects [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] The CH3 domain nucleic acid sequence of [or FS22-172] is listed in the sequence identification number:

[0140] [、131] [、149] [、122] [、158] [、166] [and 113] inside.

[0120]

[90] Nucleic acid molecules such as

[88] or

[89] , wherein the nucleic acid molecule comprises a nucleic acid sequence of a specific binding agent: (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] [The] nucleic acid sequences are listed in the sequence identification numbers: [twenty four] [、]

[33] [、]

[51] [、]

[0105] [、]

[78] [、]

[42] [、]

[60] [、]

[69] [、]

[87] [、]

[96] [and]

[0177] Inside [;] or [] (ii) [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] [or FS22-172] [The] nucleic acid sequences are listed in the sequence identification numbers:

[0142] [、133] [、151] [、124] [、160] [、168] [and 115] inside.

[0121]

[91] Nucleic acid molecules such as

[89] or

[90] , wherein the nucleic acid molecule contains a CH3 domain nucleic acid sequence, or nucleic acid sequence, that specifically binds to the following: (i) [FS22-172-003]; or (ii) [FS22-053-008]

[0122]

[92] A vector comprising a nucleic acid as described in any of

[88] to

[91] .

[0123]

[93] A recombinant host cell comprising a nucleic acid as described in any one of

[88] to

[91] or a vector as described in

[92] .

[0124]

[94] A method for producing a specific binding compound or antibody molecule as described in any of [1] to

[85] , comprising culturing recombinant host cells

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

[0125]

[95] The method of

[94] further includes isolating and / or purifying the specific binding compound or antibody molecule.

[0126]

[96] A pharmaceutical composition comprising a specific binding compound or antibody molecule as described in any of [1] to

[87] and a pharmaceutically acceptable excipient.

[0127]

[97] A specific binding compound or antibody molecule of any of [1] to

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

[0128]

[98] A method of treating a disease or disorder in an individual, comprising administering a therapeutically effective amount of a specific binding agent or antibody molecule such as [1] to

[87] to the individual.

[0129]

[99] A specific binding agent or antibody molecule as provided in

[97] or a method as in

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

[0130]

[0100] The specific binding agent or antibody molecule supplied as in

[97] or

[99] , or the method as in

[98] or

[99] , wherein the treatment method comprises administering the specific binding agent or antibody molecule combined with a second therapeutic agent to the individual. Simple Explanation of the Diagram

[0131] [picture] [1] [:picture] [1A] [、] [B] [and] [C] Displays the alignment of the CH3 domain sequences of Fcabs FS22-053, FS22-053-008, FS22-053-009, FS22-053-010, FS22-053-011, FS22-053-012, FS22-053-013, FS22-053-014, FS22-053-015, FS22-053-016, FS22-053-017, FS22-172, FS22-172-001, FS22-172-002, FS22-172-003, FS22-172-004, FS22-172-005, and FS22-172-006 and wild-type (WT) Fcab. This indicates residue numbering based on the IMGT, IMGT exons (sequential numbering), EU, and Kabat numbering systems. [picture] [1D] Showing strains FS22-053-008 to FS22-053-016 and FS22-053-017 (see) [Example] [10.1] ) percentage of sequence identity between the CH3 domain of the parent FS22-053 and the CH3 domain of the parent FS22-053. [picture] [1E] Shows the percentage of sequence identity of the CH3 domain of strains FS22-172-001 to FS22-172-006 compared to the CH3 domain of parental FS22-053. [Figure 2] shows NF-κB signaling as determined by the luminescence value of luciferase production caused by CD137 clustering and activation. In HEK hCD137 NF-κB reporter assay, when cross-linked with protein L, anti-human CD137 parental Fcabs FS22-053 and FS22-172 in the HelD1.3 pseudomAb2 format drove limited CD137 clustering and NF-κB signaling (hollow and solid circles). When cross-linked with protein L in HEK hCD137 NF-κB reporter gene analysis, the affinity-matured anti-human CD137 Fcabs FS22-053-008, -009, -011 (hollow squares, triangles, and rhombuses) and FS22-172-002, -003, -004 (solid squares, triangles, and rhombuses) in the HelD1.3 pseudomAb2 format drove stronger CD137 clustering and NF-κB signaling compared to the parent strains. When cross-linked with protein L, the positive control anti-human CD137 mAb, 20H4.9 (dotted line) showed increased luminescence and a smaller EC50 value compared to the affinity-matured anti-human CD137 Fcabs. [picture] [3] IL-2 release in T cell activation assay in the presence of anti-human CD137 Fcabs. CD137 clustering and activation driven by FS22-053-008, FS22-053-009, FS22-053-011 and FS22-172-002, FS22-172-003, FS22-172-004 in the PD-L1(S70 LALA) mAb2 format resulted in mouse interleukin-2 (mIL-2) release in DO11.10 T cell activation assay only when HEK cells overexpressing PD-L1 were crosslinked (hollow and solid black symbols). The positive control anti-human CD137 mAb, 20H4.9, showed increased mIL-2 release at progressively increasing concentrations (dotted lines), however the maximum release was significantly less than that of anti-human CD137 Fcabs. Compared to cross-linked cells (hollow and solid black symbols), all anti-human CD137 Fcabs (solid gray symbols) in the PD-L1 model mAb2 format showed significantly lower mIL-2 release in HEK cells without PD-L1 overexpression cross-linking. [picture] [4] Human IL-2 (hIL-2) release was shown in the human primary CD8+ T cell activation assay. Affinity-matured anti-human CD137 Fcabs FS22-053-008, FS22-053-009, FS22-053-011, FS22-172-002, FS22-172-003 and FS22-172-004 in the PD-L1 mAb2 format drove CD137 activation in CD8+ T cells, leading to human IL-2 release. The affinity-matured Fcabs showed better activity than the parental Fcabs FS22-053 (hollow circle) and FS22-172 (solid circle). The positive control anti-human CD137 mAb, 20H4.9 (dotted line), showed increased hIL-2 release and greater IL-2 release than Fcabs; however, the EC50 value was larger than some anti-human CD137 Fcabs, indicating that some anti-human CD137 Fcabs achieved better activity. [] [picture] [5] The NF-κB signaling was determined by the luminescence value of luciferase production caused by CD137 clustering and activation. In HEK mCD137 NF-κB reporter gene analysis, when cross-linked with protein L, the anti-mouse CD137 parental Fcab FS22m-063 in the HelD1.3 pseudomAb2 format drove CD137 clustering and NF-κB signaling (solid circles). At concentrations above 100 nM and without cross-linking with protein L, FS22m-063 drove some CD137 clustering and NF-κB signaling, but at much lower levels than when cross-linked (hollow circles). When cross-linked with protein L, the positive control anti-mouse CD137 mAb, Lob12.3, showed increased luminescence (solid squares), compared to no cross-linking (hollow squares), however, the maximum response achieved at the highest antibody concentration was significantly less than that of anti-mouse CD137 Fcab. As expected, the isotype control showed no activity. [picture] [6] Tumor volume measurements of subendothelial MC38 syngeneic tumor patterns in C57BL6 mice treated with G1-AA / 4420 (IgG control), G1-AA / S70 (PD-L1 positive control), G1-AA / Lob12.3 (CD137 positive control), a combination of G1-AA / S70 and G1-AA / Lob12.3, and FS22m-063-AA / S70 (anti-mouse CD137 Fcab FS22m-063 in PD-L1 mAb2 format). Mean tumor volume [in mm3] is shown with or without 95% confidence intervals. Compared with IgG control mice, anti-PD-L1 positive control mAb mice, and anti-CD137 positive control mice, FS22m-063-AA / S70 significantly reduced tumor growth in the MC38 syngeneic tumor pattern. Pairwise, the statistical significance of growth rate over the entire study period was demonstrated using a mixture model analysis. **** ≤ 0.0001 p-value [Figure 7] [:A] Tumor volume measurements of subendothelial MC38 syngeneic tumor patterns in C57BL / 6 mice treated with G1-AA / 4420 (IgG control), G1-AA / F2 (PD-1 positive control), G1 / Lob12.3 (CD137 positive control), a combination of G1-AA / F2 and G1-AA / Lob12.3, and FS22m-063-AA / F2 (anti-mouse CD137 Fcab FS22m-063 in PD-1 mAb2 format). Mean tumor volume [in mm3] is shown with or without 95% confidence intervals. Compared with IgG control mice, FS22m-063-AA / F2 significantly reduced tumor growth in the MC38 syngeneic tumor pattern. Pairwise, growth rate statistical significance over all study time is shown using a pooled model analysis. **** ≤ 0.0001 p-value. [B] The same data as shown in Figure 7A is presented, except that the tumor volume of each treated mouse is displayed separately. This emphasizes that, with the exception of one mouse, none of the mice treated with FS22m-063-AA / F2 showed any tumor regrowth during the study. Conversely, tumor regrowth was observed in mice treated with G1-AA / Lob12.3 and G1-AA / F2 at the end of the study. [picture] [8] Mean tumor volume measurement of subcutaneous mesothelin-positive CT26 syngeneic tumor pattern (Balb / c). Mice were treated with G1-AA / 4420 (IgG control) or FS22m-063-AA / FS28m-228 mAb². Mice treated with FS22m-063-AA / FS28m-228 showed significant inhibition of tumor growth compared with mice treated with IgG control. Mean tumor volume [in mm³] was plotted at 95% confidence intervals. This demonstrates the statistical significance of comparing growth rates over the entire study period using a mixed model analysis. **** ≤ 0.0001 p-value [picture] [9] Individual tumor volume measurements of the CT26.G10 syngeneic tumor pattern were shown, which were treated with G1-AA / HelD1.3 (human IgG1 control), FS22m-063-AA / HelD1.3 (anti-mouse CD137 Fcab in pseudomAb2 format), FS22m-063-AA / 4420 (anti-mouse CD137 Fcab in pseudomAb2 format), G1-AA / FS28m-228-010 (anti-mouse MSLN mAb), a combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN mAb), and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN mAb2). Compared with the isotype control and other treatment groups, FS22m-063-AA / FS28m-228-010 showed reduced tumor growth. [picture]

[10] Kaplan Meier survival curves in the CT26.G10 syngeneic tumor model were shown for treatment with the following drugs: G1-AA / HelD1.3 (IgG control), FS22m-063-AA / HelD1.3 (anti-mouse CD137 Fcab in mAb2 format), FS22m-063-AA / 4420 (anti-mouse CD137 Fcab in mAb2 format), G1-AA / FS28m-228-010 (anti-mouse MSLN Fab), a combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN Fab), and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN mAb2). The results showed that FS22m-063-AA / FS28m-228-010 treatment resulted in significantly improved survival in mice compared to isotype control mice or other treatment groups. [picture]

[11] The mouse IL-2 release of cross-reactive CD137 Fcabs FS22-053-014 and FS22-053-017 in the pseudo-mAb2 format was shown in the DO11.10 human CD137 T cell activation assay. Both Fcabs activated CD137 when cross-linked with protein L. The positive control anti-human CD137 mAb, 20H4.9, showed increased mIL-2 release, similar to the pseudo-mAb2 of FS22-053-017. Both anti-human CD137 Fcabs in the pseudo-mAb2 format showed negligible mIL-2 release in the absence of protein L cross-linking (hollow symbol), compared to when cross-linked (solid symbol). [picture]

[12] Mouse IL-2 release was shown in the DO11.10 mouse CD137 T cell activation assay, with mouse and human cross-reactive CD137 Fcabs FS22-053-014 and FS22-053-017 in pseudomAb2 format, when cross-linked with protein L. All FS22-053-014 and FS22-053-017 in pseudomAb2 format (HelD1.3) and anti-mouse CD137 Fcab FS22m-063, when cross-linked with protein L, activated CD137, leading to mIL-2 release. The positive control anti-mouse CD137 mAb, Lob12.3, showed increased mIL-2 release as expected. All anti-CD137 Fcabs in pseudomAb2 format showed a significant decrease in mIL-2 release without protein L cross-linking (dotted lines) compared to when cross-linked. FS22-053-017 showed lower activity in this analysis (EC50 was 8-fold lower than that of FS22-053-014), but it still showed activity in the analysis. [picture]

[13] The image shows the homology between FS22 Fcabs and a structural template containing the wild-type human IgG1 Fc CH3 domain (PDB ID 5JII). The image shows 5JII Fc ( [A]), and FS22-053-008 ( [B] ) and FS-172-003 ( [C] ) Representative structures of Fcabs. The CH3 domains of each structure have been visualized using gray band diagrams. The AB, CD, and EF loops are represented by black shading and marked accordingly. The predicted AB loop structure is highlighted with dotted lines for comparison, and the prominence caused by the PPY portion is shown, indicating its potential key role in bonding. [picture]

[14] Sensorgrams showing the binding kinetics of the mAb2-format homodimer Fcab (FS22-172-003-AA) (containing two CD137-binding CH3 domains) compared to the mAb2-format heterodimer Fcab (containing one CD137-binding CH3 domain and one wild-type CH3 domain). These sensorgrams show that both molecules bind CD137, even with only one binding strand. The dissociation rates differ, and the homodimer Fcab exhibits a much slower dissociation dissociation compared to the heterodimer Fcab. These sensorgrams demonstrate that the anti-CD137 Fcab can bind CD137 divalently. [picture]

[15] Demonstrated positive control antibody (G1-AA / 20H4.9) and isotype control antibody (G1-AA / HelD1.3) against human CD137 Fcab pseudomAb2 format (FS22-172-003-AA / HelD1.3) and cells expressing a series of CD137 locants, including negative control cell line (hCD137neg DO11.10). [A]) binding. Binding was determined by flow cytometry. Results showed that although G1-AA / 20H4.9 could bind to all cell lines expressing CD137 ( [BE]), but FS22-172-003-AA / HelD1.3 does not bind to DO11.10 cell lines that exhibit low-level CD137 ( [B]), and the fold-difference in binding compared to the positive control is inversely proportional to the amount of CD137 expressed by the cell line. Implementation

[0132] Detailed Description of Preferred Embodiments This invention relates to a specific binding agent for CD137. CD137 is also known as member 9 of the tumor necrosis factor receptor superfamily (TNFRSF9) or 4-1BB. The specific binding agent preferably binds to human CD137, more preferably human and cynomolgus monkey CD137, and even more preferably a dimeric human and cynomolgus monkey CD137. The CD137 portion bound by the specific binding agent is preferably the extracellular domain of CD137. The extracellular domain of human and cynomolgus monkey CD137 may include a sequence identification number.

[0181] [and]

[0183] The sequences listed herein or constituted therein. The specific binding agent preferably binds to CD137 expressed on the cell surface. 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 (DCs) or tumor-infiltrating lymphocytes (TILs).

[0133] The specific binding agent preferably binds specifically to CD137. The term "specific" can refer to the specific binding agent binding to its specific binding partner, in this case CD137, and other molecules will not show any significant binding. The term "specific" can also apply to the specific binding agent binding to a specific epitope carried by some antigens, such as the epitope on CD137, in which case the specific binding agent can bind to various antigens carrying that epitope. The specific binding agent preferably does not bind to CD40, OX40, and / or GITR, or does not show any significant binding.

[0134] As described in the background section above, treatment of patients with the anti-CD137 antibody urogenumab is associated with dose-limiting high-grade hepatitis. While not wishing to be bound by theory, it is believed that the hepatitis observed with urogenumab treatment may be due to the activation of T cells present in the liver, or the infiltration and accumulation of activated T cells within the patient's liver. To select molecules that reduce or eliminate hepatitis, the inventors chose Fcabs with high CD137 total binding affinity. Specifically, the inventors selected Fcabs with a higher affinity for binding to dimeric CD137 than to monomeric CD137. T cells upregulate CD137 expression during priming and activation. It is generally believed that due to the higher expression of CD137 on activated T cells, CD137 exists in a dimer, trimer, or higher-order multimeric form on the surface of these cells. Conversely, inactive T cells show low or even undetectable CD137 expression. Therefore, it is believed that CD137, to the extent that it appears on the surface of T cells, may be in monomeric form. It is also believed that Fcabs with high total binding capacity to CD137 preferentially bind to activated T cells rather than inactive T cells, such as inactive T cells present in the liver, thus exhibiting reduced or absent liver inflammation.

[0135] The specific binding target is better suited to bind to the dimer human CD137 with an affinity of 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM or 2 nM or greater.

[0136] In a preferred embodiment, the specific binding compound exhibits a higher affinity for the dimer CD137 than for the monomer CD137. In a preferred embodiment, the specific binding compound exhibits an affinity for the dimer CD137 that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 200 times higher than the specific binding compound's affinity for the monomer CD137.

[0137] For example, human CD137 can have a sequence identification number:

[0183] The sequence is listed herein. This sequence is the same regardless of whether the antigen is in monomeric or dimer form.

[0138] It has also been demonstrated that specific binding compounds from the FS22-53 and FS22-172 lineages bind to the dimeric cynomolgus macaque CD137. Binding to both cynomolgus macaque CD137 and human CD137 is advantageous because it allows for testing the efficacy and toxicity of the specific binding compounds in cynomolgus macaques before administration to humans.

[0139] In a preferred embodiment, the specific binding agent can bind to the dimeric cynomolgus macaque CD137 with an affinity (KD) 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 greater. More preferably, the specific binding agent binds to the dimeric cynomolgus macaque CD137 with an affinity (KD) of 2 nM or greater.

[0140] This specific binding compound can bind with similar affinity to both human CD137 and cynomolgus macaque CD137. This is considered advantageous for conducting efficacy and toxicity studies of this specific binding compound in cynomolgus macaques, allowing for prediction of its effects on humans.

[0141] Therefore, in a preferred embodiment, the affinity of the specific binding object to the dimer cynomolgus macaque CD137 is no more than 10 times lower or higher than the affinity of the specific binding object to the dimer human CD137, preferably no more than 5 times.

[0142] This specific binding compound can bind to dimeric mouse CD137, dimeric cynomolgus monkey CD137, and dimeric human CD137. This is considered advantageous because the same specific binding compound can be used to perform initial efficacy studies in mice, toxicity studies in cynomolgus monkeys, and clinical trials in humans, potentially simplifying the clinical pathway. Specifically, it was unexpectedly discovered that the specific binding compounds FS22-053-014 and FS22-053-017 bind to dimeric mouse, human, and cynomolgus monkey CD137.

[0143] The binding affinity of a specific binding object to a homologous antigen, such as human or cynomolgus monkey CD137, can be determined by, for example, surface plasma resonance (SPR), such as Biacore.

[0144] The term "specific binding object" describes an immunoglobulin or a fragment thereof containing a constant domain containing a CD137 antigen-binding address. When used herein, the term "specific binding object" therefore includes an antigen-binding fragment provided that the antigen-binding fragment contains a CD137 antigen-binding address located within a constant domain of the specific binding object. The constant domain can be a CH1, CH1, CH2, CH3, or CH4 domain, preferably a CH1, CH2, or CH3 domain, more preferably a CH2 or CH3 domain, and most preferably a CH3 domain. The specific binding object can be partially or fully synthetically produced.

[0145] Preferably, the specific binding compound comprises a CH2 and a CH3 domain, wherein the CH2 or CH3 domain, preferably the CH3 domain, contains a CD137 antigen-binding address. The specific binding compound is preferably a dimer of two (identical) polypeptide chains, each polypeptide chain containing a CH2 and a CH3 domain. In a preferred embodiment, the specific binding compound further comprises an immunoglobulin hinge region or a portion thereof at the N-terminus of the CH2 domain. This molecule may also be referred to herein as an antigen-binding Fc fragment or FcabTM. The hinge region can be identified by sequence numbering:

[0179] The sequences or fragments listed herein constitute or contain sequence identification numbers:

[0179] The sequence or fragment thereof listed herein. Preferably, the fragment is a sequence identification number:

[0179] The C-terminal segment of the sequence listed herein. The length of this segment may be up to 20, up to 10, up to 8, or up to 6 amino acids. The length of this segment may be at least 3, at least 4, at least 5, or at least 6 amino acids. In a preferred embodiment, the hinge region has a sequence identification number: [7] The sequences listed in the text.

[0146] In a preferred embodiment, the specific binding object is an antibody molecule, preferably a monoclonal antibody or a fragment thereof. The antibody molecule is preferably human or humanized. The antibody molecule may be an immunoglobulin G molecule, such as IgG1, IgG2, IgG3, or IgG4, preferably IgG1, IgG2, or IgG4, more preferably IgG1, or a fragment thereof.

[0147] Because antibodies can be modified in various ways, the term "antibody molecule" should be interpreted to encompass antibody fragments, derivatives, functional equivalents, and antibody homologs, whether natural or wholly or partially synthetic. An example of an antibody fragment containing a CH3 domain is the Fc domain of an antibody. An example of an antibody fragment containing both a CDR sequence and a CH3 domain is a minibody containing an scFv linked to a CH3 domain (Hu et al. (1996), Cancer Res., 56(13):3055-61).

[0148] The specific binding object includes a CD137 antigen-binding address. The CD137 antigen-binding address is located in a constant domain of the specific binding object, preferably the CH3 domain. The CD137 antigen-binding address includes one or more modified structural loops within the constant domain of the specific binding object. Engineering structural loops in the constant domain of an antibody to create antigen-binding addresses for target antigens is known in the art and described by way of example in Wozniak-Knopp G et al. (2010); WO2006 / 072620 and WO2009 / 132876.

[0149] Following a large-scale selection and affinity maturation program, the inventors isolated two sets of Fcabs that preferentially bind to the dimer rather than the monomeric human CD137. Surprisingly, all isolated Fcabs contained the sequence PPY and a 5-amino acid insertion within their respective AB ring structures. These two sets of Fcabs were independently selected from different selection activities, each using a library containing a 5-amino acid insertion within the AB ring structure. Thus, the PPY sequence was independently selected twice, indicating its importance for CD137 binding. Isolation of anti-CD137 Fcabs from the Fcab library that did not contain the 5-amino acid insertion within the AB ring structure was not pursued further, for example, because the selected Fcabs were unlikely to undergo affinity maturation. This indicates that the amino acid insertion within the AB ring structure may be important for CD137 binding. Therefore, the presence of the PPY sequence and the 5-amino acid insertion within the AB ring structure, where the PPY sequence may be selectively present in all or part of the 5-amino acid insertion, may be important for CD137 binding.

[0150] Therefore, the CD137 antigen binding site of the specific binding object may include a first and / or a second sequence, preferably a first and a second sequence, wherein the first and second sequences are respectively located in the constant domain of the specific binding object, preferably the CH3 domain, and the AB and EF structural loops.

[0151] In a preferred embodiment, the residues at positions 95 and 96 of the CH3 domain of the specific binding compound are wild-type, preferably arginine (R) and tryptophan (W), respectively. Both of these residues are located within the EF ring structure. Unless otherwise stated, the amino acid residue positions herein are numbered according to the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., 2005.

[0152] The first sequence preferably contains the sequence PPY (sequence identification number:

[10] ).

[0153] The PPY sequence may be located at positions 10 to 19 of the CH3 domain of the specific binding compound, preferably between positions 15 and 17. In a preferred embodiment, the PPY sequence is located at positions 16, 16.5, and 16.4 of the CH3 domain. Alternatively, the PPY sequence may be located at positions 16 to 17 of the CH3 domain. In an alternative preferred embodiment, the PPY sequence is located at positions 16.3, 16.2, and 16.1 of the CH3 domain. In the IMGT numbering scheme, the inserted residues are numbered according to the direction of the loop in which they are located. If the loop is "upward," the inserted residue is the number of the residue immediately preceding the insertion, and the inserted residue number is represented in ascending decimal numbers within the sequence, such as 16, 16.1, 16.2, and 16.3 in the case of three mutations after residue 16. If the loop moves "down", the inserted residue is the number of the residue immediately preceding the insertion and the inserted residue number is represented by a descending decimal number within the sequence, such as 16, 16.3, 16.2, and 16.1 in the case of three mutations after residue 16 (LeFranc et al., 2005, and LeFranc et al., 2015).

[0154] In a preferred embodiment, the AB ring structure includes an amino acid insertion. The length of the insertion can be 1 to 10, 2 to 9, 3 to 7, 4 to 6, or 5 amino acids. Preferably, the insertion length is 5 amino acids.

[0155] The insertion may be located between positions 10 and 19 of the CH3 domain of the specific union object, preferably between positions 14 and 17, and more preferably between positions 16 and 17. In a preferred embodiment, the insertion is as follows: [picture] [1] shows the location 16.5 to 16.1 of the CH3 domain in the specific bonded object.

[0156] Most of the specific binding compounds identified after affinity maturation contain a leucine (L) residue at position 97 of the CH3 domain. Many specific binding compounds also contain an aspartic acid (D) residue or a glutamic acid (E) residue at position 98 of the CH3 domain of the specific binding compound. Both of these amino acid alterations are located within the EF structural ring. These results suggest that one or both of these residues may be important for CD137 binding. Therefore, the second sequence preferably contains the sequence LD or LE, wherein the LD or LE sequence is preferably located at positions 97 and 98 of the CH3 domain of the specific binding compound.

[0157] The first and second sequences can be the first and second sequences of the following CH3 domains: specific binding objects [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] [Or FS22-172], preferably a specific bonding object. [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [Or FS22-172-006], preferably a specific combination object. [FS22-172-003] [FS22-172-002] [Or FS22-172-004], preferably a specific combination object. [FS22-172-003]

[0158] Occasionally, the first and second sequences can be the first and second sequences of the following CH3 domains: specific binding objects [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 a specific bonding object. [FS22-053-008] [FS22-053-009] [FS22-053-011] [FS22-053-017] [Or FS22-053-014], preferably a specific combination object. [FS22-053-008] [FS22-053-009] [FS22-053-011] [,] or [FS22-053-017], even better as a specific combination of objects. [FS22-053-008]. In any preferred embodiment, the first and second sequences are the first and second sequences of the following CH3 domains: specific binding objects. [FS22-053-017] or [FS22-053-014] is better suited for specific object combinations. [FS22-053-017]

[0159] Exclusivity of objects [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 The CH3 domain sequence of [FS22-172-006] is listed in the sequence identification number:

[0175] [,twenty one] [、30] [、39] [、49] [、58] [、66] [、75] [、84] [、93] [、102] [、112] [、121] [、130] [、139] [、148] [、157] [and 166] inside.

[0160] Exclusivity of objects [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] The first and second sequences of [and FS22-172-006] can be specific binding objects, respectively. [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] The sequence between positions 14 to 17 and positions 91 to 99 of the CH3 domain.

[0161] Choose any location, and combine objects with specificity. [FS22-053-008] [FS22-053-010] [FS22-053-011] [FS22-053-012] The first and second sequences of [and FS22-053-016] can be specific binding objects respectively. [FS22-053-008] [FS22-053-010] [FS22-053-011] [FS22-053-012] [and FS22-053-016] The sequence between positions 14 to 17 and positions 92 to 99 of the CH3 domain.

[0162] Exclusivity of objects The first and second sequences of [FS22-053-015] can be selectively combined objects. [FS22-053-015] The sequence between positions 14 to 17 and positions 92 to 98 of the CH3 domain.

[0163] The CD ring sequence of the specific binding object is preferably unmodified, i.e., wild-type. Therefore, the CD ring sequence preferably has a sequence identification number. [2] The sequence listed in the text. The CD ring sequence is preferably located at position 43 to 78 of the CH3 domain of the specific binding object.

[0164] The first and second sequences can be specific combination objects, respectively. [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] The complete AB and EF structural ring sequences. Determining the positions of the AB, CD, and EF structural rings in the CH3 domain sequence, for example according to the IMGT, IMGT exon, EU, or Kabat numbering system, is within the capabilities of those skilled in the art and is described in Hasenhindl et al. (2013). In a preferred embodiment, the AB, CD, and EF structural rings, according to the IMGT numbering system, are respectively located between positions 10 to 19, 42 to 79, and 91 to 102 of the CH3 domain of the specific binding object. In a preferred embodiment, the first, second, and third sequences are therefore specific binding objects. [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] The sequence between positions 10 to 19, 42 to 79, and 91 to 102 of the CH3 domain of [or FS22-172].

[0165] In a preferred embodiment, the specific combination object includes a first and / or a second, preferably a first and second sequence of the following specific combination objects: (i) [FS22-172-003], listed in sequence identification number: 141; (ii) [FS22-172-002], listed in sequence identification number: 132; (iii) [FS22-172-004], listed within sequence identification number: 150; (iv) [FS22-172-001], listed in sequence identification number: 123; (v) [FS22-172-005], listed in sequence identification number: 159; (vi) [FS22-172-006], listed in sequence identification number: 167; or (vii) [FS22-172], listed in sequence identification number: 114; wherein the first and second sequences are preferably located between positions 14 to 17 and 91 to 99 of the CH3 domain of the specific binding object, respectively.

[0166] In a more preferred embodiment, the specific combination object includes a first and / or a second, preferably a first and second sequence of the following specific combination objects: (i) [FS22-172-003] [,] are listed within sequence identification number: 141; (ii) [FS22-172-002] [,] are listed within sequence identification number: 132; (iii) [FS22-172-004] [,] are listed within sequence identification number: 150; (iv) [FS22-172-001] [,] are listed within sequence identification number: 123; (v) [FS22-172-005] [,] Listed within sequence identification number: 159; or (vi) [FS22-172-006] [,] are listed in sequence identification number: 167.

[0167] In a more preferred embodiment, the specific combination object includes a first and / or a second, preferably a first and second sequence of the following specific combination objects: (i) [FS22-172-003] [,] are listed within sequence identification number: 141; (ii) [FS22-172-002] [,] Listed within sequence identification number: 132; or (iii) [FS22-172-004] [,] are listed within sequence identification number: 150.

[0168] In an even more preferred embodiment, the specific combination object includes a first and / or a second, preferably a specific combination object listed in sequence identification number 141. The first and second sequences of [FS22-172-003].

[0169] In a preferred embodiment, the specific bonding object, particularly one comprising a first and / or a second, is preferably a specific bonding object. The specific binding objects of the first and second sequences of [FS22-172-006] may include a leucine (L) residue at position 19 of the CH3 domain of the specific binding object.

[0170] In a preferred embodiment, the specific combination object includes a first and / or a second, preferably a first and second sequence of the following specific combination objects: (i) [FS22-053-008] [,] are listed within sequence identification number: 23; (ii) [FS22-053-009] [,] are listed within sequence identification number: 32; (iii) [FS22-053-011] [,] are listed within sequence identification number: 50; (iv) [FS22-053-017] [,] are listed within sequence identification number: 104; (v) [FS22-053-014] [,] are listed within sequence identification number: 77; (vi) [FS22-053-010] [,] are listed within sequence identification number: 41; (vii) [FS22-053-012] [,] are listed within sequence identification number: 59; (viii) [FS22-053-013] [,] Listed within sequence identification number: 68; (ix) [FS22-053-015] [,] Listed within sequence identification number: 86; (x) [FS22-053-016] [,] Listed within sequence identification number: 95; [or] (xi) [FS22-053] [,] are listed in sequence identification number: 15; wherein the first and second sequences are preferably located between positions 14 to 17 and 91 to 99 of the CH3 domain of the specific binding object, respectively.

[0171] In a more preferred embodiment, the specific combination object includes a first and / or a second, preferably a first and second sequence of the following specific combination objects: (i) [FS22-053-008] [,] are listed within sequence identification number: 23; (ii) [FS22-053-009] [,] are listed within sequence identification number: 32; (iii) [FS22-053-011] [,] are listed within sequence identification number: 50; (iv) [FS22-053-017] [,] Listed within sequence identification number: 104; or (v) [FS22-053-014] [,] are listed in sequence identification number: 77.

[0172] In a more preferred embodiment, the specific binding object includes a first and / or a second, preferably a first and second sequence of the CH3 domain of the following specific binding object: (i) [FS22-053-008] [,] are listed within sequence identification number: 23; (ii) [FS22-053-009] [,] are listed within sequence identification number: 32; (iii) [FS22-053-011] [,] Listed within sequence identification number: 50; or (iv) [FS22-053-017] [,] are listed in sequence identification number: 104.

[0173] In an even more preferred embodiment, the specific combination object includes a first and / or a second, preferably a specific combination object listed in sequence identification number 23. The first and second sequences of [FS22-053-008].

[0174] As an alternative to IMGT numbering, amino acid residue positions, including the amino acid sequence, substitution, deletion and insertion positions as described herein, can be numbered according to IMGT exon numbering (also known as sequential numbering), EU numbering or Kabat numbering. [picture] [1] Showing the concordance between the IMGT numbering, IMGT exon numbering, EU numbering, and Kabat numbering of the CH3 domain residue positions. Thus, for example, when the residue positions are numbered according to the IMGT numbering scheme, this application alleges that the first sequence is located between positions 14 and 17 of the CH3 domain of the specific binding compound, and when the residue positions are numbered according to the IMGT exon numbering scheme, the first sequence is located between positions 18 and 21 of the CH3 domain, such as... [picture] As shown in [1]. Alternatively, the positions of amino acid residues in the CH3 domain, including the amino acid sequences, substitutions, deletions, and insertions in the CH3 domain as described herein, can be found in [1]. [Sequence Identification Number:] [4] The position of the wild-type CH3 domain sequence listed in the table is used to define it. [picture] [1] It also shows the concordance between the IMGT number and the wild-type CH3 domain sequence.

[0175] In a preferred embodiment, the specific binding object comprises a CH3 domain sequence that includes, has, or consists of the following: specific binding object [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] The CH3 domain sequence of [or FS22-172] is preferably a specific binding object. [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] The CH3 domain sequence of [or FS22-172-006] is preferably a specific binding object. [FS22-172-003] [FS22-172-002] The CH3 domain sequence of [or FS22-172-004] is preferably a specific binding object. [FS22-172-003] CH3 domain sequence.

[0176] In a preferred embodiment, the specific binding object comprises a CH3 domain sequence that includes, has, or consists of the following: specific binding object [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] The CH3 domain sequence of [or FS22-053] is preferably a specific binding object. [FS22-053-008] [FS22-053-009] [FS22-053-011] [、FS22-053-017] or The CH3 domain sequence of [FS22-053-014] is preferably a specific binding object. [FS22-053-008] [FS22-053-009] [、FS22-053-011] or The CH3 domain sequence of [FS22-053-017] is even better as a specific binding object. [FS22-053-008] CH3 domain sequence.

[0177] The CH3 domain of a specific binding object may selectively include an additional lysine residue (K) at the C-terminus closest to the CH3 domain sequence.

[0178] It is possible to obtain monoclonal and other antibodies and use recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. These techniques may involve introducing CDRs or variable regions into different immunoglobulins. The introduction of CDRs of one immunoglobulin into another is described, for example, in EP-A-184187, GB 2188638A, and EP-A-239400. Similar techniques can be used to introduce constant domain sequences to combine the CD137 antigen-binding address of a specific binding agent as described in this invention into a constant domain, such as the CH3 domain of another specific binding agent, thereby producing a specific binding agent whose constant domain contains a CD137 antigen-binding address. Alternatively, a fragment of the complete constant domain sequence of a specific binding agent can be replaced with the constant domain sequence of a specific binding agent as described in this invention to prepare a specific binding agent whose constant domain contains a CD137 antigen-binding address. A segment of a constant domain sequence of a specific binding object can also be replaced by a corresponding segment of a constant domain sequence of a specific binding object containing a CD137 antigen binding address, as in the present invention.

[0179] The CH2 domain of this specific binding compound may contain one or more mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII, and / or complement. The inventors hypothesize that reducing or eliminating binding to Fcγ receptors would decrease or eliminate ADCC mediated by this specific binding compound. Similarly, reducing or eliminating binding to complement is expected to decrease or eliminate CDC mediated by this specific binding compound. Mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors and / or complement are known in the art (Wang et al., 2018). Such mutations include the "LALA mutation" described in Bruhns et al., 2009, and Hezareh et al., 2001, which involves replacing leucine residues at positions 1.3 and 1.2 of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, generating α-glycosyl-free antibodies by mutating the conserved N-linked glycosylation site at the IMGT position 84.4 of the CH2 domain to alanine, glycine, or glutamic acid (N84.4A, N84.4G, or N84.4Q) is known to reduce IgG1 effector function (Wang et al., 2018). Alternatively, mutating the proline at the IMGT position 114 of the CH2 domain to alanine or glycine (P114A or P114G) is known to reduce complement activation (C1q binding) and ADCC (Idusogie et al., 2000; Klein et al., 2016). These mutations can also be combined to generate specific binding compounds with further reduced or absent ADCC or CDC activity.

[0180] Therefore, the specific binding object may include a CH2 domain, wherein the CH2 domain preferably includes: (i) Alanine residues at positions 1.3 and 1.2; and / or (ii) Alanine or glycine at position 114; and / or (iii) Alanine, glutamic acid, or glycine at position 84.4; The amino acid residues are numbered according to the IMGT numbering system.

[0181] In a preferred embodiment, the specific binding object includes a CH2 domain, wherein the CH2 domain preferably includes: (i) Alanine residues at positions 1.3 and 1.2; and / or (ii) Alanine or glycine at position 114; The amino acid residues are numbered according to the IMGT numbering system.

[0182] In another preferred embodiment, the specific binding object includes a CH2 domain, wherein the CH2 domain includes: (i) Alanine residue at position 1.3; and (ii) Alanine residue at position 1.2; The amino acid residues are numbered according to the IMGT numbering system.

[0183] For example, the CH2 field may have a sequence identification number: [6] The sequence is listed below. [LALA] In a preferred embodiment, the specificity-binding object includes a CH2 domain, wherein the CH2 domain includes: (i) Alanine residue is located at position 1.3; (ii) Alanine residue at position 1.2; and (iii) Alanine at position 114; The amino acid residues are numbered according to the IMGT numbering system.

[0184] For example, the CH2 domain could have the sequence listed in sequence identification number 5. [LALA-PA]

[0185] In a preferred embodiment, the specific bonding object comprises, has, or consists of the following: specific bonding object [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] The CH2 and CH3 domain sequences of [or FS22-172] are preferably specific binding objects. [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] The CH2 and CH3 domain sequences of [or FS22-172-006] are preferably specific binding objects. [FS22-172-003] [FS22-172-002] The CH2 and CH3 domain sequences of [or FS22-172-004] are preferably specific binding objects. [FS22-172-003] CH2 and CH3 domain sequences, wherein specific binding objects [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] The CH2 and CH3 domain sequences of [and FS22-172] are respectively [Displayed at sequence identification number 141] [、132] [、150] [、123] [、159] [、167] [and 114] Inside, starting from amino acid 7 and moving forward.

[0186] In any preferred embodiment, the exclusive bonding object comprises, has, or consists of the following: exclusive bonding object [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] The CH2 and CH3 domain sequences of [or FS22-053] are preferably specific binding objects. [FS22-053-008] [FS22-053-009] [FS22-053-011] [、FS22-053-017] or The CH2 and CH3 domain sequences of [FS22-053-014] are preferably specific binding objects. [FS22-053-008] [FS22-053-009] [、FS22-053-011] or The CH2 and CH3 domain sequences of [FS22-053-017] are preferably specific binding objects. [FS22-053-008] CH2 and CH3 domain sequences, wherein specific binding objects [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] The CH2 and CH3 domain sequences of [and FS22-053] are respectively [Displayed in sequence identification number] [twenty three] [、]

[32] [、]

[50] [、]

[0104] [、]

[77] [、]

[41] [、59] [、]

[68] [、]

[86] [、]

[95] [and]

[15] Inside, starting from amino acid 7 and moving forward.

[0187] In any preferred embodiment, the exclusive bonding object comprises, has, or consists of the following: exclusive bonding object [FS22-053-008] [FS22-053-009] [FS22-053-011] [FS22-053-017] [FS22-053-014] [FS22-053-010] [FS22-053-012] [FS22-053-013] [FS22-053-015] [FS22-053-016] [Or FS22-053] sequence, preferably a specific combination of objects. [FS22-053-008] [FS22-053-009] [FS22-053-011] [、FS22-053-017] or The sequence [FS22-053-014] is preferably a specific combination of objects. [FS22-053-008] [FS22-053-009] [、FS22-053-011] or The sequence of [FS22-053-017] is even better as a specific combination of objects. The sequence of [FS22-053-008], in which specific combination objects [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] sequence system [Respectively] Listed in [Sequence Identification Number] [twenty three] [、]

[32] [、]

[50] [、]

[0104] [、]

[77] [、]

[41] [、59] [、]

[68] [、]

[86] [、]

[95] [and]

[15] Inside.

[0188] In a preferred embodiment, in addition to the CD137 antigen-binding address located in the constant domain of the specific binding object, the specific binding object may also include one or more other antigen-binding addresses. These one or more other antigen-binding addresses preferably bind specifically to their equivalent homologous antigens.

[0189] The one or more additional antigen-binding sites may bind to CD137 or another antigen. The specific binding object can therefore be multispecific, such as a bispecific, trispecific, or tetraspecific molecule, preferably a bispecific molecule. In a preferred embodiment, the specific binding object can bind to both CD137 and the one or more additional antigens simultaneously.

[0190] Antibody molecules are known to have modular architectures containing discrete domains that can be combined in various ways to create multispecificity, such as bispecific, trispecific, or tetraspecific antibody formats. Exemplary multispecific antibody formats are described by way of example in Spiess et al. (2015) and Kontermann (2012). The specific binding objects of this invention can be used in such multispecific antibody formats. This offers additional advantages by introducing additional antigen-binding addresses into such multispecific antibody formats through the presence of antigen-binding addresses within the constant domains of the specific binding object, such as the CH3 domain.

[0191] For example, the specific binding object of the present invention may be a heterodimeric antibody molecule, such as a heterodimeric intact immunoglobulin molecule or a fragment thereof. In this case, a portion of the antibody molecule may have one or more sequences as described herein. For example, in the case of a bispecific heterodimeric antibody molecule, the specific binding object may comprise a heavy chain containing a CH3 domain as described herein, paired with a heavy chain that binds to an antigen other than CD137. Techniques for preparing heterodimeric antibodies are known in the art and include knob-into-hole (KIH) techniques, which involve engineering the CH3 domain of the antibody molecule to create “knobs” or “holes” to facilitate heterodimerization of the chain. Alternatively, heterodimeric antibody preparation may be achieved by introducing charge pairs into the antibody molecule to avoid homodimerization of the CH3 domain by electrostatic repulsion and to directly heterodimerize by electrostatic attraction. Examples of heterodimeric antibody formats include CrossMab, mAb-Fv, seed-body, and KIH IgG.

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

[0193] Other suitable multispecificity formats will be obvious to those skilled in the art.

[0194] In a preferred embodiment, the specific binding object includes a second antigen-binding address that binds to a second antigen, wherein the second antigen-binding address is preferably a CDR-based antigen-binding address. The CDR-based antigen-binding address is an antigen-binding address located within a mutable region of the antibody. The CDR-based antigen-binding address is formed by six CDRs: three light chain mutable domain (VL) CDRs and three heavy chain mutable domain (VH) CDRs.

[0195] The preparation of antibody molecules against a putative antigen and the determination of the CDR sequence of such antibody molecules are established techniques, and many suitable methods are known in this art. The CDR sequence can be determined, for example, according to Kabat et al., 1991, or the International ImMunoGeneTic Information System (IMGT) (Lefranc et al., 2015).

[0196] For example, the specific binding agent can be a mAb2 (TM) bispecific antibody. A mAb2 bispecific antibody as described herein is an IgG immunoglobulin comprising a CDR-based antigen-binding address in each of its variable regions and at least one antigen-binding address in a constant region. In the case of the specific binding agent of the present invention being in the mAb2 format, in addition to the CD137 antigen-binding address within the constant region of the specific binding agent, the specific binding agent thus also comprises a CDR-based antigen-binding address in each of its variable regions.

[0197] The three VH domain CDRs of antigen-binding sites can be located within the immunoglobulin VH domain, and the three VL domain CDRs can be located within the immunoglobulin VL domain. For example, a CDR-based antigen-binding site can be located within an antibody-mutable region.

[0198] Specific binding agents may have one or more, for example two, antigen-binding sites based on CDR- of the second antigen. Specific binding agents may therefore contain one VH and one VL domain, but preferably two VH and two VL domains, i.e., two VH / VL domain pairs, as in the case of naturally occurring IgG molecules.

[0199] In some preferred embodiments, the specific binding object can be an immunoglobulin molecule containing two variable regions, each containing a CDR-based antigen-binding address for a second antigen.

[0200] In a preferred embodiment, the antibody molecule is therefore an antibody molecule capable of binding to CD137 and a second antigen, the antibody molecule comprising: (i) The two CD137 antigen-binding sites located within the two CH3 domains of the antibody molecule; and (ii) Two antigen-binding sites based on CDR for the second antigen, each consisting of an immunoglobulin VH domain and an immunoglobulin VL domain.

[0201] In a more specific example, the antibody is a complete immunoglobulin molecule, such as a complete IgG1 molecule, which binds CD137 and a second antigen. The antibody molecule comprises: (i) The two CD137 antigen-binding sites located within the two CH3 domains of the antibody molecule; and (ii) Two CDR-based antigen-binding sites for the second antigen, each formed by an immunoglobulin VH domain and an immunoglobulin VL domain; and The immunoglobulin molecule further includes CH1, CH2 and CL domains.

[0202] CD137 activation requires CD137 clusters to accumulate on the surface of immune cells, such as T cells, which in turn stimulates intracellular signaling pathways and immune cell activation. Specific binding media bind to CD137 on the surface of immune cells. In the absence of specific binding media cross-linking, CD137 clustering may not occur, and therefore immune cell activation may not result.

[0203] The inventors have demonstrated that specific binding compounds of the FS22-53 and FS22-172 lineages do not lead to T cell activation in the absence of specific binding compound crosslinking (see [link to invention]). [Example] [5]).

[0204] As explained above, cross-linking of antibody molecules to Fcγ receptors is inefficient and cannot target specific locations, such as disease sites, because Fcγ receptor-expressing cells exist throughout the entire human body. Therefore, the second antigen bound to the second antigen binding site is preferably not an Fcγ receptor.

[0205] In a preferred embodiment, the specific binding object of the present invention thus includes a second antigen binding address that binds to a second antigen, wherein the second antigen can bind to and crosslink multiple specific binding objects.

[0206] For example, the inventors have demonstrated that when the second antigen is a multimeric molecule, the binding of the specific conjugate to the second antigen leads to or enhances T cell activation. The second antigen is therefore preferably a multimeric antigen, such as a dimer, trimer, or higher-order multimer, and is thus capable of crosslinking several specific conjugates.

[0207] The inventors have also demonstrated that, using CD137 / second antigen mAb2 molecules, when the second antigen is a surface antigen, such as a cell surface antigen, which can be a monomer or polymer and exists in high concentrations and / or clusters on surfaces such as cell surfaces, the binding of antibody molecules to the second antigen leads to or enhances T cell activation. While not wishing to be bound by theory, it is believed that the binding of antibody molecules to abundant cell surface antigens, for example, leads to high concentrations of antibody molecules bound to the cell surface, placing the antibody molecules close enough to drive CD137 clustering and immune cell activation. In a preferred embodiment, the second antigen is therefore a surface antigen present in high concentrations on surfaces such as cell surfaces.

[0208] A specific binding agent, also known as a conditional activator, comprises a second antigen-binding site as described herein that binds to a second antigen, and activates immune cells, such as T cells, only upon binding to the second antigen, or the activation activity of those immune cells is enhanced upon binding to the second antigen. This activation activity upon binding to the second antigen is independent of the binding of the specific binding agent to an Fcγ receptor and / or an external cross-linking agent, such as protein A or G or a secondary antibody, and thus the conditional activator activity of the specific binding agent allows targeting to the site where the second antigen is present. For example, in the case where the second antigen is a disease antigen, the specific binding agent can selectively activate immune cells at the disease site rather than elsewhere in the individual.

[0209] Furthermore, a specific binding agent that activates immune cells, such as T cells, only upon binding to a second antigen preferably exhibits enhanced immune cell activation activity compared to specific binding agents that rely on other mechanisms, such as cross-linking with external cross-linking agents or cross-linking via Fcγ receptor interaction. Because CD137 activation is more efficient, the specific binding agent described herein can achieve immune cell activation at lower concentrations compared to other specific binding agents.

[0210] Therefore, when the specific binding object of the present invention is cross-linked, such as by binding a second antigen, the specific binding object preferably induces a higher level of activation of immune cells, such as T cells, than when the specific binding object is not cross-linked.

[0211] The ability of antibody molecules or specific binding agents to activate T cells can be measured using T cell activation assays. T cells release IL-2 upon activation. Therefore, T cell activation assays can measure IL-2 release to determine the level of T cell activation induced by antibody molecules or specific binding agents.

[0212] For example, the ability of an antibody molecule or specific binding agent to activate T cells can be measured in a T cell activation assay by measuring the concentration of the antibody molecule or specific binding agent required to achieve the half-maximal release of IL-2 from T cells when the specific binding agent or antibody molecule is cross-linked. This is referred to as the EC50 of the antibody molecule or specific binding agent. A lower EC50 indicates a lower concentration of antibody molecule or specific binding agent required to achieve the half-maximal release of IL-2 from T cells in a T cell activation assay, and therefore the antibody molecule or specific binding agent has higher T cell activation activity. This specific binding agent or antibody molecule can be, for example, cross-linked with an anti-CH2 antibody.

[0213] In a preferred embodiment, the EC50 of the antibody molecule or specific binding agent in the T cell activation assay is within 10, 5, 4, 3, or 2 times that of FS22-172-003 / HelD1.3 (containing the LALA mutation) in the same assay, wherein FS22-172-003 / HelD1.3 (containing the LALA mutation) consists of or contains the following: the heavy chain listed in sequence identification number 145 and the light chain listed in sequence identification number 173.

[0214] In a preferred embodiment, the EC50 of the antibody molecule or specific binding agent in the T cell activation assay is within 10, 5, 4, 3, or 2 times that of FS22-053-008 / HelD1.3 (containing the LALA mutation) in the same assay, wherein FS22-053-008 / HelD1.3 (containing the LALA mutation) consists of or contains the following: the heavy chain listed in sequence identification number 27 and the light chain listed in sequence identification number 173.

[0215] For example, the antibody molecule or specific binding compound may have an EC50 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 in T cell activation assays.

[0216] In addition, or alternatively, the ability of an antibody molecule or specific binding agent to activate T cells can be determined by measuring the maximum concentration of IL-2 released by T cells in the presence of the antibody molecule or specific binding agent during a T cell activation assay, wherein the antibody molecule or specific binding agent is cross-linked.

[0217] In a preferred embodiment, in a T-cell activation assay, in the presence of the cross-linked antibody molecule or specific binding agent, the maximum concentration of IL-2 released by T cells is within 3, 2, or 1.5 times the maximum concentration of IL-2 released by T cells in the presence of FS22-053-008 / HelD1.3 (containing LALA mutation) or FS22-172-003 / HelD1.3 (containing LALA mutation).

[0218] T cell activation analysis can be T cell analysis as described herein, such as the CD8+ T cell analysis described in this embodiment, see example. [Example 5.4]

[0219] For example, a T-cell activation assay can be an IL-2 release assay based on CD8+ T cells isolated from human peripheral blood mononuclear cells (PBMCs). For example, a T-cell activation assay can include isolating human PBMCs from leukocyte depletion cones. Methods for isolating PBMCs are known in the art and are described within this embodiment. CD8+ T cells can then be isolated from the PBMCs. Methods for isolating CD8+ T cells from PBMCs are known in the art and are described within this embodiment.

[0220] CD8+ T cells can be added to a multi-well plate coated with anti-human CD3 antibody. Appropriate dilutions of the test antibody molecules or specific binding agents can be prepared and added to the wells. The T cells can then be incubated with the test antibody at 5% CO2 and 37°C for 24 hours. The supernatant can be collected and analyzed to determine the IL-2 concentration in the supernatant. The method for determining the IL-2 concentration in the solution is known in the art and described in this embodiment. A graph of human IL-2 concentration relative to the log concentration of the antibody molecule or specific binding agent can be plotted. The resulting curve can be fitted using a log (agonist) relative reaction equation.

[0221] The second antigen bound to the specific binding site of a binding agent can be an immune cell antigen or a disease antigen. Disease antigens include pathogenic antigens and tumor antigens.

[0222] The specific binding object binds to immune cell antigens and CD137, which may be present on the same immune cells or different immune cells.

[0223] Immune cell antigens can be members of the tumor necrosis factor receptor superfamily (TNFRSF) other than CD137. TNFRSF receptors are membrane-bound intercytokine receptors containing an extracellular cysteine-rich domain that binds one or more ligands of the tumor necrosis factor superfamily (TNFSF).

[0224] TNFRSF receptors can reside on the surface of immune cells. Once bound to a TNFRSF ligand, these receptors form clusters on the surface of immune cells, thereby activating them. For example, ligand-bound TNFRSF receptors can form multimers, such as trimers, or clusters of multimers. The presence of these ligand-bound TNFRSF receptor clusters stimulates intracellular signaling pathways, thus activating immune cells.

[0225] While not wanting to be bound by theory, it is believed that by binding to CD137 and the second TNFRSF receptor on the surface of immune cells, specific binding agents would cause both CD137 and the second TNFRSF receptor to cluster and activate immune cells. In other words, when specific binding agents bind to both targets, they will act as a TNFRSF receptor agonist.

[0226] 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 a reference amino acid sequence of NP_001233.1 and is encoded by a reference nucleotide sequence of NM_001242.4. CD40 (TNFRSF5: gene ID 958) has a reference amino acid sequence of NP_001241.1 and is encoded by a reference nucleotide sequence of NM_001250.5. EDA2R (TNFRSF27: gene ID 60401) has a reference amino acid sequence of NP_001186616.1 and is encoded by a reference nucleotide sequence of NM_001199687.2. EDAR (gene ID 10913) has a reference amino acid sequence of NP_071731.1 and is encoded by a reference nucleotide sequence of NM_022336.3. FAS (TNFRSF6: gene ID 355) has a reference amino acid sequence of NP_000034.1 and is encoded by a reference nucleotide sequence of NM_000043.5. LTBR (TNFRSF3: gene ID 4055) has a reference amino acid sequence of NP_001257916.1 and is encoded by a reference nucleotide sequence of NM_001270987.1. RELT (TNFRSF19L: gene ID 84957) has a reference amino acid sequence of NP_116260.2 and is encoded by a reference nucleotide sequence of NM_032871.3. TNFRSF1A (gene ID 7132) has a reference amino acid sequence of NP_001056.1 and is encoded by a reference nucleotide sequence of NM_001065.3. TNFRSF1B (gene ID 7133) has a reference amino acid sequence of NP_001057.1 and can be encoded by a reference nucleotide sequence of NM_001066.2.

[0228] TNFRSF4 (gene ID 7293) has the reference amino acid sequence NP_003318 and is encoded by the reference nucleotide sequence NM_003327. TNFRSF6B (gene ID 8771) has the reference amino acid sequence NP_003814.1 and is encoded by the reference nucleotide sequence NM_003823.3. TNFRSF8 (gene ID 943) has the reference amino acid sequence NP_001234.3 and is encoded by the reference nucleotide sequence NM_001243.4. TNFRSF10A (gene ID 8797) has the reference amino acid sequence NP_003835.3 and is encoded by the reference nucleotide sequence NM_003844.3. TNFRSF10B (gene ID 8795) has a reference amino acid sequence of NP_003833.4 and is encoded by a reference nucleotide sequence of NM_003842.4. TNFRSF10C (gene ID 8794) has a reference amino acid sequence of NP_003832.2 and is encoded by a reference nucleotide sequence of NM_003841.4. TNFRSF10D (gene ID 8793) has a reference amino acid sequence of NP_003831.2 and is encoded by a reference nucleotide sequence of NM_003840.4. TNFRSF11A (gene ID 8792) has a reference amino acid sequence of XP_011524547.1 and is encoded by a reference nucleotide sequence of XM_11526245.2. TNFRSF11B (gene ID 4982) has a reference amino acid sequence of NP_002537.3 and is encoded by a reference nucleotide sequence of NM_002546.3. TNFRSF12A (gene ID 51330) has a reference amino acid sequence of NP_057723.1 and is encoded by a reference nucleotide sequence of NM_016639.2. TNFRSF13B (gene ID 23495) has a reference amino acid sequence of NP_0036584.1 and is encoded by a reference nucleotide sequence of NM_012452.2. TNFRSF13C (gene ID 115650) has a reference amino acid sequence of NP_443177.1 and is encoded by a reference nucleotide sequence of NM_052945.3. TNFRSF14 (gene ID 8764) has a reference amino acid sequence of NP_001284534.1 and is encoded by a reference nucleotide sequence of NM_001297605.1. TNFRSF17 (gene ID 608) has a reference amino acid sequence of NP_001183.2 and is encoded by a reference nucleotide sequence of NM_001192.2.TNFRSF18 (gene ID 8784) has a reference amino acid sequence of NP_004195.2 and is encoded by a reference nucleotide sequence of NM_004186.1. TNFRSF19 (gene ID 55504) has a reference amino acid sequence of NP_001191387.1 and is encoded by a reference nucleotide sequence of NM_001204458.1. TNFRSF21 (gene ID 27242) has a reference amino acid sequence of NP_055267.1 and is encoded by a reference nucleotide sequence of NM_014452.4. TNFRSF25 (DR3: gene ID 8718), which binds to the ligand TNFSF15 (TL1A), has a reference amino acid sequence of NP_001034753.1 and is encoded by a reference nucleotide sequence of NM_001039664.1.

[0229] Alternatively, the immune cell antigen bound to the second antigen-binding site can be a molecule other than a TNFRSF member that has immune system regulatory functions, such as immune co-stimulatory molecules or inhibitory checkpoint molecules. Examples of such immunomodulatory molecules include ICOS (CD278), LAG3, PD1, PD-L1, PD-L2, B7H3, B7H4, CTLA4, TIGIT, BTLA, HVEM, T cell immunoglobulins, mucin-domain containing-3 (TIM-3), CD47, CD73, A2aR, CD200, CD200R, community-stimulating factor 1 receptor (CSF-1R), VISTA CD28, CD80, LLT1, lactose lectin 9, NKG2A, NKG2D, and KIR.

[0230] The immune cells that bear immune cell antigens can belong to any immune cell subset and can be T cells, tumor-infiltrating leukocytes (TILs), bone marrow lineage cells such as antigen-presenting cells (APCs), NK cells, and / or B cells. When the immune cell antigen is a TNFRSF receptor, the immune cells that bear the TNFRSF receptor are preferably T cells.

[0231] Arbitrarily, the second antigen-binding site can bind to disease antigens as described above. While not wishing to be bound by theory, it is believed that specific binding of the object to CD137 and disease antigens leads to T cell activation near the disease site. Activated T cells may then initiate, promote, or participate in immune responses, for example, immune responses against pathogens or cancer cells. Chen and Mellman (2013) provide an overview of the role of the immune system in recognizing and eradicating cancer cells.

[0232] In a preferred embodiment, the disease antigen is a tumor antigen. Tumor antigens are antigens primarily found in the tumor environment and are not ubiquitous elsewhere in the individual. For example, tumor antigens may be present on the surface of tumor cells or in other stromal cells within the tumor microenvironment or in biofluids near the tumor. Tumor antigens thus serve as an indicator of the location of tumor cells within the individual.

[0233] In some specific cases, tumor antigens may be antigens located on the surface of cancer cells. Preferably, tumor antigens on tumor cells are upregulated or overexpressed, while corresponding normal stromal cells from the same tissue without tumors do not express tumor antigens in large quantities.

[0234] In some specific cases, tumor antigens on stromal cells in the tumor microenvironment are upregulated or overexpressed compared to stromal cells in corresponding normal tissues without tumors.

[0235] Better tumor antigens are present on the cell surface and are not rapidly internalized.

[0236] Tumor antigens suitable for targeting by this specific binding agent can be identified using methods well known in the art. For example, a specific binding agent targeting the CD137 receptor and a tumor antigen can be used in an assay that co-cultures CD137-expressing cells and tumor antigen-expressing cells, and, for example, measures the activation of CD137-expressing cells by T-cell activation assays, proliferation assays, or cytotoxicity assays.

[0237] Cell surface tumor antigens may be tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).

[0238] Tumor antigens expressed by cancer cells may include, for example, cancer testis (CT) antigens encoded by cancer germline genes, such as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-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), and 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 and their immune fragments or variants (Simpson et al., 2005; Gure et al., 2005; Velazquez et al., 2007; Andrade et al., 2008; Tinguely et al., 2008; Napoletano et al., 2008).

[0239] Other cell surface tumor antigens include, for example, AFP, αvβ3 (vitronectin receptor), αvβ6, B cell maturation agent (BCMA), CA125 (MUC16), CD4, CD20, CD22, CD33, CD52, CD56, CD66e, CD80, CD140b, CD227 (MUC1), EGFR (HER1), EpCAM, GD3 ganglioside, HER2, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), CD5, CD19, CD21, CD25, CD37, CD30, CD33, CD45, HLA-DR, anti-idiotype, carcinoembryonic antigen (CEA), and cell adhesion molecules such as carcinoembryonic antigen-associated molecule 5. (CEACAM5), TAG-72, folate-binding protein, A33, G250, ferritin, glycolipids such as gangliosides, carbohydrates such as CA-125, IL-2 receptor, fibroblast activation protein (FAP), IGF1R, B7H3, B7H4, PD-L1, CD200, EphA2, and mesothelin or its variants. These and other cell surface tumor antigens are described in Carter et al., 2004; Scott and Renner, 2001; Cheever et al., 2009; Tai and Anderson, 2015; and Podojil and Miller, 2017.

[0240] Other tumor antigens include out-of-frame peptide-MHC complexes generated by non-AUG translation initiation mechanisms utilized by "stressed" cancer cells (Malarkannan et al., 1999).

[0241] Other tumor antigens include peptide-MHC complexes on the surface of tumor cells or cells in the tumor microenvironment, wherein the peptide-MHC complex contains a tumor-specific neoantigen peptide fragment of a mutated intracellular tumor antigen, and wherein the neoantigen peptide carries one or more tumor-specific mutations (Gubin et al., 2015). Other tumor antigens are well known in the art (see, for example, WO00 / 20581; Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge). The sequences of such tumor antigens are readily available from public databases but can also be found in WO1992 / 020356 A1, WO1994 / 005304 A1, WO1994 / 023031 A1, WO1995 / 020974 A1, WO1995 / 023874 A1 and WO1996 / 026214 A1.

[0242] Preferred tumor antigens include HER2, FAP, EpCAM, CEACAM5, CD20, CD73, PSMA, mesothelin, EphA2, IGF1R, CD200, αvβ6, BCMA, PD-L1, B7H3, B7H4, and EGFR.

[0243] In a more specific example, the tumor antigen is mesothelin (MSLN).

[0244] In any preferred specific case, the tumor antigen is PD-L1.

[0245] HER2 (ERBB2; gene ID 2064) can have a reference amino acid sequence of NP_001005862.1 and can be encoded by a reference nucleotide sequence of NM_001005862.2. FAP (gene ID 2191) can have a reference amino acid sequence of NP_001278736.1 and can be encoded by a reference nucleotide sequence of NM_001291807.1. EpCAM (gene ID 4072) can have a reference amino acid sequence of NP_002345.2 and can be encoded by a reference nucleotide sequence of NM_002354.2.

[0246] CEACAM5 (gene ID 1048) can have a reference amino acid sequence of NP_001278413.1 and can be encoded by a reference nucleotide sequence of NM_001291484.2. CD20 (MS4A1; gene ID 931) can have a reference amino acid sequence of NP_068769.2 and can be encoded by a reference nucleotide sequence of NM_021950.3. CD73 (NT5E; gene ID 4907) can have a reference amino acid sequence of NP_001191742.1 and can be encoded by a reference nucleotide sequence of NM_001204813.1. PSMA (FOLH1; gene ID 2346) can have a reference amino acid sequence of NP_001014986.1 and can be encoded by a reference nucleotide sequence of NM_001014986.1. Mesothelin (MSLN; gene ID 10232) can have a reference amino acid sequence of NP_001170826.1 and can be encoded by a reference nucleotide sequence of NM_001177355.2. EphA2 (gene ID 1969) can have a reference amino acid sequence of NP_001316019.1 and can be encoded by a reference nucleotide sequence of NM_001329090.1. IGF1R (gene ID 3480) can have a reference amino acid sequence of NP_000866.1 and can be encoded by a reference nucleotide sequence of NM_000875.4. CD200 (gene ID 4345) can have a reference amino acid sequence of NP_001004196.2 and can be encoded by a reference nucleotide sequence of NM_001004196.3. αvβ6 is a heterodimer composed of integrin subunits αV and β6. Integrin subunit αV (ITGAV; gene ID 3685) can have a reference amino acid sequence of NP_001138471.1 and can be encoded by a reference nucleotide sequence of NM_001144999.2. Integrin subunit β6 (ITGB6; gene ID 3694) can have a reference amino acid sequence of NP_000879.2 and can be encoded by a reference nucleotide sequence of NM_000888.4. BCMA (TNFRSF17; gene ID 608) can have a reference amino acid sequence of NP_001183.2 and can be encoded by a reference nucleotide sequence of NM_001192.2. PD-L1 (CD274; gene ID 29126) can have a reference amino acid sequence of NP_001254635.1 and can be encoded by a reference nucleotide sequence of NM_001267706.1. B7H3 (CD276; gene ID 80381) can have a reference amino acid sequence of NP_001019907.1 and can be encoded by a reference nucleotide sequence of NM_001024736.1.B7H4 (VTCN1; gene ID 79679) can have a reference amino acid sequence of NP_001240778.1 and can be encoded by a reference nucleotide sequence of NM_001253849.1. EGFR (gene ID 1956) can have a reference amino acid sequence of NP_001333826.1 and can be encoded by a reference nucleotide sequence of NM_001346897.1.

[0247] In other specific examples, tumor antigens can be soluble tumor antigens, such as growth factors produced by or in response to cancer cells. Soluble factors in biofluids near the tumor can be upregulated or overexpressed. Soluble tumor antigens can be polymers, such as dimers or trimers. The concentration of soluble tumor antigens present at the tumor site or tumor microenvironment may be higher than elsewhere in the individual's body. Bhome et al. (2015) describe the tumor microenvironment and associated soluble tumor antigens in more detail.

[0248] 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.

[0249] VEGF (VEGFA; gene ID 7422) has a reference amino acid sequence of NP_001020537.2 and is encoded by a reference nucleotide sequence of NM_001025366.2. HGF (gene ID 3082) has a reference amino acid sequence of NP_000592.3 and is encoded by a reference nucleotide sequence of NM_000601.5. SDF1 (CXCL12; gene ID 6387) has a reference amino acid sequence of NP_000600.1 and is encoded by a reference nucleotide sequence of NM_000609.6. TGF-β-1 (TGFB1; gene ID 7040) has a reference amino acid sequence of NP_000651.3 and is encoded by a reference nucleotide sequence of NM_000660.6. TGF-β-2 (TGFB2; gene ID 7042) can have a reference amino acid sequence of NP_001129071.1 and can be encoded by a reference nucleotide sequence of NM_001135599.3. TGF-β-3 (TGFB3; gene ID 7043) can have a reference amino acid sequence of NP_001316867.1 and can be encoded by a reference nucleotide sequence of NM_001329938.1. TGF-β-4 (LEFTY2; gene ID 7044) can have a reference amino acid sequence of NP_001165896.1 and can be encoded by a reference nucleotide sequence of NM_001172425.2.

[0250] In any preferred specific example, the disease antigen is the pathogenic antigen.

[0251] Specific binding agents that activate immune cells, such as T cells, NK cells, and / or macrophages, near the site of an infectious disease are expected to be used to treat infectious diseases. The infectious disease can be acute or persistent, but persistent is preferred.

[0252] Pathogenic antigens are preferably antigens expressed by human pathogens, such as viral, bacterial, fungal, or parasitic antigens (e.g., protozoan antigens), with viral or bacterial antigens being more preferred. Pathogenic antigens are those primarily present in the pathogen or near the site of the infectious disease, and are not ubiquitous in other parts of the individual.

[0253] For example, a pathogenic antigen can be an antigen present on the surface of a virus, bacteria, fungus, or parasite, or a soluble antigen expressed by a virus, bacteria, fungus, or parasite. The virus, bacteria, fungus, or parasite can be any virus, bacteria, fungus, or parasite mentioned elsewhere in this document.

[0254] When the pathogenic antigen is a soluble antigen, it can be upregulated or overexpressed in biological fluids near the site of the infectious disease. For example, the concentration of soluble pathogenic antigens present at or near the site of the infectious disease may be higher than in other parts of the individual's body. Soluble pathogenic antigens can be polymers, such as dimers or trimers.

[0255] Pathogenic antigens suitable for targeting by this specific binding agent can be identified using methods well known in the art. For example, a specific binding agent targeting CD137 and a pathogenic antigen can be used in an analysis that co-cultures CD137-expressing cells with a pathogen or pathogenic antigen, and, for example, to determine the activation of OX40-expressing cells by T-cell activation assays, proliferation assays, or cytotoxicity assays.

[0256] Numerous pathogenic antigens suitable for targeting with this specific binding agent are known in the art and can be selected by those skilled in the art based on the infectious disease to be treated. Examples of viral antigens include proteins p24, gp120, and gp41 expressed by human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg) expressed by hepatitis B virus (HBV), and hemagglutinin and neuraminidase expressed by influenza virus. Examples of bacterial antigens include Rv1733, Rv2389, and Rv2435n expressed by Mycobacterium tuberculosis.

[0257] The specific binding compound may also include variants of the first or second sequence disclosed herein, ABCD or EF structural loop sequences, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain and / or heavy chain sequences. Suitable variants can be obtained by sequence alteration or mutation and screening methods. In a preferred embodiment, a specific binding compound containing one or more variant sequences retains one or more multifunctional characteristics of the parental specific binding compound, such as the binding specificity and / or binding affinity for CD137. For example, a specific binding compound containing one or more variant sequences preferably binds to CD137 with the same affinity as the (parental) specific binding compound, or with a higher affinity than the (parental) specific binding compound. The parental specific binding compound is a specific binding compound that does not contain amino acid substitutions, deletions and / or insertions that have been incorporated into the variant specific binding compound.

[0258] For example, a specific binding object may comprise a first, second, or third sequence, an AB, CD, or EF structural ring sequence, a CH3 domain, a CH2 domain, an Fcab, CDR, VH domain, a VL domain, a light chain, and / or a heavy chain sequence, which 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 with the first, second, or third sequence, the AB, CD, or EF structural ring sequence, the CH3 domain, the CH2 domain, the Fcab, CDR, the VH domain, the VL domain, the light chain, or the heavy chain sequence disclosed herein.

[0259] In a preferred embodiment, the specific binding object has or includes a CH3 domain sequence, which is associated with a sequence identification number: [twenty one] [、30] [、48] [、102] [、75] [、39] [、57] [、66] [、84] [、93] [、175] [、139] [、130] [、148] [、121] [、157] [、165] [Or 112] The CH3 domain sequence listed herein 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.

[0260] In another preferred embodiment, the specific binding object has or includes a CH2 domain sequence, which is associated with a sequence identification number: [5] [or 6] The CH2 domain sequences listed have 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.

[0261] In another specific example, the specific binding object has a sequence, comprises a sequence, or consists of a sequence, the sequence being identified by a sequence number: [twenty three] [、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] The Fcab sequences listed have 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.

[0262] Sequence identity is generally defined using the GAP algorithm (Wisconsin GCG software package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithms to arrange two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used, with a gap creation penalty of 12 and a gap expansion penalty of 4. While using GAP may be preferable, other algorithms such as BLAST (using the method of Altschul et al. (1990)), FASTA (using the method of Pearson and Lipman (1988)), the Smith-Waterman algorithm (Smith and Waterman (1981)), or the earlier TBLASTN program by Altschul et al. (1990) can also be used, generally with default parameters. Specifically, the psi-Blast algorithm can be used.

[0263] A specific binding object may comprise a first, second, or third sequence, an AB, CD, or EF structural ring sequence, a CH3 domain, a CH2 domain, an Fcab, CDR, VH domain, VL domain, a light chain, or a heavy chain sequence, which, compared with the first, second, or third sequence, AB, CD, or EF structural ring sequence, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain, or heavy chain sequence disclosed herein, has one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of an amino acid residue), preferably 20 or fewer alterations, 15 or fewer alterations, 10 or fewer alterations, 5 or fewer alterations, 4 or fewer alterations, 3 or fewer alterations, 2 or fewer alterations, or 1 alteration.

[0264] In a preferred embodiment, the specific binding object may include a CH3 domain sequence, which is associated with a sequence identification number: [twenty one] [、30] [、48] [、102] [、75] [、39] [、57] [、66] [、84] [、93] [、175] [、139] [、130] [、148] [、121] [、157] [、165] [or 112] Compared to the CH3 domain sequence listed herein, it has one or more amino acid sequence alterations (addition, deletion, substitution and / or insertion of an amino acid residue), preferably 20 or fewer alterations, 15 or fewer alterations, 10 or fewer alterations, 5 or fewer alterations, 4 or fewer alterations, 3 or fewer alterations, 2 or fewer alterations or 1 alteration.

[0265] In another preferred embodiment, the specific binding object may include a CH2 domain sequence, which is associated with a sequence identification number: [5] Compared to the CH2 domain sequence listed in [or 6], it has one or more amino acid sequence alterations (addition, deletion, substitution and / or insertion of an amino acid residue), preferably 20 or fewer alterations, 15 or fewer alterations, 10 or fewer alterations, 5 or fewer alterations, 4 or fewer alterations, 3 or fewer alterations, 2 or fewer alterations or 1 alteration.

[0266] In another preferred embodiment, the specificity binding object comprises or consists of a sequence, the sequence being associated with a sequence identification number: [twenty three] [、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] Compared to the Fcab sequence listed herein, it has one or more amino acid sequence alterations (addition, deletion, substitution and / or insertion of an amino acid residue), preferably 40 or fewer alterations, 30 or fewer alterations, 20 or fewer alterations, 15 or fewer alterations, 10 or fewer alterations, 5 or fewer alterations, 4 or fewer alterations, 3 or fewer alterations, 2 or fewer alterations or 1 alteration.

[0267] In cases where the specific binding compound is comprised of a variant of the disclosed first sequence, AB ring sequence, CH3 domain, Fcab, or heavy chain sequence, the specific binding compound preferably retains the sequence PPY between positions 11 and 19, and more preferably between positions 15 and 17, of the CH3 domain. Furthermore, the specific binding compound preferably retains an insertion, preferably five amino acid insertions, between positions 16 and 17 of the CH3 domain. In another preferred embodiment, the specific binding compound preferably retains the sequence at positions 97 and 98 of the CH3 domain.

[0268] Specifically, the specific binding compound may be (or the antibody molecule may contain) a variant of the specific binding compound FS22-053, wherein the variant: (i) Compared with the sequence of the specific binding object FS22-053 disclosed herein, it includes one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of an amino acid residue), preferably 20 or fewer alterations, 15 or fewer alterations, 10 or fewer alterations, 5 or fewer alterations, 4 or fewer alterations, 3 or fewer alterations, 2 or fewer alterations, or 1 alteration; or (ii) Having a sequence identity of 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% with the specific binding object FS22-053 disclosed herein; and The specific binding compound or antibody molecule includes the sequence PPY between positions 15 and 17 of the CH3 domain of the specific binding compound or antibody molecule, and selectively includes five amino acids inserted between positions 16 and 17; and The residue number is based on the IMGT residue numbering method.

[0269] Furthermore, or alternatively, in cases where the specific binding compound is comprised of variants of the CH3 domain, CH2 and CH3 domains, Fcab, light chain, or heavy chain sequence disclosed herein, the variant preferably does not contain any amino acid alterations within the first, second, and third sequences of the AB, CD, and EF structural rings located within the CH3 domain of the specific binding compound. For example, the variant may not contain any amino acid alterations within the AB, CD, and EF structural rings of the CH3 domain of the specific binding compound.

[0270] In preferred embodiments where one or more amino acid systems are substituted by another amino acid, such substitutions may be, for example, retained substitutions as shown in the table below. In some embodiments, amino acid systems of the same category in the middle column substitute for each other, i.e., for example, a nonpolar amino acid system is substituted by another nonpolar amino acid. In some embodiments, amino acid systems in the same row in the rightmost column substitute for each other.

[0271] In some specific instances, the substitution can be functionally preservative. That is, in some specific instances, one or more functional properties (such as binding affinity) of a specific bond containing the substitution may be unaffected (or substantially unaffected) by the substitution compared to an equivalent unsubstituted specific bond.

[0272] It is also anticipated that a specific binding object includes a CD137 antigen binding site located in a constant domain of the specific binding object, preferably within the CH3 domain, and that it competes with the specific binding object of the present invention for binding to CD137, or binds to the same epitope on CD137 as the specific binding object of the present invention. Methods for determining competition between two specific binding objects for an antigen are known in the art. For example, competition between two specific binding objects for binding to an antigen can be determined using surface plasma resonance, such as Biacore. Methods for mapping the epitope bound by a specific binding object are also known in the art.

[0273] In some specific examples, the specific binding object may not contain a CDR-based antigen binding address.

[0274] Specifically, the specific binding object may not contain an antigen-binding address based on CDR- of PD-L1.

[0275] In addition, or alternatively, the specific binding object may not contain an antigen-binding site based on a CDR- of mesothelin (MSLN).

[0276] For example, the specific binding object may not contain an antigen-binding address based on CDR- that binds to PD-L1 or MSLN, wherein the specific binding object includes a site located at the specific binding object. [FS22-53-008] or The first, second, and third sequences of the AB, CD, and EF structural rings in the CH3 domain of [FS22-172-003], specifically binding objects. [FS22-53-008] or The complete AB and EF structural ring sequences of the CH3 domain of [FS22-172-003], and / or specific binding objects. [FS22-53-008] or The CH3 domain sequence of [FS22-172-003].

[0277] Specifically, the specific binding object may not contain the CDRs, VH and / or VL domains, and / or heavy and / or light chain sequences of FS22-172-003-AA / E12v2 and FS22-053-008-AA / E12v2 listed below. [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 [FS22-172-003-AA / E12v2] [and FS22-053-008-AA / E12v2 VH] [domain] EVQLVQSGAEVKRPGASVKVSCKAS [GYPFTSYG] ISWVRQAPGQG LEWMGW [ISAYSGGT] NYAQKLQGRVTMTTDTSSTAYMELRSLRS DDTAVYYC [ARDLFPTIFGVSYYYY] WGQGTLVTVSS [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 [FS22-172-003-AA / E12v2] [and FS22-053-008-AA / E12v2 VL] [Region] DIQMTQSPSTLSASVRDRVIITCRAS [QSIGNR] LAWYQHKPGKAPKL LIY [EAS] TSETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC [QQSY] [STPYT] FGQGTKLEIK [Heavy chain FS22-172-003-AA / E12v2] EVQLVQSGAEVKRPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISAYSGGTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLFPTIFGVSYYYYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA , ] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELPYIIPPYNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVGADRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG​ [Light chain FS22-172-003-AA / E12v2] DIQMTQSPSTLSASVRDRVIITC [RAS, QSIGNR , LA] WYQHKPGKAPKLLIY EAS , ] [TSET] GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPYT , ] FGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Heavy chain FS22-053-008-AA / E12v2] EVQLVQSGAEVKRPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISAYSGGTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLFPTIFGVSYYYYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA , ] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG [Light chain FS22-053-008-AA / E12v2] ​​​DIQMTQSPSTLSASVRDRVIITC [RAS, QSIGNR , LA] WYQHKPGKAPKLLIY [ EAS , ] [TSET] GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC [ QQSYSTPYT , ] FGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0278] In addition, or alternatively, the specific binding compound may not contain the CDRs and / or VH and / or VL domains of the anti-MSLN antibody FS28-256-271 listed below. [FS28-256-271] [VH] [domain] [CDRs] HCDR1 (AA) (IMGT) GFTFTHTY HCDR1 (AA) (Kabat) HTYMS HCDR2 (AA) (IMGT) ISPTYSTT HCDR2 (AA) Kabat) AISPTYSTTNYADSVKG HCDR3 (AA) (IMGT) ARYNAYHAALDY HCDR3 (AA) (Kabat) YNAYHAALDY [FS28-256-271] [] [VH] [domain] EVQLLESGGGLVQPGGSLRLSCAAS [GFTFT, HTY , ] [MS] WVRQAPGKGLEWVS [N, ISPTYSTT , NYADSVKG] RFTISRDNNKNTLYLQMNSLRAEDTAVYYC [AR, YNAYHAALDY , ] WGQGTLVTVSS [FS28 - 256 - 271 VL] [Domain] [CDRs] LCDR1 (AA) (IMGT) QSVSSSY LCDR1 (AA) (Kabat) RASQSVSSSYLA LCDR2 (AA) (IMGT) GAS LCDR2 (AA) (Kabat) GASSRAT LCDR3 (AA) (IMGT) QQTVPYPYT LCDR3 (AA) (Kabat) QQTVPYPYT[[ID=?]] [FS28 - 256 - 271] [] [VL] [Domain] EIVLTQSPGTLSLSPGERATLSC [RAS, QSVSSSY , LA] WYQQKPGQAPRLLIY GAS , ] [SRAT] GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQTVPYPYT , ] FGQGTKVEIK

[0279] Note: There seems to be an issue with the "[[ID=?]]" tag in the original. It's likely a formatting error in the input. I've left it as is in the translation for consistency with the original.​This specific binding compound can be conjugated to a bioactive molecule or a detectable label. In this case, the specific binding compound can be referred to as a conjugate. Such conjugates have applications in the treatment of diseases as described herein.

[0280] For example, bioactive molecules can be immune system regulators, such as cytokines, preferably human cytokines. For example, cytokines can be those that stimulate T cell activation and / or proliferation. Examples of cytokines suitable for use in complex specific binding compounds include IL-2, IL-10, IL-12, IL-15, IL-21, GM-CSF, and IFN-γ.

[0281] Wherever possible, bioactive molecules can act as ligand traps, such as ligand traps for cytokines like TGF-β or IL-6.

[0282] Suitable detectable markers that can be combined with specific binding objects are known in the art and include radioactive isotopes such as iodine-125, iodine-131, yttrium-90, indium-111, and uranium-99; fluorescent dyes such as luciferin, rose red, phycoerythrin, Texas Red, and cyanine dye derivatives such as Cy7 and Alexa750; chromogenic dyes such as diaminobenzidine; latex beads; enzyme markers such as horseradish peroxidase; phosphors or laser dyes with spectrally isolated absorption or emission characteristics; and chemical components such as biotin, which can be detected by binding to specific homologous detectable components, such as labeled avidin.

[0283] This specific binding compound can be complexed with a bioactive molecule or a detectable label via any suitable covalent or non-covalent bond, such as a disulfide bond or a peptide bond. In the case where the bioactive molecule is a cytokine, the cytokine can be complexed with the specific binding compound via a peptide bond connector. Suitable peptide bond connectors 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.

[0284] In some specific examples, bioactive molecules can be complexed with the specific binding object via cleavable linkers. The linker allows the bioactive molecule to be released from the specific binding object at the therapeutic site. Linkers may include amide bonds (such as peptide linkers), disulfide bonds, or hydrazones. For example, peptide linkers can be cleaved by site-specific proteases, disulfide bonds can be cleaved by a cytosol-reducing environment, and hydrazones can be cleaved by acid-mediated hydrolysis.

[0285] The complex can be a fusion protein comprising a specific binding element and a bioactive molecule. In this case, the bioactive molecule can be complexed with the specific binding element via a peptide linker or peptide bond. When the specific binding element is a multi-chain molecule, such as being or containing Fcab or mAb2, the bioactive molecule can be complexed with one or more chains of the specific binding element. For example, the bioactive molecule can be complexed with one or two heavy chains of the mAb2 molecule. Fusion proteins offer the advantages of easy production and purification, facilitating the production of clinical-grade materials.

[0286] This invention also provides isolated nucleic acid molecules or multiple nucleic acid molecules encoding specific binding objects of this invention. The preparation of this nucleic acid molecule using methods well-known in this art is not difficult for those skilled in the art.

[0287] In a preferred embodiment, the nucleic acid molecule encodes the CH3 domain of the following specific binding compounds: [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], preferably [FS22-172-003] [FS22-172-002] [Or FS22-172-004], even better is... [FS22-172-003]

[0288] In any preferred specific example, the nucleic acid molecule encodes the CH3 domain of the following specific binding targets: [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], preferably [FS22-053-008] [FS22-053-009] [FS22-053-011] [Or FS22-053-017], even better is... [FS22-053-008]

[0289] This article describes the CH3 domain sequence of such specific binding objects.

[0290] For example, a nucleic acid molecule may encode the CH3 domain of the following specific binding compounds: (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], listed in the sequence identification number respectively: [twenty two] [、]

[31] [、]

[49] [、]

[0103] [、]

[76] [、]

[40] [、]

[58] [、]

[67] [、]

[85] [、]

[94] [and]

[0176] Inside; or (ii) [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] [or FS22-172], listed in the sequence identification number respectively:

[0140] [、131] [、149] [、122] [、158] [、166] [and 113] inside.

[0291] In a preferred embodiment, the nucleic acid molecule encodes the following specific binding objects: [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], preferably [FS22-172-003] [FS22-172-002] [Or FS22-172-004], even better is... [FS22-172-003]

[0292] In a preferred specific example, the nucleic acid molecule encodes the following specific binding targets: [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], preferably [FS22-053-008] [FS22-053-009] [FS22-053-011] [,] or [FS22-053-017], even better [FS22-053-008]

[0293] For example, a nucleic acid molecule encodes the following specific binding objects: (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], listed in the sequence identification number respectively: [twenty four] [、]

[33] [、]

[51] [、]

[0105] [、]

[78] [、]

[42] [、]

[60] [、]

[69] [、]

[87] [、]

[96] [and]

[0177] Inside; or (ii) [FS22-172-003] [FS22-172-002] [FS22-172-004] [FS22-172-001] [FS22-172-005] [FS22-172-006] [or FS22-172], listed in the sequence identification number respectively:

[0142] [、133] [、151] [、124] [、160] [、168] [and 115] inside.

[0294] An isolated nucleic acid molecule can be used to represent the specific binding object of the present invention. Generally, nucleic acids in the form of recombinant vectors are provided. Another aspect of the invention thus provides vectors comprising the nucleic acids described above. Suitable vectors can be selected or constructed, containing suitable regulatory sequences, including promoter sequences, terminator fragments, polyadenylated sequences, enhancer sequences, marker genes, and other suitable sequences. Preferably, the vector contains suitable regulatory sequences to drive the expression of the nucleic acid within host cells. The vector can be a suitable plastid, virus such as a bacteriophage, or phagemid.

[0295] The nucleic acid molecules or vectors described herein can be introduced into host cells. The techniques for introducing nucleic acids or vectors into host cells are established in this art and any suitable techniques can be used. A range of host cells suitable for producing recombinant specific binding materials are known in this art, including bacterial, yeast, insect, or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NSO, or HEK cells, for example, HEK293 cells. The optimal host cell is CHO cells.

[0296] Another aspect of the present invention provides a method for producing the specific binding compound of the present invention, comprising expressing nucleic acid encoding the specific binding compound within a host cell and selectively isolating and / or purifying the specific binding compound 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 compound. Techniques for purifying recombinant specific binding compounds are well known in the art and include, for example, HPLC, FPLC, or affinity chromatography, using protein A or protein L. In some specific examples, purification may be performed using an affinity tag on the specific binding compound. The method may also comprise formulation of the specific binding compound into a pharmaceutical composition, selectively adding a pharmaceutically acceptable excipient or other substances as described below.

[0297] As explained above, CD137 is expressed on cells of the immune system, including CD8+ T cells, CD4+ T cells, Treg cells, B cells, NK cells, NKT cells, dendritic cells, and tumor-infiltrating lymphocytes (TILs). Specifically, CD137 activation has been shown to play a role in enhancing the proliferation, survival, and cytotoxic effector function of CD8+ T cells, as well as in CD8+ T cell differentiation and the maintenance of memory CD8+ T cells. While CD137 is expressed at a lower level on CD4+ T cells than on CD8+ T cells, it has also been shown to be involved in inducing the proliferation and activation of some CD4+ T cell subsets. CD137 activation has also been shown to enhance NK cell-mediated ADCC, as well as B cell proliferation, survival, and cytokine production.

[0298] Given the immune-enhancing activity of CD137, research has been conducted on CD137 agonist molecules for the treatment of cancer and chronic infections.

[0299] The specific binding material described herein can therefore be used for therapeutic applications, particularly cancer treatment. Furthermore, it is anticipated that this specific binding material can be used to treat infectious diseases, such as persistent infections.

[0300] The specific combination of the objects described herein can therefore be used in a method of treating the human or animal body. Related aspects of the invention are provided; (i) The specific binding material described herein is intended for use as a pharmaceutical agent. (ii) The specific combination object described herein is intended for use in a method of treating a disease or disorder. (iii) The specific combination of the article described herein is used in the manufacture of a pharmaceutical agent for the treatment of a disease or disorder; and, (iv) A method for treating a disease or disorder in an individual, wherein the method comprises administering a therapeutically effective amount of a specific binding object as described herein to the individual.

[0301] An individual can be a patient, preferably a human patient.

[0302] Treatment can be any treatment or therapy that achieves some desired therapeutic effect, such as inhibiting or delaying the progression of a condition, including slowing the rate of progression, stopping the rate of progression, improving the condition, curing or alleviating the condition (whether partially or completely), preventing, improving, delaying, mitigating or stopping or prolonging the survival of an individual or patient beyond what would have been expected without treatment.

[0303] This also includes treatment as a preventative measure (i.e., prophylaxis). For example, individuals suspected of having or at risk of developing or recurring a disease such as cancer can be treated as described in this article. This treatment can prevent or delay the onset or recurrence of the disease in an individual.

[0304] A treatment method as described herein may include administering at least one additional treatment to the individual, in addition to the specific binding agent. The specific binding agent described herein can therefore be administered to the individual alone or in combination with one or more other treatments. When the specific binding agent is combined with another treatment and administered to the individual, the additional treatment may be administered to the individual simultaneously, sequentially, or separately from the administration of the specific binding agent. When the additional treatment is administered simultaneously with the specific binding agent, the specific binding agent and the additional treatment may be administered to the individual as a combined preparation. For example, the additional treatment may be a known therapy or therapeutic agent for the disease to be treated.

[0305] When a specific conjugate can be administered alone, it is typically administered as a pharmaceutical composition that may contain at least one component other than the specific conjugate. Another aspect of the invention thus provides a pharmaceutical composition comprising the specific conjugate as described herein. A method of comprising incorporating a specific conjugate into a pharmaceutical composition is also provided.

[0306] Pharmaceutical compositions, in addition to specific binding agents, may contain a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other substance well known to those skilled in the art. When used herein, the term "pharmaceuticalally acceptable" refers to a compound, substance, composition, and / or dosage form that, to a sound medical judgment, is suitable for contact with the tissues of an individual (e.g., a human) without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Each carrier, excipient, etc., must also be "acceptable" in the sense of compatibility with the other components of the formulation. The exact nature of the carrier or other substance will depend on the route of administration, which may be via perfusion, injection, or any other suitable route, as described below.

[0307] For non-enteral administration, such as subcutaneous or intravenous administration, such as by injection, pharmaceutical compositions containing the specific conjugate can be in a non-enteral acceptable aqueous solution form that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can use, for example, isotonic carriers such as sodium chloride injection, Ringer's solution, and lactated Ringer's solution to prepare suitable solutions. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be used as needed, including buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexahydroquinone quaternary ammonium chloride; phenylenediamine dimethyl ammonium chloride; benzenesulfonium chloride). chloride); phenol, butanol, or benzyl alcohol; alkyl benzoates, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEENTM, PLURONICS™, or polyethylene glycol (PEG).

[0308] In some specific cases, specific binding agents in lyophilized form may be provided for reconstitution before administration. For example, lyophilized specific binding agents may be reconstituted with sterile water and mixed with saline solution before administration to an individual.

[0309] An "effective therapeutic dose" may be administered, which is sufficient to demonstrate benefit to the individual. The actual dose, rate, and timing of administration will depend on the nature and severity of the condition being treated, the specific individual being treated, the individual's clinical condition, the cause of the disorder, the site of delivery of the components, the type of specific binding agent, the method of administration, the schedule of administration, and other factors known to the physician. Prescribing treatment, such as determining the dosage, is within the responsibility of general practitioners and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated. Appropriate doses of immunoglobulins are well known in this art (Ledermann et al. (1991) Int. J. Cancer 47: 659-664; and Bagshawe et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). The specific doses shown herein or the specific doses suitable for the antibody molecule indicated in Physician's Desk Reference (2003) may be used. Regarding the antibody molecule, the therapeutically effective amount or appropriate dose of a specific binding agent can be determined by comparing its in vitro activity with its in vivo activity in animal models. Methods for extrapolating effective doses from mice and other test animals to humans are known. The exact dose will depend on several factors, including the size and location of the treatment area and the exact nature of the specific binding agent.

[0310] Typical immunoglobulin doses for systemic application range from 100 µg to 1 g, and for topical application range from 1 µg to 1 mg. A higher, faster-acting initial dose can be administered, followed by one or more lower doses. This is a single-treatment dose for adults; doses for children and infants can be adjusted proportionally, and other specific binding modalities can be adjusted in proportion to the molecular weight.

[0311] Treatment may be repeated daily, twice a week, weekly, or monthly, at the physician's discretion. An individual's treatment schedule may depend on the pharmacokinetics and pharmacodynamics of the specific binding agent, the route of administration, and the nature of the condition being treated.

[0312] Treatment can be administered periodically, with intervals ranging from approximately two weeks or longer, such as approximately three weeks or longer, approximately four weeks or longer, approximately once a month or longer, approximately five weeks or longer, or approximately six weeks or longer. For example, treatment can be administered every two to four weeks or every four to eight weeks. Appropriate preparations and routes of administration are as described above.

[0313] In a preferred embodiment, the specific binding object described herein can be used in a method for treating cancer.

[0314] Cancer is characterized by the abnormal proliferation of malignant cancer cells. In the context of specific types of cancer, such as breast cancer, this refers to the abnormal proliferation of malignant cells in the relevant tissue, such as breast tissue. A secondary cancer located in the breast but resulting from the abnormal proliferation of malignant cells in another tissue, such as ovarian tissue, will not be referred to as breast cancer but rather as ovarian cancer in this article.

[0315] Cancer can be primary or secondary. Therefore, the specific binding object described herein can be used as a method for treating cancer in an individual, wherein the cancer is a primary tumor and / or a metastatic tumor.

[0316] Cancerous tumors to be treated using the specific binding agents described herein may contain TILs expressing CD137, for example, on their cell surface. In one specific example, the tumor may have been identified as containing TILs expressing CD137. Methods for determining the expression of an antigen on a cell surface are known in the art and include, for example, flow cytometry.

[0317] For example, cancers to be treated using the specific binding agents described herein can be selected from the following groups: leukemias, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastoid leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphomas, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma; and solid cancers, such as sarcomas (e.g., soft tissue sarcoma) and skin cancers (e.g., Merkel cell carcinoma). Cancers that can cause various cancers include: melanoma, bladder cancer (such as urothelial carcinoma), brain cancer (such as glioblastoma multiforme), breast cancer, uterine / endometrial cancer, ovarian cancer (such as ovarian serous cystadenoma), prostate cancer, lung cancer (such as non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), colorectal cancer (such as colorectal adenocarcinoma), cervical cancer (such as cervical squamous cell carcinoma and cervical adenocarcinoma), liver cancer (such as hepatocellular carcinoma), head and neck cancer (such as head and neck squamous cell carcinoma), esophageal cancer, pancreatic cancer, kidney cancer (such as renal cell carcinoma), adrenal cancer, stomach cancer, testicular cancer, gallbladder and bile duct cancer (such as bile duct cancer), thyroid cancer, thymus cancer, bone cancer, and brain cancer.

[0318] In a preferred embodiment, the cancer to be treated using the specific binding device described herein is a solid cancer. More preferably, the cancer to be treated using the specific binding device described herein is a solid cancer selected from the following group: sarcoma, melanoma, bladder cancer, brain cancer, breast cancer, uterine / endometrial cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer, and stomach cancer.

[0319] In the case of cancer, treatment may include inhibiting cancer growth, including complete cancer remission and / or inhibiting cancer metastasis, as well as inhibiting cancer recurrence. Cancer growth generally refers to any of several indices representing changes in cancer reaching a more advanced form. Therefore, indices for measuring cancer growth inhibition include reduced cancer cell survival, decreased tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), delayed tumor growth, disruption of tumor vascularization, improved performance on delayed-type hypersensitivity skin tests, increased activity of anti-cancer immune cells or other anti-cancer immune responses, and reduced levels of tumor-specific antigens. Activating or enhancing an individual's immune response to cancerous tumors can improve an individual's ability to resist cancer growth, particularly the growth of cancer already present in the individual and / or reduce the tendency for cancer to grow in the individual.

[0320] In the context of cancer treatment, specific binding agents as described herein can be combined with another anticancer therapy or therapeutic agent, such as an anticancer therapy or therapeutic agent that has been proven or is expected to be suitable for the treatment of the cancer in question, and administered to the individual. For example, a specific binding agent can be combined with a chemotherapy drug, radiation therapy, immunotherapy drug, antitumor vaccine, oncolytic virus, recipient-receptor cell transfer (ACT) therapy (e.g., recipient-receptor NK cell therapy or therapy with chimeric antigen receptor (CAR) T cells, autologous tumor-infiltrating lymphocytes (TILs) or γ / δ T cells) or hormone therapy preparations and administered to the individual.

[0321] While not wishing to be bound by theory, it is believed that the specific binding agents described in this article can act as adjuvants to anticancer therapies. Specifically, it is generally believed that administering this specific binding agent in combination, for example, chemotherapy and / or radiotherapy, or in combination with an anti-tumor vaccine, to an individual will trigger a stronger anti-cancer immune response than that achieved by chemotherapy and / or radiotherapy, or an anti-tumor vaccine alone.

[0322] One or more chemotherapeutic agents intended for use in combination with specific binding agents as described herein may be selected from the group consisting of: taxanes, cytotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, β-Raf enzyme inhibitors, MEK inhibitors, c-MET inhibitors, VEGFR inhibitors, PDGFR inhibitors, alkylating agents, platinum analogs, nucleoside analogs, antifolate agents, thalidomide derivatives, anti-tumor 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; and antifolate agents include methotrexate and pemetrexed. Other chemotherapeutic agents suitable for use in this invention include defactinib, entinostat, eribulin, irinotecan, and vinblastine.

[0323] Preferred therapeutic agents that can be administered together with antibody molecules as described herein include doxorubicin, dihydroxyanthraquinone, cyclophosphamide, cisplatin, and oxaliplatin.

[0324] Radiation therapy intended for use in combination with specific binding objects as described herein may be external beam radiation therapy or brachytherapy.

[0325] Immunotherapy agents intended for use in combination with specific binding agents as described herein can be therapeutic antibody molecules, nucleic acid interleukins, or interleukin-based therapies. For example, therapeutic antibody molecules can bind to immunomodulatory molecules, such as inhibitory checkpoint molecules or immune co-stimulatory molecules, or tumor antigens, such as cell surface tumor antigens or soluble tumor antigens. Examples of immunomodulatory molecules that 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 that 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 that therapeutic antibody molecules can bind to include HER2, EGFR, CD20, and TGF-β.

[0326] The nucleic acid supplied for combining specific binding agents as described herein may be siRNA.

[0327] Interferon or interferon-based therapies may be selected from the group consisting of: IL-2, complex IL-2 precursors, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21 and type I interferon.

[0328] Antitumor vaccines for cancer treatment have been implemented clinically and discussed in detail in the scientific literature (e.g., Rosenberg, S. 2000). This primarily involves stimulating the immune system to respond to various cellular markers expressed by autologous or allogeneic cancer cells, which are used as a vaccination method, with or without granulocyte-macrophage community-stimulating factor (GM-CSF). GM-CSF elicits a strong response to antigen presentation and works particularly well when used in conjunction with this strategy.

[0329] Chemotherapy drugs, radiotherapy drugs, immunotherapy drugs, antitumor vaccines, oncolytic viruses, ACT therapy, or hormone therapy preparations are preferably used for the cancer in question, i.e., chemotherapy drugs, radiotherapy drugs, immunotherapy drugs, antitumor vaccines, oncolytic viruses, ACT therapy, or hormone therapy preparations that have been proven effective in treating the cancer in question. The selection of suitable chemotherapy drugs, radiotherapy drugs, immunotherapy drugs, antitumor vaccines, oncolytic viruses, ACT therapy, or hormone therapy preparations that have been proven effective in treating the cancer in question is within the capabilities of those skilled in the art.

[0330] Given the immune-enhancing activity of CD137, CD137 agonist molecules are expected to find applications in the treatment of infectious diseases. Therefore, in another preferred embodiment, antibody molecules as described herein can be used in a method for treating an infectious disease, such as an acute or persistent infectious disease.

[0331] While not wishing to be bound by theory, it is generally believed that CD137 agonist molecules may enhance the immune response to acute infectious diseases by inducing rapid infiltration and activation of innate immune cells, such as neutrophils and monocytes, thereby promoting the clearance of pathogens causing the acute infectious disease. Therefore, in other specific examples, antibody molecules as described herein can be used as a method for treating an acute infectious disease, such as an acute bacterial disease. In a preferred specific example, the acute infectious disease is an acute bacterial disease caused by Gram-positive bacteria, such as Listeria, Streptococcus pneumoniae, or Staphylococcus aureus.

[0332] Infectious diseases are usually cleared by the immune system, but some infections can persist for a long period of time, such as months or years, and the immune system cannot fight them. These infections are also called persistent or chronic infections.

[0333] Preferably, antibody molecules as described herein are used to treat persistent infectious diseases, such as persistent viral, bacterial, fungal, or parasitic infections, preferably persistent viral or bacterial infections.

[0334] In a preferred embodiment, persistent viral infections that are to be treated with antibody molecules as described herein are those caused by the following persistent infections: human immunodeficiency virus (HIV), Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, or varicella-zoster virus.

[0335] In a preferred embodiment, the persistent bacterial infection to be treated with the antibody molecules described herein is caused by the following persistent infections: Staphylococcus aureus, Haemophilus influenzae, Mycobacterium tuberculosis, Mycobacterium leprae, Salmonella typhi, Helicobacter pylori, Treponema pallidum, or Streptococcus pneumoniae.

[0336] CD137 efficacy has been described as beneficial in the treatment of Gram-positive bacterial infections. Therefore, in a preferred embodiment, the persistent bacterial infection to be treated with the antibody molecule described herein is a persistent Gram-positive bacterial infection. In a more preferred embodiment, the persistent bacterial infection is a persistent bacterial infection caused by Gram-positive bacteria selected from the following group: Staphylococcus aureus, Mycobacterium leprae, and Streptococcus pneumoniae.

[0337] In a preferred embodiment, persistent fungal infections that require treatment with specific binding agents as described herein are those caused by persistent infections of the following species: 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 mentagrophytes.

[0338] In a preferred embodiment, persistent parasitic infections that are to be treated with specific binding agents as described herein are those caused by persistent infections of Plasmodium, such as Plasmodium falciparum, or Leishmania, such as Leishmania donovani.

[0339] In the treatment of persistent infectious diseases, treatment may include eliminating the infection, reducing the individual's pathogenic load, and preventing recurrence of the infection. For example, treatment may include the prevention, improvement, delay, mitigation, or prevention of one or more symptoms and / or signs of persistent infection. Alternatively, treatment may include the prevention of the infectious disease.

[0340] In the treatment of infectious diseases, the specific binding agent described herein can be combined with another therapeutic agent for treating the infectious disease and administered to the individual, such as a therapeutic agent that has been shown to be suitable, or is expected to be suitable, for the treatment of the infectious disease in question. For example, the specific binding agent can be combined with an immunotherapeutic agent and administered to the individual. The immunotherapeutic agent provided for combination with the antibody molecule described herein can be a therapeutic antibody molecule. For example, the therapeutic antibody molecule can bind to receptors of the innate immune system. Examples of receptors of the innate immune system that therapeutic antibody molecules can bind to include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (e.g., RIG-I and MDA-5), and STING.

[0341] In cases where specific binders are used to prevent infectious diseases, these specific binders can be combined for use in the administration of vaccines against the pathogen in question. While not wishing to be bound by theory, it is generally believed that specific binders as described herein can act as adjuvants to vaccination. Specifically, it is thought that administering such a specific binder combination to an individual with a vaccine will trigger a greater immune response against the pathogen than the vaccine alone.

[0342] In view of the disclosure, including the following experimental examples, other aspects and specific examples of the invention will be obvious to those skilled in the art.

[0343] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0344] As used herein, “and / or” should be considered as each of the two specified features or components having or not having the specific disclosure of the other. For example, “A and / or B” should be considered as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were individually stated herein.

[0345] Unless the context otherwise requires, the descriptions and definitions of the features listed above are not limited to any particular form or specific example of the invention and are equally applicable to all forms and specific examples described.

[0346] Other aspects and examples of the invention are provided for the above aspects and examples in which the terms “composed of” or “substantially composed of” are used instead of the term “comprising”, unless the context otherwise requires.

[0347] The present invention will now be described in detail with reference to the embodiments and the drawings described above. [Example] [Example] [1] Production, characterization, and selection of human, mouse, and cynomolgus monkey antigens. 1.1 Recombinant Antigen

[0348] Members of the tumor necrosis factor receptor superfamily (TNFRSF) are known for their tendency to cluster together to form polymers when they bind to their homologous ligands (Croft, M. 2003). This aggregation tendency of their functions makes the production of soluble recombinant proteins that do not aggregate in solution—for use in in vitro selection processes such as the characterization of proteins displayed and selected by bacteriophages and yeast—challenging.

[0349] Several commercially available recombinant antigens were tested, and it was found that most were unsuitable for this selection process due to the presence of aggregates. Of those tested, only the biotinylated, human-secreted CD137, hFc-fusion protein (BPS Biosciences, catalog number 71171), hereinafter referred to as 'hCD137-hFc-Avi-BPS', exhibited sufficiently low aggregation to be suitable for selection, although success was limited (see Example 2).

[0350] Since most commercially available antigens are deemed unsuitable, the following recombinant dimers and monomeric CD137 antigens are produced in-house (see table). [1] ) For selection purposes: [Table 1]

[0351] Monomeric antigens were produced by using EcoRI-HF and BamHI-HF restriction enzymes to select DNA encoding the extracellular domain of human (sequence number: 181) or mouse CD137 (sequence number: 185) along with the Avi sequence and six C-terminal histidine residues into a modified pFUSE vector (Invivogen cat no pfuse-mg2afc2). 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 particle size separation chromatography (SEC) to ensure that the antigen was a single species and free of aggregates.

[0352] A DNA construct encoding the extracellular domain of human, mouse, or cynomolgus CD137 fused with the mIgG2a Fc domain, along with the Avi sequence, was selected and colonized into a modified pFUSE vector and transfected into HEK293-6E cells to produce a dimeric antigen. The recombinant CD137 line was purified using a MabSelect SuRe™ Protein A column (GE Healthcare, 11003494) and particle size separation chromatography (SEC) to ensure the antigen was a single species and free of aggregates.

[0353] The BirA biotin-biotin protein ligase reaction kit (Avidity LLC, BirA500) was used to biotinylate each dimer and monomeric antigen to produce a single biotinylated monomeric CD137 antigen and a dimer CD137 antigen, each labeled with two biotin molecules on each of the two monomers. 3 mg of antigen was mixed with 7.8 μl of BirA enzyme mixture to a molar ratio of 1:50 for enzyme and matrix. Additives (142 μl Biomix A, 142 μl Biomix B, 142 μBiotin) were then added according to the manufacturer's recommendations, and the reaction mixture was incubated at room temperature for two hours. The reaction mixture was immediately buffer-exchanged to DPBS (Life Technologies 14190-169) using an Amicon 30 μm filter (Merck Millipore UFC503096).

[0354] The protein was further purified by SEC to ensure the removal of the BirA enzyme and to produce a final high-quality monodisperse protein preparation free of high molecular weight aggregates. More specifically, the material was mixed with the production batch and its stability and purity were analyzed by particle size distribution high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and SEC-MALS. Intact biotinylation of the protein was confirmed by streptavidin-shift SDS-PAGE gel electrophoresis. The binding of recombinant human and mouse antigens to anti-CD137 positive control antibodies (20H4.9 (US Patent No. 7288638) and Lob12.3 (University of Southampton)) in vitro was confirmed by surface plasma resonance (SPR), and binding to DO11.10 cells expressing human and mouse CD137 ligands was confirmed by flow cytometry. Cells were incubated with the CD137 antigen for 1 hour, and cell binding was then detected using a fluorescently labeled anti-mouse Fc fragment antibody. Binding of the recombinant cynomolgus monkey antigen to DO11.10 cells (National Jewish Health) expressing the cynomolgus monkey CD137 ligand was confirmed by flow cytometry as described above. To ensure the highest possible purity of material for the selection process, thorough protein characterization of the antigen was performed to confirm the presence of protein aggregates not exceeding 2%. 1.2 Antigens expressed by cells

[0355] DO11.10 cells (National Jewish Health) are produced, representing either full-length mouse CD137 (sequence identification number: 184) or human CD137 (sequence identification number: 180), designated 'DO11.10.mCD137' and 'DO11.10.hCD137', respectively, to present membrane-bound antigens, mostly similar to the natural form, for selection and further characterization of selected Fcabs, such as... [surface] listed in [2].

[0356] The production of DO11.10 cells overexpressing the human or mouse CD137 receptor was achieved using lentiviral transduction via the Lenti-X HTX packaging system (Takara, catalog number 631249). The Lenti-X expression vector (pLVX) (Takara, catalog number 631253), containing cDNA encoding human CD137 (sequence identification number: 180) or mouse CD137 (sequence identification number: 184), was co-transfected with the Lenti-X HTX packaging mixture into the Lenti-X 293T cell line (Takara, catalog number 632180) to generate the virus. The DO11.10 cell line was then transduced using these lentiviral vectors.

[0357] The expression of human or mouse CD137 on these cells was confirmed by flow cytometry using the binding of 20H4.9 and Lob12.3 anti-CD137 positive control antibodies to the cells. Cells were incubated with the human or mouse positive control antibodies for 1 hour, and then cell binding was detected using a fluorescently indicated anti-human Fc detection antibody (Stratech Scientific Ltd, catalog number 109-546-098-JIR).

[0358] The DO11.10 cells expressing cynomolgus macaque CD137, designated 'DO11.10.cCD137', were also generated using the same lentiviral transduction methodology and used to test the cross-reactivity between anti-human CD137 Fcabs and cynomolgus macaque CD137. The expression of cynomolgus macaque CD137 was confirmed by determining the binding of the anti-CD137 positive control antibody (MOR_7480.1, US 2012 / 0237498 A1) to the cells using flow cytometry as described above. [Table 2] [Example 2] [:Anti-human CD137 Fcabs] [The Initial Choice]

[0359] To identify Fcabs that bind to human CD137 and maximize the diversity of identified binders, yeast and bacteriophage display selection activities were used. Since some non-immune cell types exhibit low CD137 expression levels, it was decided that selection would selectively target anti-human CD137 Fcabs in cells that highly express CD137, such as activated T cells. While not wishing to be bound by theory, it is assumed that cells with low or negligible CD137 expression levels are more likely to have monosomic CD137 on their cell surface, unlike activated T cells which have highly upregulated CD137 expression, with most of the protein expected to exist on the cell surface in a dimer, trimer, or higher multimeric state.

[0360] Fcab selection uses cells that overexpress CD137 or recombinant dimeric human CD137 protein to expose representative epitopes that promote binding interactions with cells highly upregulated by multimeric CD137. Furthermore, it is considered advantageous to use dimeric antigens to select divalent Fcabs, which bind eagerly to CD137 and maintain target binding for a longer period, which is presumably to promote preferential binding with cells upregulated by CD137 expression levels. In this context, monomeric recombinant CD137 is not used to prevent the selection of very high-affinity monovalent Fcab binders whose binding to CD137 would be too strong.

[0361] The goal of the selection strategy is to obtain CD137 Fcabs that preferentially bind to activated T cells and do not bind well to naive T cells that only exhibit monomeric CD137 or other cells with very low CD137 levels. By selecting CD137 Fcabs that bind divalently and preferentially to both dimer and multimer antigens compared to monomeric antigens, it is believed that off-target T cell activation and associated reduced toxicity will be reduced. Phage display

[0362] Six initial phage libraries displaying the CH3 domain of human IgG1 were used for selection via phage display. All six libraries contained randomized AB loops (containing residues at positions 14-18 according to IMGT numbers) and randomized EF loops (containing residues at positions 92-101 according to IMGT numbers). One of these libraries contained a strain with an insertion 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 numbers).

[0363] A total of 12 selection activities were performed to identify anti-human CD137 binders. In the selection process using six phage libraries, cells were expressed using either in-house hCD137-mFc-Avi antigen or commercially available hCD137-hFc-Avi-BPS antigen and / or DO11.10 hCD137 antigen. Because only one functional Fcab sequence was identified in the early selection using the hCD137-hFc-Avi-BPS recombinant antigen, the remaining rounds of recombinant CD137 selection used in-house hCD137-mFc-Avi. The initial rounds were performed using 100 nM biotinylated antigen, followed by a deselection step using 500 nM unlabeled recombinant human Fc fragment produced in-house. Phage binders were captured by magnetic beads coated with streptoavidin or neutral avidin. In cases where the selected product is detected by phage ELISA as nonspecifically binding to Fc fragments (A450-630nm > 0.4), a further deselection step is introduced to incubate the (in-house produced) biotinylated Fc fragments with the phage product. Non-Fc-binding fragments are then separated by magnetically capturing biotinylated Fc-binding phages.

[0364] To find binders that bind to the membrane-bound native conformation of CD137, cellular selection was performed as follows: phage product lines from recombinant antigen selection were incubated with DO11.10 cells lacking human CD137 to reject unwanted binders, such as those that bind nonspecifically to cells. Next, the phage was incubated with 1 × 10⁷ DO11.10.hCD137 cells, and the binders were dissolved by trypsin digestion and then propagated for the next round. A third round followed a similar approach, increasing the selective pressure by reducing the number of DO11.10.hCD137 cells to 5 × 10⁶.

[0365] After each round of selection, phage ELISAs were performed to determine antigen-specific phage enrichment. 96-well strepto-biotinylated plates were coated with 1 μg / ml biotinylated antigen and incubated overnight. After blocking the plates with 4% Marvell PBS, 50 μl of 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 the plate was washed four times with 0.1% Tween PBS and four times with PBS. Anti-M13 phage-HRP complex antibody was then added to detect phages bound to the immobilized antigen. The phage solution was discarded by inverting the plate, and the plate was washed four times with 0.1% Tween PBS and four times with PBS. TMB microperoxidase solution was added to each well, allowing up to 30 minutes for color development. The reaction was terminated with 1M sulfuric acid, and OD450–630 was read using a microtitrator. Specific hits are defined as those that exhibit a signal intensity at least 4 times higher than the negative control, i.e., PBS or negative non-binding phages such as wild-type CH3, and a binding intensity at least 10 times higher than the biotinylated recombinant Fc fragment.

[0366] Cell selection products from rounds 2 and 3 were used to perform phage fluorescence-induced cell sorting (phage FACS) analysis. In short, 2 × 10⁵ cells were transferred to a round-bottom microtiter plate. Phage supernatant was added to the cells and incubated at 4 °C for 1 hour. Cells were then washed twice with ice-cold PBS 2% BSA buffer, resuspended in 100 μl of a solution of anti-M13 antibody and FITC-conjugated goat anti-mouse IgG F(ab') fragment, and incubated on ice for 1 hour. After three PBS washes, cells were analyzed by flow cytometry. For phage FACS, specific hits were defined as cells exhibiting a geometric mean fluorescence intensity (MFI) at least 10 times higher than the binding signal of recombinant CD137 and at least 4 times higher than the background signal defined by the negative control (PBS).

[0367] 3230 phage strains were screened for binding to the dimeric recombinant hCD137 antigen using phage ELISA, with recombinant Fc as a negative control. 1140 phage strains were screened for specific binding to DO11.10.hCD137 cells using phage FACS. Individual hits were then sequenced, and the resulting 76 unique sequences were assigned an Fcab strain identification symbol and subselectively colonized into a pTT5 expression vector (National Research Council of Canada) containing a HelD1.3 IgG1 heavy chain expression cartridge for expression of Fcab strains in mAb2 format (see Example 3.1). Yeast display

[0368] Four initial yeast libraries displaying the CH1 to CH3 domains of human IgG1 were used for selection via yeast display. All four libraries contained a randomized AB loop (containing residues at positions 14 to 18 according to IMGT numbers) and a randomized EF loop (containing residues at positions 92 to 101 according to IMGT numbers) in the CH3 domain. Both of these libraries further contained five amino acid residues inserted at position 16 of the AB loop in the CH3 domain (residues at positions 16.5 to 16.1 according to IMGT numbers).

[0369] Selection of four yeast libraries was performed using either in-house hCD137-mFc-Avi antigen or commercially available hCD137-hFc-Avi-BPS antigen. While similar to phage selection, the commercial antigen was discontinued after early rounds of selection and replaced with the in-house antigen. For each library, the first round of selection was performed using magnetic cell separation (MACS). After library growth, induction, and a deselection step using 1.25 μM unlabeled human or mouse Fc, cells were incubated with 250 or 300 nM biotinylated recombinant antigen at a density of 1 × 10¹⁰ cells. Yeast conjugates were isolated by adding streptoacidin magnetic beads and using a MACS LS column (Miltenyi Biotech 130-042-401). Subsequent selection rounds utilize fluorescently indicated antibodies to detect bound antigens (anti-biotin-APC (Miltenyi Biotech 130-090-856), streptavidin-APC (BD Biosciences 349024), or neutral avidin-DyLight-488 (Thermo Fisher 22832), correctly folded IgG architecture (anti-human IgG CH2 domain-FITC (Biorad AbD Serotec (MCA647F)), or the performance of aga2-IgG constructs (anti-Xpress (Life Technologies R91025), and anti-mouse IgG-FITC (Sigma F2653-.5ML)), via fluorescence-excited cell sorting (FACS) on a FACS-Aria II instrument (BD). The procedure was performed on a bioscience database. Whenever possible, one of the antigenic markers and structural markers was used together to normalize the binding strength signal of Fcab expression on the yeast surface. Unstained and control yeast populations were used to set up the following classification circles: yeast cells were plotted on FSC-A and SSC-A maps, FSC-W and FSC-H maps to distinguish between monomorphic and multimorphic or budding yeast cells, and FITC-A and APC-A maps to detect double positivity for Fcab binding.

[0370] The antigen concentration used in each round was determined empirically by utilizing the antigen concentration used in previous rounds for product quality control. If binding enrichment increased (>5-fold) compared to previous rounds, the antigen concentration was reduced by 1:2 or 1:3; otherwise, it remained unchanged. This method was also followed to determine how many rounds of selection were needed and whether the selection branches were considered exhausted (if diversity decreased to only a few dominant sequences or if antigen binding did not increase after 2 rounds).

[0371] The 2784 yeast monocultures identified in the library selection process were individually screened as follows: After each selection round, single yeast cell lines were spotted onto SDCAA agar plates using a FACS-Aria II (BD Biosciences) instrument and screened using flow cytometry antigen binding assays: monocultures were grown until a diameter of 2 mm was reached, and then transferred to deep-well plates containing 600 μl of SDCAA liquid medium. Cultures were grown at 30 °C with shaking at 1000 rpm overnight. The expression of the aga2-IgG protein architecture was induced by replacing the growth medium with SGRCAA induction medium at an optical density of 1 (OD600=1). Cells were incubated with biotinylated recombinant human antigen or mouse Fc fragment to distinguish yeast cell lines binding the Fc portion of recombinant hCD137 antigen. Yeast cell lines binding either protein were labeled using streptavidin-APC. Anti-CH2-FITC antibody was also used as a structural IgG marker. To analyze the screening results, a graph of Fc fragment binding was plotted, and any strains binding biotinylated Fc were discarded. Under the selection set using unstained and negative control samples, cells with more than 0.2% APC fluorescence positivity were defined as bound.

[0372] The selection process was repeated under different antigen concentrations and conditions, such as increasing the induction temperature, decreasing the strictness of the selection process, or reducing the number of rounds, in order to increase the number of successful candidates. Sequencing of successful candidates showed that product diversity was quite low, and only nine Fcab strains with unique sequences were identified: FS22-053, FS22-172, FS22-173, FS22-174, FS22-175, FS22-176, FS22-177, FS22-178, and FS22-179. [Example 3] [:From the initial selective effect of anti-human CD137 Fcabs] [Characteristics] 3.1 Preparation of anti-human CD137 Fcabs in "mock" mAb2 format

[0373] A pseudo-mAb2 antibody composed of IgG1 molecules was produced, comprising 76 anti-human CD137 Fcab strains isolated from bacteriophages and 9 strains selected from yeast to allow for the characterization of Fcabs in mAb2 format. The pseudo-mAb2 was prepared by replacing the corresponding region of the CH3 domain of the anti-hen egg white lysozyme antibody HelD1.3 with a portion of the CH3 domain of the Fcabs, including the AB, CD, and EF loops. The production of the HelD1.3 antibody is described in Tello et al. (1993). The heavy and light chain sequences of antibody HelD1.3 are shown in SEQ ID 186 and 173, respectively. The pseudo-mAb2 molecule was produced by transiently expressing it in HEK293-6E cells. To assess the amount of protein produced, the IgG protein content was quantified using the Octet QKe platform of PALL and a protein A quantitative biosensor (18-5021) via biolayer interferometry. Protein lines were purified using protein A affinity chromatography on a mAb SelectSure column. Fifty-three phage-derived CD137 mAb² proteins showed levels below the detection threshold and were therefore deemed unsuitable for further analysis. Thirty-two mAb² lines were purified using a 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, FS22-164, FS22-165, FS22-176, FS22-177, FS22-178, FS22-179, FS22-171, FS22-172, FS22-173, FS22-179, FS22-170, FS22-171, FS22-172, FS22-173, FS22-179 ... 22-174, 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.

[0374] For the purpose of comparing performance levels, some early Fcabs exhibiting soluble Fcabs (containing truncated hinges) were also selected and cultured in HEK293-6E cells and purified using mAb Select SuRe protein A columns. Interestingly, some Fcabs were found to have significantly better biophysical behavior and produced better yields in the pseudo-mAb2 format than soluble Fcabs. The initial strain FS22-053 was such a case, producing very low yields of soluble Fcabs, but the yield improved 25-fold when exhibiting the pseudo-mAb2 format, resulting in more strains available for characterization. 3.2 Recombinant antigen binding via BLI

[0375] The binding of 31 purified pseudomAb² molecules (excluding strain FS22-175) to human recombinant antigens was tested using a single-point binding assay on the Octet QKe platform and biolayer interferometry. A streptavidin BLI biosensor (PALL 18-5021) was used to capture biotinylated hCD137-mFc-Avi antigen (PALL) at 10 μg / ml in kinetic buffer. The sensor was then immersed for 240 seconds in wells containing purified mAb² diluted 1:1 with the same kinetic buffer, followed by 240 seconds in wells containing 1× kinetic buffer. Based on the BLI reaction of superbuffered buffer and wild-type IgG1 control HelD1.3 mAb (G1 / HelD1.3), the binding targets were classified into 12 mAb² non-binding and 19 binding CD137-coated sensors (FS22-007, FS22-033, FS22-042, FS22-049, FS22-050, FS22-052, FS22-053, FS22-054, FS22-169, FS22-172, FS22-173, FS22-174, FS22-179, FS22-180, FS22-181, FS22-183, FS22-187, FS22-194, FS22-195). 3.3 Activity of the selected anti-CD137 pseudomAb2 in the human NF-κB reporter gene analysis

[0376] Multimerization and clustering are essential for TNFR signaling (Bitra et al., 2017). When CD137 interacts with its homologous ligand, CD137L, it clusters and activates the NF-κB signaling pathway. Agonist molecules mimic the ligand, driving CD137 clustering and activation, thereby activating the NF-κB signaling pathway. Some agonist antibodies are known to inherently induce CD137 clustering upon binding, such as urogenumab; however, other antibodies require additional cross-linking by the antibody itself to induce CD137 clustering, such as utomemab (Fisher et al., 2012). This cross-linking is known to be induced in vivo by Fcγ receptors on effector cells, although it is inefficient and may occur at sites far from the site of therapeutic interest. Since dose-limiting toxicity has been associated with the treatment of some anti-CD137 antibodies, it was decided to select anti-CD137 binding Fcabs that do not inherently possess agonizing ability, and only those requiring additional cross-linking to induce CD137 clustering. Therefore, an analysis was developed that can detect the activation of the NF-κB signaling pathway in cells when CD137 on the cell surface is clustered by cross-linked antibodies, but exhibits very low activity when the antibody is not cross-linked. This analysis was then used to test the agonizing activity of 27 anti-CD137 Fcab strains in pseudomAb2 format and 6 anti-CD137 Fcab strains in anti-CD20 mAb² format, regardless of whether these Fcabs bind recombinant antigens according to BLI.

[0377] In the analysis, protein L was used as a cross-linking agent to drive the cross-linking of pseudomAb2 via its Fab moiety and to measure NF-κB activation.

[0378] The cDNA encoding human CD137 (SEQ ID 180) was subselected into the pMSCV-neomycin vector (Takara Clontech, Cat. 634401) using EcoRI-HF and XhoI restriction enzyme. Retroviral particles were produced using the RetroPack PT67 cell line (Clontech, Cat. 631510) following the manufacturer's procedures. This retrovirus was then used to transduce HEK.FRT.luc cells, which were previously produced by transducing the Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) with a lentivirus containing the Qiagen Cignal Lenti NFkB reporter gene (luc) (Qiagen, cat no. 336851) containing an NF-κB-sensitive promoter controlling luciferase expression. HEK.FRT.luc.hCD137 cells were used to screen for pseudomAbs containing CD137 binders identified by selection.

[0379] Various pseudomAbs were prepared using DPBS (Life Technologies, 14190169) and diluted 2 µM. Further dilutions were then performed using reporter gene cell culture medium (DMEM (Gibco, Cat. 61965-026); 10% FCS (Gibco, Cat. 10270-106); 1x penicillin-streptomycin (PennStrep) (Gibco, Cat. 15140-122); blastomycin 15 µg / ml (Melford Laboratories Ltd. Cat. B1105); puromycin 5 µg / ml (Life technologies, Cat. A11113803); genomiconazole 100 µg / ml (InvivGen, Cat. 11006-33-0); and geneticin 500 µg / ml (Life Technologies, Cat. 10131-027). Protein L (Life Technologies, ...) was used. 21189) was used as an artificial cross-linking agent and mixed with mAb² molecules at a molar ratio of 1:4. After incubation for 24 hours, cells were treated with 100 µl of Promega Bio-Glo™ luciferase assay kit (Promega cat no. G7941) according to the manufacturer's instructions, and luminescence at an integration time of 0.5 seconds was measured using a disc reader and Gen5 software, BioTek. The luminescence value is used as a measure of the luciferase produced in response to the activation of the NF-κB signaling pathway of CD137 clusters induced by cross-linked Fcabs. A graph of luminescence value versus log Fcab concentration was plotted, and the resulting curve was fitted using GraphPad Prism with a log (activator) vs. reaction equation. []

[0380] A protein L cross-linked luciferase signal was considered a hit if it showed at least a 10-fold increase compared to the uncross-linked state. These strains were used to determine if they could induce CD137 clustering and subsequent activation of downstream signaling pathways. Of all tested strains, FS22-053 and FS22-172 showed a 10-fold increase in luciferase upon cross-linking, although the EC50 of either could not be determined. Both were selected for further characterization using the DO11.10 T cell activation assay. Surprisingly, no activity was observed in the remaining strains under cross-linking conditions. Although BLI binds to the CD137 target, this may indicate that they bind to unrelated epitopes on CD137, or that the affinity of these strains is insufficient to bind CD137 strongly enough to initiate the NF-κB signaling cascade.

[0381] In summary, although more than 30 Fcabs were tested, only two Fcabs (FS22-053 and FS22-172) were identified from the initial selection. These two Fcabs exhibited the desired function when cross-linked and had very little activity when not cross-linked, as shown in the NF-κB reporter gene analysis. 3.4 Activity of the selected anti-CD137 pseudomAb2 in DO11.10 T cell activation analysis

[0382] T cell activation and downstream signaling are triggered by the aggregation of CD137 agonist molecules on activated T cells, leading to, but not limited to, IL-2 production. Since FS22-053 and FS22-172 have been confirmed to be active in NFKB reporter gene assays, their ability to activate CD137 was tested using a T cell activation assay. A DO11.10 T cell activation assay was developed using DO11.10 T cells engineered to overexpress human CD137 and assessing T cell activation by measuring IL-2 release.

[0383] DO11.10 T cells (National Jewish Health) were transduced using lentiviral vectors designed to overexpress mouse or human CD137, as described above. This DO11.10 T cell activation assay tested the following strains as well as FS22-053 and FS22-172: FS22-007, FS22-033, FS22-042, FS22-049, FS22-050, FS22-052, and FS22-054 (all in “pseudo” HelD1.3 mAb2 format). mAb2 with or without recombinant protein L (Life Technologies, 21189) cross-linking agent, or a 20H4.9 positive control mAb, was diluted and added to DO11.10.hCD137 cells that had been coated overnight with 0.1 µg / ml anti-CD3 antibody (breed 17A2, BioLegend, 100208). After 18 hours of incubation, the supernatant was collected and analyzed using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86) following the manufacturer's instructions. The absorbance was read at 450 nm using a plate reader and Gens software (BioTek). The absorbance at 450 nm was subtracted from the absorbance at 570 nm (corrected). A standard curve for interleukin concentration was calculated using a four-parameter logistic curve fitting (Gens software, BioTek). Plotting mlL-2 concentrations vs. the log concentration of mAb2 or the baseline mAb, and fitting the resulting curves with GraphPad Prism using the log (activator) vs. reaction equation.

[0384] In this analysis, strains FS22-053 and FS22-172 showed significantly enhanced activity upon cross-linking with protein L. The activity of FS22-053 was 126 nM without cross-linking and 21 nM with cross-linking (a 6-fold improvement), while the activity of FS22-172 was 950 nM without cross-linking and 44 nM with cross-linking (a 22-fold improvement). As a result, both strains were selected for affinity maturation. Furthermore, although strain FS22-033 showed no activity in this analysis, it was also selected for affinity maturation because it is believed to likely bind the recombinant antigen to a different epitope, as it did not compete with strains FS22-053 and FS22-172 for binding in the BLI binding analysis (data not shown). It is generally believed that the improved affinity of this strain for CD137 may also lead to improved functional activity (see Example 4.1). 3.5 Preparation of anti-human CD137 Fcabs in CD137 / CD20 mAb2 format

[0385] A panel of anti-CD137 Fcabs (FS22-053, FS22-175, FS22-176, FS22-177, FS22-178, FS22-179) was produced to allow for the characterization of Fcabs in a more biologically relevant mAb2 format. This mAb2 was prepared by replacing a portion of the CH3 domain of the Fcab, including the AB, CD, and EF loops, with the corresponding region of the CH3 domain of the anti-CD20 2F2 strain (from US 8,529,902 B2) to produce a mAb2 binding to both CD137 and CD20. These CD137 / CD20 mAb2s were produced by transiently expressing them in HEK293-6E cells and purified using mAb Select SuRe protein A columns. 3.6 Activity of anti-CD137 / CD20 mAb2 in DO11.10 T cell activation analysis

[0386] Example 3.4 describes a T-cell activation assay in which the anti-CD137 pseudomAb² line uses protein L cross-linking. This setup provides a reliable and reproducible way to screen a considerable number of molecules, although the higher-order structures formed by the cross-linking of antibodies and mAb2 are suboptimal and do not represent the physiological environment. A more biologically relevant setup is one where mAb² molecules are cross-linked via their Fab arms to targets present within the biological system. This cell-based in vitro system optimizes the presentation of antibodies and mAb2 through Fab binding to the cell membrane, driving antibody clustering, which in turn increases the affinity of the CD137 Fcab arm for its targets on T cells.

[0387] CD20+ Daudi cells (ATCC CCL-213) were seeded at a 1:1 ratio with DO11.10.hCD137 cells used in Example 3.4 in 96-well round-bottom dishes. Six colony lines producing CD137 / CD20 mAb² from Example 3.5 were tested in this DO11.10 T cell activation assay. mAb2 or a dilution of a positive control mAb, with or without recombinant protein L (Life Technologies, 21189) cross-linking agent, was prepared and added to DO11.10.hCD137 and Daudi cells that had been coated overnight with 0.1 µg / ml anti-CD3 antibody (colony 17A2, BioLegend, 100208). After 18 hours of incubation, the supernatant was collected and analyzed using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86) following the manufacturer's instructions. Using a plate reader and Gens software, BioTek read the plates at 450 nm. The absorbance value at 450 nm was subtracted from the absorbance value at 570 nm (corrected). A standard curve for intercytokine concentration was calculated using a four-parameter logarithmic curve fitting (Gens software, BioTek). A graph was plotted of mlL-2 concentration vs. the logarithmic concentration of mAb2 or the baseline mAb, and the resulting curve was fitted using GraphPad Prism with a log (activator) vs. reaction equation. Of all the strains tested, FS22-053 was the only one to show 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).

[0388] Examples 3.4 and 3.6 show that FS22-053 can drive the clustering and activation of CD137 on the surface of DO11.10 T cells when cross-linked by protein L or by cell-based cross-linking via Fab and another target (HelD1.3 and CD20) in the tested mAb² format. [Example 4] [:Anti-human Fcab] [Of] [Mature Affinity]

[0389] As mentioned earlier, three strains were selected for affinity maturation. Based on their functional characteristics (FS22-053 and FS22-172) as equal to NF-κB reporter gene analysis and DO11.10 T cell activation analysis, it was considered that they were bound to different CD137 regions (FS22-033). 4.1 Affinity Maturity of FS22-033

[0390] Two yeast strains and two bacteriophage display libraries were constructed from strain FS22-033. One library was constructed by randomizing five residues in the AB loop of the CH3 domain using the ELLA primer, and the other library was constructed by randomizing five residues in the EF loop of the CH3 domain. The ELLA primer specified the codons used for each amino acid and their relative abundance within the mixture. Only cysteine ​​was excluded from the mixture, and no other amino acids were biased.

[0391] For the phage libraries FS22-033AB and FS22-033EF, affinity-mature strains underwent a two-round selection process: the first round used 200 nM hCD137-mFc-Avi, and the second round used 10 nM hCD137-mFc-Avi. Ninety-six strains from each selection product were screened by phage ELISA. This screening identified 24 unique strains (FS22-033-001 to FS22-033-024), all of which produced pseudomAb² containing HelD1.3. Modified binding kinetics were then tested using BLI as described in Example 3.2. In this analysis, none of these 24 affinity-mature strains performed better than the parental FS22-033 strain, and therefore no further selection was performed. Regarding the yeast strains FS22-033 AB and FS22-033 EF, a three-round selection process was performed as follows: Rounds 1 and 2 used 300 nM hCD137-mFc-Avi, followed by Round 3 using 300 nM hCD137-mFc-Avi, 300 nM cynomolgus monkey CD137-mFc-Avi, or 300 nM hCD137-Avi-his. The 1056 strains from Rounds 2 and 3 were sequenced. All identified unique strains were screened for improved antigen-binding activity compared to the parental FS22-033 strains using 30 nM human dimeric recombinant antigen and antigen-binding flow cytometry analysis. Strains were also graded based on the percentage of APC+ cells correlated with binding strength. The five best strains (FS22-033-025, FS22-033-026, FS22-033-027, FS22-033-028, FS22-033-029) were subsequently produced as HelD1.3 mAb² (as described in Example 3.1) and their binding to the human dimer recombinant antigen was tested by BLI as previously described. However, none of these strains showed improved kinetic profiles compared to the parental FS22-033 strain, and therefore these strains were not further developed. 4.2 Affinity Maturation of FS22-053 and FS22-172

[0392] Four yeast display libraries were constructed from Fcab strains FS22-053 and FS22-172. Seven residues (at positions 15-16.1 according to IMGT) were randomized within the AB loop of the CH3 domain of each strain using an ELLA primer, having the same trinucleotide distribution as described in Example 4.1, to produce libraries FS22-053 AB and FS22-172 AB. Five residues (at positions 92-94 and 97-98 according to IMGT) were randomized within the EF loop of the CH3 domain using an ELLA primer to produce libraries FS22-053 EF and FS22-172 EF.

[0393] For libraries FS22-053 AB and FS22-053 EF, and FS22-172 AB and FS22-172 EF, selection was performed on three or four rounds of yeast libraries to select affinity-mature strains using either the dimeric hCD137-mFc-Avi antigen or the monomeric hCD137-Avi-His antigen. The monomeric and dimeric antigens were used alternately to ensure that the strains retained affinity for the antigens and did not bind solely through total binding. The use of monomeric or dimeric antigens, and the antigen concentrations used in flow cytometry during each round, were determined empirically based on whether enrichment of the monomeric or dimeric antigens was observed in previous rounds. Whenever possible, higher-affinity selection than the parental selection was used to isolate strains with affinity comparable to the parental molecules. Selection pressure was increased to 1 nM of the dimeric antigen. During each selection round, individual strains were spotted onto agar plates to assess the progress of selection. Each strain was individually grown and induced, and then its binding and structural parameters were determined by flow cytometry using the biotinylated dimeric antigen and anti-CH2 structural marker as described above. This screening cascade was followed to allow for determination of the success of the selection process based on strain samples from the selected product and to allow for early screening of individual strains that could subsequently produce soluble proteins.

[0394] The binding of a total of 1152 yeast monocultures to biotinylated recombinant antigens was screened using antigen-binding flow cytometry as described above. Selection from the FS22-053EF library resulted in the enrichment of 138 unique loop sequences. Similarly, 30 unique loop sequences were isolated from the FS22-172AB library. Neither the FS22-053AB nor the FS22-172EF libraries contained any monocultures that showed any improvement in binding compared to their parental monocultures. Sequence analysis across the best-binding monocultures from the FS22-053EF and FS22-172AB libraries revealed a conserved PPY sequence pattern within the AB loop. Since this sequence is retained after affinity maturation, and both individual Fcabs lineages independently selected this sequence, it is likely important for CD137 epitope binding. Furthermore, the conserved LE or LD sequence patterns within the EF loop of the CH3 domain in the FS22-053 and FS22-172 lineages suggest that this amino acid portion within the EF loop is necessary for binding modification.

[0395] To assess the progress of selection and whether recombinant recombinant analysis of mutated AB and EF loops between affinity-mature colonies was necessary, the top five unique colonies from the FS22-053 EF library (FS22-053-008, FS22-053-009, FS22-053-010, FS22-053-011, FS22-053-012) and the top six unique colonies from the FS22-172 AB library (FS22-172-001, FS22-172-002, FS22-172-003, FS22-172-004, FS22-172-005, FS22-172-006) were graded by specific binding to 10 nM human antigen dimer (more than 30% APC-positive cells in flow cytometry combined with analysis). All were analyzed in the same manner using 10 nM human antigen dimer. During screening for nM dimer human antigens, more than 10% of APC-positive cells were produced as pseudo mAb2 (HelD1.3) and mimic mAb2 (PD-L1) mAb² to evaluate the functional and kinetic improvements of the randomization loop. [Example 5] [:present"] [fake”mAb, 2 , ] [Format Affinity Maturity Anti-Human CD137 Fcabs] [Of] [Construction, Representation, and Characterization] 5.1 Construction of Anti-human CD137 Fcabs in “fake” and simulated mAb2 format

[0396] Sixteen affinity-mature colonies (FS22-053-001 to FS22-053-016) derived from parental colony FS22-053, and six colonies (FS22-172-001 to FS22-172-006) derived from parental colony FS22-172, were prepared in mAb2 format. Colonies FS22-053-001 to FS22-053-007 were not further processed because their mAb2 formats did not exhibit levels suitable for downstream purification for further testing and characterization. The remaining colonies were found to have at least 95% sequence identity in their CH3 domains compared to the CH3 sequences of the parental colonies from which they were derived. Percentage sequence similarity matrices were generated by comparing the positions of each amino acid with those of reference sequences (parental colonies FS22-053 or FS22-172). (Figure 1D shows the percentage of sequence identity of the CH3 domain of strains FS22-053-008 to FS22-053-016 and FS22-053-017 (see Example 10.1) compared to the CH3 domain of the parental FS22-053. Figure 1E shows the percentage of sequence identity of the CH3 domain of strains FS22-172-001 to FS22-172-006 compared to the CH3 domain of the parental FS22-053.)

[0397] "Pseudo" mAb2 antibodies containing HelD1.3-presenting anti-human CD137 Fcabs were prepared for further characterization of affinity-matured Fcabs in mAb² format. These mAbs² were prepared as described in Example 3.1.

[0398] Also produced were mimic mAbs containing anti-human CD137 Fcabs and a PD-L1 binding Fab region (strain YW243.55.S70 from US 8,217,149 B2). These were prepared using a method similar to that described in Example 3.1, by replacing the corresponding Fcab region with a portion of the CH3 domain of the anti-PD-L1 binding antibody, including AB, CD, and EF loops. These PD-L1 mimic mAbs contained an LALA mutation in the CH2 domain (AA). It is known that introducing an LALA mutation into the CH2 domain of human IgG1 reduces Fcγ receptor binding (Bruhns, P., et al. (2009) and Hezareh M., et al. (2001)).

[0399] CD137 / HelD1.3 and CD137-AA / PD-L1 mAb² were produced by transient expression in HEK293-6E cells and purified using mAb Select SuRe protein A column. 5.2 Activity of human Fcabs in pseudomAb2 format in human NF-κB reporter gene cell analysis

[0400] [Table 3] The functional activity of the pseudomAb2 (HelD1.3) format affinity-matured anti-human CD137 Fcabs listed was tested using the same NF-κB luciferase assay described in Example 3.3. Emissions at an integration time of 0.5 seconds were measured using a disc reader and Gen5 software, BioTek. The results of this analysis are shown in... [surface] [3] and in Figure 2. As expected, none of the Fcabs showed activity in the absence of protein L crosslinking (-XL). All affinity-mature CD137 Fcabs showed a significant improvement over the parental CD137 Fcabs, and although they were positive in this analysis, it was impossible to calculate their EC50 values ​​(see Example 3.3). When crosslinked with protein L (+XL), FS22-053-008 and FS22-172-003 showed the best activity from each family with the lowest EC50. [Table 3] N / A - Not applicable because the signal is too low to allow EC50 determination. 5.3 Activity of human Fcabs with affinity in a mimicking mAb2 format in human DO11.10 T cell activation assay

[0401] The functional activity of mature human Fcabs with affinity in the mimicry mAb2 (PD-L1 LALA) format listed in [Table 4] was tested using DO11.10 T cell activation analysis, similar to the analysis described in Example 3.6.

[0402] HEK.mPD-L1 cells were produced by subselecting the cDNA encoding mouse PD-L1 (sequence identification number: 188) into the pcDNA5FRT vector (Life Technologies) using the KpnI and NotI restriction sites, and then transfecting the vector into Flp-In T-REx 293 cell line (Life Technologies, R780-07) using liposome 2000 (Life Technologies, 11668-019). The cell line was grown in DMEM containing 10% FBS, 100 µg / ml hygromycin B (Melford Laboratories Ltd, Z2475), and 15 µg / ml blastomycin (Melford Laboratories Ltd, B1105) for 3-4 weeks until a stable transfected cell population was formed. These communities were expanded in the presence of 1 µg / ml doxycycline (Sigma Aldrich, D9891) and PD-L1 performance was tested using a PE-based anti-mouse PD-L1 (MIH5) antibody (BD Biosciences, 558091).

[0403] Cells were isolated using cell dissociation buffer, washed once with PBS, and cultured at 2 x 10⁵ cells per well in a 96-well dish. The cells were then incubated with antibody diluted 1:20 in PBS at 4 °C for 1 hour. Cells were washed once with PBS and measured using an Accuri C6 cell counter (BD Biosciences), and data were analyzed using FlowJoX. Mouse PD-L1 expression was reconfirmed.

[0404] In Example 5.1, 15 lineages producing CD137 / PD-L1 mAb² were tested using the DO11.10 T cell activation assay. A dilution of mAb² or a positive control mAb was prepared and added to DO11.10.hCD137 (7.5 x 10³ cells per well) and HEK.mPD-L1 cells (2 x 10⁴ cells per well) or DO11.10.hCD137 (7.5 x 10³ cells per well) and HEK cells (2 x 10⁴ cells per well) that had been coated overnight with 0.1 µg / ml anti-CD3 antibody (lineage 17A2, BioLegend, 100208) in a flat-bottomed 96-well dish, which had not been transduced to express mPD-L1 (2 x 10⁴ cells per well). After 18 hours of incubation, the supernatant was collected and analyzed using a mouse IL-2 ELISA kit (eBioscience, 88-7024-86) following the manufacturer's instructions. Using a plate reader and Gens software, BioTek read the plates at 450 nm. The absorbance value at 450 nm was subtracted from the absorbance value at 570 nm (correction). A standard curve for intercytokine concentration was calculated using a four-parameter logarithmic curve fit (Gens software, BioTek). A graph was plotted of mlL-2 concentration vs. the logarithmic concentration of mAb2 or the baseline mAb, and the resulting curve was fitted using GraphPad Prism with a log (activator) vs. reaction equation. T cell activation was detected by measuring IL-2 release.

[0405] The results of this analysis are shown in [surface] [4] and [picture] [3] Within. Without cross-linking via PD-L1 expression cell binding ( Without cell-based XL column, no T cell activity was observed. Once cross-linked, all mAb2s exhibited potent T cell activity, as indicated by high IL-2 release levels and sub-nanomolar EC50 values. The positive control anti-human CD137 mAb, G1-AA / 20H4.9, showed increased mIL-2 release at progressively increasing concentrations; however, the maximum release was significantly less than that of anti-human CD137 Fcabs. All strains except FS22-053-009 and FS22-172-005 had EC50 values ​​less than 0.3 nM, meaning they were either better than the positive control or as good. The smallest E was observed to be the largest, a measure of maximum T cell activation and possibly associated with greater in vivo T cell antitumor activity, at 7758 pg / ml, which was higher than the positive control. [Table 4] N / A - Not applicable because the signal is too low to allow EC50 determination. 5.4 Primary Human CD8+ T Cell Activation Analysis

[0406] Example 5.3 demonstrated that Fcabs activate CD137 activity on HEK cells overexpressing CD137. Primary human T cell assays are required to test the activity of Fcabs on cells that have not been engineered to overexpress CD137. Activated cytotoxic CD8+ T cells are responsible for directly killing cancer cells and express CD137 on their cell surface (Ye et al., 2014). CD137 clustering 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 assess the ability of Fcabs (in mAb2 format, details below) to drive CD137 clustering and subsequent downstream signaling. CD8+ T cell activation was determined based on IL-2 release.

[0407] To isolate T cells, peripheral blood mononuclear cells (PBMCs) are isolated from platelet-donated byproducts, leukocyte-depleted cytoplasm. In short, the contents of the leukocyte-depleted cytoplasm are washed with PBS and coated onto a Ficoll (Sigma-Aldrich, 1440-02) gradient. PBMCs are isolated by centrifugation, and cells that have not crossed the Ficoll gradient are recovered. PBMCs are further washed with PBS, and the remaining red blood cells are dissolved by adding 10 ml of 1X erythrocyte lysis buffer (eBioscience, 00-4300-54) following the manufacturer's instructions. CD8+ T cells are isolated from the PBMCs present in the extract using CD8+ T cell isolation kit II (Miltenyi Biotec Ltd, 130-096-495) according to the manufacturer's instructions.

[0408] Incubation with anti-CD3 antibody was used as the first signal to drive the initiation of T cell activation. 96-well flat-bottomed tissue culture dishes were coated overnight at 4 °C with 8 µg / ml anti-CD3 antibody (Cultured strain UCHT1, R&D Systems, MAB100-SP) in PBS. The dishes were then washed three times with 200 µl PBS.

[0409] Regarding cell-based crosslinking of human CD137 Fcabs with affinity maturing in a PD-L1 mimicking mAb2 format, HEK293 cells (HEK.hPD-L1) that overexpress hPD-L1 were produced, essentially as described in Example 5.3, but via subselective proliferation of cDNA encoding human PD-L1 (sequence identification number: 187) instead of mouse PD-L1. HEK.hPD-L1 cell lines were cultured at 2 x 10⁵ cells per well in 96-well flat-bottomed dishes coated with anti-CD3 antibody (8 µg / ml). The medium consisted of 100 µl of T cell culture medium (RPMI medium (Life Technologies, 61870-044) plus 10% FBS (Life Technologies), 1X penicillin-streptomycin (Life Technologies, 15140122), 1 mM sodium pyruvate (Gibco, 11360-070), 10 mM Hepes (4-hydroxyethylpiperazine ethanesulfonic acid) (Sigma-Aldrich, H0887), 2 mM L-glutamic acid (Sigma-Aldrich, G7513), and 50 µM 2-mercaptoethanol (Gibco, M6250)). Once HEK.hPD-L1 cells or HEK cells that were not transduced to express hPD-L1 had adhered after 4 hours of incubation, all T cell culture medium was removed and replaced with 100 µl of T cell culture medium containing 5.0 x 10⁵ cells / ml, resulting in 5.0 x 10⁴ cells / well.

[0410] mAb2 was started at 500 nM, diluted to a final 2X concentration with T cell culture medium, and titrated 1:3. 100 µl of mAb2 was added and titrated to a total analytical volume of 200 µl and a 1X concentration of antibody.

[0411] Positive control anti-D137 antibody (G1-AA / 20H4.9) and negative control isotype IgG antibody (G1-AA / HelD1.3) were each diluted to 500 nM, 2X final concentration, in T-cell medium containing 500 nM crosslinking agent (anti-human CH2, strain MK1A6 (Jefferis et al., 1985 and Jefferis et al., 1992), produced in-house) and titrated 1:3. 100 µl of the diluted positive control antibody / crosslinking agent mixture or negative control IgG antibody / crosslinking agent mixture was added to the cells to a total analytical volume of 200 µl and a 1X antibody concentration.

[0412] The analysis was performed by incubation at 5% CO2 and 37°C for 72 hours. Supernatants were collected and analyzed using the Human IL-2 ELISA Ready-SET-Go! kit (eBioscience, Cat. 88-7025-88) following the manufacturer's instructions. The absorbance was read at 450 nm using a plate reader and Gen5 software (BioTek). The absorbance at 450 nm was subtracted from the absorbance at 630 nm (correction). A standard curve for interleukin concentration was calculated using a four-parameter logarithmic curve fitting (Gen5 software, BioTek). A graph of human IL-2 (hIL-2) concentration vs. logarithmic antibody concentration was plotted, and the resulting curve was fitted using GraphPad Prism with a log (agonist) vs. reaction equation.

[0413] [surface] [5] The EC50 value and maximum IL-2 release response observed in T cell activation assays were tested using cell-based crosslinking in the presence of mature Fcab strains with affinity in the PD-L1 mimicry mAb2 format. The positive control, anti-human CD137 mAb, 20H4.9, showed increased hIL-2 release and an EC50 of 0.5 nM when crosslinked with anti-hCH2 antibody. All strains were active in this analysis, with most showing good potency and sub-nanomolar EC50s. mAb2 containing Fcabs FS22-053-007, FS22-053-008, FS22-053-010, FS22-053-011, FS22-053-012, FS22-172-003, FS22-172-004, and FS22-172-005 elicited the largest T-cell responses and the smallest EC50s in the range of 0.19 to 0.49 nM. Subpopulations of mAb2 not cross-linked with PD-L1-expressing HEK cells (containing Fcabs FS22-053-008, FS22-053-011, FS22-053-014, FS22-173-003, and FS22-172-004) were also tested and showed no activity in this analysis, as expected. This confirmed the activity observed in the NF-κB and DO11.10 T cell activation assays. Figure 4 shows a representative graph of IL-2 release in the T cell activation assays of FS22-053-007-AA / PD-L1, FS22-053-008-AA / PD-L1, FS22-172-002-AA / PD-L1, FS22-172-003-AA / PD-L1, and FS22-172-004-AA / PD-L1 mAb2. [Table] 5 5.5 Determining the specificity of anti-human CD137 Fcabs by surface plasma resonance (SPR)

[0414] The specificity of anti-human CD137 Fcabs compared to other related TNFSFR family members was tested. Eight Fcabs in pseudomAb2 (HelD1.3) format were tested and their binding to other human TNFSFR receptors: CD40, OX40, and GITR was measured using a Biacore T200 (GE Healthcare) SPR assay. Human CD40, GITR, and OX40 were coated onto a Biacore CM5 chip (GE Healthcare, CAT no. 29149603) with an amine conjugate (amine conjugate kit, GE Healthcare, BR-1000-50) to approximately 1000 RU. Anti-human CD137 Fcabs (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 starting at 1 μM in HBS-EP+ buffer (BR100669) and injected at 30 µl / min for 3 min, followed by allowing dissociation in buffer for 4 min. The wafer was regenerated by injecting 10 mM glycine at pH 2.5 at a rate of 30 µl / min for 12 s. Antibodies specific to different TNFRSF members were used as positive controls to confirm Biacore wafer coating. Data were subtracted from double references and analyzed using BIAevaluation 3.2 software. Fcabs did not bind to any of the tested TNFRSF receptors, demonstrating their specificity for CD137. As a result, off-target binding by Fcabs was not expected. 5.6 Anti-human CD137 Fcabs in pseudomAb2 format binding affinity to human, cynomolgus monkey, and mouse CD137, according to SPR

[0415] The affinity of anti-human CD137 Fcabs (FS22-053-008, FS22-053-011, FS22-053-014, FS22-172-004, FS22-172-004) for human, cynomolgus (cynomolgus monkey), and mouse CD137 was measured using SPR to determine whether the Fcabs were suitable for testing in animal studies. An anti-human Fab capture system, following the manufacturer's recommendations (GE Healthcare, Human Fab Capture Kit, #28958325), was fixed to all four flow chambers of a CM5 S-series wafer (GE Healthcare #BR-1005-30) to achieve an average surface density of 6000 RU. Various mAbs were captured to approximately 150 RU by injecting a 3 μg / ml mAb² solution diluted in HBS-EP+ buffer (GE Healthcare #BR1006-69) at 30 µl / min for 60 seconds. Subsequently, different concentrations of human, cynomolgus monkey, or mouse CD137 antigens (unbiotinylated human, cynomolgus monkey, or mouse CD137-mFc-Avi or human CD137-Avi-His) prepared in HBS-EP+ buffer were flowed over the wafer at 60 μl / min for 3 min, followed by a 10-minute dissociation period. After each antigen concentration, 10 mM glycine (pH 2.1) was injected at 30 μl / min for 30 seconds to regenerate the wafer. HBS-EP+ buffer was injected before the highest and after the lowest antigen concentrations for reference subtraction, and one concentration was randomly repeated twice. A 1:1 Langmuir model was used to fit the binding kinetics to generate the equilibrium binding constant (KD) for each sample. Data analysis was performed using BiaEvaluation software version 3.2. Results are shown in […]. [surface] [6] Inside.

[0416] Results analysis showed improved binding to both human and cynomolgus monkey CD137 in all mature strains compared to their respective parent molecules. The binding affinity to the monomeric human CD137 antigen was at least 100-fold weaker than that to the dimeric human and cynomolgus monkey Fc fusion antigen. As discussed in Example 2, the selection of FcAbs that preferentially bind to the dimeric CD137 compared to the monomeric form confirms the success of this selection strategy. This dynamic behavior makes it less likely for these FcAbs to bind to the minimum quasi-monomer CD137 expressed on unstimulated T cells, thus reducing the risk of hepatotoxicity or systemic toxicity associated with some anti-CD137 monoclonal antibody therapies.

[0417] The data also shows that anti-human CD137 Fcabs bind to cynomolgus monkey dimer CD137 with an affinity comparable to that of human CD137.

[0418] The ability of Fcabs to bind to mouse dimeric CD137 was also tested. None of these strains showed a stronger binding affinity than the mouse antigen (e.g., [surface] [6] As shown, N / A indicates no KD (which cannot be calculated), except for strain FS22-053-014, which was surprisingly found to have a KD of 24 nM against mouse antigen. This was unexpected because mouse CD137 and human CD137 share less than 57% sequence homology. [Table 6] N / A - Not applicable because the signal is too low to allow KD determination. 5.7 Determination of Fcab binding valence when using heterodimer and homodimer Fcabs

[0419] Fcabs typically contain two homodimeric Fc chains and antigen-binding addresses within the CH3 domain. Because these antigen-binding addresses within the two CH3 domains are very close, the binding valence of the Fcabs is tested to determine whether these CH3 domains can independently bind CD137. Using a knob-into-hole mutation (knob: T22W, hole: T22S L24A Y66V) (Atwell S et al., 1997), heterodimeric Fcabs containing a single antigen-binding CH3 domain were constructed by combining an FS22-172-003 chain and a wild-type Fc chain. As a result, each heterodimer contains one CH3 (sequence identification number: 139) from FS22-172-003 on one chain and one wild-type CH3 (sequence identification number: 4) on the other. Fcabs were prepared in mAb2 format.

[0420] SPR binding assays were used to compare the heterodimer Fcab with the homodimer FS22-172-003 (containing two antigen-binding CH3 domains). In this experiment, the monomeric human CD137-mFc-Avi was preferred over the dimeric CD137 antigen. To compensate for... [Example] [5.6] The weaker binding of the monomeric antigen described herein resulted in the human CD137-mFc-Avi lineage being immobilized on a CM5 wafer at a high density of 450 RU. Heterogeneous or homodimeric Fcabs were injected and flowed over the immobilized antigen. [picture]

[14] The results showed that the heterodimer Fcab containing only one CD137-binding CH3 domain could still bind to the antigen even under suboptimal conditions. The dissociation rate of the heterodimer Fcab was observed to be significantly faster than that of the homodimer Fcab. These results confirm that FS22-172-003 Fcab can bind to CD137 via one or both CH3 domains and confirm that... The selection strategy described in [Example 2] is successful because Fcab can bivalently connect to its target. 5.8 Binding of anti-CD137 Fcabs to cells with different CD137 expression levels

[0421] like [Example] [2] As described, Fcabs that bind to CD137 are selected to preferentially bind to cells with increased CD137 expression levels. The bivalent and thermally cleavable binding system of FS22-172-003 is achieved through... [Example] [5.6] SPR confirmed that FS22-172-003 exhibits high total binding capacity and stronger binding to the dimer CD137 compared to the monomer CD137.

[0422] like [Example] [3.5] describes the production of a series of DO11.10 cells exhibiting different CD137 levels. To determine the relative CD137 level for each cell line, antibody binding capacity (ABC) was determined according to the manufacturer's procedure (Quantum™ Simply Cellular® #816 Bangs Labs). Each cell line was classified by CD137 level after background subtraction: high hCD137 (ABC: 1,206,283), intermediate hCD137 (ABC: 404,597), intermediate / low hCD137 (ABC: 143,065), low hCD137 (ABC: 14,208), and negative hCD137 (ABC: 0). []

[0423] The binding assays for anti-human CD137 Fcab (FS22-172-003-AA / HelD1.3) in pseudomAb2 format, positive control antibody (G1-AA / 20H4.9), or isotype control (G1-AA / HelD1.3) with each cell type as described above were performed as follows: DO11.10 cells were harvested from T175 cell culture flasks, centrifuged at 1200 rpm for 3 min, and resuspended at 2 x 10⁶ cells / ml in ice-cold FACS buffer consisting of DPBS (Life Technologies, 14190169) and 1% BSA (Sigma-Aldrich, A7906). 50 µl of each antibody was seeded in a 96-well V-bottom dish (Costar, 3894). All tested antibodies were diluted in 120 µl of FACS buffer. DO11.10 cells were then centrifuged, the supernatant was removed, and the cells were resuspended in 100 µl of antibody dilution and incubated at 4 °C for 45 min. Cells were washed twice with 150 µl of FACS buffer and resuspended in 100 µl of 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 min. Cells were washed once with 150 µl of FACS buffer, then washed with 150 µl of DPBS and resuspended in 150 µl of DPBS containing 1:10,000 DAPI (Biotium, 40043) and read using BDCantoII or iQue (Intellicyt). FlowJo v10 was used to analyze the data to determine the geometric mean of the PE signal of viable cells in each well.

[0424] like [picture]

[15] As shown, compared with the positive control G1-AA / 20H4.9, FS22-172-003-AA / HelD1.3 showed stronger binding to cells with higher CD137 levels. [Figure 15D]: CD137int and [Figure 15E]: High CD137 levels), and cells that do not bind CD137 at very low levels ( [Figure 15B]: CD137 low and [Figure 15C]: CD137int / low). In contrast, the positive control G1-AA / 20H4.9 binds better to both the dimer and monomeric antigens, and its KD with either the dimer or monomeric antigen differs by less than 10 times, unlike FS22-172-003-AA / HelD1.3, whose binding to the monomeric CD137 antigen is 200 times weaker than that to the dimer antigen. [Example] As described in [5.6]. [Example 6] [:Anti-mouse CD137 Fcabs] [Initial Selection]

[0425] To test the activity of anti-CD137 Fcabs in a live mouse model, Fcabs that specifically bind to mouse CD137 were produced and characterized. Phage display

[0426] The initial phage library of six human IgG1 CH3 domain-expressing Fcabs previously used to select Fcabs that bind to human CD137 was used to select Fcabs that bind to mouse CD137, with recombinant mouse dimer CD137 or cells expressing full-length mouse CD137 used as antigens.

[0427] Selection using six phage libraries employed the in-house mCD137-mFc-Avi antigen and DO11.10 cells expressing mCD137 (DO11.10.mCD137). A simple selection protocol was followed, in which all three selection rounds were performed using 100 nM biotinylated antigen, followed by a deselection step using 500 nM of unlabeled recombinant human Fc fragment. Binders were captured using magnetic beads coated with streptavidin or neutral avidin. Additionally, selection of DO11.10.mCD137 cells expressing mouse CD137 also used the first-round product binding to the recombinant antigen. In short, the phage product was incubated with DO11.10 cells lacking mouse CD137 to discard unwanted binders, such as those that bind nonspecifically to cells. Next, the phage was incubated with 1 × 10⁷ DO11.10.mCD137 cells. The cells were then digested with trypsin to lyse the conjugates and then propagated for the second round of selection. The third round followed a similar approach, increasing the selection pressure by reducing the number of DO11.10.mCD137 cells to 5 × 10⁶.

[0428] All Round 3 recombinant antigen products (576 strains) and all Round 3 selection products (576 strains) were screened by phage ELISA (as described above) and cell binding to DO11.10.mCD137 cells. Regarding ELISA, most strains showed high antigen-binding signal intensity (OD450 > 1). Therefore, strains showing less than a 10-fold increase in antigen binding compared to mouse-Fc binding were discarded. Regarding cell binding, a FITC MFI higher than 5 × 10⁵ was considered positive. The three tests in the phage library were performed poorly, and many strains showed non-specific binding to DO11.10.mCD137 cells and the recombinant antigen. The 34 Fcab strains that hit were subselected and produced as HelD1.3 mAb² as described in Example 3.1. Yeast display

[0429] The four initial yeast libraries displaying the CH1 to CH3 domains of human IgG1, previously used to select Fcabs that bind to human CD137, were used to select Fcabs that bind to mouse CD137.

[0430] A total of 53 individual rounds of selection were performed to identify anti-mouse CD137 binders. Recombinant, dimerized, biotinylated mouse CD137 (mCD137-mFc-Avi) antigen, produced in-house, was used to select binders from an initial yeast library. In short, the first round of binders was selected by incubating the initial library with 300 nM of recombinant antigen and deselecting with 2.5 μM of unlabeled mouse IgG2a Fc fragment. The product was separated using MACS and streptoacidin magnetic beads. Three rounds of FACS selection were performed, as described in Example 2, using 300 nM of recombinant antigen and 1.5 μM of mouse Fc for selection and deselection, respectively.

[0431] Single strains from rounds 2, 3, and 4 were speckled on agar plates. To determine product diversity, at least 96 strains from each selected product were sequenced. 126 unique strains (50 from round 3 of one library, 48 from round 3 of another library, and 18 from round 4 of the remaining libraries) were screened for binding to the recombinant antigen using flow cytometry. Strains showing positive APC fluorescence channels (greater than 10%) when incubated with the recombinant antigen and less than 0.2% when incubated with recombinant mFc were considered hits. [Example 7] [From the initial choice] [anti] [Mouse CD137 Fcabs] [Characteristics] 7.1 Determining the specificity of anti-mouse CD137 Fcab based on BLI

[0432] The specificity of anti-mouse CD137 Fcabs in the HelD1.3 “pseudo” mAb2 format to mouse CD137 was tested, and the binding of Fcabs to other mouse TNFRSF receptors (CD40, OX40, GITR) was measured using the Octet QKe system according to BLI. Streptomycin biosensors (PALL ForteBio 18-5021) were coated with 10 ng / μl of mouse CD40, GITR, and OX40 receptors (all obtained from R&D Systems and biotinylated using the EZ-Link Sulfo-NHS-SS-Biotin kit from Thermoscientific #21328). The anti-mouse CD137 Fcabs in the pseudomAb2 format were diluted 1:1 with kinetic buffer (PALL 18-1092) to a final concentration of at least 1 μM. The antigen-coated sensors were immersed in mAb² solution for 180 seconds, followed by 1 × kinetic buffer for 180 seconds. Antibodies against each TNFRSF receptor were used as positive controls. Fcab strains FS22m-055, FS22m-063, FS22m-066, FS22m-075, FS22m-135, FS22m-055, FS22m-063, and FS22m-066 did not bind to any of the tested TNFRSF receptors, thus demonstrating their specificity for mouse CD137. 7.2 Activity of mouse Fcabs in pseudomAb2 format in mouse NF-κB reporter cells

[0433] HEK.FRT.luc cells expressing the mouse CD137 sequence (sequence identification number: 184) were produced following the same methodology described previously in Example 3.3. mAbs containing previously selected anti-mouse CD137 Fcabs were generated using this cell line, HEK.FRT.luc.mCD137, screened according to the method described in Example 3.3. Fifty-six mAbs were tested, of which 29 were positive for NF-κB activity. The validity of the analysis was confirmed by the use of Lob12.3 (G1AA / Lob12.3) containing the LALA-mutant human IgG1 Fc as a positive control anti-mouse CD137 mAb, which showed increased luminescence. HelD1.3, also containing the LALA-mutant human IgG1 Fc, was used in this analysis as a negative control human IgG isotype to exclude interference from human IgG pseudofabs. EC50s were calculated where possible, and mAbs whose activity did not reach the plateau region were ignored to facilitate the display of typical S-shaped activity kinetics. mAbs were graded based on the fold change in EC50 and activity upon cross-linking with protein L. FS22m-063 was selected based on its optimal EC50 (1.44 nM) and the highest fold change in activity (27-fold) upon cross-linking. Figure 5 shows that in HEK mCD137 NF-κB reporter gene analysis, the anti-mouse CD137 Fcab FS22m-063, in the HelD1.3 pseudo-mAb2 format, drives CD137 clustering and NF-κB signaling when cross-linked with protein L. [] [Example 8] [:FS22m-063 Fcab(] [presented as FS22m-063-AA / PD-L1 mAb, 2 , )] [Anti-tumor activity in vivo]

[0434] It has been demonstrated that FS22m-063 Fcab can drive CD137 clustering and activation in vitro, and we hope to test its ability to activate CD137 in vivo. 8.1 Preparation of FS22m-063 Fcab in mAb2 format for in vivo mouse testing.

[0435] The method used in Example 7.1 to produce a simulated mAb2 was similar to that used to prepare mAb2 containing Fab regions specific to anti-mouse CD137 Fcab, FS22m-063 and PD-L1, and the in vivo antitumor activity was tested using the MC38 syngeneic mouse tumor model.

[0436] Control: G1-AA / Lob12.3, G1-AA / S70, G1-AA / 4420.

[0437] The control antibody system for in vivo experiments is produced by linking the variable heavy region of the anti-PD-L1 antibody S70 (strain YW243.55. S70 is from US 8,217,149 B2) to the constant region of human IgG1 (G1m17) containing the LALA mutation, and the variable light region from the S70 antibody is linked to the human κ J- region and the human constant region (Lm1). mAb2 is produced by replacing the CH3 domain of the modified construct described above with FS22m-063 and is named 'FS22m-063-AA / S70'. 8.2 Activity of FS22m-063-AA / S70 mAb2 in MC38 syngeneic tumor model

[0438] Allogeneic mouse models are accepted as suitable rodent systems for testing the antitumor effects of inhibitory therapeutic targets and have been widely used to validate the development of human therapies. This experiment used the MC38 allogeneic tumor model because MC38 tumors are known to be highly immunogenic and responsive to anti-CD137 antibody monotherapy (Kocak et al., 2006) and exhibit PD-L1 expression (Juneja et al., 2017).

[0439] Female C57BL / 6 mice (The Jackson Laboratory), aged 9–10 weeks and weighing between 17.92 and 23.89 g, rested for one week before the start of the study. All animals were micro-chipped and given unique identification tags. Each group consisted of 12 mice. MC38 colon cancer cell lines (National Cancer Institute, USA) were initially amplified, stored, and pre-screened for pathogens and confirmed to be pathogen-free. Each animal received a subcutaneous injection of 1 x 10⁶ cells in 100 µl of serum-free medium (Dulbecco's Modified Eagle Medium) on its right side. Seven days after tumor cell inoculation, mice without tumors at this point were removed from the study.

[0440] FS22m-063-AA / S70 mAb2 and control antibodies (G1-AA / Lob12.3 (CD137 positive control), G1-AA / S70 (positive control PD-L1), G1-AA / 4420 (isotype control)) were administered intraperitoneally to mice at a fixed concentration of 20 µg per dose, prepared in DPBS + 1 mM arginine + 0.05 Tween 80. Mice received either mAb2 molecules or control antibodies via intraperitoneal (IP) injection at 200 µl on days 7, 9, and 11 post-tumor inoculation. Accurate tumor measurements were performed, and any drug administrations were administered on appropriate days as discussed, with close monitoring of the mice for the remainder of the study. Tumor volume was measured using a diameter to determine the longest and shortest axes of the tumor. Tumor volume was calculated using the following formula: LX (S2) / 2 Where L = longest axis; S = shortest axis

[0441] As shown in Figure 6, FS22m-063-AA / S70 mAb2 showed significant tumor growth inhibition in mice compared to mice treated with any control antibody. Mixed-model analysis compared all groups, and the growth rate showed paired statistical significance throughout the entire study period. [surface] As shown in [7], surprisingly, all mice treated with FS22m-063-AA / S70 mAb2 were tumor-free 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 either PD-L1 or CD137 antibody alone. [Table 7] [:MC38] [Same gene] [No tumors in each treatment group in tumor mode] [Mouse]

[0442] Studies have shown that CD137 exerts its effects in mice with fully functioning immune systems, presumably as a result of PD-L1 cross-linking, likely due to increased cytotoxic activity of CD8+ T cells within tumors, leading to reduced tumor growth. [Example 9] [:FS22m-063 Fcab(] [presented as FS22m-063-AA / PD-1 mAb, 2 , )] [Anti-tumor activity in vivo]

[0443] It has been demonstrated that FS22m-063 Fcab can drive CD137 clustering and activation in vitro. It is hoped that it can be tested to determine its ability to activate CD137 in vivo via another Fab target, in this case a target that exists only on immune cells, PD-1. 9.1 Preparation of FS22m-063 Fcab in mAb2 format for in vivo mouse testing.

[0444] mAb2 containing specific Fab regions against mouse CD137 Fcab, FS22m-063, and PD-1 was prepared using a similar methodology to that used in Example 7.1 to produce the mimic mAb2, and its in vivo antitumor activity was tested using the MC38 syngeneic mouse tumor model.

[0445] The control antibodies (G1 / Lob12.3, G1-AA / F2, G1-AA / 4420) for in vivo experiments are produced by linking the variable heavy region of the anti-PD-1 antibody F2 (the PD1-F2 strain is from WO 2004 / 056875 A1) to the constant region of human IgG1 (G1m17) containing the LALA mutation, and the variable light region from the F2 antibody is linked to the human κ J- region and the human constant region (Lm1). mAb2 is produced by replacing the CH3 domain of the modified construct described above with FS22m-063 and is named 'FS22m-063-AA / F2'. 9.2 Activity of FS22m-063-AA / F2 mAb2 in MC38 syngeneic tumor models

[0446] This experiment used the MC38 syngeneic tumor model as described in Example 8.2, along with the following biases: Female C57BL / 6 mice (Charles River) aged 9-11 weeks were dissected with micro-notches and given unique identification marks. Each group consisted of 12 mice. Each animal received a subcutaneous injection of 1 x 10⁶ MC38 colon cancer cells in 100 µl of serum-free culture medium on its right dorsal side.

[0447] FS22m-063-AA / F2 mAb2 and control antibodies (G1-AA / Lob12.3 (CD137 positive control), G1-AA / F2 (positive control PD-1), G1-AA / 4420 (isotype control)) were administered intraperitoneally to mice at a fixed concentration of 20 µg per dose, prepared in DPBS + 1 mM arginine + 0.05 Tween 80. Mice received either mAb2 molecules or control antibodies via intraperitoneal (IP) injection at the time of the first dose, when the tumor volume reached 50-60 mm3 (day 0), and on days 2 and 4. Tumors were measured as described in Example 8.2.

[0448] As shown in Figures 7A and 7B, FS22m-063-AA / F2 mAb2 showed a highly significant inhibition of tumor growth in mice treated with the isotype control antibody and the positive control PD-1 antibody. [surface] As shown in [8], surprisingly, 10 out of 12 mice treated with FS22m-063-AA / F2 mAb2 were tumor-free at the end of the study, compared to only 7 out of 12 mice treated with a combination of anti-PD-1 and anti-CD137 antibodies (G1-AA / F2 + G1-AA / Lob12.3). [Table 8]

[0449] Studies have shown that CD137 is effective in mice with fully functioning immune systems, presumably due to cross-linking via PD-1 linkage and additional PD-1 blockade. This is likely due to increased cytotoxic activity of CD8+ T cells within the tumor, leading to reduced tumor growth.

[0450] It also demonstrates that when CD137 Fcabs are in mAb2 format, they can cross-link with immune cell targets through the binding of Fab arms, leading to CD137 clustering and activation. [Example 10] [:mCD137 / MSLN mAb, 2 , ] [Anti-tumor activity in vivo]

[0451] mAb² containing FS22m-063 Fcab and PD-L1 Fab has been proven. [Example 8] The aim was to test the efficacy of CD137 Fcabs in mAb2 format, which are cross-linked with a tumor-specific antigen (TSA), in this case mesothelin (MSLN), via their Fab arms. This tumor-targeting approach is expected to be advantageous in localizing T cell activation within the tumor microenvironment, as the cross-linking of mAb2 and therefore the CD137 efficacy will only occur at the site of MSLN expression.

[0452] A syngeneic mouse tumor model expressing mouse MSLN was constructed. CT26 colon cancer cells (ATCC, CRL-2638) expressing full-length mouse mesothelin (sequence identification number: 189) were produced using pcDNA3.1 vector(+) (Thermo Fisher Scientific, catalog number V79020) via lipofection (Liposome 3000, Thermo Fisher Scientific, catalog number L3000008). Following the manufacturer's procedures, the CT26 cell line was transfected with the pcDNA3.1 vector containing mouse MSLN cDNA. Stable transfection was achieved using genimycin as the selection antibiotic (600 μg / ml) in complete medium (RPMI, 10% FBS).

[0453] The expression of mouse MSLNs on CT26 cells was confirmed by flow cytometry using the positive control antibody MOR6626 (WO 2009 / 068204 A1). Specifically, cells were incubated with the positive control antibody for 1 hour, and cell binding was detected using a fluorescently labeled anti-human IgG detection antibody (Stratech Scientific Ltd, catalog number 109-546-098-JIR). A population of proliferating strains was expanded and subsequently analyzed using the same flow cytometry procedure to determine relative phenotypic levels. One proliferating strain was then selected and designated CT26.G10.

[0454] In vivo confirmation of CT26.G10 tumor growth was achieved. Eight- to ten-week-old female Balb / c mice (Charles River) were surgically incised and given unique identification tags. Each group consisted of 17 mice, and each animal received a subcutaneous injection of 1 x 10⁵ cells in 100 µl of serum-free medium via the left dorsal side. [Example] [8] describes using a caliper to measure tumor volume three times a week. [Example] [8] The study was conducted in accordance with the regulations of the UK Home Office.

[0455] At the end of the study, tissue samples were collected, and the presence of membrane-bound mesothelin was confirmed by immunohistochemical staining of formalin-fixed paraffin-embedded (FFPE) tumor tissue as follows: 4µm FFPE tissue sections were dewaxed and antigen was extracted at 97°C using a low pH 6.1 method (Dako PT Link), followed by peroxidase and protein blocking, and then incubation with a 1µg / ml concentration of primary anti-mesothelin antibody (LifeSpan Biosciences, catalog number LS-C407883). Anti-mesothelin antibodies were detected using the labeled polymer-HRP anti-rabbit secondary reagent and DAB (3,3'-diaminobenzidine) chromogenic endpoint (Dako EnVision+ System).

[0456] To evaluate the efficacy of Fcab FS22m-063, the following molecules or combinations were tested in vivo: anti-MSLN FS28m-228-010 antibody with a human IgG1 isotype and LALA mutation (G1-AA / FS28m-228-010), Fcab in two "pseudo" mAb2 formats (FS22m-063-AA / HelD1.3 and FS22m-063-AA / 4420), a combination of FS28m-228-010 antibody and LALA-mutant pseudo-CD137 mAb2 (G1-AA / FS28m-228-010 + FS22m-063-AA / HelD1.3), human isotype control antibody (G1-AA / HelD1.3), and finally, CD137 / MSLN mAb² with LALA mutation. (FS22m-063-AA / FS28m-228-010).

[0457] Balb / c female mice (Charles River), aged 8-10 weeks and weighing 20-25 g each, were acclimatized to their new environment for one week before the study began. All animals were micro-notched and given unique identification tags. Each group consisted of 20 mice, except for FS22m-063-AA / 4420 (n=10 mice). The CT26.G10 colon cancer cell line was expanded to generate a cell bank. Each animal received a subcutaneous injection of 1 x 10⁵ cells in 100 µl of serum-free medium on its left side. Mice without tumors 12 days after tumor cell inoculation were removed from the study.

[0458] Each antibody was prepared in 200 µg doses (~10 mg / kg) and injected intraperitoneally (IP) into mice. Additionally, FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 were each prepared in 200 µg doses (~10 mg / kg) for the combination groups. Mice were administered 200 µl doses on days 12, 14, and 16 post-tumor inoculation (q2dx3). Tumor volume was measured three times weekly using a diameterer, and mice were closely monitored. Study endpoints were determined based on tumor volume, mouse condition, and humane endpoints.

[0459] like [picture] As shown in [9], FS22m-063-AA / FS28m-228-010 mAb2 significantly inhibited tumor growth compared with the G1-AA / HelD1.3 isotype control. [surface] [9] Shows a pairwise comparison of tumor growth rates in all treatment groups over the entire study period using mixed model analysis to compare all groups with the G1-AA / HelD1.3 isotype control.

[0460] [Table 9] [Comparing all groups with G1-AA / HelD1.3 using a mixed model analysis] [Isotype control, tumor growth] [in pairs] [Comparison results.] NS p≥0.05;* p>0.05;** p>0.01;*** p>0.001;**** p>0.0001

[0461] At the end of the study, all animals with tumors measured at or below 62.5 mm³ were counted as fully responsive animals (see [reference]). [surface]

[10] ). In the last 35% of the animals treated with anti-CD137 / MSLN mAb2, there was a complete response to treatment, compared with 0% in the G1-AA / HelD1.3 isotype control, FS22m-063-AA / HelD1.3, FS22m-063-AA / 4420, and the combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010. [Table 10] [:CT26.G10] [Same gene] [Tumor Pattern] [At the end of the study] [No tumors] [Mouse] The number and percentage of [[] [Tumor ≤ 62 mm3]

[0462] Survival analysis ( [picture]

[10] [and Table 11]) shows that FS22m-063-AA / FS28m-228-010 mAb2 induced a significant survival benefit compared to G1-AA / HelD1.3 antibody, while the fractions (G1-AA / FS28m-228-010, FS22m-063-AA / HelD1.3) or G1-AA / FS28m-228-010+FS22m-063-AA / 4420 did not show a survival advantage. Furthermore, FS22m-063-AA / FS28m-228-010 mAb2 resulted in an improved median survival 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 plus G1-AA / FS28m-228-010 (29 days). [Table 11] [:CT26.G10] [Same gene] [Median survival time of animals treated with various compounds in tumor models, and results of paired statistical analysis () [Logarithmic rank]. NS p≥0.05;* p>0.05;** p>0.01;*** p>0.001;**** p>0.0001

[0463] These data suggest that mAb2 cross-linking via MSLN can drive the efficacy against CD137 within tumors; the bispecific antibody is superior to targeting CD137 and / or MSLN alone (and even in combination), resulting in significantly improved survival in tumor-bearing mice. Fcab FS22m-063-AA / HelD1.3 or FS22m-063-AA / 4420 (in pseudo-mAb2 format) without any MSLN-targeted Fab showed no intrinsic activity in this study. [Example 11] [:Gain the ability to combine rodent and human CD137] [of Fcab] [The role of selection]

[0464] Since it was surprising to find that some Fcabs that bind to human CD137 also bind to mouse CD137 (see Example 5.5 on the specificity of binding to human, mouse and cynomolgus monkey CD137), it was decided to find out whether these strains could be improved. 11.1: Site-specific modification to remove potential sequence liability within the FS22-053-014 strain.

[0465] The mouse-human cross-reactive strain FS22-053-014 was selected because it was confirmed by SPR to bind the mouse dimeric CD137 antigen (see Example 5.5). However, further sequence analysis revealed a potential sequence responsibility that led to post-translational aspartic acid isomerization of the EF loop in its CH3 domain, creating a DG moiety due to the Q98D mutation along with the wild-type G99 position. Other affinity-mature strains within the FS22-053 lineage (see Example 5.5) [surface]

[12] ) Instead, it contains Q98E modification at the same position within the EF loop. The Q98D strain was mutated to Q98E using the QuickChange II site-directed mutagenesis kit (Agilent, catalog number 200523) according to the manufacturer's recommendations, and its resulting strain was FS22-053-017. The following [surface]

[12] Shows the LE portion that frequently occurs in the EF loop of the CH3 domain of strains FS22-053-008, FS22-172-003 and FS22-172-004, and the DG portion of the EF loop of the CH3 domain of strain FS22-053-014. [Table 12] [Example 12] [:] [Mutant FS22-053-017 Fcab] [Cultural strain] [Characteristics] 12.1 Activity of FS22-053-017 Fcab in pseudomAb2 format in human CD137 DO11.10 T cell activation assay

[0466] The Fcab strain FS22-053-017 was subselected and presented in the HelD1.3 “pseudo” mAb2 format. Human CD137 DO11.10 T cell activation analysis was then performed as described in Example 3.4, and compared with the FS22-053-014 strain, which also presented in the HelD1.3 mAb² format. G1-AA / 20H4.9 was used as an anti-CD137 positive control, and G1-AA / D1.3 as an IgG control. mAb² was tested with or without protein L cross-linking or with protein L cross-linking at a 1:4 ratio. [Table 13] N / A - Not applicable because the signal is too low to allow EC50 determination.

[0467] [Table 13] and [picture]

[12] The results showed that FS22-053-17 Fcab and FS22-053-014 Fcab were in pseudo-mAb2 format. Both showed comparable activity when cross-linked with protein L in the same DO11.10 T cell activation assay. Therefore, the mutation induction did not negatively affect functional activity. Neither of the two pseudo-mAb2 format strains showed activity without cross-linking. []

[0468] As expected, the positive control anti-CD137 was active upon cross-linking, while the IgG control was inactive, regardless of whether cross-linking was present or not. 12.2 Activity of the pseudomAb2 mutant FS22-053-017 Fcab in mouse CD137 DO11.10 T cell activation assay

[0469] The mouse CD137 DO11.10 T cell activation assay, as described in Example 3.4, was used to compare the FS22-053-017 colony (in HelD1.3 pseudomAb2 format) against the mouse CD137-binding Fcab colony FS22m-063 (also in HelD1.3 pseudomAb2 format) and the parental FS22-053-014 colony (in HelD1.3 pseudomAb2 format). mAb² molecules were cross-linked with protein L at a molar ratio of 4:1 (mAb2: protein L).

[0470] As expected, measured by IL-2 release, all tested molecules showed activity when cross-linked with protein L, but no activity when not cross-linked. FS22m-063, which binds to mouse CD137, was selected as having the best activity in the analysis when cross-linked, with an EC50 of 0.39 nM. Both FS22-053-14 and FS22-053-017 showed activity in the analysis, indicating that their function was not lost due to mutation, although FS22-053-017 showed a slight loss of activity and an EC50 8 times worse than FS22-053-14 when cross-linked with protein L. [picture]

[12] CD137 Fcabs FS22-053-014 and FS22-053-017, which exhibited mature human and mouse cross-reactivity in the HelD1.3 pseudomAb2 format, and anti-mouse CD137 Fcab FS22m-063, activated CD137 and led to the release of mIL-2 when cross-linked with protein L in the DO11.10 T cell activation assay. [Table 14] N / A - Not applicable because the signal is too low to allow EC50 determination. 12.3 Bonding Dynamics of FS22-053-017

[0471] The equilibrium dissociation constant (KD) of Fcab strain FS22-053-017 and strain FS22-053-014 were compared using SPR on a BIAcore T200 system. The hCD137-mFc-Avi antigen was used to immobilize anti-human Fab molecules on a CM5 wafer to a surface density between 9,000 and 11,000 response units (RU). Antibody was diluted with HBS-EP buffer to 4 µg / ml and captured by anti-Fab molecules at a flow rate of 30 μl / s. Eight different concentrations of hCD137-mFc antigen—200 nM; 66.67 nM; 22.22 nM (including secondary concentrations); 7.41 nM; 2.47 nM; 0.82 nM; 0.27 nM; and 0.091 (diluted with HBS-EP+ buffer)—were passed over the captured mAb².

[0472] Different methods were used to determine the binding kinetics of mCD137-mFc-Avi. Mouse PD-L1-mFc-Avi was immobilized on a CM5 wafer at a surface density of 200 RUs. mAb² containing FS22-053-014 and FS22-053-017 Fcabs was used to capture the immobilized mPD-L1 protein at a flow rate of 60 μl / s using an anti-mouse PD-L1 Fab (S70) system diluted with HBS-EP buffer to 7.5 μg / ml. Eight different concentrations of mCD137-mFc antigen were flowed over the captured mAb²: 600 nM; 200 nM; 66.67 nM (including secondary concentrations); 22.2 nM; 7.1 nM; 2.47 nM; and 0.82 nM (diluted with HBS-EP+ buffer).

[0473] Data analysis was performed using Biacore T200 evaluation software. The curves with the blank flow chamber subtracted were fitted using a 1:1 Langmuir combination model with mass transfer, with constants and R-values ​​set to zero locally. Results are summarized in... [surface]

[15] Both Fcab strains showed kinetic profiles very similar to human antigens, thus demonstrating that the Q98D to Q98E mutation had no negative effect on binding to human antigens. The 2-fold decrease in binding strength to mouse antigens was also consistent with... [Example] The functional data shown in [11.2] is consistent. [Table 15] [Example 13] [PPY] [Regarding antigens] [Involvement in Combination () [keep] [PPY] [Partial alanine scan]

[0474] like [Example] [5] As described, the PPY sequence portion was identified within the AB loop of two independently selected Fcabs, and this portion was conserved across all affinity-mature strains analyzed. Therefore, it is desirable to understand the involvement of this portion in binding CD137 and in the overall protein structure. Alanine scanning is a common biotechnique used to determine the importance of a specific residue based on protein-protein interactions. In short, each of the three amino acids is replaced sequentially and then together with an alanine residue. Alanine is considered chemically inert, non-bulky, and therefore unlikely to aid in binding. 13.1 Production of mutant strains for alanine scanning

[0475] The mutant strains were produced through site-directed mutagenesis of the parent strains (FS22-172-003 and FS22-053-008). The positions of the amino acid substitutions within the AB ring are summarized in... [surface]

[16] Within, all other residues retain their respective parents. The mAb variant then follows as [Example] [3] The mutant was temporarily expressed in HEK293-6E cells and purified using mAb Select SuRe protein A column. The mutant yield was similar to that of the parent, suggesting that alanine substitution has no effect on protein production. [Table 16] 13.2 Binding kinetics of mutant strains

[0476] Binding kinetics of two parental strains and eight mutant strains were compared using the Octet Qke system from ForteBio. Dimeric biotinylated hCD137-mFc-Avi lines were captured at 10 μg / ml on streptoacidin sensors, and their interactions with individual mAbs at 200 nM were analyzed. All FS22-172-003 mutant strains showed impaired binding of mAbs to the dimeric antigen compared to the parental strains. Strains FS22-172-003_AAA and FS22-172-003_APY lost all binding, while strains FS22-172-003_PPA and FS22-172-003_PAY retained some binding, although significantly reduced compared to the parents (5.5 and 4.4 times lower response units, respectively). Binding to FS22-053-008 was also affected, with variants PAY and AAA losing all binding, and variants APY and PPA showing reduced binding to the antigen and slower association analysis compared to the parental FS22-053-008 strain. These data suggest that the PPY moiety is important for the binding of both mAb² and CD137 antigen. 13.3 Homologous Simulation Method

[0477] Given that the PPY portion involves binding interactions, computer simulations to model Fcabs to evaluate the predicted protein structure of the CH3 domain provide rich information. The Chemical Computing Group ULC (MOE) software suite version 2019.0101 was used to perform structural homology simulations and subsequent conformational searches. The Fc region of structure 5JII from the protein database [PDB] was used as the structural template for the Fcab regions of FS22-172-003 and FS22-053-008. Since there are insertions of the selected structural templates into the AB and EF loop regions, de novo loop searches and residue branching optimization were applied to generate the AB and EF loop structures. The generated homology simulations were performed based on geometric criteria including backbone bond lengths, angles, dihedral angles, and palmitometry, minimizing energy and scoring. The score-based structural homology simulations were used as the basis for conformational sampling, which employed the LowModeMD simulation method implemented within MOE.

[0478] [picture]

[13] Intra-homology simulations showed that the AB and EF rings of the two Fcabs adopted different configurations compared to template 5JII, and that the PPY moiety played a role in the core of the AB ring within the CH3 domain. The PPY moiety also mediated the interaction that led to the stability of the AB ring configuration, through interactions within the ring and between the ring and other structures. Configuration searches performed using LowModeMD simulations also highlighted that the tyrosine residues of the PPY moiety interact with the Q3 (IMGT) and D12 (IMGT) of the CH3 domain in the same chain, which may be stabilizing the AB ring. Putting together the results shown in Example 13.2, this suggests that the PPY moiety is important for the binding of both FS22-172-003 and FS22-053-008 to CD137.

[0479] [Sequence List] [WT Fcab CH3] [Acid sequence of domain-structured ring] WT Fcab AB loop – RDELTKNQ (Sequence identification number: 1) WT Fcab CD ring – SNGQPENNY (Sequence identification number: 2) WT Fcab EF ring – DKSRWQQGNV (Sequence identification number: 3) [WT Fcab CH3] [Acid sequence of the domain()] [Sequence Identification Number: 4)] Draw the bottom lines for circles AB, CD, and EF. GQPREPQVYTLPPS [RDELTKNQ] VSLTCLVKGFYPSDIAVEWE [SNGQPENNY] KTTPPVLDSDGSFFLYSKLTV [DKSRWQQGNV]FSCSVMHEALHNHYTQKSLSLSPG [With LALA-PA] [mutation] [of Fcab CH2] [Acid sequence of the domain()] [Sequence Identification Number: 5] LALA-PA mutation marks the bottom line APE [AA] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL [A] APIEKTISKAK [There's LALA] [mutation] [of Fcab CH2] [Acid sequence of the domain()] [Sequence Identification Number: 6)] LALA mutation sets a bottom line APE [AA] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK [Truncation] [of] [Fcab] [Amino acid sequence of the hinge region] [(] [Sequence Identification Number:] [7)] TCPPCP [have] [LALA] [Mutated] [WT Fcab] [Amino acid sequence () [Sequence Identification Number: 8)] Hinge region (underlined), CH2 region (bold), CH3 region (italic), LALA mutation (bold and underlined) [TCPPCP] [APE, AA, GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [none] [LALA] [Mutated] [WT Fcab] [Amino acid sequence () [Sequence Identification Number: 9] Hinge area (underlined), CH2 area (bold), and CH3 area (italic). [TCPPCP] [APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG [PPY] [part] [Amino acid sequence] [(] [Sequence Identification Number:] [10)] PPY [There's LALA] [Mutated] [Fcab FS22-033] [Amino acid sequence] [(] [Sequence Identification Number:] [11)] Hinge region (underlined), CH2 region (bold), CH3 region (italic), LALA mutation (bold and underlined) [TCPPCP] [APE, AA , GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDEYFEQEVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVARHRWQLGNVFSCSVMHEALHNHYTQKSLSLSPG [none] [LALA] [Mutated] [Fcab FS22-033] [Amino acid sequence () [Sequence Identification Number: 12] Hinge area (underlined), CH2 area (bold), and CH3 area (italic). [TCPPCP] [APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDEYFEQEVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVARHRWQLGNVFSCSVMHEALHNHYTQKSLSLSPG [have] [LALA] [Mutant FS22-033 / HelD1.3] [fake mAb, 2 , ] The amino acid sequence of the heavy chain of [[] [Sequence Identification Number: 13] VH domain (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYFEQEVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVARHRWQLGNVFSCSVMHEALHNHYTQKSLSLSPG [None] [LALA] [Mutated FS22-033 / HelD1.3] [Pseudo mAb, 2 , ] [amino acid sequence of the heavy chain of (] [Sequence ID No.: 14)] VH domain (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYFEQEVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVARHRWQLGNVFSCSVMHEALHNHYTQKSLSLSPG [There's LALA] [Mutant Fcab FS22-053] [Amino acid sequence () [Sequence Identification Number: 15] Hinge region (underlined), CH2 region (bold), CH3 region (italic), LALA mutation (bold and underlined) [TCPPCP] [APE, AA , GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVYYNRWQDGNVFSCSVMHEALHNHYTQKSLSLSPG [none] [LALA] [Mutated] [Fcab FS22-053] [Amino acid sequence () [Sequence Identification Number: 16] Hinge area (underlined), CH2 area (bold), and CH3 area (italic). [TCPPCP] [APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVYYNRWQDGNVFSCSVMHEALHNHYTQKSLSLSPG [have] [LALA] [Mutant FS22-053 / HelD1.3] [fake mAb, 2 , ] The amino acid sequence of the heavy chain of [[] [Sequence Identification Number: 17] VH domain (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVYYNRWQDGNVFSCSVMHEALHNHYTQKSLSLSPG [None] [LALA] [Mutated FS22-053 / HelD1.3] [Pseudo mAb, 2 , ] [amino acid sequence of the heavy chain of (] [Sequence ID No: 18)] VH region (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVYYNRWQDGNVFSCSVMHEALHNHYTQKSLSLSPG [Fcab FS22-053-008 CH3] [Acid sequence of domain-structured ring sequence] FS22-053-008 First Sequence – NPPYLFS (Sequence Identification Number: 19) FS22-053-008 Second Sequence – DYWRWLE (Sequence Identification Number: 20) [Fcab FS22-053-008 CH3] [Acid sequence of the domain] [(] [Sequence Identification Number:] [twenty one)] The first and second sequences are marked with a bottom line. GQPREPQVYTLPPSRDEL [NPPYLFS] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV [DYWRWLE] GNVFSCSVMHEALHNHYTQKSLSLSPG [Fcab FS22-053-008 CH3] [Nucleic acid sequence of the domain] [(] [Sequence Identification Number:] [twenty two)] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTACTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCGCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGT [There's LALA] [Mutated] [Fcab FS22-053-008] [Amino acid sequence () [Sequence Identification Number: 23] Hinge region (underlined), CH2 region (bold), CH3 region (italic), LALA mutation (bold and underlined) [TCPPCP] [APE, AA , GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG [have] [LALA] [Mutated] [Fcab FS22-053-008] [Nucleic acid sequence of] [Sequence ID: 24)] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT [None] [LALA] [Mutated] [Fcab FS22-053-008] [Amino acid sequence of] [Sequence ID: 25)] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic) [TCPPCP] [APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG [none] [LALA] [Mutated] [Fcab FS22-053-008] [nucleic acid sequence()] [Sequence Identification Number: 26] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACTGGAGGTGGCTGGAAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT [has] [LALA] [mutated FS22-053-008 / HelD1.3] [pseudo mAb, 2 , ] [amino acid sequence of the heavy chain of (] [Sequence ID No.: 27)] VH region (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG [None] [LALA] [Mutated FS22-053-008 / HelD1.3] [Pseudo mAb, 2 , ] [amino acid sequence of the heavy chain (] [Sequence ID No.: 28)] VH region (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYWRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG [Fcab FS22-053-009 CH3] [Acid sequence of domain-structured ring sequence] FS22-053-009 First Sequence – NPPYLFS (Sequence Identification Number: 19) FS22-053-009 second sequence – EHTRWLD (Sequence identification number: 29) [Fcab FS22-053-009 CH3] [Acid sequence of the domain] [(] [Sequence Identification Number:] [30)] The first and second sequences are marked with a bottom line. GQPREPQVYTLPPSRDEL [NPPYLFS] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV [EHTRWLD] GNVFSCSVMHEALHNHYTQKSLSLSPG [Fcab FS22-053-009 CH3] [domain] [nucleic acid sequence()] [Sequence Identification Number: 31] GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAACATACTAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT [There's LALA] [Mutated] [Fcab FS22-053-009] [Amino acid sequence () [Sequence Identification Number: 32] Hinge region (underlined), CH2 region (bold), CH3 region (italic), LALA mutation (bold and underlined) [TCPPCP] [APE, AA , GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVEHTRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG [have] [LALA] [Mutated] [Fcab FS22-053-009] [Nucleic acid sequence of (] [Sequence identification number: 33)] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAACATACTAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT [None] [LALA] [Mutated Fcab FS22-053-009] [Amino acid sequence of (] [Sequence identification number: 34)] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic) [TCPPCP] [APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVEHTRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG [none] [LALA] [Mutated] [Fcab FS22-053-009] [nucleic acid sequence()] [Sequence Identification Number: 35] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAACATACTAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT [has] [LALA] [mutated FS22-053-009 / HelD1.3] [pseudo mAb, 2 , ] [amino acid sequence of the heavy chain of (] [Sequence ID No.: 36)] VH region (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVEHTRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG [None] [LALA] [Mutated FS22-053-009 / HelD1.3] [pseudo mAb, 2 , ] [amino acid sequence of the heavy chain of (] [Sequence ID No.: 37)] VH region (underlined) [QVQLQESGPGLVRPSQTLSLTCTVSGTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSS] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVEHTRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG [Fcab FS22-053-010 CH3] [Acid sequence of domain-structured ring sequence] FS22-053-010 First Sequence – NPPYLFS (Sequence Identification Number: 19) FS22-053-010 second sequence – DYMRWLD (Sequence identification number: 38) [Fcab FS22-053-010 CH3] [Acid sequence of the domain()] [Sequence Identification Number: 39] The first and second sequences are marked with a bottom line. GQPREPQVYTLPPSRDEL [NPPYLFS] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV [DYMRWLD] GNVFSCSVMHEALHNHYTQKSLSLSPG [Fcab FS22-053-010 CH3] [domain] [nucleic acid sequence()] [Sequence Identification Number: 40] GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACATGAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT [There's LALA] [Mutant Fcab FS22-053-010] [Amino acid sequence () [Sequence Identification Number: 41] Hinge region (underlined), CH2 region (bold), CH3 region (italic), LALA mutation (bold and underlined) [TCPPCP] [APE, AA , GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYMRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG [have] [LALA] [Mutated] [Fcab FS22-053-010] [nucleic acid sequence()] [Sequence Identification Number: 42] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATTACATGAGGTGGCTGGATGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT [None] [LALA] [Mutated Fcab FS22-053-010] [Amino acid sequence of (] [Sequence identification number: 43)] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italicized) [TCPPCP] [APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK] GQPREPQVYTLPPSRDELNPPYLFSNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDYMRWLDGNVFSCSVMHEALHNHYTQKSLSLSPG [none] [LALA] [Mutated] [Fcab FS22-053-010] [nucleic acid sequence()] [Sequence Identification Number: 44] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAACCCGCCGTACCTGTTCTCTAAC...

Claims

1. A specific binding object that binds to CD137 and includes a CD137 antigen binding site located in a CH3 domain of the specific binding object, wherein the specific binding object includes the CH3 domain sequences of the following specific binding objects: (i) the CH3 domain sequence of FS22-172-003, listed in sequence identification number 139; (ii) the CH3 domain sequence of FS22-172-002, listed in sequence identification number 130; (iii) the CH3 domain sequence of FS22-172-004, listed in sequence identification number 148; (iv) the CH3 domain sequence of FS22-172-001, listed in sequence identification number 121; (v) the CH3 domain sequence of FS22-172-005, listed in sequence identification number 157; (vi) The CH3 domain sequence of FS22-172-006 is listed under sequence identification number 165; (vii) The CH3 domain sequence of FS22-172 is listed under sequence identification number 112; (viii) The CH3 domain sequence of FS22-053-008 is listed under sequence identification number 21; (ix) The CH3 domain sequence of FS22-053-009 is listed under sequence identification number 30; (x) The CH3 domain sequence of FS22-053-011 is listed under sequence identification number 48; (xi) The CH3 domain sequence of FS22-053-017 is listed under sequence identification number 102; (xii) The CH3 domain sequence of FS22-053-014 is listed under sequence identification number 75; (xiii) The CH3 domain sequence of FS22-053-010 is listed at sequence identification number 39; (xiv) The CH3 domain sequence of FS22-053-012 is listed at sequence identification number 57; (xv) The CH3 domain sequence of FS22-053-013 is listed at sequence identification number 66; (xvi) The CH3 domain sequence of FS22-053-015 is listed at sequence identification number 84; (xvii) The CH3 domain sequence of FS22-053-016 is listed at sequence identification number 93; or (xviii) The CH3 domain sequence of FS22-053 is listed at sequence identification number 175.

2. The specific combination object as claimed in claim 1, wherein the specific combination object includes specific combination objects 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- The sequences 017, FS22-053-014, FS22-053-010, FS22-053-012, FS22-053-013, FS22-053-015, FS22-053-016 or FS22-053 are listed in the sequence identification numbers: 141, 132, 150, 123, 159, 167, 114, 23, 32, 50, 104, 77, 41, 59, 68, 86, 95 and 15 respectively.

3. The specific combination object as claimed in claim 1, wherein the specific combination object comprises: (i) the CH3 domain sequence of the specific combination object FS22-172-003, FS22-053-008 or FS22-053-017, which are respectively listed in sequence identification number: 139, sequence identification number: 21 and sequence identification number: 102; or (ii) the sequence of the specific combination object FS22-172-003, FS22-053-008 or FS22-053-017, which are respectively listed in sequence identification number: 141, sequence identification number: 23 and sequence identification number:

104.

4. The specific combination object as claimed in claim 3, wherein the specific combination object comprises: (i) the CH3 domain sequence of the specific combination object FS22-172-003 or FS22-053-008, which are listed in sequence identification number: 139 and sequence identification number: 21, respectively; or (ii) the sequence of the specific combination object FS22-172-003 or FS22-053-008, which are listed in sequence identification number: 141 and sequence identification number: 23, respectively.

5. The specific binding object as claimed in claim 4, wherein the specific binding object comprises: (i) the CH3 domain sequence of the specific binding object FS22-172-003, which is listed in sequence identification number: 139; or (ii) the sequence of the specific binding object FS22-172-003, which is listed in sequence identification number:

141.

6. A specific binding object as claimed in any of claims 1 to 5, wherein the CH3 domain contains a lysine residue (K) at the C-terminus closest to the sequence of the CH3 domain.

7. The specific binding object of claim 1, wherein the specific binding object further comprises a CDR-based antigen-binding site.

8. The specific binding object of claim 7, wherein (i) the specific binding object is an antibody molecule; and / or (ii) the CDR-based antigen binding site binds a second antigen selected from one of the following groups: an immune cell antigen, a tumor antigen, and a pathogenic antigen.

9. A specific binding object as claimed in claim 1, wherein the specific binding object has been modified to reduce or cancel the binding of the specific binding object to one or more Fcγ receptors.

10. A nucleic acid molecule that encodes a specific binding object as described in any of claims 1 to 9.

11. A recombinant host cell comprising nucleic acid molecules as claimed in claim 10.

12. A method for producing a specific binding article as claimed in any one of claims 1 to 9, comprising culturing a recombinant host cell as claimed in claim 11 under conditions for producing the specific binding article.

13. Use of a specific combination of any one of claims 1 to 9 in the preparation of a medicament for treating a person’s cancer or infectious disease.

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